WO2011145085A2 - Novel antibodies and methods of use for the treatment and diagnosis of cancer - Google Patents

Novel antibodies and methods of use for the treatment and diagnosis of cancer Download PDF

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Publication number
WO2011145085A2
WO2011145085A2 PCT/IB2011/052228 IB2011052228W WO2011145085A2 WO 2011145085 A2 WO2011145085 A2 WO 2011145085A2 IB 2011052228 W IB2011052228 W IB 2011052228W WO 2011145085 A2 WO2011145085 A2 WO 2011145085A2
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fragment
antibody
monoclonal antibody
isolated
antigen binding
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PCT/IB2011/052228
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French (fr)
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WO2011145085A3 (en
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Itai Benhar
Yeshayahu Yakir
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Procognia (Israel) Ltd
Ramot At Tel Aviv University Ltd
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Publication of WO2011145085A2 publication Critical patent/WO2011145085A2/en
Publication of WO2011145085A3 publication Critical patent/WO2011145085A3/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3023Lung
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046Stomach, Intestines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622Single chain antibody (scFv)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/02Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins

Definitions

  • compositions comprising substantially
  • Protein glycosylation can affect protein folding, intracellular trafficking and localization, the rate of degradation and can determine their organizational framework within the cytoplasm, on the membrane and extracellularly.
  • Abnormal glycosylation is one of the hallmarks of the cancer cell and is associated with tumor invasion, metastasis and is involved in all stages of tumor progression. Alterations of cell surface carbohydrates are often observed as a result of malignant transformation and can be detected in the earliest stages of malignant transformation.
  • the often-observed association between changes in tumor cell glycosylation and prognosis and survival of cancer patients suggests that alterations in tumor cell glycosylation patterns are an important part of tumor progression toward more malignant phenotype and metastatic phase (Dube, D.H. & Bertozzi, C.R. Nat. Rev. Drug Discov. 4, 477-488 (2005), Fuster, M.M. & Esko, J.D. Nat. Rev.
  • carcinoembryonic antigen CEA
  • carcinoembryonic antigen-related proteins PSA
  • CD24 carcinoembryonic antigen-related proteins
  • CA125 ovarian cancer marker
  • glycosylation changes and modifications that occur in malignant cells can take a
  • sialic acid 9-O-acetylation either can be up- regulated in melanoma cells.
  • a particularly interesting phenomenon is the aberrant expression of Neu5Gc in human tumor cells.
  • This sialic acid differs from the common sialic acid N-acetylneuraminic acid (Neu5Ac) by the addition of a single oxygen atom.
  • Ne5Ac N-acetylneuraminic acid
  • adults do not express significant levels of Neu5Gc on their normal cells, and they mount an immune response to this epitope when infused with Neu5Gc-containing animal serum.
  • ST6GALNAC5 specifically mediates brain metastasis of breast cancer.
  • the expression of ST6GALNAC5 in breast cancer cells enhances their adhesion to brain endothelial cells and their passage through the blood-brain barrier, which highlights the role of cell-surface glycosylation in organ-specific metastasis (Nature, June 2009).
  • GALNT12 polypeptide N- acetylgalactosaminyltransferase 12
  • glycan molecules associated with tumor biomarkers that preferentially appear in cancers are of clinical importance as serum tumor markers, as target for in- vivo molecular imaging and serve as a target for cancer therapeutics.
  • lung cancer Among the major types of cancer for which better diagnostics and treatments are required are lung cancer and colorectal cancer.
  • the two major categories of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Less common cancers of the lung are known as carcinoids, cylindromas, and certain sarcomas. Cancers in the lung may have metastasized from other primary sites, such as the breast, thyroid, or colon.
  • Non-small cell lung cancers are categorized into three types: squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma. These separate types are grouped together because, in early stages before the cancers have spread, they all can be treated surgically.
  • Tumors formed from squamous cells are usually found in the centre of the lung, either in a major lobe or in one of the main airway branches. They may grow to large sizes and form cavities in the lungs. When a squamous cell cancer metastasizes, it may reach the bone, adrenal glands, liver, small intestine, and brain. Squamous cell carcinoma is nearly always caused by smoking and used to be the most common cancer. It still makes up between 25% and 40% of all lung cancers.
  • Adenocarcinomas usually arise from the mucus-producing cells in the lung and in the
  • Adenocarcinoma of the colorectal and NSCLC adenocarcinoma are the predominant lung and colon cancer-.
  • adenocarcinoma is usually a slow- growing cancer, but can be difficult to detect because the disease typically involves the periphery of the lung, resulting in fewer early symptoms than cancers that develop centrally, near the airways.
  • signs of the disease do occur, they may include painful breathing, shortness of breath, wheezing, and a persistent cough.
  • lung adenocarcinoma has already metastasized by the time any symptoms develop, resulting in an overall five year survival rate associated with the disease that is less than 20 percent.
  • Secondary tumors most commonly form in the opposite lung, the brain, spinal cord, bones, liver, and adrenal glands. Additional symptoms related to tumor growth in these or other areas of the body sometimes develop before signs of the primary tumor.
  • Bronchoalveolar lung cancer is a subtype of adenocarcinoma. It develops as a layer of column-like cells on the lung and spreads through the airways, causing great volumes of sputum. This cancer also is increasing in incidence.
  • Large Cell Carcinoma Large cell carcinoma, which makes up about 10% to 20% of lung cancers, includes cancers that cannot be identified under the microscope as squamous cell cancers or adenocarcinomas.
  • Detection of lung cancer at an early stage is necessary for successful therapy and improved survival rates.
  • Numerous potential DNA biomarkers such as hypermethylations of the promoters and mutations in K-ras, p53, and protein biomarkers; carcinoembryonic antigen (CEA), CYFRA21-1, plasma kallikrein Bl (KLKB1), Neuron- specific enolase, EGFR, etc. have been discovered as lung cancer biomarkers.
  • CEA carcinoembryonic antigen
  • CYFRA21-1 CYFRA21-1
  • KLKB1 plasma kallikrein Bl
  • Neuron- specific enolase EGFR
  • Kim et al. (Cancer Res 2007; 67: 7431- 7438) proposed four genes (CBLC, CYP24A, AKR1B10, and ALDH3A1) to be potential biomarkers for non-small-cell lung cancer patients.
  • Two genes (CBLC and CYP24A1) are particularly promising. With respect to the histopathologic aspects, these genes were expressed in both adenocarcinoma and squamous cell carcinoma.
  • CBLC is a member of the Cbl family of multidomain signaling proteins with a tyrosine kinase binding domain and a RING finger domain. It is recruited to the epidermal growth factor (EGF) receptor (EGFR) on EGF stimulation and increases ubiquitination of EGFR, thereby down-regulating EGFR signaling. Mutations in the EGFR gene have been reported in non-small-cell lung cancer patients, especially in patients with adenocarcinoma, women, nonsmokers, and East Asians.
  • CYP24A1 is a member of the cytochrome P450 superfamily of enzymes involved in drugmetabolism and synthesis of cholesterol, steroids, and other lipids.
  • Lectins are protein or glycoprotein substances, usually of plant origin, of non- immunoglobulin nature, capable of specific recognition of and reversible binding to, carbohydrate moieties of complex glycoconjugates without altering the covalent structure of any of the recognized glycosyl ligands.
  • This group includes monovalent lectins (i.e. bacterial and plant toxins). These lectins bind to sugar moieties in cell walls or membranes and thereby change the physiology of the membrane to cause agglutination, mitosis, or other biochemical changes in the cell. Most lectins studied to date are multimeric, consisting of non-covalently associated subunits.
  • a lectin may contain two or more of the same subunit, such as Con A, or different subunits, such as Phaseolus vulgaris agglutinin.
  • Lectins are tools to explore a myriad of biological structures and processes. Because of the specificity that each lectin has toward a particular carbohydrate structure, even
  • oligosaccharides with identical sugar compositions can be distinguished or separated. Some lectins will bind only to structures with mannose or glucose residues, while others may recognize only galactose residues. Some lectins require that the particular sugar be in a terminal non-reducing position in the oligosaccharide, while others can bind to sugars within the oligosaccharide chain. The affinity between a lectin and its receptor may vary a great deal due to small changes in the carbohydrate structure of the receptor. All of these unique and desirable properties of lectins enable them to discriminate between structures and to isolate a glycoconjugate, cell or virus from a mixture or to study one process among several. Since virtually all biological membranes and cell walls contain glycoconjugates, all living organisms can be studied with lectins. Therefore, carbohydrate residues of the membrane glycoproteins can be detected using lectins due to their binding specificity to carbohydrates.
  • lectins themselves can be toxic to humans.
  • certain lectins found in food such as phytohaemagglutnin (kidney bean lectin, found in high concentrations in undercooked or raw kidney beans), may have toxic effects when ingested.
  • Other lectins are deadly when administered, such as ricin (Ricinus communis agglutinin), which is a
  • hemagglutinin limits their use as a therapeutic or diagnostic agent.
  • the present invention is directed to compositions comprising an isolated protein, which is optionally and preferably an immune molecule, optionally comprising one or more of substantially purified antibodies, monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, and uses of such antibodies and fragments thereof.
  • antibody as used herein may also optionally encompass isolated proteins and isolated immune molecules.
  • Immune molecules including without limitation antibodies and antibody mimetics that specifically recognize one or more glyco-epitopes on a single glycomolecule or multiple glycomolecules are disclosed in accordance with some embodiments.
  • Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • the glycomolecule is recognized by a saccharide-binding agent, which is any agent that binds specifically to a carbohydrate-portion of a glycomolecule.
  • Suitable saccharide-binding agents include, e.g., lectins or modified variants such as non-toxic variants of lectins, antibodies and antibody mimetics that recognize carbohydrate-containing epitopes, and carbohydrate-modifying enzymes, such as glycosidases.
  • Lectins are proteins isolated from plants that bind saccharides.
  • lectin also encompasses saccharide-binding proteins from animal species (e.g. "mammalian lectins").
  • lectins examples include lectins isolated from the following plants: Conavalia ensiformis, Anguilla anguilla, Tritium vulgaris, Datura stramonium, Galnthus nivalis, Maackia amurensis, Arachis hypogaea, Sambucus nigra, Erythtina cristagalli, Sambucis nigra, Erythrina cristagalli, Lens culinaris, Glycine max, Phaseolus vulgaris Allomyrina dichotoma, Dolichos biflorus, Lotus tetragonolobus, Ulex europaeus, and Ricinus commurcis.
  • glycosidases include a- Galactosidase, (3-Galactosidase, N-acetylhexosaminidase, a-mannosidase, ⁇ -mannosidase, and a-Fucosidase.
  • composition comprising isolated antibodies and isolated monoclonal antibody fragments or antigen binding portions or fragments thereof in a method of treating a disease or disorder, for example cancer or other proliferative disorder.
  • a diagnostic kit and method comprising isolated antibodies, isolated monoclonal antibody fragments or antigen binding portions or fragments thereof for in vitro and in vivo diagnosis of a disease or disorder, for example cancer or other proliferative disorder, and methods of use thereof.
  • kits comprising isolated antibodies, isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, for therapeutic use and/or diagnostic use, e.g., for use in in vitro and in vivo diagnostics to identify a cancer in a subject.
  • cancer comprises one or more of non-small cell lung carcinoma (NSCLC), colorectal cancer, breast cancer, adenocarcinomas and other solid tumors.
  • NSCLC non-small cell lung carcinoma
  • colorectal cancer breast cancer
  • adenocarcinomas other solid tumors.
  • the present invention relates to monoclonal
  • antibodies, fragments, antigen binding portions or fragments thereof which displace lectin binding on human and/or mouse tumor associated carbohydrate antigens expressed on cancer cells.
  • the inventors have used a lectin-displacement assay to identify monoclonal antibodies and complementary determining regions (CDRs) which bind to tumor associated carbohydrate antigens expressed on cancer cells.
  • CDRs complementary determining regions
  • NSCLC non small cell lung cancer
  • SCLC small cell lung cancer
  • carcinoids cylindromas
  • sarcomas certain sarcomas.
  • Cancers of the lung also include lung cancers which have metastasized from other primary sites, such as the breast, thyroid, or colon.
  • Non- small cell lung cancers are categorized into three types: squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma.
  • squamous cell carcinoma also called epidermoid carcinoma
  • adenocarcinoma adenocarcinoma
  • large cell carcinoma a non-limiting example of such lung cancers.
  • bronchoalveolar lung cancer a non-limiting example of such lung cancers.
  • the cancer may optionally be related to the abnormal proliferative growth in of any one or more of the following organs and tissues: lung, bone, pancreatic, skin, head or neck, eye, uterus, ovary, rectum, anal region, stomach, colon, breast, fallopian tubes, endometrium, cervix, vagina, vulva, lymph including Hodgkin's and non-Hodgkin's and lymphocytic lymphomas, esophagus, small intestine, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, urethra, penis, prostate, blood including chronic or acute leukemia, bladder, kidney, the central nervous system (CNS) including spinal axis tumors, brain stem glioma; and pituitary.
  • organs and tissues include lung, bone, pancreatic, skin, head or neck, eye, uterus, ovary, rectum, anal region, stomach, colon, breast, fallopian tubes, endo
  • a non-limiting aspect of the present invention provides a new process for the preparation of substantially purified and isolated antibodies that are biologically active, effective and functional and which optionally bind to the same antigens and epitopes on glycoproteins which also bind to one or more lectin molecules.
  • the present invention relates to an immune molecule which is capable of interacting with a human and/or mouse glyco-epitope, of which non-limiting examples are antibodies, e.g. monoclonal antibodies, or isolated monoclonal antibody fragments, or antigen binding portions or fragments thereof which bind to human and/or mouse glyco-epitope, such as a glycoprotein for example, with an affinity (KD) of at least 10 ⁇ 6 M for the Fab fragment.
  • a human and/or mouse glyco-epitope of which non-limiting examples are antibodies, e.g. monoclonal antibodies, or isolated monoclonal antibody fragments, or antigen binding portions or fragments thereof which bind to human and/or mouse glyco-epitope, such as a glycoprotein for example, with an affinity (KD) of at least 10 ⁇ 6 M for the Fab fragment.
  • KD affinity
  • K D describes the binding characteristics of an epitope and antibody
  • potency describes the effectiveness of the antibody itself as a function of the antibody.
  • a relatively low K D does not automatically mean a high potency.
  • antibodies can have a relatively low K D and a high potency (e.g., they bind well and alter the function strongly), a relatively high K D and a high potency (e.g., they don't bind well but have a strong impact on function), a relatively low K D and a low potency (e.g., they bind well, but not in a manner effective to alter a particular function) or a relatively high K D and a low potency (e.g., they simply do not bind to the target well).
  • high potency means that there is a high level of inhibition with a low concentration of antibody.
  • an antibody is potent or has a high potency when its IC 5 o is a small value, for example, 130-110, 110-90, 90-60, 60-30, 30-25, 25-20, 20-15, or less pM.
  • the term "selectively binds" in reference to an antibody does not mean that the antibody only binds to a single substance. Rather, it denotes that the K D of the antibody to a first substance is less than the K D of the antibody to a second substance. Antibodies that exclusively bind to an epitope only bind to that single epitope.
  • these antibodies bind to the glyco-epitope by displacing or blocking lectin
  • the immune molecule may optionally comprise an isolated protein which interacts with a human and/or mouse glyco-epitope that optionally comprises a tumor associated carbohydrate antigen.
  • the tumor associated carbohydrate antigen may optionally comprise a glycoprotein. Such interaction may optionally occur through at least one of binding, neutralizing and/or displacing another molecule bound to the glyco-epitope.
  • glyco-epitope is associated with a cell, optionally neutralizing by the immune
  • cell quiescence it is meant that the cell is induced to return to and/or to remain in a nonproliferative state.
  • a “neutralizing antibody” is an antibody molecule that is able to eliminate or
  • a neutralizing antibody as used herein is capable of eliminating or significantly reducing a glycomolecule function, such as, for example, binding of a glycomolecule to another protein or glycomolecule.
  • a neutralizing antibody will reduce a glycomolecule function by 1-10, 10-20, 20-30, 30-50, 50-70, 70-80, 80-90, 90-95, 95-99, 99-100%.
  • the isolated protein which may optionally
  • isolated immune molecule comprising any type of isolated immune molecule, including but not limited to antibodies, e.g., monoclonal antibodies, or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof may optionally cross-react with a human glyco-epitope, such as a human glycoprotein for example, with an EC50 of less than 20 mg/kg when administered to a subject.
  • antibodies e.g., monoclonal antibodies, or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof may optionally cross-react with a human glyco-epitope, such as a human glycoprotein for example, with an EC50 of less than 20 mg/kg when administered to a subject.
  • a biological sample taken from a subject where the biological sample, is for example, a bodily fluid or secretion including but not limited to seminal plasma, blood, serum, urine, prostatic fluid, seminal fluid, semen, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid, sputum, saliva, milk, peritoneal fluid, pleural fluid, cyst fluid, broncho alveolar lavage, lavage of the reproductive system and/or lavage of any other part of the body or system in the body, and stool or a tissue sample (for example, any type of tissue sample).
  • a bodily fluid or secretion including but not limited to seminal plasma, blood, serum, urine, prostatic fluid, seminal fluid, semen, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid, sputum, saliva, milk, peritoneal fluid, pleural fluid, cyst fluid, bron
  • the term may also optionally encompass samples of in vivo cell culture constituents.
  • the sample can optionally be diluted with a suitable eluant before contacting the sample to isolated antibodies and isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, thereby diagnosing a disease or disorder, for example cancer or other proliferative disorder.
  • a method for disease detection in vivo in the body of a subject comprising administering isolated antibodies and isolated monoclonal antibody fragments or antigen binding portions or fragments thereof to the body of the subject, and then detecting the location of the isolated antibodies and isolated monoclonal antibody fragments or antigen binding portions or fragments thereof within the body of the subject for example, optionally through a ligand or tag attached thereto.
  • the ligand or tag may optionally comprise polyethylene glycol (PEG), a nanoparticle or particles, or a label.
  • the nanoparticle or particles may optionally comprise one or more of solid nanospheres microencapsulated with different dye compounds and/or magnetic properties, which may optionally be prepared from a variety of functional polymeric materials, e.g. polyacrolein, polyglutaraldehyde, polymethyl a-(hydroxymethyl) acrylate,
  • uniform magnetic nanoparticles which may optionally comprise one or more of organic-inorganic hybrid particles composed of cores from micron-sized uniform polystyrene particles and shells from magnetite- silica nanospheres of approximately 30 nm diameter; and/or biodegradable, non-toxic, magnetic metal oxide (i.e. Fe304) nanoparticles of very narrow size distribution in sizes ranging from approximately 20 nm up to 0.5 microns; or non-magnetic and magnetic silica hollow micron-sized particles.
  • biodegradable, non-toxic, magnetic metal oxide i.e. Fe304
  • a label can be a fluorescent label or a bioluminescent label or any label known by one of ordinary skill in the art for in vivo imaging.
  • detection is performed through computer tomography (CT), magnetic resonance imaging (MRI) and spectroscopy (MRS), single photon emission computed tomography (SPECT), and positron emission tomography (PET).
  • CT computer tomography
  • MRI magnetic resonance imaging
  • MRS spectroscopy
  • SPECT single photon emission computed tomography
  • PET positron emission tomography
  • isolated nucleic acid molecules encoding the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof of the invention as well as an expression vector comprising at least one copy of said nucleic acid molecules.
  • One aspect of the present invention relates to host cells comprising an expression vector encoding and expressing an antibody, e.g., a hybridoma secreting an isolated antibody, e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, as well as transgenic non-human animals having a genome comprising said isolated nucleic acid molecule and/or the expression vector are also disclosed.
  • an antibody e.g., a hybridoma secreting an isolated antibody, e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, as well as transgenic non-human animals having a genome comprising said isolated nucleic acid molecule and/or the expression vector are also disclosed.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof of the present invention, and a pharmaceutically acceptable carrier.
  • a further embodiment of the invention concerns the use of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition, for the preparation of a medication for inhibiting the proliferation of a cell, e.g., cancer cell which the antibodies bind to, or for promoting cell death of the cells, e.g., cancer cells, to which the isolated antibodies bind to.
  • a cell e.g., cancer cell which the antibodies bind to
  • promoting cell death of the cells e.g., cancer cells
  • Another embodiment of the invention comprises the use of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition, in the manufacture of a medicament for the treatment of cancer in a subject.
  • isolated antibodies e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition
  • the present invention concerns a
  • kits comprising at least an effective amount of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, together with instructions for use.
  • isolated antibodies e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, together with instructions for use.
  • FCS Fetal calf serum
  • DMEM Dulbecco's modified Eagle's medium
  • PBS Phosphate buffered saline
  • BSA bovine serum albumin
  • HRP horse radish peroxidase
  • DAPI 4',6'-diamidino-2-phenylindole (DNA stain)
  • PNA Peanut (Arachis hyposaea)
  • Figures 1A-1C shows micrographs of immunohistochemical staining of cells
  • Figure 1A shows staining of normal human fibroblasts with the different scFv fragments.
  • Figure IB shows staining of NSCLC A549 cells with scFv fragments.
  • Figure 1C shows staining of NSCLC human tumor specimens with scFv fragments.
  • Figure 2 shows quantitative binding of scFv fragments to NSCLC A549 cells.
  • Figure 3A-3B shows survival of cells after scFvl treatment.
  • Figure 3A shows survival of
  • NSCLC A549 cells after treating the cells with scFvl fragments and 0.08uM antiMBP, while
  • Figure 3B is a histogram represents the survival of the cells after treatment with scFvland three different concentration of antiMBP (0.08uM, 0.165uM and 0.42uM) according to at least some embodiments of the present invention.
  • Figure 4A-40 shows immunohistochemical staining of different cancerous colorectal tissue, obtained as described above.
  • Figures 5A-5B shows immunostaining of paraffin embedded colorectal specimens from two human patients with scFvs8, demonstrating that the cancerous tissue is specifically bound while normal tissue is not bound.
  • Figure 6A-6D shows that staining intensity increases closer to tumor area in colorectal specimens (human patients), thereby indicating that relatively low levels of tumor tissue can be detected.
  • Figure 7 shows binding of scFv fragments to LS-174-T cells.
  • Figure 8 shows immunohistochemistry binding to such cells as for Figure 7.
  • Figure 9 shows the binding intensity of scFv8-IO-NIR to A549 cell line.
  • Figure 10 shows the binding of scFv8 to human colon cancer specimen and adjacent
  • Figures 11A-11B show images of colons taken from two mice with colonic tumors after administration of scFv8-HAS-NIR.
  • Panel (A) in Figures 11A and 11B show fluorescent imaging of the removed colon; the green dots are the tumors area stained with the probe.
  • Panel (B) in Figures 11A and 11B show the removed colon photo in white light.
  • Panel (C) in Figures 11A and 11B show the translation of fluorescent intensity into red and blue color whereas red shows areas of most intense fluorescence, and blue shows areas of least intense fluorescence.
  • Figures 12A-12C show scFv competition with lectins.
  • Figure 12A shows scFvl competition with lectins.
  • Figure 12B shows scFv8 competition with lectins.
  • Figure 13 shows a table of competition of scFvs with lectins and other targets.
  • Figure 14 shows a table of immunohistochemistry (ICH) of lung cancer specimens
  • nucleotide bases and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1-6 show the nucleotide sequences of the single-chain variable fragment
  • scFv of 6 different isolated antibodies.
  • the specific sequences correspond to the following SEQ ID NOs: SEQ ID NO:l - scFv # 1; SEQ ID NO:2 - scFv # 8; SEQ ID NO:3 - scFv # A7; SEQ ID NO:4 - scFv # C2; SEQ ID NO: 5 - scFv # G2; and SEQ ID NO:6 - scFv # H2.
  • SEQ ID NO: 7-12 - show the amino acid sequences of the scFv of 6 different isolated
  • the specific sequences correspond to the following: SEQ ID NO:7 - scFv#l; SEQ ID NO:8 - scFv #8; SEQ ID NO:9 - scFv#A7; SEQ ID NO: 10 - scFv#C2; SEQ ID NO: 11 - scFv#G2; SEQ ID NO: 12 - scFv#H2.
  • Table 1 shows SEQ ID NOs: 13-40 which are CDRs (CDRl, CRD2 and CDR3) according to at least some embodiments of the present invention.
  • SEQ ID NO 8 - scFv Cannot be assigned Cannot be assigned DPDPYGSESFRLFGTFDY #8: VH (SEQ ID NO: 19)
  • SEQ ID NO: 10 SGDMG AITTGGGSPNYADSVKG DEGMVGATYFDH scFv#C2: VH (SEQ ID NO:23) (SEQ ID NO:24) (SEQ ID NO:25)
  • SEQ ID NO: 11 SGSSSNIDSSPVN LINERPS QVWDSISDHWV scFv#G2: VL (SEQ ID NO;32) (SEQ ID NO:33) (SEQ ID NO:34)
  • scFv#A7 VH (SEQ ID NO:35) (SEQ ID NO:36) (SEQ ID NO:37)
  • VH of scFv #8 is not a canonical VH sequence since the sequence does not align in its middle part with other VHs.
  • Framework 1 is canonical almost all the way to CDR1, then the sequence is shuffled (source of shuffling unknown) and returns to canonical VH a little before CDR3.
  • the sequence is an intact open reading frame and seems to produce a functional protein. Therefore, CDRs 1 and 2 cannot be assigned.
  • peptidic or “peptidic chain” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • amino acid residue means any amino acid residue known to those skilled in the art. This encompasses naturally occurring amino acids (including for instance, using the three-letter code, Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val), as well as rare and/or synthetic amino acids and derivatives thereof (including for instance Aad, Abu, Acp, Ahe, Aib, Apm, Dbu, Des, Dpm, Hyl, McLys, McVal, Nva, and the like).
  • Said amino acid residue or derivative thereof can be any isomer, especially any chiral
  • isomer e.g. the L- or D- isoform.
  • amino acid derivative it is meant any amino acid derivative as known in the art.
  • amino acid derivatives include residues derivable from natural amino acids bearing additional side chains, e.g. alkyl side chains, and/or heteroatom substitutions.
  • an antibody e.g., monoclonal antibody preparation is one in which the protein is more pure than the protein in its natural environment within a cell.
  • a preparation of antibody e.g., a monoclonal antibody is purified such that the protein represents at least 50% of the total protein content of the preparation.
  • substantially pure refers to material that is at least 50% pure, preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure and most preferably 99% pure, or with greater purity.
  • an "isolated antibody”, as used herein, is intended to refer to an antibody which is
  • the antibody is a lectin displacing antibody
  • it is an isolated antibody that specifically binds to a glycoprotein in such a way as to displace a lectin molecule already bound, where the antibody is substantially free of antibodies that specifically bind antigens other than the glycoprotein to which the lectin is bound.
  • An isolated antibody that specifically binds to an epitope present on a glycoprotein may, however, have cross-reactivity to other related antigens, e. g., from other species (e. g., glycoproteins from other cancer cells, or other species homologues).
  • an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • a combination of "isolated" monoclonal antibodies having different specificities are combined in a well defined composition.
  • disease refers to a pathological condition of a part, organ, or system of an organism resulting from various causes, such as infection, genetic defect, or environmental stress, and characterized by an identifiable group of signs or symptoms.
  • subject refers to patients of human or other vertebrates in particular mammal and includes any individual it is desired to examine or treat using the methods according to the present invention. However, it will be understood that “patient” does not automatically imply that symptoms or diseases are present.
  • the term “patient” preferably refers to a human in need of treatment, e.g., to treat cancer.
  • the mammal is classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, monkeys etc.
  • the mammal is a human.
  • the vertebrate is classified as a vertebrate, including birds, amphibians and fishes.
  • the vertebrate is a human.
  • treatment refers to therapeutic treatment of a disease or disorder in a subject.
  • the term treatment also refers to prophylactic or preventive measures.
  • Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a subject to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder.
  • treatment or “treating” herein encompasses curative treatment, preventive treatment as well as palliative treatment, more specifically palliative treatment and curative treatment.
  • phrases "pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a patient in particular to a human.
  • the expression "effective amount” is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results, preventing or attenuating symptoms resulting from the disease, decreasing the dose of other medicaments required to treat the disease.
  • An effective amount can be administered in one or more administrations of the active substance.
  • the active substance is a molecular composition inducing a biological activity when interacting with a glyco-epitope. This includes a carbohydrate epitope or a mixed carbohydrate-protein epitope.
  • the term "receptor” refers to a structure on the surface of a cell (or inside a cell) that selectively receives and binds a specific molecule which affects the activities of the cell.
  • the term "antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to elicit the production of antibodies capable of binding to an epitope of that antigen.
  • An antigen may have one or more epitopes.
  • tumor- associated carbohydrate antigen refers to a carbohydrate moiety that is linked to the cell membrane of a tumor cell either directly or via another molecule such as a protein or lipid.
  • the carbohydrate can be a polysaccharide as well as a molecule to which a polysaccharide is linked (e.g., by a covalent bond) to a second molecule.
  • the tumor- associated carbohydrate antigen may be a lectin, a glycosaminoglycan, a glycoprotein or a glycolipid.
  • Carbohydrate antigens are conjugated to lipids and proteins via a process known as glycosylation.
  • carbohydrate binding molecule refers to a molecule
  • a lectin including a lectin, antibody or a fragment thereof, a peptide, a peptide analog and a small organic and inorganic molecule to bind to a carbohydrate moiety associated with the cell membrane wherein that binding depends on the specific structure and properties of the molecule.
  • antibody refers to a protein which may, for example, be produced by the immune system that protects the organism against an antigen. But, as used herein, the term encompasses not only intact monoclonal antibodies but also fragments thereof, single chains, mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, as well as antibodies produced by mammalian or bacterial cells that carry antibody coding DNA sequences, and also recombinant antibodies that are made in transgenic animals carrying the genes coding for the recombinant antibodies.
  • antibodies are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • native antibodies and immunoglobulins refer to
  • heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light (L) chains and two identical heavy (H) chains.
  • Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e. g., effector cells) and the first component (Clq) of the classical complement system.
  • agonist refers to a molecule, e.g., a drug, ligand or antibody which binds to a receptor and activates it, producing a pharmacological response (e.g. contraction, relaxation, secretion, enzyme activation, etc.).
  • agonist refers to a molecule, e.g., a drug, ligand or antibody which increases the biological activity by at least about 10% or more than 10% of the receptor to which it is an agonist to.
  • mAb monoclonal antibody
  • monoclonal antibody composition refers to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • epitope means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • antigen-binding portion of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a glyco-epitope and/or or retains the ability to displace a bound molecule from a glyco-epitope, such as a bound lectin molecule as a non-limiting example. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding examples include (i) a Fab fragment, a monovalent fragment consisting of the V L, V H , CL and CHI domains; (ii) a F (ab)' 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and CHI domains; (iv) a Fv fragment consisting of the VL and V H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR); and (vii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains.
  • a Fab fragment a monovalent fragment consisting of the V L, V H , CL and CHI domains
  • a F (ab)' 2 fragment a
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Huston et al (1988) Proc. Natl. Acad. Sc USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • fragments refers to sequences sharing at least 10% amino acids in length with the respective sequence of the intact or full length antibody, e.g., monoclonal antibodies (native). These sequences can be used as long as they exhibit the same properties as the native sequence from which they derive.
  • a fragment can be at least 6 amino acids in length, and can be, for example, at least 8, at least 10, at least 14, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 25 amino acids or greater than 25 amino acids from the full length protein from which the fragment was derived.
  • the term fragment encompasses at least 6, 10, 20, 50, 100, 250, 500 amino acids from the full length protein from which the fragment was derived.
  • Exemplary fragments include C-terminal truncations, N-terminal truncations, or truncations of both C- and N-terminals (e.g., deletions of 1, 2, 3, 4, 5, 8, 10, 15, 20, 25, 40, 50, 75, 100 or more amino acids deleted from the N-termini, the C-termini, or both).
  • these sequences share more than 70%, preferably more than 80%, in particular more than 90% amino acids in length with the respective sequence the intact or full length antibody, e.g., monoclonal antibodies.
  • fragments when used in reference to fragments of monoclonal antibodies, or monoclonal antibody fragments or antigen binding portions or fragments usually refers to a portion of at least 2, or at least about 5, or at least about 6, or at least about 8, or at least about 10 or more consecutive amino acids of the epitope binding region of an antibody.
  • a fragment includes at least 2, or at least about 5, or at least about 6, or at least about 8, or at least about 10 or more consecutive amino acids of the epitope binding region of an antibody selected from the group consisting of SEQ ID NO: 7- 12.
  • a fragment is a CDR region of at least 3 consecutive amino acids from any of the group consisting of SEQ ID NO: 7-12.
  • a fragment is a CDR region selected from any and a combination of CDRs listed in Table 1, e.g., SEQ ID NO: 13-40.
  • the fragment is a functional fragment, where a "functional fragment” as used in the context of a "functional fragment of an antibody” refers to a fragment of the antibody that mediates the same effect as the full length antibody, e.g., specifically binds to the same antigen with the same, or a greater affinity as compared to the full length antibody.
  • a funtional fragment is a CDR region of at least 3 consecutive amino acids from any of the group consisting of SEQ ID NO: 7-12.
  • a functional fragment is a CDR region selected from any and a combination of CDRs listed in Table 1, e.g., SEQ ID NO: 13-40.
  • useful fragments include, but are not limited to: a CDR region, especially a CDR3 region of the heavy or light chain; a variable domain of a heavy or light chain; a portion of an antibody chain or just its variable region including two CDRs; and the like.
  • useful functional fragments include, but are not limited at least one or any combination of CDRs from the same antibody, e.g., selected from SEQ ID NOs; 13- 15, or SEQ ID NOs: 16-18, or SEQ ID NOs: 20-22, or SEQ ID NOs: 23-25, or SEQ ID NOs: 26-28, or SEQ ID NOs: 29-31, or SEQ ID NOs: 32-34, or SEQ ID NOs: 35-37, or SEQ ID NOs: 38-40.
  • Suitable antibodies are immunologically functional immunoglobulins.
  • immunologically functional immunoglobulin fragment refers to a polypeptide fragment that is optionally and preferably capable of immunologically interacting with a glyco-epitope. Such interaction optionally and preferably comprises specifically binding to a glyco-epitope and/or specifically displacing a glycomolecule, such as a lectin for example, bound to a glyco-epitope and/or specifically neutralizing a glyco-epitope, wherein "neutralizing” optionally also encompasses inducing cell death and/or apoptosis and/or cell quiescence.
  • a fragment of such an antibody can bind specifically to and/or modulate the biological activity of a glycomolecule, such as a glycoprotein for example.
  • a glycomolecule such as a glycoprotein for example.
  • an immunologically functional immunoglobulin fragment specifically binds to the glyco-epitope at the same location, or in close proximity to the same location as the location on the glyco-epitope is bound by the glycomolecule such as a lectin for example.
  • the Cys residues are highly conserved in CDR regions.
  • the CDR regions of the Light-chain and Heavy- chains of SEQ ID NO: 7-12 can be determined and are useful in the method and compositions herein to produce specific glyco-epitope binding and/or glycomolecule displacing antibodies.
  • the Rosetta Antibody server at world- wide- web:
  • antibody.graylab.jhu.edu can be used to predict CDR in antibody sequences.
  • Residues before generally Ile-Tyr can also be: Val-Tyr, Ile-Lys, Ile-Phe
  • Residues typically Leu-Glu-Trp-Ile-Gly, but a number of variations
  • the present invention concerns a method for producing one or more antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof.
  • an antibody can also be produced by the method comprising the steps of: (a) producing an antigen of a glycomolecule, or a fragment, or a fusion protein thereof, of any species, e.g., human and/or vertebrate species
  • an antigen used to produce an antibody is human, or mouse, or from another mammalian species, or from another vertebrate species.
  • the antigen used to produce an antibody is a cell line
  • the antigen is naturally expressing full length antigen or a fragment of the antigen, or a fusion protein of the antigen and another protein, or the antigen is part of a virus-like particle.
  • the antigen used to produce the antibody is expressed in a cell line syngenic with mice of step b), or the antigen used to produce antibodies is fused to the Fc portion of an IgG.
  • the antigen used to produce antibodies is human or mouse antigen fused to the Fc portion of human IgGl.
  • an antibody, or fragment thereof such as single chain Fv can be obtained by selecting antibody sequences by phage display on the antigen of step a).
  • the binding assays of step e) is carried out by applying
  • step e) is carried out by direct or capture ELISA.
  • antibodies can be purified, for example by protein A or G affinity chromatography or by protein L, anti-mouse IgG antibody-based affinity chromatography, ion exchange, ethanol or ammonium sulfate precipitation and the like.
  • an immunogen e.g., an antigen used to produce lectin- displacing antibodies
  • immunizing an animal are well-known in the art (Kohler and Milstein 1975 Nature 256:495-497; Brown et al.
  • SEQ ID NOS: 1-6 relate to the nucleotide sequences of the single-chain human antibody fragments (scFv) of 6 different isolated antibodies.
  • the specific sequences correspond to the following SEQ ID NOs: SEQ ID NO:l - scFv # 1; SEQ ID NO:2 - scFv # 8; SEQ ID NO:3 - scFv # A7; SEQ ID NO:4 - scFv # C2; SEQ ID NO: 5 - scFv # G2; and SEQ ID NO:6 - scFv # H2.
  • SEQ ID NOS: 7-12 The amino acid sequences thereto are shown in SEQ ID NOS: 7-12, which relates to the amino acid sequences of the scFv of 6 different isolated antibodies.
  • the specific sequences correspond to the following: SEQ ID NO:7 - scFv#l; SEQ ID NO:8 - scFv #8; SEQ ID NO:9 - scFv#A7; SEQ ID NO: 10 - scFv#C2; SEQ ID NO: 11 - scFv#G2; SEQ ID NO: 12 - scFv#H2.
  • the in vitro test was performed by contacting NSCLC cells with the antibodies in the media and analyzing cell survival over a period of time, as disclosed in the Examples.
  • antibodies e.g., sc-Fvl and sc-Fv8 decreased cell survival of NSCLC cells.
  • This in vitro assay is certainly a standard test, which can be easily performed by one of ordinary skill in the art, e.g., by scientists without experience in the field, and in a variety of different cell types and cancer cell types.
  • an isolated antibody e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof obtainable by the above-described process and wherein said isolated antibody, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof bind to human and/or mouse glyco- epitope with an affinity constant (KD) of at least 10 "6 M for the Fab fragment, and which can optionally displace a bound glycomolecule from the glyco-epitope with an affinity constant of at least this amount.
  • KD affinity constant
  • an antibody or fragment or antigen binding portion thereof as disclosed herein specifically binds with a high affinity to the glyco-epitope.
  • binding refers to antibody binding to a predetermined antigen.
  • the antibody binds with a affinity constant (KD) of 10 ⁇ 6 M or less for the Fab fragment, and binds to the predetermined antigen with a KD that is at least ten-fold less than its KD for binding to a non-specific antigen (e. g., BSA, casein) other than the KD.
  • KD affinity constant
  • an antibody recognizing an antigen and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
  • Kassoc or "Ka”, as used herein, is intended to refer to the association rate of a particular antibody- antigen interaction, whereas the term “KdiS” or “Kd” is intended to refer to the dissociation rate of a particular antibody- antigen interaction.
  • high affinity for an IgG antibody refers to an affinity constant (KD) for the Fab fragment of at least about 10 "6 M, at least about 10 "7 M, at least about 10 "8 M, at least about 10 "9 M, at least about 10 "10 M, at least about 10 "11 M, or at least about 10 "12 M or greater, e. g., up to 10 "1 3 M or 10 "14 M or greater.
  • KD affinity constant
  • the isolated antibody e.g., monoclonal antibodies or isolated
  • monoclonal antibody fragments or antigen binding portions or fragments thereof comprise a polypeptide having an amino acid sequence selected from any of the group according to any of SEQ ID NOs 7-12, and or any combination thereof.
  • the isolated antibody preferably specifically binds to a glyco-epitope and/or specifically displaces a glycomolecule bound to such an epitope and/or neutralizes such a glyco-epitope.
  • the antibody is a lectin- displacing antibody, e.g., lectin-displacing monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof binds to a glycoprotein at the same epitope or region where a lectin would bind.
  • conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups:
  • amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (5) confer or modify other physicochemical or functional properties on such polypeptides.
  • the isolated antibody e.g., monoclonal antibody according to the present invention is not limited to the whole molecule, and may be a fragment of the antibody or the modified product thereof, as long as it still retains one or more of following properties: specifically binds to a glyco-epitope and/or specifically displaces a glycomolecule bound to such an epitope and/or neutralizes such a glyco-epitope.
  • the antibody or fragment or modified product thereof may optionally displace a lectin bound to a glycoprotein and retain the capacity of binding to the glycoprotein at the region where the lectin typically binds.
  • Multivalent, preferably bivalent, antibody and a monovalent antibody are included
  • the fragment of an antibody include Fab, F(ab)'2, Fv, Fab/c having one Fab and a complete Fc, and a single chain Fv (scFv) wherein the Fv of the H- chain or the L-chain is ligated with an appropriate linker.
  • an antibody fragment is synthesized by treating the antibody with an enzyme such as papain, pepsin or ficin, or genes encoding these antibody fragments are constructed, the genes are introduced into expression vectors, and the genes are then expressed by appropriate host cells (see e g, Rousseaux, J et al, Methods in Enzymology (1989) 121, 663-669, and Bird, R E et al, TIBTECH (1991)9, 132-137).
  • an enzyme such as papain, pepsin or ficin
  • genes encoding these antibody fragments are constructed, the genes are introduced into expression vectors, and the genes are then expressed by appropriate host cells (see e g, Rousseaux, J et al, Methods in Enzymology (1989) 121, 663-669, and Bird, R E et al, TIBTECH (1991)9, 132-137).
  • Fab refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
  • Fv refers to a minimum antibody fragment that contains a complete antigen-recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region generally comprises amino acid residues from a "complementarity determining region” or "CDR” (e.g. residues 24-34 (LI), 50-62 (L2), and 89-97 (L3) in the light chain variable domain and 31-55 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • CDR complementarity determining region
  • residues from a "hypervariable loop” e.g. residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26- 32 ((HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • the term "framework Region” or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
  • CDRs complementarity determining regions
  • the CDRs of immunological receptors are the most variable part of the receptor protein, giving receptors their diversity, and are carried on six loops at the distal end of the receptor's variable domains, three loops coming from each of the two variable domains of the receptor.
  • epitopope is used to refer to binding sites for antibodies on antigens.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • glyco-epitope refers to a binding site for an antibody on a glycomolecule.
  • glycomolecule refers to a protein comprising a
  • glycomolecule can includes carbohydrate-containing proteins (glycoproteins) or glycolipids, and free polysaccharides.
  • Glycoproetins include, e.g., fetuin, al Acid GP, and tPA.
  • scFv is obtained by linking the H-chain V-region and the L-chain V-region of
  • the H-chain V-region and the L-chain V-region are linked via a linker, or preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883).
  • the H-chain V-region and the L-chain V-region in scFv may be derived from any of those described as antibodies in this specification.
  • As a peptide linker to link the V-regions for example, any single-stranded peptide comprising 12 to 19 amino acid residues is used.
  • the isolated antibodies e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g., the antibodies, e.g.
  • monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof may present an antibody heavy chain selected among: IgG, IgM, IgA, IgE, single chain antibody and other immunoglobulin-derived constructs or non antibody binding proteins.
  • the non antibody binding proteins comprise adnectins (fibronectin-based reagents), Affibody (protein A-based reagents), DARPins (ankyrin-based reagents), avimers (cysteine rich cell surface receptor proteins), anticalins (lipocalin-derived reagents), and nucleotide-based reagents and the like (see for example Nutall & Walsh 2008 Curr Op Pharmacol 8:609).
  • the isolated antibodies e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof may present a IgG antibody heavy chain
  • the latter may be selected among: IgGl, IgG2, IgG3 or IgG4, mutated IgGl that is no longer recognized by FcR; mutated IgG4 sequence that no longer undergoes heavy chain swapping; mutated IgG to modify glycosylation; PEGylated IgG and the like. It is acknowledged that all possible "isotype switching" known to the person skilled in the art may be envisioned in the context of the present invention.
  • isotype switching refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.
  • V NAR which are lamprey-derived single domain antibodies may be advantageously used.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Human light chains are classified as kappa ( ⁇ ) and lambda ( ⁇ ) light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • an intact antibody has two binding sites. Except in bifunctional or bispecific
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. (See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992)). Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab', and Fv).
  • the present invention further provides at least one isolated antibody, e.g., a
  • fragments of (d) wherein the fragments are binding glycoproteins to displace a bound lectin; and (f) biological active variants or modifications of amino acid sequences specified in (a) or (b) or (c) or (d) or (e) having the properties of fragments of (d) or (e).
  • sequence identity/similarity has its ordinary meaning in the field.
  • identity or percent “identity” in the context of two or more polypeptide sequences, refer to two or more sequences that are the same, or have a specified percentage of amino acid residues that are the same (i.e., at least 70% identity, preferably at least 75%, 80%, 85%, 90%, even more preferably at least 95% or 98% or even 99% identity over a specified region), when compared and aligned for maximum correspondence. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.
  • Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.
  • Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (e.g., human and mouse sequences), compared to species more distantly related (e.g., human and C. elegans sequences).
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
  • NCBI National Center for Biological Information
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, 99% sequence identity.
  • homologs When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.
  • Homologs of the disclosed antibodies e.g., monoclonal antibodies are typically characterized by possession of at least 70%, preferably of at least 95%, and more preferably of at least 98% sequence identity sequence identity counted over the full-length alignment with the disclosed amino acid sequences using the NCBI Blast 2.0, or using the manual alignment as described above.
  • Proteins with even greater similarity to the antibodies e.g., monoclonal antibody sequences will show increasing percentage identities when assessed by this method, such as at least 75%, 80%, 85%, 90%, 95% or even 98% sequence identity.
  • sequence identity When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or even 98% depending on their similarity to the reference sequence.
  • sequence identity ranges are provided for guidance only; it is possible that strongly significant homologs could be obtained that fall outside the ranges provided.
  • the present invention also includes variants of the antibodies, e.g., monoclonal antibodies.
  • variants or derivatives or equivalents of the antibodies, e.g., monoclonal antibody sequences refer to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide that is amino acid sequences that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles.
  • the amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Typically, such variants possess at least 90%, preferably at least 95%, and very particularly preferably at least 98%, sequence identity with the native sequence.
  • variants which are particularly preferred in this connection are replacement variants which typically contain less than 10, preferably less than 5, and very particularly preferably less than 3, replacements as compared with the respective disclosed sequences.
  • antibodies e.g., monoclonal antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen- dependent cellular cytotoxicity.
  • an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • Variants, derivatives and equivalents of an antibody useful in the compositions and methods as disclosed herein have substantially the same, or a greater biological activity than the antibody they are derived from.
  • the biological activity of an antibody can be determined by one of ordinary skill in the art, for example, using an assay such as that disclosed herein in the Examples, such as an in vitro assay to assess the ability of the derivative, variant or equivalent thereof to specifically bind to a glyco-epitope and/or specifically displace a glycomolecule bound to such an epitope and/or neutralize such a glyco- epitope.
  • the assay may optionally assess the ability of the derivative, variant or equivalent thereof to displace a lectin bound to a
  • glycoprotein as compared to the antibody it is derived from, or alternatively, the ability of the derivative, variant or equivalent of the lectin-displacing antibody to induce cell death as compared to the lectin-displacing antibody it is a variant thereof.
  • a variant, derivative or equivalent of an antibody useful in the compositions and methods as disclosed herein has at least about the same, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%, or at least about 100% of the biological activity than the antibody they are derived from, or at least about 1.5-fold, or at least about 2-fold, or at least about 2.5-fold, or at least about 5-fold, or at least about 10-fold greater biological activity than the antibody they are derived from.
  • a fragment of an antibody useful in the compositions and methods as disclosed herein is a functional fragment, and comprises at least one or two or more CDR's as disclosed in Table 1.
  • a fragment of an antibody for use in the methods and compositions as disclosed herein comprises at least 3 consecutive amino acids from any of the CDR regions selected from any, or a combination of CDRs listed in Table 1, e.g., SEQ ID NO: 13-40.
  • a fragment of an antibody useful in the compositions and methods as disclosed herein comprises at least one or any combination of CDRs from the same antibody, e.g., selected from SEQ ID NOs; 13-15 (CDRs of scFv#l, VH), or SEQ ID NOs: 16-18 (CDRs of scFv#l, VL (Kappa)), or SEQ ID NO; 19 (CDR of scFv#8, VH), or SEQ ID NOs: 20-22 (CDRs of scFv#8, VL, kappa), or SEQ ID NOs: 23-25 (CDRs of scFv#2, VH), or SEQ ID NOs: 26-28 (CDRs of scFv#C2, VL), or SEQ ID NOs: 29-31 (CDRs of scFv#G2, VH), or SEQ ID NOs: 32-34 (CDRs of scFv#G2, VL), or SEQ ID NOs:
  • a fragment of an antibody useful in the compositions and methods as disclosed herein comprises at least one or any combination of CDRs from the same antibody, e.g., selected from SEQ ID NOs; 13-18 (CDRs of scFv#l), or SEQ ID NO; 19-22 (CDRs of scFv#8), or SEQ ID NOs: 23-28 (CDRs of scFv#2), or SEQ ID NOs: 29-34 (CDRs of scFv#G2) or SEQ ID NOs: 35-34 (CDRs of scFv#A7, VH).
  • glycosylation of an antibody, or antibody variant, derivative or functional fragment thereof can be altered to, for example, increase the affinity of the antibody for antigen.
  • carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such a glycosylation may increase the affinity of the antibody for antigen.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery.
  • Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express antibodies, e.g., monoclonal antibodies of the invention to thereby produce an antibody with altered glycosylation.
  • the frucose residues of the antibody may be cleaved off using a fucosidase enzyme.
  • Another modification of the antibodies herein that is contemplated by the invention is pegylation.
  • An antibody can be pegylated to, for example, increase the
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is a glycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al, which are incorporated herein in their entirety by reference.
  • an antibody e.g., monoclonal antibodies of the present invention do not contain asparagine isomerism sites.
  • a deamidation or isoaspartic acid effect may occur on N-G or D-G sequences, respectively. The deamidation or isoaspartic acid effect results in the creation of isoaspartic acid which decreases the stability of an antibody by creating a kinked structure off a side chain carboxy terminus rather than the main chain.
  • antibodies are selected that do not rapidly degrade. Fragmentation of an antibody, e.g., monoclonal antibody may be measured using capillary electrophoresis (CE) and MALDI-MS, as is well understood in the art (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32). In another preferred embodiment, antibodies are selected that have minimal aggregation effects. Aggregation may lead to triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable with aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less and even more preferably 5% or less. Aggregation may be measured by several techniques well known in the art, including size-exclusion column (SEC) high performance liquid chromatography (HPLC), and light scattering to identify monomers, dimers, trimers or multimers.
  • SEC size-exclusion column
  • HPLC high performance liquid chromatography
  • an antibody e.g., a lectin-displacing monoclonal antibody would have binding specificities that would function to induce cell death, as lectin
  • an isolated nucleic acid molecule encoding the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof as defined above.
  • isolated antibodies e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof as defined above.
  • Specific examples of such nucleotide sequences are given as SEQ ID NOs 1-6.
  • nucleic acid molecule is intended to include DNA
  • a nucleic acid molecule may be single- stranded or double- stranded, but preferably is double- stranded DNA.
  • artificially altered gene recombinant antibodies such as chimeric antibodies or humanized antibodies can be used for, for example, lowering heteroantigenicity against a human.
  • altered antibodies can be produced using a known method.
  • Chimeric antibodies can e.g., be obtained by ligating the DNA encoding the
  • Humanized antibodies are also referred to as reshaped human antibodies, which are prepared by grafting an antibody CDR (complementarity determining region) of a mammal other than a human, such as a mouse, to the CDR of a human antibody.
  • CDR complementarity determining region
  • humanized antibody is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
  • Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a non-human monoclonal antibody prepared as described above.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the non- human hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques.
  • murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al, which are incorporated herein in their entirety by reference).
  • murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al, which are incorporated herein in their entirety by reference).
  • an antibody of the invention is a human monoclonal antibody.
  • scFv molecules are their monovalent interaction with target antigen.
  • scFv-based structures A number of multivalent scFv-based structures has been engineered, including for example, miniantibodies, dimeric miniantibodies, minibodies, (scFv) 2 , diabodies and triabodies. These molecules span a range of valence (two to four binding sites), size (50 to 120 kDa), flexibility and ease of production.
  • Single chain Fv antibody fragments (scFvs) are predominantly monomeric when the VH and VL domains are joined by, polypeptide linkers of at least 12 residues. The monomer scFv is thermodynamically stable with linkers of 12 and 25 amino acids length under all conditions.
  • the noncovalent diabody and triabody molecules are easy to engineer and are produced by shortening the peptide linker that connects the variable heavy and variable light chains of a single scFv molecule.
  • the scFv dimers are joined by amphipathic helices that offer a high degree of flexibility and the miniantibody structure can be modified to create a dimeric bispecific (DiBi) miniantibody that contains two miniantibodies (four scFv molecules) connected via a double helix.
  • DiBi dimeric bispecific
  • Gene-fused or disulfide bonded scFv dimers provide an intermediate degree of flexibility and are generated by straightforward cloning techniques adding a C-terminal Gly4Cys sequence.
  • scFv-CH3 minibodies are comprised of two scFv molecules joined to an IgG CH3 domain either directly (LD minibody) or via a very flexible hinge region (Flex minibody). With a molecular weight of approximately 80 kDa, these divalent constructs are capable of significant binding to antigens.
  • the Flex minibody exhibits impressive tumor localization in mice. Bi- and tri-specific multimers can be formed by association of different scFv molecules. Increase in functional affinity can be reached when Fab or single chain Fv antibody fragments (scFv) fragments are complexed into dimers, trimers or larger aggregates.
  • the most important advantage of multivalent scFvs over monovalent scFv and Fab fragments is the gain in functional binding affinity (avidity) to target antigens.
  • High avidity requires that scFv multimers are capable of binding simultaneously to separate target antigens.
  • the gain in functional affinity for scFv diabodies compared to scFv monomers is significant and is seen primarily in reduced off-rates, which result from multiple binding to two or more target antigens and to rebinding when one Fv dissociates.
  • scFv molecules associate into multimers, they can be designed with either high avidity to a single target antigen or with multiple specificities to different target antigens.
  • Antibodies conjugated with moieties that improve their properties are also contemplated for the instant invention.
  • antibody conjugates with PEG that increases their half-life in vivo can be used for the present invention.
  • Immune libraries are prepared by subjecting the genes encoding variable antibody fragments from the B
  • lymphocytes of naive or immunized animals or patients to PCR amplification include lymphocytes of naive or immunized animals or patients to PCR amplification.
  • Combinations of oligonucleotides which are specific for immunoglobulin genes or for the immunoglobulin gene families are used.
  • Immunoglobulin germ line genes can be used to prepare semisynthetic antibody repertoires, with the complementarity-determining region of the variable fragments being amplified by PCR using degenerate primers.
  • the phage-display technique can be used to increase the affinity of antibody fragments, with new libraries being prepared from already existing antibody fragments by random, codon-based or site-directed mutagenesis, by shuffling the chains of individual domains with those of fragments from naive repertoires or by using bacterial mutator strains.
  • SCID-hu mouse for example the model developed by Genpharm, can be used to produce antibodies, or fragments thereof.
  • a new type of high avidity binding molecule termed a "peptabody" has been created by harnessing the effect of multivalent interaction is contemplated.
  • ligands and/or chimeric inhibitors can be targeted to tissue- or tumor- specific targets by using bispecific antibodies, for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target.
  • bispecific antibodies for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target.
  • Ab anti-ligand antibody
  • molecular conjugates of antibodies can be used for production of recombinant bispecific single-chain Abs directing ligands and/or chimeric inhibitors at cell surface molecules.
  • each conjugate includes a targeting moiety, for example, a different antibody.
  • Each antibody is reactive with a different target site epitope (associated with the same or a different target site antigen).
  • the different antibodies with the agents attached accumulate additively at the desired target site.
  • Antibody-based or non-antibody- based targeting moieties can be employed to deliver a ligand or the inhibitor to a target site.
  • a natural binding agent for an unregulated or disease associated antigen is used for this purpose.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA., IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • An example of anti-chondroitin sulfate antibody production is provided in the Examples section below.
  • Anti-glyco-epitope antibodies can be prepared by methods as commonly known in the art, and as described in the Examples section below. Briefly, one or more homogeneous glycomolecules or polypeptides comprsing the glycol-epitope can be conjugated to bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). The glycomolecule-conjugates were then dialyzed, and the protein concentrations were determined. Mice were immunized with the glycomolecule-conjugate and boosted five times over a period of 2 months. Spleen cells of the mice were fused to a myeloma cell line, and multiclonal cell lines were then screened via ELISA analysis.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • Clones specific for antibodies to the desired glyco-epitope and with absorbance values greater than 1.0 were kept for subsequent expansion.
  • Single cell clones can then screened via ELISA, and clones specific for the desired glyco-epitope and with absorbance values greater than 1.0 were analyzed by dot blot analysis.
  • Antibodies to a glyco- epitope preferably are able to bind specifically to a single subtype of glycomolecule.
  • the genetic material that encodes an antibody that specifically binds to a specific glyco- epitope can be isolated, and that material can be introduced into a suitable expression vector and thereafter transfected into host cells.
  • anti-glyco-epitope antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell, such as a CHO cell.
  • Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference). The transformation procedure used depends upon the host to be transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
  • ATCC American Type Culture Collection
  • Cell lines of particular preference are selected through determining which cell lines have high expression levels and produce antibodies with anti-glycomolecule binding properties.
  • the invention also includes functional equivalents of the antibodies described in this specification.
  • Functional equivalents have binding characteristics that are comparable to those of the antibodies, and include, for example, chimerized, humanized and single chain antibodies as well as fragments thereof. Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319, European Patent Application No. 239,400; PCT
  • Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the invention. "Substantially the same" as applied to an amino acid sequence is defined herein as a sequence with at least 80%, preferably at least about 90%, and more preferably at least about 95% sequence identity to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 2448 (1988).
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al. 1984 Proc Natl Acad Sci USA 81:6851-6855; Neuberger et al. 1984 Nature 312:604-608; Takeda et al. 1985 Nature 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimerized antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human.
  • Humanized antibodies preferably have constant regions and variable regions other than the complement determining regions (CDRs) derived substantially or exclusively from the corresponding human antibody regions and CDRs derived substantially or exclusively from a mammal other than a human.
  • CDRs complement determining regions
  • Functional equivalents also include single-chain antibody fragments, also known as
  • Single-chain antibody fragments of the present invention are shown in the Examples herein, and are recombinant polypeptides which bind glycol-epitopes. These fragments contain at least one fragment of an antibody variable heavy-chain amino acid sequence (VH) tethered to at least one fragment of an antibody variable light-chain sequence (VL) with or without one or more interconnecting linkers.
  • VH antibody variable heavy-chain amino acid sequence
  • VL antibody variable light-chain sequence
  • Such a linker may be a short, flexible peptide selected to assure that the proper three-dimensional folding of the (VL) and (VH) domains occurs once they are linked so as to maintain the target molecule binding- specificity of the whole antibody from which the single-chain antibody fragment is derived.
  • the carboxyl terminus of the (VL) or (VH) sequence may be covalently linked by such a peptide linker to the amino acid terminus of a complementary (VL) and (VH) sequence.
  • Single-chain antibody fragments may be generated by molecular cloning, antibody phage display library or similar techniques. These proteins may be produced either in eukaryotic cells or prokaryotic cells, including bacteria.
  • Single-chain antibody fragments contain amino acid sequences having at least one of the variable or complementarity determining regions (CDR's) of the whole antibodies described in this specification, but are lacking some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute a major portion of the structure of whole antibodies. Single-chain antibody fragments may therefore overcome some of the problems associated with the use of antibodies containing a part or all of a constant domain. For example, single-chain antibody fragments tend to be free of undesired interactions between biological molecules and the heavy-chain constant region, or other unwanted biological activity.
  • single-chain antibody fragments are considerably smaller than whole antibodies and may therefore have greater capillary permeability than whole antibodies, allowing single-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently. Also, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. Furthermore, the relatively small size of single-chain antibody fragments makes them less likely to provoke an immune response in a recipient than whole antibodies.
  • Functional equivalents further include fragments of antibodies that have the same, or comparable binding characteristics to those of the whole antibody.
  • Antibody fragments which recognize specific epitopes can be generated by known techniques.
  • fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be constructed (Huse et al., 1989 Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • the antibody fragments contain all six complement determining regions of the whole antibody, although fragments containing fewer than all of such regions, such as three, four or five CDRs, are also functional.
  • the functional equivalents may be or may combine members of any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
  • Single chain antibodies can be formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient.
  • fully human antibodies can be generated through the introduction of human antibody function into a rodent so that the rodent produces fully human antibodies.
  • “human” and “fully human” antibodies can be used interchangeably herein. The term “fully human” can be useful when distinguishing antibodies that are only partially human from those that are completely, or fully human.
  • Fully human antibodies can be made by any methods known in the art.
  • One method for generating fully human antibodies is through the use of XENOMOUSETM.
  • strains of mice which have been engineered to contain 245 kb and 190 kb- sized germline configuration fragments of the human heavy chain locus and kappa light chain locus. See Green et al. Nature Genetics 7:13-21 (1994).
  • the XENOMOUSETM strains are available from Abgenix, Inc.
  • minilocus an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus.
  • V H genes one or more DH genes
  • J H genes one or more J H genes
  • a mu constant region preferably a gamma constant region
  • an expression vector comprising at least one copy of a nucleic acid molecule as described above, for example, an expression vector comprising at least one, or any combination of nucleic acid sequences SEQ ID NO: 1-6, or a fragment thereof.
  • vector as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e. g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.
  • non- episomal mammalian vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, “expression vectors").
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e. g. replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e. g. replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the invention also concerns host cells comprising and expressing the above
  • recombinant host cell (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • Recombinant host cells include, for example, CHO cells and lymphocytic cells.
  • vectors suitable for expression of recombinant antibodies are commercially available.
  • the vector may, e. g. be a bare nucleic acid segment, a carrier-associated nucleic acid segment, a nucleoprotein, a plasmid, a virus, a viroid, or a transposable element.
  • Host cells known to be capable of expressing functional immunoglobulins include, e. g.: mammalian cells such as Chinese Hamster Ovary (CHO) cells; COS cells; myeloma cells, such as NSO and SP2/0 cells; insect cells, bacteria such as Escherichia coli; yeast cells such as Saccharomyces cerevisiae; and other host cells.
  • mammalian cells such as Chinese Hamster Ovary (CHO) cells; COS cells; myeloma cells, such as NSO and SP2/0 cells
  • insect cells bacteria such as Escherichia coli
  • yeast cells such as Saccharomyces cerevisiae
  • other host cells e.g., CHO cells are used by many researchers given their ability to effectively express and secrete immunoglobulins.
  • NSO cells are one of the preferred types of host cells useful in the present invention.
  • Host cells are transformed following techniques that are known to the person skilled in the art.
  • a "transformed” cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation
  • nucleic acid molecule encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, calcium phosphate precipitation, and particle gun acceleration.
  • Another embodiment of the present invention relates to a hybridoma, e.g., a cell secreting at least one antibody as disclosed herein, e.g., a monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention.
  • a hybridoma e.g., a cell secreting at least one antibody as disclosed herein, e.g., a monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention.
  • prepared hybridomas which produce monoclonal antibodies can be passage-cultured in a standard culture solution, or can be stored for a long period in liquid nitrogen.
  • hybridomas involves culturing the hybridomas and obtaining monoclonal antibodies in the culture supernatant according to a standard method.
  • the former method is suitable for the mass production of antibodies.
  • a monoclonal antibody that can be used in the present invention can be a
  • recombinant monoclonal antibody that is prepared by cloning the antibody gene from the hybridoma, incorporating the gene into an appropriate vector, introducing the vector into a host, and then causing the host to produce the recombinant monoclonal antibodies by genetic engineering techniques (e.g., see Vandamme, A. M. et al., Fur. J. Biochem, (1990) 192, 767- 775, 1990).
  • transgenic animal or plant can also be used to produce a recombinant antibody.
  • transgenic non-human animal having a genome comprising the isolated nucleic acid molecule and/or the expression vector according to the present invention.
  • the present invention concerns a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutically composition comprises at least a therapeutically effective quantity or amount of the substantially purified and isolated agonist antibodies, e.g., antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention.
  • the isolated antibodies e.g., monoclonal antibodies or isolated monoclonal
  • antibody fragments or antigen binding portions or fragments thereof described herein as a "compound” may be administered with a physiologically acceptable carrier.
  • a physiologically acceptable carrier is a formulation to which the compound can be added to dissolve it or otherwise facilitate its administration.
  • An important factor in choosing an appropriate physiologically acceptable carrier is selecting a carrier in which the compound remains active or the combination of the carrier and the compound produces an active compound.
  • the pharmaceutically acceptable carrier can be a solvent or dispersion medium.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of emulsifying agents such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, such as Tween 20.
  • the pharmaceutical composition may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, pharmaceutically-acceptable antioxidants and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents, pharmaceutically-acceptable antioxidants and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol,
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention may also be envisioned.
  • a “pharmaceutically acceptable salt” refers to a salt that retains the desired
  • composition of the invention is
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,
  • any route of administration of at least one isolated antibody e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention are intravenously, intramuscularly and intraperitoneally.
  • modes of delivery are by injection and infusion.
  • Injectable forms may include sterile aqueous solutions or dispersions. Furthermore, form of sterile powders may be prepared for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability.
  • the pharmaceutical composition can be formulated to be stable under the conditions of manufacture and storage; thus, in some embodiments, the composition is prepared to prevent the contaminating action of
  • microorganisms such as bacteria and fungi.
  • Parenteral administration may be prepared as solutions or suspensions of the
  • a suitable surfactant can be included such as, for example, hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.
  • any convenient pharmaceutical media may be any convenient pharmaceutical media.
  • oral liquid preparations such as suspensions, elixirs and solutions
  • carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to form oral solid preparations such as powders, capsules and tablets.
  • oral solid preparations such as powders, capsules and tablets.
  • tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed.
  • tablets may be coated by standard aqueous or non-aqueous techniques.
  • a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the combined components in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent.
  • a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • Effective dosage regimens are adjusted to provide the optimum desired response (e. g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the isolated antibodies e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical composition of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • a dosage of a pharmaceutical composition as disclosed herein is in the amount for inducing death of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 100%, or any integer between 10-100% of the cancer cells.
  • the selected dosage level depends upon a variety of
  • pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the salt or amide thereof, the route of administration, the time of administration, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
  • a physician or veterinarian can start doses of the compounds of the invention- employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a composition of the invention is that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends upon the factors described above.
  • administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target.
  • the effective daily dose of a therapeutic composition can be administered as two; three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the isolated antibodies e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention to be administered alone, it is preferable to administer the latter as a pharmaceutical formulation (composition).
  • compositions of the present invention for the treatment of a disease or disorder, e.g., cancer such as NSCLC described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages can easily be titrated to optimize safety and efficacy by physician of ordinary skill in the art.
  • a disease or disorder e.g., cancer such as NSCLC described herein
  • Treatment dosages can easily be titrated to optimize safety and efficacy by physician of ordinary skill in the art.
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually about 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1 to 10 mg/kg.
  • a treatment regime entails administration once per day or once per week or once per two weeks or once a month or once every 3 to 6 months.
  • two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
  • an antibody can be administered to a subject on multiple occasions. Intervals can be repeated single dosages, for example, bidaily, daily, weekly, biweekly, monthly or yearly. In some embodiments, intervals can also be irregular as indicated by measuring blood levels of antibodies in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 0.001-1000 ⁇ g/ml. Alternatively, an antibody can be administered as a sustained release formulation, in which case less frequent administration is required.
  • human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • the dosage ranges of the isolated antibodies may vary with the administration routes, as well as the state of the patient (age, sex, body weight, extent of the disease etc.).
  • an isolated antibody e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof is administered to a patient in need thereof at a dosage unit from 0.1 mg/kg of body weight to 100 mg/kg of body weight.
  • the inventors have demonstrated that the administration (for example application, injection, delivery, contact etc.) of the pharmaceutical composition comprising antibodies according to at least some embodiments of the present invention results in an improved therapeutic effect in particular in the treatment of cancer, e.g., a decrease in the number of cancer cells, or an increase in the induced cell death of cancer cells, or a cancer cell population.
  • the present invention also concerns the use of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment of cancer as described herein and preferably for treatment of an epithelial cell cancer, including but not limited to the abnormal proliferative growth in of any one or more of the following organs and tissues: lung, bone, pancreatic, skin, head or neck, eye, uterus, ovary, rectum, anal region, stomach, colon, breast, fallopian tubes, endometrium, cervix, vagina, vulva, lymph including Hodgkin's and non-Hodgkin's and lymphocytic lymphomas, esophagus, small intestine, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, urethra, penis, prostate, blood including chronic or acute leuk
  • Non-limiting examples of specific cancers include lung cancer, colorectal
  • adenocarcinomas epithelial ovarian cancer
  • pancreatic cancer including endocrine and exocrine pancreatic cancers, the latter including pancreatic ductal adenocarcinomas
  • lung cancer include non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), carcinoids, cylindromas, and certain sarcomas.
  • Non-small cell lung cancers may optionally include any of squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma.
  • squamous cell carcinoma also called epidermoid carcinoma
  • adenocarcinoma adenocarcinoma
  • large cell carcinoma adenocarcinoma
  • bronchoalveolar lung cancer a non-limiting example of such lung cancers.
  • modulating refers to the effect induced by a molecule which interacts with a specific receptor to modify or control its biological activity.
  • modulation or “modulate” refers to an increase or decease in biological activity, as disclosed herein.
  • the half-lives of immunoglobulins are measured in days to weeks. Not surprisingly, the presence of monoclonal antibodies of the invention was still detectable in the serum one week following injection. The long in vivo half-life of the antibodies opens the possibility to expose patients such as or humans to these therapeutic agents for extended periods of time. Therefore this long term treatment brings unexpected therapeutic benefit, even after the postnatal period.
  • a further aspect of the present invention also concerns a method of treating cancer in a patient in need thereof.
  • the method of treating cancer comprises administering to the patient at least one isolated antibody, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof in an amount which is therapeutically effective.
  • monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention for the manufacture of a medicament for the prevention of cancer.
  • the present invention pertains to a pharmaceutical kit comprising at least an effective amount of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, together with instructions for use and in particular instructions directed to the treatment of cancer, e.g., NSCLC.
  • the isolated antibodies e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention.
  • the pharmaceutical kit according to the present invention may comprise a container comprising at least said isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof.
  • the kit comprises a container and a label or package insert on or
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the composition is used for treating the condition of choice, such as cancer, such as NSCLC.
  • kits may further comprise a second (or third)
  • a pharmaceutically-acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • Antibodies as disclosed herein that cross-react with mouse and human glyco- epitopes such as glycoproteins can also cross-react with glyco-epitopes of most mammalian species, and also with glyco-epitopes of other vertebrate species. If the antibodies do not cross- react with glyco-epitopes of a given mammalian or vertebrate species, it is possible to generate them according to the process described in this application by immunizing mice with a glyco- epitope that corresponds to that of the species of interest.
  • an isolated antibody e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, for the manufacture of a medicament for use in veterinary applications in any mammal or vertebrate species.
  • the medicament can be utilized in utero (for mammals) or in ovo (for birds, reptiles, egg-layer fishes, etc%), or in young animals, or in adult animals.
  • the treatment can be used to modulate the development of cells or tissues of ectodermal origin, such as hair, feathers, scales, horns, claws, beaks, teeth, skin, sweat glands, sebaceous glands, larynx and trachea mucus-producing cells, Meibomian glands, preputial glands, mammary glands and salivary glands,
  • the treatment can be used to temporarily or permanently modify hair, scale or feathers color and/or morphology.
  • the use of the anti-glyco-epitope antibodies or antibody mimetic in a medicament for the treatment of a cancer in a subject is contemplated.
  • the medicament can contain a therapeutically effective amount of the antibody.
  • the amount of a anti-glyco-epitope antibodies or antibody mimetic in the medicament is sufficient so that at least one beneficial result is observed, e.g., a lessening of a symptom.
  • the amount that is administered removes all of the symptoms of the cancer.
  • the amount is sufficient so that the level of a biomarker of cancer decreases in a subject after the medicament has been administered.
  • the amount of the antibody administered is about 0.001 to 1000, 0.1 to 100, 0.5 to 50, 1 to 10, 1, 3, or 10 mg of antibody/kg of subject.
  • the actual amount of the antibody can depend upon the particular disorder (e.g., type of cancer, grade of cancer, if it is malignant or non-malignant etc.), the method of administration, the frequency of administration, the desired result, the characteristics of the patient, and the characteristics of the antibody.
  • the use of the antibody in the preparation or manufacture of a medicament can involve any of the disclosed antibodies in any amount, sufficient to treat the particular condition it is directed to.
  • a medicament is prepared with one of the single chain anti-glyco-epitope antibodies selected from the group consisting of sc-Fv#l antibody, sc-Fv#8 antibody, sc-Fv#A7 antibody, sc- Fv#C2 antibody, sc-Fv#G2 antibody, and sc-Fv#H2 antibody.
  • the nature of the disorder can play a role in the amount, frequency, and method of administration.
  • relatively larger amounts, more potent antibodies, and/or more frequently administered doses of the antibody can be required.
  • the amount of antibody required for treatment can be relatively less.
  • lower amounts of the antibody can be beneficial compared to the amount required for subjects that are naturally allergic.
  • increased amounts of the antibody, as well as increased frequency of administration can be advantageous.
  • the exact amount can readily be determined by one of skill in the art, in light of the present disclosure.
  • One of skill in the art will further appreciate other factors and how to adjust the administration of the antibody accordingly.
  • the isotype of an anti-glyco-epitope antibody can be switched, for example to take advantage of a biological property of a different isotype.
  • isotypes of antibodies that are capable of the same, including, without limitation, the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgGl, and human IgG3. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather, the antibody as generated can possess any isotype and the antibody can be isotype switched thereafter using conventional techniques that are well known in the art.
  • Such techniques include the use of direct recombinant techniques (see e.g., U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418), among others.
  • an anti-glyco-epitope antibody as discussed herein is a mouse antibody.
  • the anti-glyco-epitope antibodies discussed herein can be human antibodies. If an antibody possessed desired binding to the glyco-epitope, it could be readily isotype switched to generate a human IgM, human IgGl, or human IgG3 isotype, while still possessing the same variable region (which defines the antibody's specificity and some of its affinity). Such molecule would then be capable of fixing complement and participating in CDC.
  • a myeloma or other cell line is prepared that possesses a heavy chain with any desired isotype and another myeloma or other cell line is prepared that possesses the light chain.
  • Such cells can, thereafter, be fused and a cell line expressing an intact antibody can be isolated.
  • Biologically active antibodies that bind glyco-epitopes are preferably used in a sterile pharmaceutical preparation or formulation to reduce the activity of glycomolecule.
  • Anti-glyco- epitope antibodies preferably possess adequate affinity to potently interfere with the binding of glycoprotein to another entity, e.g., protein or glycoprotein.
  • the antibody formulation is preferably sterile. This is readily accomplished by any method know in the art, for example by filtration through sterile filtration membranes. The modality of antibody administration is in accord with known methods.
  • Antibody mimetics are organic or inorganic compounds that, like antibodies, can
  • antibody mimetics encompass, but are not limited to nucleic acids, peptides or proteins, and in some embodiments, have a molar mass of about 3 to 20 kDa. Some antibody mimetics have an antibody-like beta-sheet structure. Antibody mimetics have several common advantages over antibodies, which include, but is not limited to, increased solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. Antibody mimetics are being developed as therapeutic and diagnostic agents (see Gebauer et al., "Engineered protein scaffolds as next-generation antibody therapeutics". 2009, Curr Opin Chem Biol.). In some embodiments, the antibody for use in the methods and compositions as disclosed herein can be any antibiotic mimetic as disclosed in Table 3.
  • a glycomoleeule b d g entity is an AFFIBODY®.
  • Affibody® Molecules mimic monoclonal antibodies, Compared to antibodies, the most striking dissimilarity of Affibody® Molecules is the small size,
  • Affibody® Molecules have a molecular weight of 6kDa, compared to the molecular weight of antibodies, which is 150kDa. In spite of its small size, the binding site of Affibody® Molecules is similar to that of an antibody.
  • Affibody® Molecules over antibodies are: (i) their small size, (ii) he simple structure of the molecules (iii) its robust physical properties; able to withstand a broad range of analytical conditions, including extreme pH and elevated temperature (iv) its ability to fold correctly intracellularly (iv) Conjugation or directed coupling to matrices are facilitated by the unique C-terminal cysteine.
  • Affibody® Molecules have highly competitive properties for applications within affinity purification, sample preparation and protein detection.
  • Affibodies are available commercially, for example from the sweedish company Affibody AB,
  • Affilins are a type of antibody mimetic that are genetically engineered proteins with the ability to selectively bind antigens. They are structurally derived from one of two proteins, gamma-B crystallin or ubiquitin, both occurring in humans. Affilins are constructed by modification of near- surface amino acids of these proteins and isolated by display techniques such as phage display. They resemble antibodies in their affinity to antigens but not in structure, which makes them a type of antibody mimetic. Affilins are being developed as potential new biopharmaceutical drugs.
  • artificial proteins with the ability to selectively bind antigens. They are structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaea domain. By randomizing the amino acids on the binding surface of Sac7d and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards various proteins.
  • Affitins are antibody mimetics and are being developed as an alternative to antibodies as tools in biotechnology. They have also been used as specific inhibitors for various enzymes, (see Krehenbrink, et al., (2008). "Artificial binding proteins (Affitins) as probes for conformational changes in secretin PulD". J. Mol Biol., 383 (5): 1058-68.
  • Anticalins are a type of antibody mimetic and are artificial proteins that are able to bind to antigens, either to proteins or to small molecules (Skerra A "Alternative binding proteins: anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities". FEBS J.2008: 275(11): 2677-83. They are not structurally related to antibodies, which makes them a type of antibody mimetic. Instead, they are derived from human lipocalins which are a family of naturally binding proteins. Anticalins are being used in lieu of monoclonal antibodies, but are about eight times smaller with a size of about 180 amino acids and a mass of about 20 kDa.
  • Anticalins have better tissue penetration than antibodies and are stable at temperatures up to 70 °C. Unlike antibodies, they can be produced in bacterial cells like E. coli in large amounts. While antibodies and most other antibody mimetics can only be directed at macromolecules like proteins, anticalins are able to selectively bind to small molecules as well. Characteristic for anticalins is their barrel structure formed by eight antiparallel ⁇ -strands pairwise connected by loops and an attached a-helix. The main structure of anticalins is identical to wild type lipocalins. Conformational deviations are primarily located in the four loops reaching in the ligand binding site. Mutagenesis of amino acids at the binding site allows for changing the affinity and selectivity.
  • Avimers typically consist of two or more peptide sequences of 30 to 35 amino acids each, connected by linker peptides.
  • the individual sequences are derived from A domains of various membrane receptors and have a rigid structure, stabilised by disulfide bonds and calcium.
  • Each A domain can bind to a certain epitope of the target protein.
  • the combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity (hence the name).
  • the domains of avimers useful herein can be directed against glyco- epitopes on different target glycomolecules. This approach is similar to the one taken in the development of bispecific monoclonal antibodies.
  • the plasma half-life of an anti- interleukin 6 avimer could be increased by extending it with an anti-immunoglobulin G domain. (Silverman, et al., (2005). "Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains". Nature Biotech 23 (12): 1556.
  • Avimers with two or three domains can bind to their targets glycomolecules in sub- nanomolar concentrations. They have improved heat stability compared with antibodies, but limited plasma half-life because of their smaller size. Half-life can be increased by binding them to antibodies.
  • Avimers can be produced in a library.
  • a library theoretically containing up to 1023
  • a domains targeting the desired glycolmolecule or glycol-epitope are selected with display techniques such as phage display.
  • the most promising species are linked to a second A domain via a short linker peptide, forming a new library. This process can be repeated several times, yielding avimers with an increasing number of domains.
  • DARPins an acronym for designed ankynn repeat proteins
  • DARPins are derived from natural ankyrin proteins and consist of at least three, usually four or five repeat motifs of these proteins. Their molecular mass is about 14 or 18 kDa (kilodaltons) for four- or five-repeat DARPins, respectively.
  • DARPins are derived from naturally occurring ankyrin proteins, a protein class that is mediating high-affinity protein-protein interactions in nature.
  • DARPins are composed of repetitive structural units forming a stable protein domain with a large potential target interaction surface. Typically, DARPins are composed of four or five repeats, corresponding to the average size of natural ankyrin repeat protein domains.
  • Monobodies also known as adnectins, are a type of antibody mimetic and are genetically engineered proteins that are able to bind to antigens. Despite their name, monobodies are not structurally related to antibodies.
  • Monobodies consist of 94 amino acids and have a molecular mass of about 10 kDa, fifteen times smaller than an IgG type antibody and comparable to the size of a single variable domain of an antibody. They are based on the structure of human fibronectin, more specifically on its tenth extracellular type III domain. This domain has a structure similar to antibody variable domains, with seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three
  • Monobodies lack binding sites for metal ions and the central disulfide bond.
  • Monobodies with specificity for different proteins can be tailored by modifying the loops BC (between the second and third beta sheets) and FG (between the sixth and seventh sheets) (Koide A et al., "The fibronectin type III domain as a scaffold for novel binding proteins”. J. Mol. Biol.
  • carcinomas Cancers that originate in epithelial tissue (cellular tissue that lines cavities such as the stomach or lung) are called carcinomas.
  • Epithelial tumors have a common biological profile and mechanism, such that antibodies or fragments thereof which are suitable for detection or treatment of one type of epithelial cancer, such as lung cancer, may also be suitable for other types of cancers.
  • TRAF4 protein overexpression was evaluated whether TRAF4 protein overexpression exists in other cancer types. Immunohistochemistry analysis of tumor samples from 623 patients with 20 different tumor types showed that TRAF4 was overexpressed in 268 tumors (43%), including 82 of 137 lung adenocarcinomas (60%). TRAF4 protein overexpression was limited to cancer cells and the subcellular localization was consistently cytoplasmic in a large majority of cases.
  • biomarkers related to the oncogenic process that are ubiquitously expressed by most malignancies with high sensitivity and specificity for conventional histopathologic evaluation by targeting genes whose expression is critical for invasion, metastatization, and cell survival.
  • Non-limiting examples of such other types of cancers include colorectal
  • adenocarcinomas epithelial ovarian cancer
  • pancreatic cancer including endocrine and exocrine pancreatic cancers, the latter including pancreatic ductal adenocarcinomas
  • prostate cancer pancreatic cancer
  • embodiments of the present invention may optionally be tested according to a suitable assay as is known in the art for the above cancers.
  • antibodies Beside the therapeutic use of antibodies, for example for the treatment of diseases, such as cancer, these antibodies also constitute invaluable analytical tools. Most of these antibodies show cross-reactivity with mouse and human cancer cells. Monoclonal antibodies, according to at least some embodiments of the invention are also able to detect human, rat, rabbit and dog cancer cells. These antibodies were successfully tested in flow cytometry and Western blotting applications. The use of these reagents in immunohistochemistry is of real importance as a diagnostic tool, both for in vitro and in vivo diagnostic methods.
  • the antibodies as disclosed herein for use in diagnosis may optionally feature a ligand or tag.
  • the ligand or tag may optionally comprise polyethylene glycol (PEG), a nanoparticle or particles, or a label.
  • PEG polyethylene glycol
  • the label can be detected in vitro and/or in vivo.
  • detecting the binding of an antibody as disclosed herein to a cell in a biological sample e.g., a serum or tissue sample is useful for in vitro diagnostic assays.
  • the detection of the binding of an antibody as disclosed herein to a cell present in a subject is useful for in vivo diagnostic assays.
  • Radio-imaging methods may optionally be used for in vivo diagnosis according to at least some embodiments of the present invention. These methods include but are not limited to, computer tomography (CT), magnetic resonance imaging (MRI) and spectroscopy (MRS), single photon emission computed tomography (SPECT), and positron emission tomography (PET). All of these techniques are non-invasive, and can be used to detect and/or measure a wide variety of tissue events and/or functions, such as detecting cancerous cells for example. Unlike PET, SPECT can optionally be used with two labels simultaneously. SPECT has some other advantages as well, for example with regard to cost and the types of labels that can be used. For example, US Patent No.
  • In vivo fluorescence imaging resembles fluorescence microscopy in that both use a low-light camera and appropriate filters to collect fluorescence emission light from samples.
  • the objects for imaging are typically the whole-body of small animals instead of cells in culture dishes or on slides. This extension into the in vivo setting allows visualization of biology in its intact and native physiological state.
  • NIR fluorophores small-molecule fluorophores and quantum dot (QD) nanoparticles
  • QD quantum dot
  • This type of molecule can be efficiently used to visualize and investigate in vivo molecular targets because most tissues generate little NIR fluorescence.
  • the most common organic NIR fluorophores are polymethines. Among them, pentamethine and heptamethine cyanines comprising
  • particles may optionally comprise one or more of solid nanospheres microencapsulated with different dye compounds and/or magnetic properties, which may optionally be prepared from a variety of functional polymeric materials, e.g. polyacrolein, polyglutaraldehyde, polymethyl a-(hydroxymethyl) acrylate,
  • uniform magnetic nanoparticles which may optionally comprise one or more of organic-inorganic hybrid particles composed of cores from micron-sized uniform polystyrene particles and shells from magnetite- silica nanospheres of approximately 30 nm diameter; and/or biodegradable, non-toxic, magnetic metal oxide (i.e. Fe 3 0 4 ) nanoparticles of very narrow size distribution in sizes ranging from approximately 20 nm up to 0.5 microns; or non-magnetic and magnetic silica hollow micron-sized particles.
  • Such nanoparticles may optionally be prepared as described for example with regard to US Patent No. 6,103,379, which is hereby incorporated by reference as if fully set forth herein.
  • any of the above described antibodies and fragments thereof may optionally be used in combination with an antibody, fragment thereof or other agent capable of detecting the presence of a known diagnostic marker to form a diagnostic panel.
  • diagnostic markers which may optionally be combined in the diagnostic panel are given below.
  • HCG Human chorionic gonadotropin
  • CA 19-9 This marker is associated with cancers in the colon, stomach, and bile duct. Elevated levels of CA 19-9 may indicate advanced cancer in the pancreas, but it is also associated with noncancerous conditions, including gallstones, pancreatitis, cirrhosis of the liver, and cholecystitis.
  • CA 15-3 are also associated with cancers of the ovary, lung, and prostate, as well as noncancerous conditions such as benign breast or ovarian disease, endometriosis, pelvic inflammatory disease, and hepatitis. Pregnancy and lactation also can raise CA 15-3 levels.
  • NSE Neuron- specific enolase
  • a heavy chain variable domain sequence comprising 1, 2, or 3 of the heavy chain CDR amino acid sequences described herein can be screened against a library of light chain variable domain sequences to obtain antibodies that bind human and/or mouse glyco-epitopes such as glycoproteins which are the binding sites for lectins.
  • a light chain variable domain sequence comprising 1, 2, or 3 of the light chain CDRs described herein can be screened against a library of heavy chain variable domain sequences to obtain antibodies that bind human and/or mouse glyco-epitopes such as glycoproteins which are the binding sites for lectins.
  • this methodology can be used to humanize any known antibody.
  • a non-human variable domain sequence can be screened against human variable domain sequences and then the identified human variable domain sequences screened against a second set of human variable domain sequences.
  • fusion protein refers to a polypeptide which comprises protein domains from at least two different proteins.
  • a fusion protein may comprise an antigen-binding portion or fragment of an antibody and a non-antibody protein.
  • a wide variety of ligands or tags can be coupled (i.e. linked) with the antibodies described herein.
  • the antibodies of the invention can be conjugated to either other peptides or other molecules to tailor, for example, the
  • the antibodies as disclosed herein can be coupled to form a fusion protein with a molecule or peptide to provide an additional functionality to the antibody, for example, enhanced therapeutic function such as coupling to a toxin.
  • conjugation with ligands and tags is discussed in reference to antibodies herein, it is to be understood that antibody fragments and antigen binding portions and fragments are also amenable to conjugation with ligands and tags.
  • an antibody as disclosed herein can be fused with another molecule, for example, an antibody can be fused to at least one or more additional molecules, for example for therapeutic use and/or diagnostic use of antibodies.
  • an isolated antibody e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof can be fused or conjugated to different proteins or molecules, for example to produce, but not limited to, bi-specific antibodies, antibody-drug conjugates (ADCs), antibody-radioisotope conjugates (e.g., radioimmunoconjugates), antibody-toxin fusion proteins (e.g., immunotoxins), antibody-enzyme fusion proteins for prodrug activation in Antibody Directed Enzyme Prodrug Therapy (ADEPT), and antibodies used for targeting gene delivery or drug-delivery systems.
  • ADCs antibody-drug conjugates
  • antibody-radioisotope conjugates e.g., radioimmunoconjugates
  • an isolated antibody e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof can be fused or conjugated to different ligands, including but not limited to, naturally occurring molecules, in some embodiments, a ligand can be a recombinant or synthetic molecules.
  • Exemplary ligands include, but are not limited to, polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG] 2 , polyvinyl alcohol (PVA), polyurethane, poly(2- ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidom
  • psoralen mitomycin C
  • porphyrins e.g., TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g., EDTA
  • lipophilic molecules e.g, steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimeth
  • biotin e.g., biotin
  • transport/absorption facilitators e.g., naproxen, aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of
  • vitamins e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal
  • vitamin cofactors e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal
  • vitamin cofactors e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal
  • vitamin cofactors e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal
  • vitamin cofactors e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin
  • Ligands can be used for any number of reasons including, but no limited to,
  • a targeting ligand can provide enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment.
  • a PK modulating ligand can modulate pharmacokinetics of an antibody in vivo.
  • the antibody of the invention is conjugated with a label/tag, such as a fluorescent label or a biotin label.
  • a label/tag such as a fluorescent label or a biotin label.
  • the ligand can be attached to the antibody via a linker that can be is easily cleavable under the appropriate conditions.
  • Such conditions can include acid or basic pH, heating, sonication, enzymatic cleavage, and the like.
  • label refers to a composition capable of producing a detectable signal indicative of the presence of a target. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein. For example, the antibody can be labeled with a detectable tag which can then be detected using an antibody specific to the label.
  • Exemplary fluorescent labels include, but are not limited to, Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester, Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red,
  • Allophycocyanin an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7.
  • Alexa Fluor 350 Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alex
  • the ligands can be conjugated, either directly or through a linker, to the N-terminal, C-terminal, or the amino acid side chains of the heavy and/or light chain of the antibody.
  • a ligand can be present on an amino acid when said amino acid is incorporated into the antibody heavy and/or light during synthesis.
  • the ligand can be incorporated via coupling to a "precursor" amino acid after said "precursor" amino acid has been incorporated into the antibody heavy and/or light chain.
  • a ligand having an electrophilic group e.g., a pentafluorophenyl ester or aldehyde group, can be conjugated to the N-terminal of heavy and/or light chain of the antibody.
  • a monomer having a chemical group suitable for taking part in Click Chemistry reaction can be incorporated, e.g., an azide or alkyne group.
  • a ligand having complementary chemical group e.g., an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.
  • the covalent linkages between the antibody and a ligand are mediated by a linker. This linker can be cleavable linker or non-cleavable linker, depending on the application.
  • a "cleavable linker” refers to linkers that are capable of cleavage under various conditions. Conditions suitable for cleavage can include, but are not limited to, pH, UV irradiation, enzymatic activity, temperature, hydrolysis, elimination and substitution reactions, redox reactions, and thermodynamic properties of the linkage. In some embodiments, a cleavable linker can be used to release the antibody after transport to the desired target. The intended nature of the conjugation or coupling interaction, or the desired biological effect, will determine the choice of linker group.
  • non-peptide linker means an organic moiety that
  • Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR 1 , C(O),
  • alkenylarylalkenyl alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl,
  • alkynylarylalkynyl alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,
  • alkynylheteroarylalkyl alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
  • alkylheterocyclylalkyl alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,
  • alkenylheterocyclylalkyl alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
  • alkynylheterocyclylalkyl alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S(O), S0 2 , N(R 1 ) 2 , C(O), cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R 1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • the two parts of the compound can be linked together by providing on each part of the molecule complementary chemical functionalities that undergo a coupling reaction.
  • linkers can be non-covalent coupling of two parts of a
  • non-covalent coupling can be achieved through, for example, ionic interactions, H-bonding, van der Waals interactions and affinity of one molecule for another.
  • each part of the compound can be conjugated with a moiety that has complementary to another moiety that is conjugated to the second part of the compound.
  • complementary coupling is the
  • biotin/avidin coupling Other examples include but are not limited to affinity of an
  • oligonucleotide for its complementary strand, receptor/ligand binding, aptamer/ligand binding and antibody/antigen binding.
  • One conjugation strategy is the biotin- sandwich method (Davis, et al., Proc. Natl.
  • a peptide is biotinylated and bound to biotinylated ligand using tetravalent streptavidin as a linker.
  • the peptide may be coupled to the 15 amino acid sequence of an acceptor peptide for biotinylation
  • the fusion proteins can be made by incorporating the extra sequences at the N- or the C-terminus of the peptide.
  • the acceptor peptide sequence allows site- specific biotinylation by the E. coli enzyme biotin ligase (BirA; Chen, et al., Nat. Methods 2:99-104 (2005)).
  • a ligand peptide can be similarly biotinylated for conjugation with a peptide described herein. Many commercial kits are available for biotinylating proteins.
  • Non-peptidyl ligands agents can also be conjugated with biotin using methods well known in the art for conjugating biotin to non-peptide molecules, e.g. small organic molecules. In order to prevent steric interference between the biotin/avidin groups and the peptides or the ligands, a spacer may be included between them. [00322]
  • the linkers and linking methods described herein can also be used for linking together heavy chain and light chain of an antibody, two or more Fv domains, and fragments thereof.
  • chemical moieties are defined and referred to throughout can be univalent chemical moieties (e.g., alkyl, aryl, etc.) or multivalent moieties under the appropriate structural circumstances clear to those skilled in the art.
  • alkyl refers to saturated non-aromatic hydrocarbon chains that may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include without limitation methyl, ethyl, propyl, allyl, or propargyl), which may be optionally inserted with N, O, S, SS, S0 2 ,C(0), C(0)0, OC(O), C(0)N or NC(O).
  • C C 6 indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it.
  • alkenyl refers to an alkyl that comprises at least one double bond.
  • alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl and the like.
  • aryl refers to monocyclic, bicyclic, or tricyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
  • Examplary aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12
  • bicyclic or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
  • O, N, or S e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively
  • Examplary heteroaryl groups include, but are not limited to, pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, and the like.
  • cyclyl refers to saturated and partially
  • unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted.
  • exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and the like.
  • heterocyclyl refers to a nonaromatic 5- 8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent.
  • Examplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
  • optionally substituted means that the specified group or moiety, such as an alkyl, aryl group, heteroaryl group and the like, is unsubstituted or is substituted with one or more (typically 1-4 substituents) independently selected from the group of substituents listed below in the definition for "substituents" or otherwise specified.
  • substituted refers to a group “substituted” on an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, acyl, amino group at any atom of that group.
  • Suitable substituents include, without limitation, halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkylthio, CF 3 , N- morphilino, phenylthio, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.
  • substituent can itself be optionally substituted.
  • two substituents, together with the carbons to which they are attached to can form a
  • halo refers to any radical of fluorine, chlorine, bromine or iodine.
  • acyl refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,
  • acyl groups include, but are not limited to, (Cp C 6 )alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t- butylacetyl, etc.), (C 3 - C6)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5 - carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl)
  • alkanoyl e.g., formyl, acetyl, propionyl, butyryl,
  • alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be any one of the groups described in the respective definitions.
  • alkoxy refers to an -O-alkyl radical.
  • aminoalkyl refers to an alkyl substituted with an amino
  • mercapto refers to an -SH radical.
  • thioalkoxy refers to an -S-alkyl radical.
  • haloalkyl refers to an alkyl group having one, two, three or more
  • halogen atoms attached thereto are attached thereto.
  • exemplary haloalkyl groups incude are not limited to chloromethyl, bromoethyl, trifluoromethyl, and the like.
  • scFvs described in the following examples were expressed in Escherichia coli bacteria in the form of maltose-binding protein-scFv fusion proteins (MBP-scFv as described in Bach, H., Mazor, Y., Shaky, S., Shoham-Lev, A., Berdichevsky, Y., Gutnik, D.L. and
  • the lectins used in methods include plant lectins such as Peanut Agglutinin (PNA), Ulex europaeus agglutinin (UEA), Soybean agglutinin (SBA), Dolichos biflorus (DBA), lotus tetragonolobus (LTL), Vicia villosa lectin (VVA), Maackia amurensis lectin (MAA) and Phaseolus vulgaris agglutinin (PHA); and animal lectins including Helix aspersa agglutinin (HA A) and Helix pomatia agglutinin (HP A).
  • PNA Peanut Agglutinin
  • UDA Ulex europaeus agglutinin
  • SBA Soybean agglutinin
  • DBA Dolichos biflorus
  • LTL lotus tetragonolobus
  • VVA Vicia villosa lectin
  • MAA Maackia
  • Immunohistochemistry was performed as follows. Normal human fibroblasts and for A549 NSCLC cells, 3xl0 6 cells per sample, were seeded on glass cover slip in 6 wells plate. 24 hours later, immunohistochemistry protocol was applied as describe in methods, using scFv fragments. The staining was detected via peroxidase protocol.
  • Tissue sections were deparaffinized and rehydrated to water.
  • Cell Viability Assay was performed on NSCLC A549 cell line. Cells were seeded in 96 well plates (2x103 cells/well) in 10% FCS growing DMEM media. Following 24 hours, cells were treated with scFv # 1 Fragment complexed with mouse anti MBP to form bivalent complexes in serum free medium for 6 hours. The growth medium was adjusted to 10% FCS. Cell viability was determined by methylene blue assay following 95 hours treatment.
  • MB methylene blue
  • the cells were washed with tap water and cell-bound dye was eluted with 200 ⁇ (microliter) 0.1M HC1. Eluted methylene blue was read in an ELISA plate reader at 595 nm. Methylene blue binds DNA in basic solution, and it is extracted in acidic condition. Staining of DNA by Methylene Blue correlates with cell survival.
  • Tissue sections were deparaffinized and rehydrated to water.
  • Table 4 represents the isolated scFv fragments as having binding properties to A549 cells as the indicated lectin. Each fragment was analyzed for its competition binding with the specified lectin and corresponding competing sugar molecule: [00380] Table 4: example of the isolated scFv fragments according to their lectin selection
  • Figure 1 shows micrographs of immunohistochemical staining of cells and specimens with scFv fragments as follows.
  • Figure 1A shows staining of normal human fibroblasts with the different scFv fragments.
  • Figure IB shows staining of NSCLC A549 cells with scFv fragments.
  • Figure 1C shows staining of NSCLC human tumor specimens with scFv fragments.
  • Figure 2 shows quantitative binding of scFv fragments to NSCLC A549 cells.
  • Figure 3A shows survival of NSCLC A549 cells after treating the cells with MBP- scFvl fragments and 0.08uM antiMBP
  • Figure 3B represents the survival of the cells after treatment with scFvland three different concentration of antiMBP (0.08uM, 0.165uM and
  • the scFv fragments demonstrate specific and strong cell growth inhibition of these cancer cells by actually reducing cell survival. These results suggest that these human scFv fragments or their F(ab), F(ab)2, full human derived antibody or any molecule that is based on these scFv fragments can be use for therapeutics to treat human cancer diseases.
  • This Example relates to specific binding of scFv fragments according to at least some embodiments of the present invention to cancerous tissue samples as opposed to normal (non-cancerous) tissue samples.
  • Paraffin embedded and fixed tissue samples were provided on slides in sections of about 5-10 ⁇ , including samples of both tumors and normal tissue from affected individuals, were obtained from the pathology department at a local hospital, under Helsinki regulations and approval.
  • the mounted sections of tissue then underwent blocking and staining using the ACUITY® advanced biotin free HRP polymer detection system (SIG-32904) kit from CO VANCE Inc (USA), according to the manufacturer's instructions.
  • ACUITY advanced reagent 1 serum blocker
  • MBP Methyl Binding Protein
  • Mouse anti MBP antibodies (1:1000 dilution, previously described) were added to slides, followed by incubation for 30 min at RT, followed by three washes with PBS. The slides were than incubated with ACUITY advanced reagent 2 (boost) 20min at RT followed by three washes with PBS. For specific binding detection, the slides were further incubated for 15min with ACUrfY advanced reagent 3 (HRP polymer).
  • ELISA binding assay Cells (A549 or LS-174-T) were plated in each well of 96 wells plate. The next day, cells were fixed with 4% formaldehyde lh and washed twice with PBS, cells were incubated with 3% H202 30min then blocked with 1% BSA, follow by incubation with scFvs or scFvs-IO-NIR, lh at room temperature. Cells were washed with PBSxl, then incubated with mouse anti MBP (1:1000), washed and incubated with goat anti mouse -HRP (1:1000). After washing the cells. TMB substrate was added. Reaction was stopped with 0.5M H2S04 read at OD 450nm.
  • Table 5 presents the binding intensity of all 5 scFvs to NSCLC specimens and normal adjacent lung specimens as evaluated after immunohistochemical staining and to normal adjacent lung tissue. Intensity of staining was described as weak (+), moderate (++), strong (+++) or non- stained (-). Each of the tested specimens were positively stained with at list one of the scFvs. The staining pattern with all five scFvs was different from patient to patient. The binding of the scFvs to the normal lung specimens was very weak as shown.
  • Adenocarcinoma de 1 1 1 02 ⁇ - -
  • each of the immune molecules scFvl, scFv8, scFvG2, scFvC2 and scFvH2 bound to at least certain non- small cell lung cancer samples, with little to no binding to the adjacent "normal" tissues.
  • each of the immune molecules scFvl, scFv8, scFvG2, scFvC2 and scFvH2 for diagnosis and optionally also treatment of non- small cell lung cancer, as well as combinations thereof.
  • a non-limiting optional example of such a combination is the combination of scFvl and scFvG2.
  • FIG. 4 shows example for immunohistochemical staining tumor array (TMA) contain samples from 32 colorectal patients and breast cancer with scFvs.
  • TMA tumor array
  • Figure 5 shows immunostaining of paraffin embedded colorectal specimens from two human patients with scFv8, demonstrating that the cancerous tissue is specifically bound while normal tissue is not bound.
  • Figure 6 shows that normal apparent tissue found close to the tumor is stained while normal tissue far from the tumor is not stained indicating that the scfvs may detect cancer progression in a very early stage. Staining was performed on paraffin embedded tumor specimens, immunostained with scFv8. The photographs are identified as follows: photograph no. 4 is of tissue taken from the tumor area, photograph no. 3 is of tissue taken from the peripheral area of the tumor, photograph no. 2 is of tissue taken near the tumor area and photograph no. 1 is of tissue taken far from the tumor.
  • Table 7 shows binding of four scFv fragments, scFvV8, scFvG2, scFvC2 and scFvH2 (described below as V8, G2, C2 and H2, respectively) to human colorectal and adjacent normal control specimens through immunohistochemistry staining. The results show that 91% (out of 12 tested) of colorectal cancer specimens were stained with scFv8, while 75% of the normal tissues were not stained.
  • Table 7 binding to human colorectal and adjacent normal control specimens.
  • each of the immune molecules scFv8, scFvG2, scFvC2 and scFvH2 bound to at least certain colorectal cancer samples, with little to no binding to the adjacent "normal" tissues.
  • scFvG2 alone bound to nearly all cancer samples, while those samples to which it did not bind were generally bound by scFv8. Therefore according to at least some embodiments of the present invention, there is provided use of each of the immune molecules scFv8, scFvG2, scFvC2 and scFvH2 for diagnosis and optionally also treatment of colorectal cancer, as well as combinations thereof.
  • a non-limiting optional example of such a combination is the combination of scFv8 and scFvG2.
  • Figure 7 shows binding of scFv fragments to LS-174-T cells. Such binding is necessary in order to use scFv fragments as a bio-probe in animal model. All scFv fragments bound to LS-174-T cell line as demonstrated through the ELISA assay (the assay was performed as described in Example 1). scFv8 binding intensity is two fold higher.
  • Figure 8 shows immunohistochemistry binding to such cells as for Figure 7. Again, the results confirm that all fragments can be used in a mouse experimental model.
  • scFv8-Iron Oxide NIR (scFv8-IO-NIR) conjugant also binds specifically to colon adenocarcinoma, as indicated by the specific binding to colon adenocarcinoma specimen in compare to adjacent normal control.
  • Figure 9 shows the binding intensity of scFv8-IO-NIR to A549 cell line.
  • the resultant peroxidase activity was measured at OD 450nm.
  • the graph presents the relative values of scFv8 at concentration of lOug/ml and lug/ml and scFv8-IO-NIR in dilution of 1/2 1/4 and 1/8 to control secondary antibody.
  • Figure 10 shows the binding of scFv8 to human colon cancer specimen and adjacent normal control as indicated by immunohistochemistry (C- normal adjacent, D- colon adenocarcinoma), and binding of scFv8-IO-NIR to the same patient specimen (A- light scanning of normal adjacent and adenocarcinoma specimen B- 720/780nm scanning of normal adjacent and adenocarcinoma specimen.
  • Table 8 shows the extent to which staining of normal tissue occurred with scFv8. staining of normal tissue with scFv8
  • This Example relates to in vivo experiments performed with the antibody fragments according to at least some embodiments of the present invention.
  • CDl-Nude mice were implanted with 2*10 ⁇ 6 cells/30 micro-liter LS-174T cells in the colon. These cells induced expression of orthotropic human colon cancer tumors. 14-17 days post transplantation, mice were used for the in- vivo/ex- vivo experiments described herein, with the immunoreactive fragment scFv8, conjugated to albumin and to a dye fluorescing in the near infrared spectrum.
  • HAS(human serum albumin )-NIR (near infrared)- scFv8 in a concentration of lOOug/ml was inserted into the lumen of anesthetized mice and was incubated for 20 minutes. The lumen was washed with 5ml lxPBS, and sacrificed 3h after the final wash. The colon was then removed and imaged by using a fluorescence scanner (Odyssey).
  • FIG. 11 shows images of colons taken from mice with colonic tumors after administration of scFv8-HAS-NIR.
  • Results are shown from two mice, designated as animals 62 and 63.
  • the immunoreactive molecules, such as scFv8, according to at least some embodiments of the present invention are suitable for imaging, for example of tumors, whether performed in vivo or of ex vivo tissue.
  • each lectin PNA, SBA and UEA were used for a self competition (data not shown). Indeed each lectin competed with itself but not with other lectins or BSA, except SBA which showed competition with itself and with PNA.
  • the target glycans of scFvl are N-acetylglucosamine, Galactose and Fucose.
  • the target glycans of scFv8 are N-acetylglucosamine and Fucose.
  • the target glycans of scFvH2 are Fucose and N-acetylglucosamine.
  • a method of treatment of cancer in a subject comprising determining if the cancer expresses a glyco-molecule which binds to N-acetylglucosamine, in which case the cancer may optionally be treated with one or more of scFvl, scFv8 or scFvH2; binds to fucose, in which case the cancer may optionally be treated with one or more of scFv8 or scFvH2; and/or binds to galactose in which case the cancer may optionally be treated with scFvl.
  • the above immune molecules may optionally be used for diagnosis of cancer according to the above binding.

Abstract

The present invention is related to monoclonal antibodies, fragments, antigen binding portions or fragments thereof, for specific binding to human and/or mouse tumor associated carbohydrate antigens expressed on cancer cells, e.g., human colorectal adenocarcinma, non-small cell lung carcinoma (NSCLC). These antibodies can be used to induce selective cell death in cancer cells and for the treatment of cancer, e.g., NSCLC and/or in the diagnosis of cancers expressing tumor associated carbohydrate antigens in-vitro and in-vivo.

Description

NOVEL ANTIBODIES AND METHODS OF USE FOR THE TREATMENT AND DIAGNOSIS
OF CANCER
FIELD OF THE INVENTION
[0001] The present invention is directed generally to compositions comprising substantially
purified antibodies, monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, uses of such antibodies and fragments thereof and methods of production thereof.
BACKGROUND OF THE INVENTION
[0002] As vital constituents of all living systems, carbohydrates are involved in recognition,
adherence, motility, and signaling processes. Protein glycosylation can affect protein folding, intracellular trafficking and localization, the rate of degradation and can determine their organizational framework within the cytoplasm, on the membrane and extracellularly.
Glycosylation of lipids was found to affect membrane rigidity and the function of membrane proteins such as growth factor receptors and integrins. Additionally, carbohydrate chains on proteins and lipids seems to play an important role in cell-to-cell interactions and interactions of cells with extra-cellular matrix and soluble molecules (Kornfeld, 1980).
[0003] Abnormal glycosylation is one of the hallmarks of the cancer cell and is associated with tumor invasion, metastasis and is involved in all stages of tumor progression. Alterations of cell surface carbohydrates are often observed as a result of malignant transformation and can be detected in the earliest stages of malignant transformation. The often-observed association between changes in tumor cell glycosylation and prognosis and survival of cancer patients suggests that alterations in tumor cell glycosylation patterns are an important part of tumor progression toward more malignant phenotype and metastatic phase (Dube, D.H. & Bertozzi, C.R. Nat. Rev. Drug Discov. 4, 477-488 (2005), Fuster, M.M. & Esko, J.D. Nat. Rev. Cancer 5, 526-542 (2005)) Recent results on cancer-associated glycans, indicated that the repressed transcription of some glycan genes by epigenetic silencing during early carcinogenesis, and the transcriptional induction of some other glycan genes by tumor hypoxia accompanying cancer progression at locally advanced stages, are two major factors determining cancer-associated glycan expression (Kannagi et al. Cancer Sci. 2010 Mar;101(3):586-93. Epub 2009 Dec 4). Glycans are very unique and advantageous as marker molecules because they are capable of
12987770.1 reflecting epigenetic silencing in their structures. Transcriptional induction of some glycan genes by tumor hypoxia at the later stages produces further glycan modifications, such as an unusual increase of the N-glycolyl sialic acid residues in the glycan molecules. The entire process of malignant transformation thus creates abnormal glycans, whose structures reveal the effects of both epigenetic silencing and tumor hypoxia. Glycans are often covalently bound to well-known cancer-associated proteins, such as CD44v, MUC protein family,
carcinoembryonic antigen (CEA), carcinoembryonic antigen-related proteins, PSA, CD24, ovarian cancer marker CA125 and others.
[0004] The glycosylation changes and modifications that occur in malignant cells can take a
variety of forms (in the Essentials of Glycobiology, 2nd edition Edited by Ajit Varki, Richard D Cummings, Jeffrey D Esko, Hudson H Freeze, Pamela Stanley, Carolyn R Bertozzi, Gerald W Hart, and Marilynn E Etzler. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.)
[0005] The classic reports of increased size of tumor cell glycopeptides have been convincingly explained by an increase in β1-6 branching of N-glycans, which results from an increased expression of GlcNAc transferase V (GlcNAcT-V). Clinical specimens of some human tumors show an increase in staining with the plant lectin L-PHA, which is known to preferentially recognize N-glycans bearing the GlcNAcT-V product. Most convincingly, transfection of GlcNAcT-V cDNA into cultured cells causes a visually obvious transformed phenotype associated with colony formation in soft agar, increased cell spreading, enhanced invasiveness through membranes, and tumorigenic behavior by previously nontumorigenic cells.
[0006] Changes in the amount, linkage, and acetylation of sialic acids in tumor cells was been observed.
[0007] This increase in sialylation is often manifested as specific increases in a2-6-linked sialic acids attached to outer lactosamine (Gaipi-4GlcNAc units) or to inner GalNAc-O-Ser/Thr units on O-glycans. Apart from the amount and linkage of sialic acids, there can also be significant changes in their modifications. Sialic acid 9-O-acetylation either can be up- regulated in melanoma cells.
[0008] A particularly interesting phenomenon is the aberrant expression of Neu5Gc in human tumor cells. This sialic acid differs from the common sialic acid N-acetylneuraminic acid (Neu5Ac) by the addition of a single oxygen atom. Adult humans do not express significant levels of Neu5Gc on their normal cells, and they mount an immune response to this epitope when infused with Neu5Gc-containing animal serum.
[0009] Significant changes in content of proteoglycans have been reported in the stroma
surrounding tumors, and it is suggested that these alterations can support tumor growth as well as progression and invasion. The levels of decorin, a leucine-rich proteoglycan capable of regulating matrix assembly and cell proliferation, are markedly elevated in the stroma of colon carcinomas.
[0010] Alpha2,6-sialyltransferase ST6GALNAC5 specifically mediates brain metastasis of breast cancer. The expression of ST6GALNAC5 in breast cancer cells enhances their adhesion to brain endothelial cells and their passage through the blood-brain barrier, which highlights the role of cell-surface glycosylation in organ-specific metastasis (Nature, June 2009).
[0011] Both the somatic and germ-line mutations in the gene encoding for polypeptide N- acetylgalactosaminyltransferase 12 (GALNT12) have been identified in individuals with colon cancer. Genetic defects in the O-glycosylation pathway in part underlie aberrant glycosylation in colon cancers and contribute to the development of a subset of these malignancies (PNAS, August 2009).
[0012] Many other alterations in glycosylation forms of proteins in various tissue cancers and specific proteins were observed to be specific to cancer cells and contribute to cancer phenotype and cancer progression.
[0013] There is general agreement that the early events in the evolution of neoplasia involve
alterations in oncogenes, tumor suppressor genes, mutator genes, and apoptosis-related pathways. Subsequent tumor growth, invasion, and metastasis involve the survival of the fittest cells, and it is therefore likely that the highly selective changes seen in tumor cell glycosylation have the greatest functional consequences in these later stages. Unfortunately, the intrinsic genetic instability and cellular heterogeneity of advanced tumors have previously made it difficult to determine the functional consequences of specific glycosylation changes. However, in most cancers, it is invasion and metastasis that finally kill the patient.
[0014] Identification and characterization of protein glycosylation and modification on cancer cells is expected to eventually contribute to the determination of cancer specificity and functional relevancy in cancer progression. Therefore, glycan molecules associated with tumor biomarkers that preferentially appear in cancers are of clinical importance as serum tumor markers, as target for in- vivo molecular imaging and serve as a target for cancer therapeutics. [0015] Among the major types of cancer for which better diagnostics and treatments are required are lung cancer and colorectal cancer. The two major categories of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Less common cancers of the lung are known as carcinoids, cylindromas, and certain sarcomas. Cancers in the lung may have metastasized from other primary sites, such as the breast, thyroid, or colon.
[0016] Non-small cell lung cancers are categorized into three types: squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma. These separate types are grouped together because, in early stages before the cancers have spread, they all can be treated surgically.
[0017] Tumors formed from squamous cells are usually found in the centre of the lung, either in a major lobe or in one of the main airway branches. They may grow to large sizes and form cavities in the lungs. When a squamous cell cancer metastasizes, it may reach the bone, adrenal glands, liver, small intestine, and brain. Squamous cell carcinoma is nearly always caused by smoking and used to be the most common cancer. It still makes up between 25% and 40% of all lung cancers.
[0018] Adenocarcinomas usually arise from the mucus-producing cells in the lung and in the
colorectal; about two-thirds of adenocarcinomas develop in the outer regions of the lung, while one-third develops centrally. Adenocarcinoma of the colorectal and NSCLC adenocarcinoma are the predominant lung and colon cancer-. In the lung, adenocarcinoma is usually a slow- growing cancer, but can be difficult to detect because the disease typically involves the periphery of the lung, resulting in fewer early symptoms than cancers that develop centrally, near the airways. When signs of the disease do occur, they may include painful breathing, shortness of breath, wheezing, and a persistent cough. Oftentimes, lung adenocarcinoma has already metastasized by the time any symptoms develop, resulting in an overall five year survival rate associated with the disease that is less than 20 percent. Secondary tumors most commonly form in the opposite lung, the brain, spinal cord, bones, liver, and adrenal glands. Additional symptoms related to tumor growth in these or other areas of the body sometimes develop before signs of the primary tumor.
[0019] Bronchoalveolar lung cancer is a subtype of adenocarcinoma. It develops as a layer of column-like cells on the lung and spreads through the airways, causing great volumes of sputum. This cancer also is increasing in incidence. [0020] Large Cell Carcinoma: Large cell carcinoma, which makes up about 10% to 20% of lung cancers, includes cancers that cannot be identified under the microscope as squamous cell cancers or adenocarcinomas.
[0021] Detection of lung cancer at an early stage is necessary for successful therapy and improved survival rates. Numerous potential DNA biomarkers such as hypermethylations of the promoters and mutations in K-ras, p53, and protein biomarkers; carcinoembryonic antigen (CEA), CYFRA21-1, plasma kallikrein Bl (KLKB1), Neuron- specific enolase, EGFR, etc. have been discovered as lung cancer biomarkers. Despite extensive studies thus far, few are turned out to be useful in clinic. Even those used in clinic do not show enough sensitivity, specificity and reproducibility for general use.
[0022] Kim et al. (Cancer Res 2007; 67: 7431- 7438) proposed four genes (CBLC, CYP24A, AKR1B10, and ALDH3A1) to be potential biomarkers for non-small-cell lung cancer patients. Two genes (CBLC and CYP24A1) are particularly promising. With respect to the histopathologic aspects, these genes were expressed in both adenocarcinoma and squamous cell carcinoma.
[0023] CBLC is a member of the Cbl family of multidomain signaling proteins with a tyrosine kinase binding domain and a RING finger domain. It is recruited to the epidermal growth factor (EGF) receptor (EGFR) on EGF stimulation and increases ubiquitination of EGFR, thereby down-regulating EGFR signaling. Mutations in the EGFR gene have been reported in non-small-cell lung cancer patients, especially in patients with adenocarcinoma, women, nonsmokers, and East Asians. CYP24A1 is a member of the cytochrome P450 superfamily of enzymes involved in drugmetabolism and synthesis of cholesterol, steroids, and other lipids.
[0024] Lectins are protein or glycoprotein substances, usually of plant origin, of non- immunoglobulin nature, capable of specific recognition of and reversible binding to, carbohydrate moieties of complex glycoconjugates without altering the covalent structure of any of the recognized glycosyl ligands. This group includes monovalent lectins (i.e. bacterial and plant toxins). These lectins bind to sugar moieties in cell walls or membranes and thereby change the physiology of the membrane to cause agglutination, mitosis, or other biochemical changes in the cell. Most lectins studied to date are multimeric, consisting of non-covalently associated subunits. A lectin may contain two or more of the same subunit, such as Con A, or different subunits, such as Phaseolus vulgaris agglutinin. [0025] Lectins are tools to explore a myriad of biological structures and processes. Because of the specificity that each lectin has toward a particular carbohydrate structure, even
oligosaccharides with identical sugar compositions can be distinguished or separated. Some lectins will bind only to structures with mannose or glucose residues, while others may recognize only galactose residues. Some lectins require that the particular sugar be in a terminal non-reducing position in the oligosaccharide, while others can bind to sugars within the oligosaccharide chain. The affinity between a lectin and its receptor may vary a great deal due to small changes in the carbohydrate structure of the receptor. All of these unique and desirable properties of lectins enable them to discriminate between structures and to isolate a glycoconjugate, cell or virus from a mixture or to study one process among several. Since virtually all biological membranes and cell walls contain glycoconjugates, all living organisms can be studied with lectins. Therefore, carbohydrate residues of the membrane glycoproteins can be detected using lectins due to their binding specificity to carbohydrates.
[0026] Lectin-binding studies are valuable for disease diagnosis and comparative purposes, for example to detect variations of lectins profile binding between normal and pathologic conditions of given tissues, different regions in the same organ, or homologous regions in specimens of different age, sex or species. Nonetheless, lectins lack the binding specificity of antibodies and may in fact bind to a number of carbohydrate epitopes with varying degrees of affinity.
[0027] Furthermore, lectins themselves can be toxic to humans. For example, certain lectins found in food, such as phytohaemagglutnin (kidney bean lectin, found in high concentrations in undercooked or raw kidney beans), may have toxic effects when ingested. Other lectins are deadly when administered, such as ricin (Ricinus communis agglutinin), which is a
hemagglutinin. Thus, the non-specific binding of lectins, or their toxicity, limits their use as a therapeutic or diagnostic agent.
SUMMARY OF THE INVENTION
[0028] The present invention, at least some embodiments, is directed to compositions comprising an isolated protein, which is optionally and preferably an immune molecule, optionally comprising one or more of substantially purified antibodies, monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, and uses of such antibodies and fragments thereof.
[0029] The term "antibody" as used herein may also optionally encompass isolated proteins and isolated immune molecules.
[0030] Immune molecules, including without limitation antibodies and antibody mimetics that specifically recognize one or more glyco-epitopes on a single glycomolecule or multiple glycomolecules are disclosed in accordance with some embodiments. Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
[0031] In some embodiments, the glycomolecule is recognized by a saccharide-binding agent, which is any agent that binds specifically to a carbohydrate-portion of a glycomolecule.
Suitable saccharide-binding agents include, e.g., lectins or modified variants such as non-toxic variants of lectins, antibodies and antibody mimetics that recognize carbohydrate-containing epitopes, and carbohydrate-modifying enzymes, such as glycosidases.
[0032] Lectins are proteins isolated from plants that bind saccharides. For the purpose of this application, the term "lectin" also encompasses saccharide-binding proteins from animal species (e.g. "mammalian lectins"). Examples of lectins include lectins isolated from the following plants: Conavalia ensiformis, Anguilla anguilla, Tritium vulgaris, Datura stramonium, Galnthus nivalis, Maackia amurensis, Arachis hypogaea, Sambucus nigra, Erythtina cristagalli, Sambucis nigra, Erythrina cristagalli, Lens culinaris, Glycine max, Phaseolus vulgaris Allomyrina dichotoma, Dolichos biflorus, Lotus tetragonolobus, Ulex europaeus, and Ricinus commurcis. Other biologically active compounds such as cytokines, chemokines and growth factors also bind glycomolecules, and hence, for the purposes of the present invention are considered to be lectins. Examples of glycosidases include a- Galactosidase, (3-Galactosidase, N-acetylhexosaminidase, a-mannosidase, β-mannosidase, and a-Fucosidase.
[0033] According to other embodiments of the present invention, there is provided a
pharmaceutical composition comprising isolated antibodies and isolated monoclonal antibody fragments or antigen binding portions or fragments thereof in a method of treating a disease or disorder, for example cancer or other proliferative disorder. [0034] According to still other embodiments of the present invention, there are provided a diagnostic kit and method comprising isolated antibodies, isolated monoclonal antibody fragments or antigen binding portions or fragments thereof for in vitro and in vivo diagnosis of a disease or disorder, for example cancer or other proliferative disorder, and methods of use thereof. Another aspect of the present invention provides kits comprising isolated antibodies, isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, for therapeutic use and/or diagnostic use, e.g., for use in in vitro and in vivo diagnostics to identify a cancer in a subject.
[0035] Optionally cancer comprises one or more of non-small cell lung carcinoma (NSCLC), colorectal cancer, breast cancer, adenocarcinomas and other solid tumors.
[0036] According to at least some embodiments, the present invention relates to monoclonal
antibodies, fragments, antigen binding portions or fragments thereof, which displace lectin binding on human and/or mouse tumor associated carbohydrate antigens expressed on cancer cells. For example, the inventors have used a lectin-displacement assay to identify monoclonal antibodies and complementary determining regions (CDRs) which bind to tumor associated carbohydrate antigens expressed on cancer cells.
[0037] Other aspects of the present invention relate to compositions, methods and use of the
antibodies to induce selective cell death of cancer cells and for the treatment of a cancer in a subject, e.g., NSCLC (non small cell lung cancer) and/or for use in the diagnosis of a cancer expressing tumor associated carbohydrate antigens. Non-limiting examples of such cancers include non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), carcinoids, cylindromas, and certain sarcomas. Cancers of the lung also include lung cancers which have metastasized from other primary sites, such as the breast, thyroid, or colon.
[0038] Non- small cell lung cancers are categorized into three types: squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma. Another non- limiting example of such lung cancers includes bronchoalveolar lung cancer.
[0039] According to other embodiments of the present invention, the cancer may optionally be related to the abnormal proliferative growth in of any one or more of the following organs and tissues: lung, bone, pancreatic, skin, head or neck, eye, uterus, ovary, rectum, anal region, stomach, colon, breast, fallopian tubes, endometrium, cervix, vagina, vulva, lymph including Hodgkin's and non-Hodgkin's and lymphocytic lymphomas, esophagus, small intestine, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, urethra, penis, prostate, blood including chronic or acute leukemia, bladder, kidney, the central nervous system (CNS) including spinal axis tumors, brain stem glioma; and pituitary.
[0040] A non-limiting aspect of the present invention provides a new process for the preparation of substantially purified and isolated antibodies that are biologically active, effective and functional and which optionally bind to the same antigens and epitopes on glycoproteins which also bind to one or more lectin molecules.
[0041] In one embodiment, the present invention relates to an immune molecule which is capable of interacting with a human and/or mouse glyco-epitope, of which non-limiting examples are antibodies, e.g. monoclonal antibodies, or isolated monoclonal antibody fragments, or antigen binding portions or fragments thereof which bind to human and/or mouse glyco-epitope, such as a glycoprotein for example, with an affinity (KD) of at least 10~6 M for the Fab fragment.
[0042] While KD describes the binding characteristics of an epitope and antibody, "potency" describes the effectiveness of the antibody itself as a function of the antibody. A relatively low KD does not automatically mean a high potency. Thus, antibodies can have a relatively low KD and a high potency (e.g., they bind well and alter the function strongly), a relatively high KD and a high potency (e.g., they don't bind well but have a strong impact on function), a relatively low KD and a low potency (e.g., they bind well, but not in a manner effective to alter a particular function) or a relatively high KD and a low potency (e.g., they simply do not bind to the target well). In one embodiment, high potency means that there is a high level of inhibition with a low concentration of antibody. In one embodiment, an antibody is potent or has a high potency when its IC5o is a small value, for example, 130-110, 110-90, 90-60, 60-30, 30-25, 25-20, 20-15, or less pM.
[0043] The term "selectively binds" in reference to an antibody does not mean that the antibody only binds to a single substance. Rather, it denotes that the KD of the antibody to a first substance is less than the KD of the antibody to a second substance. Antibodies that exclusively bind to an epitope only bind to that single epitope.
[0044] The term "and/or" denotes 1) including all of the relevant options, 2) including only one (or a subset) of a number of alternative options, 3) including both of the previous descriptions 1) or 2), and 4) including only one of the previous descriptions (1) or 2)).
[0045] Optionally, these antibodies bind to the glyco-epitope by displacing or blocking lectin
binding. The immune molecule may optionally comprise an isolated protein which interacts with a human and/or mouse glyco-epitope that optionally comprises a tumor associated carbohydrate antigen. The tumor associated carbohydrate antigen may optionally comprise a glycoprotein. Such interaction may optionally occur through at least one of binding, neutralizing and/or displacing another molecule bound to the glyco-epitope.
[0046] If the glyco-epitope is associated with a cell, optionally neutralizing by the immune
molecule induces cell death, cell quiescence or cell apoptosis (programmed cell death). By "cell quiescence" it is meant that the cell is induced to return to and/or to remain in a nonproliferative state.
[0047] A "neutralizing antibody" is an antibody molecule that is able to eliminate or
significantly reduce an effector function of a target antigen to which it binds. Accordingly, a "neutralizing" antibody as used herein is capable of eliminating or significantly reducing a glycomolecule function, such as, for example, binding of a glycomolecule to another protein or glycomolecule. In one embodiment, a neutralizing antibody will reduce a glycomolecule function by 1-10, 10-20, 20-30, 30-50, 50-70, 70-80, 80-90, 90-95, 95-99, 99-100%.
[0048] According to at least some embodiments, the isolated protein, which may optionally
comprise any type of isolated immune molecule, including but not limited to antibodies, e.g., monoclonal antibodies, or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof may optionally cross-react with a human glyco-epitope, such as a human glycoprotein for example, with an EC50 of less than 20 mg/kg when administered to a subject.
[0049] According to at least some embodiments of the present invention, there is provided a
diagnostic assay for disease detection in a biological sample taken from a subject (e.g,. a patient), where the biological sample, is for example, a bodily fluid or secretion including but not limited to seminal plasma, blood, serum, urine, prostatic fluid, seminal fluid, semen, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid, sputum, saliva, milk, peritoneal fluid, pleural fluid, cyst fluid, broncho alveolar lavage, lavage of the reproductive system and/or lavage of any other part of the body or system in the body, and stool or a tissue sample (for example, any type of tissue sample). The term may also optionally encompass samples of in vivo cell culture constituents. The sample can optionally be diluted with a suitable eluant before contacting the sample to isolated antibodies and isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, thereby diagnosing a disease or disorder, for example cancer or other proliferative disorder. [0050] According to other embodiments of the present invention, there is provided a method for disease detection in vivo in the body of a subject, comprising administering isolated antibodies and isolated monoclonal antibody fragments or antigen binding portions or fragments thereof to the body of the subject, and then detecting the location of the isolated antibodies and isolated monoclonal antibody fragments or antigen binding portions or fragments thereof within the body of the subject for example, optionally through a ligand or tag attached thereto. The ligand or tag may optionally comprise polyethylene glycol (PEG), a nanoparticle or particles, or a label.
[0051] In some embodiments, the nanoparticle or particles may optionally comprise one or more of solid nanospheres microencapsulated with different dye compounds and/or magnetic properties, which may optionally be prepared from a variety of functional polymeric materials, e.g. polyacrolein, polyglutaraldehyde, polymethyl a-(hydroxymethyl) acrylate,
polychloromethylstyrene, cellulose and silica; uniform magnetic nanoparticles which may optionally comprise one or more of organic-inorganic hybrid particles composed of cores from micron-sized uniform polystyrene particles and shells from magnetite- silica nanospheres of approximately 30 nm diameter; and/or biodegradable, non-toxic, magnetic metal oxide (i.e. Fe304) nanoparticles of very narrow size distribution in sizes ranging from approximately 20 nm up to 0.5 microns; or non-magnetic and magnetic silica hollow micron-sized particles.
[0052] In some embodiments, one can use any label known by persons of ordinary skill in the art, a label can be a fluorescent label or a bioluminescent label or any label known by one of ordinary skill in the art for in vivo imaging. In some embodiments, detection is performed through computer tomography (CT), magnetic resonance imaging (MRI) and spectroscopy (MRS), single photon emission computed tomography (SPECT), and positron emission tomography (PET).
[0053] Also disclosed are the isolated nucleic acid molecules encoding the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof of the invention as well as an expression vector comprising at least one copy of said nucleic acid molecules.
[0054] One aspect of the present invention relates to host cells comprising an expression vector encoding and expressing an antibody, e.g., a hybridoma secreting an isolated antibody, e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof, as well as transgenic non-human animals having a genome comprising said isolated nucleic acid molecule and/or the expression vector are also disclosed.
[0055] Furthermore, the present invention relates to a pharmaceutical composition comprising the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof of the present invention, and a pharmaceutically acceptable carrier.
[0056] A further embodiment of the invention concerns the use of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition, for the preparation of a medication for inhibiting the proliferation of a cell, e.g., cancer cell which the antibodies bind to, or for promoting cell death of the cells, e.g., cancer cells, to which the isolated antibodies bind to.
[0057] Another embodiment of the invention comprises the use of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition, in the manufacture of a medicament for the treatment of cancer in a subject.
[0058] Finally, the present invention, according to at least some embodiments, concerns a
pharmaceutical kit comprising at least an effective amount of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, together with instructions for use.
[0059] Abbreviations: FCS: Fetal calf serum; DMEM: Dulbecco's modified Eagle's medium;
PBS: Phosphate buffered saline; BSA: bovine serum albumin; HRP: horse radish peroxidase; DAPI: 4',6'-diamidino-2-phenylindole (DNA stain); PNA: Peanut (Arachis hyposaea)
Agglutinin; DBA: Dolichos biflorus Agglutinin; UEA: Ulex europeaus Agglutinin; SBA: soybean (Glycine max) Agglutinin ; RCA: Ricin communis Agglutinin; PHA-L: Phaseolus vulgaris-L Agglutinin; WGA: Wheat germ (triticum vulgaris ) Agglutinin; MAA: Maackia amurensis Agglutinin; LICA: Ligand induced cell apoptosis agent; RT: room temperature.
BRIEF DESCRIPTION OF THE FIGURES
[0060] Figures 1A-1C shows micrographs of immunohistochemical staining of cells and
specimens with scFv fragments as follows. Figure 1A shows staining of normal human fibroblasts with the different scFv fragments. Figure IB shows staining of NSCLC A549 cells with scFv fragments. Figure 1C shows staining of NSCLC human tumor specimens with scFv fragments.
[0061] Figure 2 shows quantitative binding of scFv fragments to NSCLC A549 cells.
[0062] Figure 3A-3B shows survival of cells after scFvl treatment. Figure 3A shows survival of
NSCLC A549 cells after treating the cells with scFvl fragments and 0.08uM antiMBP, while
Figure 3B is a histogram represents the survival of the cells after treatment with scFvland three different concentration of antiMBP (0.08uM, 0.165uM and 0.42uM) according to at least some embodiments of the present invention.
[0063] Figure 4A-40 shows immunohistochemical staining of different cancerous colorectal tissue, obtained as described above.
[0064] Figures 5A-5B shows immunostaining of paraffin embedded colorectal specimens from two human patients with scFvs8, demonstrating that the cancerous tissue is specifically bound while normal tissue is not bound.
[0065] Figure 6A-6D shows that staining intensity increases closer to tumor area in colorectal specimens (human patients), thereby indicating that relatively low levels of tumor tissue can be detected.
[0066] Figure 7 shows binding of scFv fragments to LS-174-T cells.
[0067] Figure 8 shows immunohistochemistry binding to such cells as for Figure 7.
[0068] Figure 9 shows the binding intensity of scFv8-IO-NIR to A549 cell line.
[0069] Figure 10 shows the binding of scFv8 to human colon cancer specimen and adjacent
normal control as indicated by immunohistochemistry (C- normal adjacent, D- colon adenocarcinoma), and binding of scFv8-IO-NIR to the same patient specimen (A- light scanning of normal adjacent and adenocarcinoma specimen B- 720/780nm scanning of normal adjacent and adenocarcinoma specimen
[0070] Figures 11A-11B show images of colons taken from two mice with colonic tumors after administration of scFv8-HAS-NIR. Panel (A) in Figures 11A and 11B show fluorescent imaging of the removed colon; the green dots are the tumors area stained with the probe. Panel (B) in Figures 11A and 11B show the removed colon photo in white light. Panel (C) in Figures 11A and 11B show the translation of fluorescent intensity into red and blue color whereas red shows areas of most intense fluorescence, and blue shows areas of least intense fluorescence. [0071] Figures 12A-12C show scFv competition with lectins. Figure 12A shows scFvl competition with lectins. Figure 12B shows scFv8 competition with lectins. Figure 12C
shows scFvH2 competition with lectins.
[0072] Figure 13 shows a table of competition of scFvs with lectins and other targets.
[0073] Figure 14 shows a table of immunohistochemistry (ICH) of lung cancer specimens and
comparison between lectins binding and scFvs binding.
SEQUENCE LISTING
[0074] The nucleic and amino acid sequences listed in the accompanying sequence listing are
shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
[0075] SEQ ID NO: 1-6 show the nucleotide sequences of the single-chain variable fragment
(scFv) of 6 different isolated antibodies. The specific sequences correspond to the following SEQ ID NOs: SEQ ID NO:l - scFv # 1; SEQ ID NO:2 - scFv # 8; SEQ ID NO:3 - scFv # A7; SEQ ID NO:4 - scFv # C2; SEQ ID NO: 5 - scFv # G2; and SEQ ID NO:6 - scFv # H2.
[0076] SEQ ID NO: 7-12 - show the amino acid sequences of the scFv of 6 different isolated
antibodies. The specific sequences correspond to the following: SEQ ID NO:7 - scFv#l; SEQ ID NO:8 - scFv #8; SEQ ID NO:9 - scFv#A7; SEQ ID NO: 10 - scFv#C2; SEQ ID NO: 11 - scFv#G2; SEQ ID NO: 12 - scFv#H2.
[0077] Table 1 shows SEQ ID NOs: 13-40 which are CDRs (CDRl, CRD2 and CDR3) according to at least some embodiments of the present invention.
CDRl CDR2 CDR3
SEQ ID NO: 7 - SNYMH AINSNGDSTYYPDTVKD CHLSRLYDLGSHPPYYFDF scFv#l: VH (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15)
SEQ ID NO: 7 - RASQSVSSGYLA AASSRAT QQYYGSPWT
scFv#l: VL (Kappa) (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18)
SEQ ID NO 8 - scFv Cannot be assigned Cannot be assigned DPDPYGSESFRLFGTFDY #8: VH (SEQ ID NO: 19)
SEQ ID NO 8 - scFv RASQSVTSSSLA GASARAT QQYYDWPLT
#8: VL (Kappa) (SEQ ID NO:20) (SEQ ID NO:21) (SEQ ID NO:22)
SEQ ID NO: 10 - SGDMG AITTGGGSPNYADSVKG DEGMVGATYFDH scFv#C2: VH (SEQ ID NO:23) (SEQ ID NO:24) (SEQ ID NO:25)
SEQ ID NO: 10 - SGSNSNIGYSQVS EVSKRPS QSYDSSLNSYI scFv#C2: VL (SEQ ID NO:26) (SEQ ID NO:27) (SEQ ID NO:28)
(Lambda)
SEQ ID NO: 11 - NFVMN RINTDGTSTNYADSVTG SPYTIFGVVHFDY scFv#G2: VH (SEQ ID NO:29) (SEQ ID NO: 30) (SEQ ID NO:31)
SEQ ID NO: 11 - SGSSSNIDSSPVN LINERPS QVWDSISDHWV scFv#G2: VL (SEQ ID NO;32) (SEQ ID NO:33) (SEQ ID NO:34)
(Lambda)
SEQ ID NO: 9 - EYGMN GISWNSDTIDYGDSVKG RQYYAMDV
scFv#A7: VH (SEQ ID NO:35) (SEQ ID NO:36) (SEQ ID NO:37)
SEQ ID NO: 9 - SGNSGSIASNSVQ DNDKRPS SSYTSSSTLVV
scFv#A7: VL (SEQ ID NO:38) (SEQ ID NO:39) (SEQ ID NO:40)
(Lambda)
[0078] * VH of scFv #8 is not a canonical VH sequence since the sequence does not align in its middle part with other VHs. Framework 1 is canonical almost all the way to CDR1, then the sequence is shuffled (source of shuffling unknown) and returns to canonical VH a little before CDR3. The sequence is an intact open reading frame and seems to produce a functional protein. Therefore, CDRs 1 and 2 cannot be assigned.
DETAILED DESCRIPTION OF THE INVENTION
[0079] Section headings are provided below for the purpose of description only and without any intention of being limiting in any way.
[0080] As used herein, the following definitions are supplied in order to facilitate the
understanding of the present invention.
[0081] The terms "A" or "an" means "at least one" or "one or more."
[0082] The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components.
[0083] As used herein, the terms "protein", "polypeptide", "polypeptidic", "peptide" and
"peptidic" or "peptidic chain" are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
[0084] The terms "Amino acid residue" means any amino acid residue known to those skilled in the art. This encompasses naturally occurring amino acids (including for instance, using the three-letter code, Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val), as well as rare and/or synthetic amino acids and derivatives thereof (including for instance Aad, Abu, Acp, Ahe, Aib, Apm, Dbu, Des, Dpm, Hyl, McLys, McVal, Nva, and the like).
[0085] Said amino acid residue or derivative thereof can be any isomer, especially any chiral
isomer, e.g. the L- or D- isoform.
[0086] By amino acid derivative, it is meant any amino acid derivative as known in the art. For instance, amino acid derivatives include residues derivable from natural amino acids bearing additional side chains, e.g. alkyl side chains, and/or heteroatom substitutions.
[0087] The term "purified" does not require absolute purity; rather, it is intended as a relative term.
Thus, for example, an antibody, e.g., monoclonal antibody preparation is one in which the protein is more pure than the protein in its natural environment within a cell. Preferably, a preparation of antibody, e.g., a monoclonal antibody is purified such that the protein represents at least 50% of the total protein content of the preparation.
[0088] As used herein, the expression "substantially pure" refers to material that is at least 50% pure, preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure and most preferably 99% pure, or with greater purity.
[0089] An "isolated antibody", as used herein, is intended to refer to an antibody which is
substantially free of other antibodies having different antigenic specificities. As a non-limiting example, if the antibody is a lectin displacing antibody, then it is an isolated antibody that specifically binds to a glycoprotein in such a way as to displace a lectin molecule already bound, where the antibody is substantially free of antibodies that specifically bind antigens other than the glycoprotein to which the lectin is bound. An isolated antibody that specifically binds to an epitope present on a glycoprotein may, however, have cross-reactivity to other related antigens, e. g., from other species (e. g., glycoproteins from other cancer cells, or other species homologues). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies having different specificities are combined in a well defined composition.
[0090] The term "disease", as used herein, refers to a pathological condition of a part, organ, or system of an organism resulting from various causes, such as infection, genetic defect, or environmental stress, and characterized by an identifiable group of signs or symptoms. [0091] The term "subject" refers to patients of human or other vertebrates in particular mammal and includes any individual it is desired to examine or treat using the methods according to the present invention. However, it will be understood that "patient" does not automatically imply that symptoms or diseases are present. As used herein, the term "patient" preferably refers to a human in need of treatment, e.g., to treat cancer.
[0092] The term "Mammal" as used herein for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably, the mammal is a human.
[0093] The term "Vertebrate" as used herein for purposes of treatments refers to any animal
classified as a vertebrate, including birds, amphibians and fishes. Preferably, the vertebrate is a human.
[0094] The term "treatment" as used herein refers to therapeutic treatment of a disease or disorder in a subject. In some embodiments, the term treatment also refers to prophylactic or preventive measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, a subject to be treated herein may have been diagnosed as having the disorder or may be predisposed or susceptible to the disorder. Thus the term "treatment" or "treating" herein encompasses curative treatment, preventive treatment as well as palliative treatment, more specifically palliative treatment and curative treatment.
[0095] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a patient in particular to a human.
[0096] The expression "effective amount" is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results, preventing or attenuating symptoms resulting from the disease, decreasing the dose of other medicaments required to treat the disease. An effective amount can be administered in one or more administrations of the active substance. For the purpose of this invention, the active substance is a molecular composition inducing a biological activity when interacting with a glyco-epitope. This includes a carbohydrate epitope or a mixed carbohydrate-protein epitope.
[0097] In the present invention, the term "receptor" refers to a structure on the surface of a cell (or inside a cell) that selectively receives and binds a specific molecule which affects the activities of the cell. [0098] The term "antigen" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to elicit the production of antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.
[0099] The term "tumor- associated carbohydrate antigen" refers to a carbohydrate moiety that is linked to the cell membrane of a tumor cell either directly or via another molecule such as a protein or lipid. Thus, the carbohydrate can be a polysaccharide as well as a molecule to which a polysaccharide is linked (e.g., by a covalent bond) to a second molecule. The tumor- associated carbohydrate antigen may be a lectin, a glycosaminoglycan, a glycoprotein or a glycolipid. Carbohydrate antigens are conjugated to lipids and proteins via a process known as glycosylation.
[00100] As used herein, the term "carbohydrate binding molecule" refers to a molecule
including a lectin, antibody or a fragment thereof, a peptide, a peptide analog and a small organic and inorganic molecule to bind to a carbohydrate moiety associated with the cell membrane wherein that binding depends on the specific structure and properties of the molecule.
[00101] The term "antibody", as used herein, refers to a protein which may, for example, be produced by the immune system that protects the organism against an antigen. But, as used herein, the term encompasses not only intact monoclonal antibodies but also fragments thereof, single chains, mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, as well as antibodies produced by mammalian or bacterial cells that carry antibody coding DNA sequences, and also recombinant antibodies that are made in transgenic animals carrying the genes coding for the recombinant antibodies.
[00102] The term "antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas. [00103] The term "native antibodies and immunoglobulins" as used herein refer to
heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Chothia et al. J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985); Chothia et al., Nature 342: 877-883 (1989)).
[00104] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[00105] The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e. g., effector cells) and the first component (Clq) of the classical complement system.
[00106] The term "agonist" refers to a molecule, e.g., a drug, ligand or antibody which binds to a receptor and activates it, producing a pharmacological response (e.g. contraction, relaxation, secretion, enzyme activation, etc.). In some embodiments, the term agonist refers to a molecule, e.g., a drug, ligand or antibody which increases the biological activity by at least about 10% or more than 10% of the receptor to which it is an agonist to.
[00107] The terms "monoclonal antibody" (mAb) or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
[00108] The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."
[00109] The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a glyco-epitope and/or or retains the ability to displace a bound molecule from a glyco-epitope, such as a bound lectin molecule as a non-limiting example. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F (ab)' 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR); and (vii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Huston et al (1988) Proc. Natl. Acad. Sc USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
[00110] The term "fragments" as used herein refers to sequences sharing at least 10% amino acids in length with the respective sequence of the intact or full length antibody, e.g., monoclonal antibodies (native). These sequences can be used as long as they exhibit the same properties as the native sequence from which they derive. In some embodiments, a fragment can be at least 6 amino acids in length, and can be, for example, at least 8, at least 10, at least 14, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 25 amino acids or greater than 25 amino acids from the full length protein from which the fragment was derived. In some embodiments, the term fragment encompasses at least 6, 10, 20, 50, 100, 250, 500 amino acids from the full length protein from which the fragment was derived. Exemplary fragments include C-terminal truncations, N-terminal truncations, or truncations of both C- and N-terminals (e.g., deletions of 1, 2, 3, 4, 5, 8, 10, 15, 20, 25, 40, 50, 75, 100 or more amino acids deleted from the N-termini, the C-termini, or both). Preferably these sequences share more than 70%, preferably more than 80%, in particular more than 90% amino acids in length with the respective sequence the intact or full length antibody, e.g., monoclonal antibodies. In some embodiments, the term "fragments" as used herein, when used in reference to fragments of monoclonal antibodies, or monoclonal antibody fragments or antigen binding portions or fragments usually refers to a portion of at least 2, or at least about 5, or at least about 6, or at least about 8, or at least about 10 or more consecutive amino acids of the epitope binding region of an antibody. In some embodiments, a fragment includes at least 2, or at least about 5, or at least about 6, or at least about 8, or at least about 10 or more consecutive amino acids of the epitope binding region of an antibody selected from the group consisting of SEQ ID NO: 7- 12. In some embodiments, a fragment is a CDR region of at least 3 consecutive amino acids from any of the group consisting of SEQ ID NO: 7-12. In some embodiments, a fragment is a CDR region selected from any and a combination of CDRs listed in Table 1, e.g., SEQ ID NO: 13-40.
[00111] In some embodiments, the fragment is a functional fragment, where a "functional fragment" as used in the context of a "functional fragment of an antibody" refers to a fragment of the antibody that mediates the same effect as the full length antibody, e.g., specifically binds to the same antigen with the same, or a greater affinity as compared to the full length antibody. In some embodiments, a funtional fragment is a CDR region of at least 3 consecutive amino acids from any of the group consisting of SEQ ID NO: 7-12. In some embodiments, a functional fragment is a CDR region selected from any and a combination of CDRs listed in Table 1, e.g., SEQ ID NO: 13-40.
[00112] In the case of an antibody, e.g., monoclonal antibody according to at least some embodiments of the present invention, useful fragments include, but are not limited to: a CDR region, especially a CDR3 region of the heavy or light chain; a variable domain of a heavy or light chain; a portion of an antibody chain or just its variable region including two CDRs; and the like. In some embodiments, useful functional fragments include, but are not limited at least one or any combination of CDRs from the same antibody, e.g., selected from SEQ ID NOs; 13- 15, or SEQ ID NOs: 16-18, or SEQ ID NOs: 20-22, or SEQ ID NOs: 23-25, or SEQ ID NOs: 26-28, or SEQ ID NOs: 29-31, or SEQ ID NOs: 32-34, or SEQ ID NOs: 35-37, or SEQ ID NOs: 38-40.
[00113] Suitable antibodies, e.g., monoclonal antibody, or fragments of the invention are immunologically functional immunoglobulins. The term "immunologically functional immunoglobulin fragment" as used herein refers to a polypeptide fragment that is optionally and preferably capable of immunologically interacting with a glyco-epitope. Such interaction optionally and preferably comprises specifically binding to a glyco-epitope and/or specifically displacing a glycomolecule, such as a lectin for example, bound to a glyco-epitope and/or specifically neutralizing a glyco-epitope, wherein "neutralizing" optionally also encompasses inducing cell death and/or apoptosis and/or cell quiescence. In some embodiments, a fragment of such an antibody can bind specifically to and/or modulate the biological activity of a glycomolecule, such as a glycoprotein for example. In some embodiments, an immunologically functional immunoglobulin fragment specifically binds to the glyco-epitope at the same location, or in close proximity to the same location as the location on the glyco-epitope is bound by the glycomolecule such as a lectin for example.
[00114] In some embodiments, one can identify CDRs, e.g., CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3 in an antibody sequence based on amino acid sequence identification, as shown in Tables 2A-2F. Typically, the Cys residues are highly conserved in CDR regions. Thus, in some embodiments, the CDR regions of the Light-chain and Heavy- chains of SEQ ID NO: 7-12 can be determined and are useful in the method and compositions herein to produce specific glyco-epitope binding and/or glycomolecule displacing antibodies. In alternative embodiments, the Rosetta Antibody server (at world- wide- web:
"antibody.graylab.jhu.edu" can be used to predict CDR in antibody sequences.
[00115] Table 2A: CDR-L1
Figure imgf000023_0001
[00116] Table 2B: CDR-L2 Start always 16 residues after the end of LI
Residues before generally Ile-Tyr, but in some embodiments can also be: Val-Tyr, Ile-Lys, Ile-Phe
Length always 7 residues (except NEW (7FAB) which has a deletion in this region)
[00117] Table 2C: CDR-L3
Figure imgf000024_0001
Length 7 to 11 residues
[00118] Table 2D: CDR-H1
Figure imgf000024_0002
Chothia definition excludes the last 4 residues
[00119] Table 2E: CDR-H2
Start always 15 residues after the end of Kabat / AbM definition) of CDR-H1
Residues typically Leu-Glu-Trp-Ile-Gly, but a number of variations
before
Residues after Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala
Length E abat definition 16 to 19 residues;
AbM (and recent Chothia) definition ends 7 residues earlier
[00120] Table 2F: CDR-H3
Start always 33 residues after end of CDR-H2 (always 2 after a Cys)
Residues always Cys-XXX-XXX (typically Cys-Ala-Arg)
before
Residues after always Trp-Gly-XXX-Gly
Length 3 to 25 residues
[00121] In one embodiment, the present invention concerns a method for producing one or more antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof. In some embodiments, such an antibody can also be produced by the method comprising the steps of: (a) producing an antigen of a glycomolecule, or a fragment, or a fusion protein thereof, of any species, e.g., human and/or vertebrate species
(b) immunizing a rodent, e.g., mice with the antigen of a glycomolecule, or a fragment, or a fusion protein thereof
(c) detection of specifically binding, neutralizing or displacing antibodies in the serum of the mice
(d) producing hybridomas between lymph node cells from the mice and myeloma cells to produce antibodies.
In some embodiments, an antigen used to produce an antibody is human, or mouse, or from another mammalian species, or from another vertebrate species.
[00122] In some embodiments, the antigen used to produce an antibody is a cell line
naturally expressing full length antigen or a fragment of the antigen, or a fusion protein of the antigen and another protein, or the antigen is part of a virus-like particle.
[00123] In some embodiments, the antigen used to produce the antibody is expressed in a cell line syngenic with mice of step b), or the antigen used to produce antibodies is fused to the Fc portion of an IgG.
[00124] In some embodiments, the antigen used to produce antibodies is human or mouse antigen fused to the Fc portion of human IgGl.
[00125] As an alternative to steps b), c) and d), an antibody, or fragment thereof such as single chain Fv can be obtained by selecting antibody sequences by phage display on the antigen of step a).
[00126] In some embodiments, the binding assays of step e) is carried out by applying
visualizing methods comprising ELISA, dot blot, Western blot, RIA, immunoprecipitation, flow cytometry, fluorescence microscopy, electron microscopy, confocal microscopy, calorimetry, surface plasmon resonance, test of Ouchterlony, complement-mediated lysis of red blood cells, antibody-dependent cell cytotoxicity and the like. More preferably, the binding assays of step e) is carried out by direct or capture ELISA.
[00127] In particular, antibodies can be purified, for example by protein A or G affinity chromatography or by protein L, anti-mouse IgG antibody-based affinity chromatography, ion exchange, ethanol or ammonium sulfate precipitation and the like. [00128] Methods for preparing an immunogen (e.g., an antigen used to produce lectin- displacing antibodies) and immunizing an animal are well-known in the art (Kohler and Milstein 1975 Nature 256:495-497; Brown et al. 1981 J Immunol 127:539-46; Brown et al., 1980 J Biol Chem 255:4980-83; Yeh et al., 1976 Proc Natl Acad Sci USA 76:2927-31; Yeh et al., 1982 Int J Cancer 29:269-75; Kozbor et al., 1983 Immunol Today 4:72; Cole et al., 1985 Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96; U.S. Pat. No.
4,816,567; Clackson, et al., 1991 Nature 352:624-628; Marks, et al., 1991 J Mol Biol 222:581- 597).
[00129] Antibody sequences according to at least some embodiments of the present
invention are those of SEQ ID NOS: 1-6, which relate to the nucleotide sequences of the single-chain human antibody fragments (scFv) of 6 different isolated antibodies. The specific sequences correspond to the following SEQ ID NOs: SEQ ID NO:l - scFv # 1; SEQ ID NO:2 - scFv # 8; SEQ ID NO:3 - scFv # A7; SEQ ID NO:4 - scFv # C2; SEQ ID NO: 5 - scFv # G2; and SEQ ID NO:6 - scFv # H2.
[00130] The amino acid sequences thereto are shown in SEQ ID NOS: 7-12, which relates to the amino acid sequences of the scFv of 6 different isolated antibodies. The specific sequences correspond to the following: SEQ ID NO:7 - scFv#l; SEQ ID NO:8 - scFv #8; SEQ ID NO:9 - scFv#A7; SEQ ID NO: 10 - scFv#C2; SEQ ID NO: 11 - scFv#G2; SEQ ID NO: 12 - scFv#H2.
[00131] Applicants have demonstrated in the Examples that in vitro screening was
performed. The in vitro test was performed by contacting NSCLC cells with the antibodies in the media and analyzing cell survival over a period of time, as disclosed in the Examples. In some instances, antibodies, e.g., sc-Fvl and sc-Fv8 decreased cell survival of NSCLC cells. This in vitro assay is certainly a standard test, which can be easily performed by one of ordinary skill in the art, e.g., by scientists without experience in the field, and in a variety of different cell types and cancer cell types.
[00132] According to another embodiment of the present invention, there is provided an isolated antibody, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof obtainable by the above-described process and wherein said isolated antibody, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof bind to human and/or mouse glyco- epitope with an affinity constant (KD) of at least 10"6 M for the Fab fragment, and which can optionally displace a bound glycomolecule from the glyco-epitope with an affinity constant of at least this amount. In some embodiments, an antibody or fragment or antigen binding portion thereof as disclosed herein specifically binds with a high affinity to the glyco-epitope.
[00133] As used herein, "specific binding" refers to antibody binding to a predetermined antigen. Typically, the antibody binds with a affinity constant (KD) of 10~6 M or less for the Fab fragment, and binds to the predetermined antigen with a KD that is at least ten-fold less than its KD for binding to a non-specific antigen (e. g., BSA, casein) other than the
predetermined antigen or a closely-related antigen. The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen".
[00134] The term "Kassoc" or "Ka", as used herein, is intended to refer to the association rate of a particular antibody- antigen interaction, whereas the term "KdiS" or "Kd" is intended to refer to the dissociation rate of a particular antibody- antigen interaction. The term "high affinity" for an IgG antibody refers to an affinity constant (KD) for the Fab fragment of at least about 10"6 M, at least about 10"7 M, at least about 10"8 M, at least about 10"9 M, at least about 10"10 M, at least about 10"11 M, or at least about 10"12 M or greater, e. g., up to 10"1 3M or 10"14 M or greater. However, "high affinity" binding can vary for other antibody isotypes.
[00135] Preferably, the isolated antibody, e.g., monoclonal antibodies or isolated
monoclonal antibody fragments or antigen binding portions or fragments thereof, according to at least some embodiments of the present invention, comprise a polypeptide having an amino acid sequence selected from any of the group according to any of SEQ ID NOs 7-12, and or any combination thereof. The isolated antibody preferably specifically binds to a glyco-epitope and/or specifically displaces a glycomolecule bound to such an epitope and/or neutralizes such a glyco-epitope. For example, optionally and without limitation, the antibody is a lectin- displacing antibody, e.g., lectin-displacing monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof binds to a glycoprotein at the same epitope or region where a lectin would bind.
[00136] Modifications and Variations
[00137] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Conservative amino acid substitutions are herein defined as exchanges within one of the following five groups:
I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly
II. Polar, positively charged residues: His, Arg, Lys
III. Polar, negatively charged residues: and their amides: Asp, Asn, Glu, Gin
IV. Large, aromatic residues: Phe, Tyr, Trp
V. Large, aliphatic, nonpolar residues: Met, Leu, lie, Val, Cys. (see, e.g., Creighton, Proteins (1984)).
[00138] According to certain embodiments, amino acid substitutions are those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (5) confer or modify other physicochemical or functional properties on such polypeptides.
[00139] The isolated antibody, e.g., monoclonal antibody according to the present invention is not limited to the whole molecule, and may be a fragment of the antibody or the modified product thereof, as long as it still retains one or more of following properties: specifically binds to a glyco-epitope and/or specifically displaces a glycomolecule bound to such an epitope and/or neutralizes such a glyco-epitope. For example, optionally and without limitation, the antibody or fragment or modified product thereof may optionally displace a lectin bound to a glycoprotein and retain the capacity of binding to the glycoprotein at the region where the lectin typically binds.
[00140] Multivalent, preferably bivalent, antibody and a monovalent antibody are included Examples of the fragment of an antibody include Fab, F(ab)'2, Fv, Fab/c having one Fab and a complete Fc, and a single chain Fv (scFv) wherein the Fv of the H- chain or the L-chain is ligated with an appropriate linker. Specifically, an antibody fragment is synthesized by treating the antibody with an enzyme such as papain, pepsin or ficin, or genes encoding these antibody fragments are constructed, the genes are introduced into expression vectors, and the genes are then expressed by appropriate host cells (see e g, Rousseaux, J et al, Methods in Enzymology (1989) 121, 663-669, and Bird, R E et al, TIBTECH (1991)9, 132-137).
[00141] Digestion of antibodies with the enzyme papain results in two identical antigen- binding fragments, known also as "Fab" fragments, and a "Fc" fragment, having no antigen- binding activity but having the ability to crystallize. Digestion of antibodies with the enzyme pepsin results in the a F(ab')2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab')2 fragment has the ability to crosslink antigen.
[00142] The term "Fab" as used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
[00143] The term "Fv" as used herein refers to a minimum antibody fragment that contains a complete antigen-recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent
association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
[00144] The term "hypervariable region" or "HV" as used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. residues 24-34 (LI), 50-62 (L2), and 89-97 (L3) in the light chain variable domain and 31-55 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26- 32 ((HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). [00145] The term "framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
[00146] The term "complementarity determining regions" or "CDRs" when used herein refers to parts of immunological receptors that make contact with a specific ligand and determine its specificity. The CDRs of immunological receptors are the most variable part of the receptor protein, giving receptors their diversity, and are carried on six loops at the distal end of the receptor's variable domains, three loops coming from each of the two variable domains of the receptor.
[00147] The term "epitope" is used to refer to binding sites for antibodies on antigens.
Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
[00148] The term "glyco-epitope" as used herein refers to a binding site for an antibody on a glycomolecule.
[00149] The term "glycomolecule" as used herein refers to a protein comprising a
saccharide moiety or one or more polysaccharides. For example, a glycomolecule can includes carbohydrate-containing proteins (glycoproteins) or glycolipids, and free polysaccharides. Glycoproetins include, e.g., fetuin, al Acid GP, and tPA.
[00150] scFv is obtained by linking the H-chain V-region and the L-chain V-region of
antibodies. In the scFv, the H-chain V-region and the L-chain V-region are linked via a linker, or preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H-chain V-region and the L-chain V-region in scFv may be derived from any of those described as antibodies in this specification. As a peptide linker to link the V-regions, for example, any single-stranded peptide comprising 12 to 19 amino acid residues is used.
[00151] According to an embodiment of the invention, the isolated antibodies, e.g.,
monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof may present an antibody heavy chain selected among: IgG, IgM, IgA, IgE, single chain antibody and other immunoglobulin-derived constructs or non antibody binding proteins.
[00152] As used herein, "isotype" refers to the antibody class (e. g., IgM, IgA, IgE or IgG) that is encoded by heavy chain constant region genes. [00153] Optionally and as non-limiting examples, the non antibody binding proteins comprise adnectins (fibronectin-based reagents), Affibody (protein A-based reagents), DARPins (ankyrin-based reagents), avimers (cysteine rich cell surface receptor proteins), anticalins (lipocalin-derived reagents), and nucleotide-based reagents and the like (see for example Nutall & Walsh 2008 Curr Op Pharmacol 8:609).
[00154] When the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof may present a IgG antibody heavy chain the latter may be selected among: IgGl, IgG2, IgG3 or IgG4, mutated IgGl that is no longer recognized by FcR; mutated IgG4 sequence that no longer undergoes heavy chain swapping; mutated IgG to modify glycosylation; PEGylated IgG and the like. It is acknowledged that all possible "isotype switching" known to the person skilled in the art may be envisioned in the context of the present invention.
[00155] As used herein, "isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.
[00156] Among the list of antibody-based scaffolds, VNAR, which are lamprey-derived single domain antibodies may be advantageously used.
Antibody Structure
[00157] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa (κ) and lambda (λ) light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. (See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)), incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.
[00158] Thus, an intact antibody has two binding sites. Except in bifunctional or bispecific
antibodies, the two binding sites are the same. [00159] The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
[00160] A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. (See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992)). Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab', and Fv).
[00161] The present invention further provides at least one isolated antibody, e.g., a
monoclonal antibody or an isolated monoclonal antibody fragment or an antigen binding portions or fragments thereof, comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequences of any of SEQ ID NOs 7-12.
(b) amino acid sequences that differ from those specified in (a) by one or more conservative amino acid substitutions;
(c) amino acid sequences having at least 95% sequence identity to the sequences specified in (a), or (b);
(d) biological active fragments of amino acid sequences specified in (a) or (b) or (c) specifically binding to a glyco-epitope and/or displacing a glyco-molecule bound to the glyco-epitope and/or neutralizing the glyco-epitope;
(e) optionally the fragments of (d) wherein the fragments are binding glycoproteins to displace a bound lectin; and (f) biological active variants or modifications of amino acid sequences specified in (a) or (b) or (c) or (d) or (e) having the properties of fragments of (d) or (e).
[00162] The term "sequence identity/similarity" has its ordinary meaning in the field. The terms "identical" or percent "identity" in the context of two or more polypeptide sequences, refer to two or more sequences that are the same, or have a specified percentage of amino acid residues that are the same (i.e., at least 70% identity, preferably at least 75%, 80%, 85%, 90%, even more preferably at least 95% or 98% or even 99% identity over a specified region), when compared and aligned for maximum correspondence. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (e.g., human and mouse sequences), compared to species more distantly related (e.g., human and C. elegans sequences).
[00163] Methods of alignment of sequences for comparison are well known in the art.
Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:23744, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol Biol. 215:403-10, 1990, presents a detailed
consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site. Homologs of the disclosed antibodies, e.g., monoclonal antibodies are typically characterized by possession of at least 70%, preferably of at least 95%, and more preferably of at least 98% sequence identity sequence identity counted over the full-length alignment with the disclosed amino acid sequences using the NCBI Blast 2.0, or using the manual alignment as described above. Proteins with even greater similarity to the antibodies, e.g., monoclonal antibody sequences will show increasing percentage identities when assessed by this method, such as at least 75%, 80%, 85%, 90%, 95% or even 98% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or even 98% depending on their similarity to the reference sequence. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is possible that strongly significant homologs could be obtained that fall outside the ranges provided.
64] The present invention also includes variants of the antibodies, e.g., monoclonal antibodies. The term "variants" or derivatives or equivalents of the antibodies, e.g., monoclonal antibody sequences refer to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide that is amino acid sequences that vary from the native sequence by conservative amino acid substitutions, whereby one or more amino acids are substituted by another with same characteristics and conformational roles. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. Typically, such variants possess at least 90%, preferably at least 95%, and very particularly preferably at least 98%, sequence identity with the native sequence. Variants which are particularly preferred in this connection are replacement variants which typically contain less than 10, preferably less than 5, and very particularly preferably less than 3, replacements as compared with the respective disclosed sequences.
[00165] In addition or alternative to modifications made within the framework or CDR
regions, antibodies, e.g., monoclonal antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen- dependent cellular cytotoxicity. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
[00166] Variants, derivatives and equivalents of an antibody useful in the compositions and methods as disclosed herein have substantially the same, or a greater biological activity than the antibody they are derived from. In some embodiments, the biological activity of an antibody can be determined by one of ordinary skill in the art, for example, using an assay such as that disclosed herein in the Examples, such as an in vitro assay to assess the ability of the derivative, variant or equivalent thereof to specifically bind to a glyco-epitope and/or specifically displace a glycomolecule bound to such an epitope and/or neutralize such a glyco- epitope. For example, optionally and without limitation, the assay may optionally assess the ability of the derivative, variant or equivalent thereof to displace a lectin bound to a
glycoprotein as compared to the antibody it is derived from, or alternatively, the ability of the derivative, variant or equivalent of the lectin-displacing antibody to induce cell death as compared to the lectin-displacing antibody it is a variant thereof.
[00167] In some embodiments, a variant, derivative or equivalent of an antibody useful in the compositions and methods as disclosed herein has at least about the same, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%, or at least about 100% of the biological activity than the antibody they are derived from, or at least about 1.5-fold, or at least about 2-fold, or at least about 2.5-fold, or at least about 5-fold, or at least about 10-fold greater biological activity than the antibody they are derived from.
[00168] In some embodiments, a fragment of an antibody useful in the compositions and methods as disclosed herein is a functional fragment, and comprises at least one or two or more CDR's as disclosed in Table 1. In some embodiments, a fragment of an antibody for use in the methods and compositions as disclosed herein comprises at least 3 consecutive amino acids from any of the CDR regions selected from any, or a combination of CDRs listed in Table 1, e.g., SEQ ID NO: 13-40. In some embodiments, a fragment of an antibody useful in the compositions and methods as disclosed herein comprises at least one or any combination of CDRs from the same antibody, e.g., selected from SEQ ID NOs; 13-15 (CDRs of scFv#l, VH), or SEQ ID NOs: 16-18 (CDRs of scFv#l, VL (Kappa)), or SEQ ID NO; 19 (CDR of scFv#8, VH), or SEQ ID NOs: 20-22 (CDRs of scFv#8, VL, kappa), or SEQ ID NOs: 23-25 (CDRs of scFv#2, VH), or SEQ ID NOs: 26-28 (CDRs of scFv#C2, VL), or SEQ ID NOs: 29-31 (CDRs of scFv#G2, VH), or SEQ ID NOs: 32-34 (CDRs of scFv#G2, VL), or SEQ ID NOs: 35-37 (CDRs of scFv#A7, VH), or SEQ ID NOs: 38-40 (CDRs of scFv#A7, VH).
[00169] In some embodiments, a fragment of an antibody useful in the compositions and methods as disclosed herein comprises at least one or any combination of CDRs from the same antibody, e.g., selected from SEQ ID NOs; 13-18 (CDRs of scFv#l), or SEQ ID NO; 19-22 (CDRs of scFv#8), or SEQ ID NOs: 23-28 (CDRs of scFv#2), or SEQ ID NOs: 29-34 (CDRs of scFv#G2) or SEQ ID NOs: 35-34 (CDRs of scFv#A7, VH).
[00170] In some embodiments, glycosylation of an antibody, or antibody variant, derivative or functional fragment thereof can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such a glycosylation may increase the affinity of the antibody for antigen. Such an approach is described in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al, which are incorporated herein in its entirety by reference
[00171] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such
carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express antibodies, e.g., monoclonal antibodies of the invention to thereby produce an antibody with altered glycosylation. Alternatively, the frucose residues of the antibody may be cleaved off using a fucosidase enzyme.
[00172] Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the
biological (e.g., serum) half life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a glycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al, which are incorporated herein in their entirety by reference.
[00173] In another embodiment, an antibody, e.g., monoclonal antibodies of the present invention do not contain asparagine isomerism sites. A deamidation or isoaspartic acid effect may occur on N-G or D-G sequences, respectively. The deamidation or isoaspartic acid effect results in the creation of isoaspartic acid which decreases the stability of an antibody by creating a kinked structure off a side chain carboxy terminus rather than the main chain.
[00174] In another embodiment, antibodies useful in the methods and compositions as
disclosed herein are selected that do not rapidly degrade. Fragmentation of an antibody, e.g., monoclonal antibody may be measured using capillary electrophoresis (CE) and MALDI-MS, as is well understood in the art (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32). In another preferred embodiment, antibodies are selected that have minimal aggregation effects. Aggregation may lead to triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies are acceptable with aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less and even more preferably 5% or less. Aggregation may be measured by several techniques well known in the art, including size-exclusion column (SEC) high performance liquid chromatography (HPLC), and light scattering to identify monomers, dimers, trimers or multimers.
[00175] Some additional unanticipated results are in favor of the non obviousness nature of the present invention. For example, but not limited to, Applicants have demonstrated that antibodies of the invention were in fact able to inhibit growth of cancer cells, specifically NSCLC cells.
[00176] It was not expected that an antibody, e.g., a lectin-displacing monoclonal antibody would have binding specificities that would function to induce cell death, as lectin
displacement from the glycoprotein by an antibody could be due to binding to the antibody to the lectin, (and thus steriometrically affecting the lectin-glycoprotein binding site) rather than the antibody competitively binding to the glycoprotein at the same site as where the lectin binds. Thus it is not obvious that a lectin-displacing antibody would serve necessarily the same function as the lectin to promote apoptosis of the cell to which it binds. Applicants results were surprising because there is no simple way of controlling antigen specificity of antibodies (unless using short antigen fragments covering the desired region of antigen specificity; with the risk of losing conformation determinants).
[00177] According to other embodiments of the present invention, there is provided an isolated nucleic acid molecule encoding the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof as defined above. Specific examples of such nucleotide sequences are given as SEQ ID NOs 1-6.
[00178] The term "nucleic acid molecule", as used herein, is intended to include DNA
molecules and RNA molecules. A nucleic acid molecule may be single- stranded or double- stranded, but preferably is double- stranded DNA.
[00179] In addition to the above described monoclonal antibodies, artificially altered gene recombinant antibodies such as chimeric antibodies or humanized antibodies can be used for, for example, lowering heteroantigenicity against a human. These altered antibodies can be produced using a known method.
[00180] Chimeric antibodies can e.g., be obtained by ligating the DNA encoding the
antibody V-region to a DNA encoding a human antibody C-region, incorporating the product into an expression vector, and then introducing the vector into a host to cause the host to produce the antibodies. Using this known method, chimeric antibodies, e.g., monoclonal antibodies useful in the present invention can be obtained. [00181] Humanized antibodies are also referred to as reshaped human antibodies, which are prepared by grafting an antibody CDR (complementarity determining region) of a mammal other than a human, such as a mouse, to the CDR of a human antibody. The general gene recombination technique thereof is also known (see European Patent Application Publication EP 125023 and WO 96/02576, or any one of their US counterparts, such as e g US 6,068,040, which are incorporated herein in their entirety by reference).
[00182] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.
[00183] Chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a non-human monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the non- human hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al, which are incorporated herein in their entirety by reference). To create a humanized monoclonal antibody, murine CDR regions can be inserted into a human framework using methods known in the art (see e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al, which are incorporated herein in their entirety by reference). In a some embodiments, an antibody of the invention is a human monoclonal antibody.
[00184] One limitation of scFv molecules is their monovalent interaction with target antigen.
One of the easiest methods of improving the binding of a scFv to its target antigen is to increase its functional affinity through the creation of a multimer. Association of identical scFv molecules to form diabodies, triabodies and tetrabodies can comprise a number of identical Fv modules. These reagents are therefore multivalent, but monospecific. The association of two different scFv molecules, each comprising a VH and VL domain derived from different parent Ig will form a fully functional bispecific diabody. A unique application of bispecific scFvs is to bind two sites simultaneously on the same target molecule via two (adjacent) surface epitopes. These reagents gain a significant avidity advantage over a single scFv or Fab fragments. A number of multivalent scFv-based structures has been engineered, including for example, miniantibodies, dimeric miniantibodies, minibodies, (scFv)2, diabodies and triabodies. These molecules span a range of valence (two to four binding sites), size (50 to 120 kDa), flexibility and ease of production. Single chain Fv antibody fragments (scFvs) are predominantly monomeric when the VH and VL domains are joined by, polypeptide linkers of at least 12 residues. The monomer scFv is thermodynamically stable with linkers of 12 and 25 amino acids length under all conditions. The noncovalent diabody and triabody molecules are easy to engineer and are produced by shortening the peptide linker that connects the variable heavy and variable light chains of a single scFv molecule. The scFv dimers are joined by amphipathic helices that offer a high degree of flexibility and the miniantibody structure can be modified to create a dimeric bispecific (DiBi) miniantibody that contains two miniantibodies (four scFv molecules) connected via a double helix. Gene-fused or disulfide bonded scFv dimers provide an intermediate degree of flexibility and are generated by straightforward cloning techniques adding a C-terminal Gly4Cys sequence. scFv-CH3 minibodies are comprised of two scFv molecules joined to an IgG CH3 domain either directly (LD minibody) or via a very flexible hinge region (Flex minibody). With a molecular weight of approximately 80 kDa, these divalent constructs are capable of significant binding to antigens. The Flex minibody exhibits impressive tumor localization in mice. Bi- and tri-specific multimers can be formed by association of different scFv molecules. Increase in functional affinity can be reached when Fab or single chain Fv antibody fragments (scFv) fragments are complexed into dimers, trimers or larger aggregates. The most important advantage of multivalent scFvs over monovalent scFv and Fab fragments is the gain in functional binding affinity (avidity) to target antigens. High avidity requires that scFv multimers are capable of binding simultaneously to separate target antigens. The gain in functional affinity for scFv diabodies compared to scFv monomers is significant and is seen primarily in reduced off-rates, which result from multiple binding to two or more target antigens and to rebinding when one Fv dissociates. When such scFv molecules associate into multimers, they can be designed with either high avidity to a single target antigen or with multiple specificities to different target antigens. Multiple binding to antigens is dependent on correct alignment and orientation in the Fv modules. For full avidity in multivalent scFvs target, the antigen binding sites must point towards the same direction. If multiple binding is not sterically possible then apparent gains in functional affinity are likely to be due the effect of increased rebinding, which is dependent on diffusion rates and antigen concentration. Antibodies conjugated with moieties that improve their properties are also contemplated for the instant invention. For example, antibody conjugates with PEG that increases their half-life in vivo can be used for the present invention. Immune libraries are prepared by subjecting the genes encoding variable antibody fragments from the B
lymphocytes of naive or immunized animals or patients to PCR amplification. Combinations of oligonucleotides which are specific for immunoglobulin genes or for the immunoglobulin gene families are used. Immunoglobulin germ line genes can be used to prepare semisynthetic antibody repertoires, with the complementarity-determining region of the variable fragments being amplified by PCR using degenerate primers. These single-pot libraries have the advantage that antibody fragments against a large number of antigens can be isolated from one single library. The phage-display technique can be used to increase the affinity of antibody fragments, with new libraries being prepared from already existing antibody fragments by random, codon-based or site-directed mutagenesis, by shuffling the chains of individual domains with those of fragments from naive repertoires or by using bacterial mutator strains. 85] Alternatively, a SCID-hu mouse, for example the model developed by Genpharm, can be used to produce antibodies, or fragments thereof. In one embodiment, a new type of high avidity binding molecule, termed a "peptabody" has been created by harnessing the effect of multivalent interaction is contemplated. A short peptide ligand was fused via a semirigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In a preferred embodiment of this invention, ligands and/or chimeric inhibitors can be targeted to tissue- or tumor- specific targets by using bispecific antibodies, for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target. To avoid the limitations of chemical conjugates, molecular conjugates of antibodies can be used for production of recombinant bispecific single-chain Abs directing ligands and/or chimeric inhibitors at cell surface molecules. Alternatively, two or more active agents and or inhibitors attached to targeting moieties can be administered, wherein each conjugate includes a targeting moiety, for example, a different antibody. Each antibody is reactive with a different target site epitope (associated with the same or a different target site antigen). The different antibodies with the agents attached accumulate additively at the desired target site. Antibody-based or non-antibody- based targeting moieties can be employed to deliver a ligand or the inhibitor to a target site. Preferably, a natural binding agent for an unregulated or disease associated antigen is used for this purpose. [00186] Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983 Immunology Today 4:72; Cole et al., 1983 Proc Natl Acad Sci USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985 in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA., IgD and any subclass thereof. The hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. An example of anti-chondroitin sulfate antibody production is provided in the Examples section below.
[00187] Anti-glyco-epitope antibodies can be prepared by methods as commonly known in the art, and as described in the Examples section below. Briefly, one or more homogeneous glycomolecules or polypeptides comprsing the glycol-epitope can be conjugated to bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). The glycomolecule-conjugates were then dialyzed, and the protein concentrations were determined. Mice were immunized with the glycomolecule-conjugate and boosted five times over a period of 2 months. Spleen cells of the mice were fused to a myeloma cell line, and multiclonal cell lines were then screened via ELISA analysis. Clones specific for antibodies to the desired glyco-epitope and with absorbance values greater than 1.0 were kept for subsequent expansion. Single cell clones can then screened via ELISA, and clones specific for the desired glyco-epitope and with absorbance values greater than 1.0 were analyzed by dot blot analysis. Antibodies to a glyco- epitope preferably are able to bind specifically to a single subtype of glycomolecule.
[00188] The genetic material that encodes an antibody that specifically binds to a specific glyco- epitope can be isolated, and that material can be introduced into a suitable expression vector and thereafter transfected into host cells. Thus, anti-glyco-epitope antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding particular antibodies can be used for transformation of a suitable mammalian host cell, such as a CHO cell. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference). The transformation procedure used depends upon the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
[00189] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. Cell lines of particular preference are selected through determining which cell lines have high expression levels and produce antibodies with anti-glycomolecule binding properties.
[00190] The invention also includes functional equivalents of the antibodies described in this specification. Functional equivalents have binding characteristics that are comparable to those of the antibodies, and include, for example, chimerized, humanized and single chain antibodies as well as fragments thereof. Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319, European Patent Application No. 239,400; PCT
Application WO 89/09622; European Patent Application 338,745; and European Patent Application EP 332,424.
[00191] Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the invention. "Substantially the same" as applied to an amino acid sequence is defined herein as a sequence with at least 80%, preferably at least about 90%, and more preferably at least about 95% sequence identity to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 2448 (1988).
[00192] In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al. 1984 Proc Natl Acad Sci USA 81:6851-6855; Neuberger et al. 1984 Nature 312:604-608; Takeda et al. 1985 Nature 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Chimerized antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human.
[00193] Humanized forms of the antibodies can be made by methods known in the art, for
example by substituting the complementarity determining regions of, for example, a mouse antibody, into a human framework domain, e.g., see PCT Pub. No. W092/22653. Humanized antibodies preferably have constant regions and variable regions other than the complement determining regions (CDRs) derived substantially or exclusively from the corresponding human antibody regions and CDRs derived substantially or exclusively from a mammal other than a human.
[00194] Functional equivalents also include single-chain antibody fragments, also known as
single-chain antibodies (scFvs). Single-chain antibody fragments of the present invention are shown in the Examples herein, and are recombinant polypeptides which bind glycol-epitopes. These fragments contain at least one fragment of an antibody variable heavy-chain amino acid sequence (VH) tethered to at least one fragment of an antibody variable light-chain sequence (VL) with or without one or more interconnecting linkers. Such a linker may be a short, flexible peptide selected to assure that the proper three-dimensional folding of the (VL) and (VH) domains occurs once they are linked so as to maintain the target molecule binding- specificity of the whole antibody from which the single-chain antibody fragment is derived. Generally, the carboxyl terminus of the (VL) or (VH) sequence may be covalently linked by such a peptide linker to the amino acid terminus of a complementary (VL) and (VH) sequence. Single-chain antibody fragments may be generated by molecular cloning, antibody phage display library or similar techniques. These proteins may be produced either in eukaryotic cells or prokaryotic cells, including bacteria.
[00195] Single-chain antibody fragments contain amino acid sequences having at least one of the variable or complementarity determining regions (CDR's) of the whole antibodies described in this specification, but are lacking some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute a major portion of the structure of whole antibodies. Single-chain antibody fragments may therefore overcome some of the problems associated with the use of antibodies containing a part or all of a constant domain. For example, single-chain antibody fragments tend to be free of undesired interactions between biological molecules and the heavy-chain constant region, or other unwanted biological activity. Additionally, single-chain antibody fragments are considerably smaller than whole antibodies and may therefore have greater capillary permeability than whole antibodies, allowing single-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently. Also, antibody fragments can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production. Furthermore, the relatively small size of single-chain antibody fragments makes them less likely to provoke an immune response in a recipient than whole antibodies.
[00196] Functional equivalents further include fragments of antibodies that have the same, or comparable binding characteristics to those of the whole antibody. Antibody fragments which recognize specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989 Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. In some embodiments, the antibody fragments contain all six complement determining regions of the whole antibody, although fragments containing fewer than all of such regions, such as three, four or five CDRs, are also functional. Further, the functional equivalents may be or may combine members of any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
[00197] Techniques described for the production of single chain antibodies (U.S. Pat. No.
4,946,778; Bird 1988 Science 242:423-426; Huston et al. 1988 Proc Natl Acad Sci USA 85:5879-5883; and Ward et al. 1989 Nature 334:544-546) can be adapted to produce single chain antibodies against a gene encoding a glyco-epitope. Single chain antibodies can be formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
Human Antibodies and Humanization of Antibodies
[00198] Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, fully human antibodies can be generated through the introduction of human antibody function into a rodent so that the rodent produces fully human antibodies. Unless specifically identified herein, "human" and "fully human" antibodies can be used interchangeably herein. The term "fully human" can be useful when distinguishing antibodies that are only partially human from those that are completely, or fully human.
[00199] Fully human antibodies can be made by any methods known in the art. One method for generating fully human antibodies is through the use of XENOMOUSE™. strains of mice which have been engineered to contain 245 kb and 190 kb- sized germline configuration fragments of the human heavy chain locus and kappa light chain locus. See Green et al. Nature Genetics 7:13-21 (1994). The XENOMOUSE™ strains are available from Abgenix, Inc.
(Fremont, Calif.).
[00200] In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal.
[00201] Vectors and Methods of Production
[00202] According to at least some embodiments of the present invention, there is provided an expression vector comprising at least one copy of a nucleic acid molecule as described above, for example, an expression vector comprising at least one, or any combination of nucleic acid sequences SEQ ID NO: 1-6, or a fragment thereof.
[00203] The term "vector" as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e. g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e. g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").
[00204] In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e. g. replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
[00205] The invention also concerns host cells comprising and expressing the above
described expression vector. The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, CHO cells and lymphocytic cells.
[00206] Expression vectors and host cells suitable for expression of recombinant antibodies and humanized antibodies are well known in the art. The following references are
representative of methods and vectors suitable for expression of recombinant immunoglobulins which may be utilized in carrying out the present invention: Weidle et al., Gene, 51: 21-29 (1987); Dorai et al., J. Immunol., 13 (12): 4232- 4241 (1987) ; De Waele et al., Eur. J.
Biochem. , 176: 287-295 (1988); Colcher et al., Cancer Res. , 49: 1738-1745 (1989); Wood et al. , J. Immunol., 145 (9): 3011-3016 (1990); Bulens et al., Eur. J. Biochem. , 195: 235-242 (1991); Beldsington et al., Biol. Technology, 10: 169 (1992); King et al., Biochem. J. , 281: 317-323 (1992); Page et al., Biol. Technology, 9: 64 (1991) ; King et al., Biochem. J., 290: 723-729 (1993) ; Chaudhary et al., Nature, 339: 394-397 (1989) ; Jones et al. , Nature, 321: 522-525 (1986) ; Morrison and Oi, Adv. Immunol., 44: 65-92 (1989) ; Benhar et al., Proc. Natl. Acad. Sci. USA, 91: 12051-12055 (1994); Singer et al. , J. hnunol., 150: 2844-2857 (1993); Couto et al., Hybridoma, 13 (3): 215-219 (1994); Queen et al. , Proc. Natl. Acad. Sci. USA, 86 : 10029-10033 (1989); Caron et al., Cancer Res., 52: 6761-6767 (1992); Coloura et al, J. Immunol. Meth. , 152: 89-109 (1992). Moreover, vectors suitable for expression of recombinant antibodies are commercially available. The vector may, e. g. be a bare nucleic acid segment, a carrier-associated nucleic acid segment, a nucleoprotein, a plasmid, a virus, a viroid, or a transposable element.
[00207] Host cells known to be capable of expressing functional immunoglobulins include, e. g.: mammalian cells such as Chinese Hamster Ovary (CHO) cells; COS cells; myeloma cells, such as NSO and SP2/0 cells; insect cells, bacteria such as Escherichia coli; yeast cells such as Saccharomyces cerevisiae; and other host cells. Of these, CHO cells are used by many researchers given their ability to effectively express and secrete immunoglobulins. NSO cells are one of the preferred types of host cells useful in the present invention.
[00208] Host cells are transformed following techniques that are known to the person skilled in the art. A "transformed" cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation
encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, calcium phosphate precipitation, and particle gun acceleration.
[00209] Another embodiment of the present invention relates to a hybridoma, e.g., a cell secreting at least one antibody as disclosed herein, e.g., a monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention.
[00210] In some embodiments, prepared hybridomas which produce monoclonal antibodies can be passage-cultured in a standard culture solution, or can be stored for a long period in liquid nitrogen.
[00211] One example of a method employed to obtain monoclonal antibodies from the
hybridomas involves culturing the hybridomas and obtaining monoclonal antibodies in the culture supernatant according to a standard method. The former method is suitable for the mass production of antibodies.
[00212] A monoclonal antibody that can be used in the present invention can be a
recombinant monoclonal antibody that is prepared by cloning the antibody gene from the hybridoma, incorporating the gene into an appropriate vector, introducing the vector into a host, and then causing the host to produce the recombinant monoclonal antibodies by genetic engineering techniques (e.g., see Vandamme, A. M. et al., Fur. J. Biochem, (1990) 192, 767- 775, 1990).
[00213] In addition to the above host cell, a transgenic animal or plant can also be used to produce a recombinant antibody.
[00214] According to at least some embodiments of the present invention, there is provided a transgenic non-human animal having a genome comprising the isolated nucleic acid molecule and/or the expression vector according to the present invention.
[00215] Pharmaceutical compositions
[00216] In another aspect, the present invention concerns a pharmaceutical composition comprising the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention and a pharmaceutically acceptable carrier. According to the present invention, the pharmaceutically composition comprises at least a therapeutically effective quantity or amount of the substantially purified and isolated agonist antibodies, e.g., antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention.
[00217] The isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal
antibody fragments or antigen binding portions or fragments thereof described herein as a "compound" may be administered with a physiologically acceptable carrier. A physiologically acceptable carrier is a formulation to which the compound can be added to dissolve it or otherwise facilitate its administration. An important factor in choosing an appropriate physiologically acceptable carrier is selecting a carrier in which the compound remains active or the combination of the carrier and the compound produces an active compound. Usually, the pharmaceutically acceptable carrier can be a solvent or dispersion medium. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical composition of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of emulsifying agents such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, such as Tween 20. [00218] The pharmaceutical composition may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, pharmaceutically-acceptable antioxidants and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
[00219] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[00220] Pharmaceutically acceptable salts of the isolated antibodies, e.g., monoclonal
antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention may also be envisioned.
[00221] A "pharmaceutically acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any undesired toxicological effects (see e. g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).
[00222] In another embodiment, a pharmaceutical composition of the invention is
formulated for parenteral, intravenous, oral, subcutaneous, intradermal, intramuscular or topical, administration.
[00223] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion.
[00224] In some embodiments, any route of administration of at least one isolated antibody, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention are intravenously, intramuscularly and intraperitoneally. In some embodiments, modes of delivery are by injection and infusion.
[00225] Injectable forms may include sterile aqueous solutions or dispersions. Furthermore, form of sterile powders may be prepared for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical composition can be formulated to be stable under the conditions of manufacture and storage; thus, in some embodiments, the composition is prepared to prevent the contaminating action of
microorganisms such as bacteria and fungi.
[00226] Parenteral administration may be prepared as solutions or suspensions of the
combined components in physiological solution. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.
[00227] In preparing oral dosage forms, any convenient pharmaceutical media may be
employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like may be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets may be coated by standard aqueous or non-aqueous techniques.
[00228] A tablet may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants.
[00229] Compressed tablets may be prepared by compressing, in a suitable machine, the combined components in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent.
[00230] Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
[00231] Effective dosage regimens are adjusted to provide the optimum desired response (e. g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
[00232] Regardless of the route of administration selected, the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical composition of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
[00233] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. For example, where the desired therapeutic response is to treat cancer by eliminating cancer cells, and/or inducing cell death of cancer cells, a dosage of a pharmaceutical composition as disclosed herein is in the amount for inducing death of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 100%, or any integer between 10-100% of the cancer cells.
[00234] In some embodiments, the selected dosage level depends upon a variety of
pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the salt or amide thereof, the route of administration, the time of administration, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors. [00235] A physician or veterinarian can start doses of the compounds of the invention- employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention is that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition can be administered as two; three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention to be administered alone, it is preferable to administer the latter as a pharmaceutical formulation (composition).
[00236] Effective doses of the compositions of the present invention, for the treatment of a disease or disorder, e.g., cancer such as NSCLC described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages can easily be titrated to optimize safety and efficacy by physician of ordinary skill in the art.
[00237] For administration with an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually about 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1 to 10 mg/kg. In some embodiments, a treatment regime entails administration once per day or once per week or once per two weeks or once a month or once every 3 to 6 months. In some methods, two or more monoclonal antibodies with different binding specificities, or alternatively, two or more different antibodies, e.g., antibodies which have different specificities and/or functionalities, for example and without limitation, antibodies to either different lectins or antibodies which displace the same lectin from different glycoproteins are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. In some embodiments, an antibody can be administered to a subject on multiple occasions. Intervals can be repeated single dosages, for example, bidaily, daily, weekly, biweekly, monthly or yearly. In some embodiments, intervals can also be irregular as indicated by measuring blood levels of antibodies in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 0.001-1000 μg/ml. Alternatively, an antibody can be administered as a sustained release formulation, in which case less frequent administration is required.
[00238] Dosage and frequency vary depending on the half-life of the antibody in the patient.
In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
[00239] The dosage ranges of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof according to the present invention may vary with the administration routes, as well as the state of the patient (age, sex, body weight, extent of the disease etc.). Ideally, an isolated antibody, e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof is administered to a patient in need thereof at a dosage unit from 0.1 mg/kg of body weight to 100 mg/kg of body weight.
[00240] Surprisingly, the inventors have demonstrated that the administration (for example application, injection, delivery, contact etc.) of the pharmaceutical composition comprising antibodies according to at least some embodiments of the present invention results in an improved therapeutic effect in particular in the treatment of cancer, e.g., a decrease in the number of cancer cells, or an increase in the induced cell death of cancer cells, or a cancer cell population.
[00241] The present invention, according to at least some embodiments, also concerns the use of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment of cancer as described herein and preferably for treatment of an epithelial cell cancer, including but not limited to the abnormal proliferative growth in of any one or more of the following organs and tissues: lung, bone, pancreatic, skin, head or neck, eye, uterus, ovary, rectum, anal region, stomach, colon, breast, fallopian tubes, endometrium, cervix, vagina, vulva, lymph including Hodgkin's and non-Hodgkin's and lymphocytic lymphomas, esophagus, small intestine, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, urethra, penis, prostate, blood including chronic or acute leukemia, bladder, kidney, the central nervous system (CNS) including spinal axis tumors, brain stem glioma; and pituitary.
[00242] Non-limiting examples of specific cancers include lung cancer, colorectal
adenocarcinomas, epithelial ovarian cancer, pancreatic cancer (including endocrine and exocrine pancreatic cancers, the latter including pancreatic ductal adenocarcinomas) and prostate cancer. Non-limiting examples of lung cancer include non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), carcinoids, cylindromas, and certain sarcomas.
[00243] Non-small cell lung cancers may optionally include any of squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma. Another non- limiting example of such lung cancers includes bronchoalveolar lung cancer.
[00244] In the present invention, the term "modulating" refers to the effect induced by a molecule which interacts with a specific receptor to modify or control its biological activity. In some embodiments, "modulation" or "modulate" refers to an increase or decease in biological activity, as disclosed herein.
[00245] The half-lives of immunoglobulins are measured in days to weeks. Not surprisingly, the presence of monoclonal antibodies of the invention was still detectable in the serum one week following injection. The long in vivo half-life of the antibodies opens the possibility to expose patients such as or humans to these therapeutic agents for extended periods of time. Therefore this long term treatment brings unexpected therapeutic benefit, even after the postnatal period.
[00246] Alternatively, a further aspect of the present invention also concerns a method of treating cancer in a patient in need thereof. According to the present invention, the method of treating cancer comprises administering to the patient at least one isolated antibody, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof in an amount which is therapeutically effective.
[00247] It is also another aspect of the invention to use the isolated antibodies, e.g.,
monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, for the manufacture of a medicament for the prevention of cancer.
[00248] Additionally, the present invention pertains to a pharmaceutical kit comprising at least an effective amount of the isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, together with instructions for use and in particular instructions directed to the treatment of cancer, e.g., NSCLC.
[00249] The pharmaceutical kit according to the present invention may comprise a container comprising at least said isolated antibodies, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof.
[00250] Generally, the kit comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer, such as NSCLC.
[00251] Alternatively, or additionally, a kit may further comprise a second (or third)
container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
[00252] Antibodies as disclosed herein that cross-react with mouse and human glyco- epitopes such as glycoproteins can also cross-react with glyco-epitopes of most mammalian species, and also with glyco-epitopes of other vertebrate species. If the antibodies do not cross- react with glyco-epitopes of a given mammalian or vertebrate species, it is possible to generate them according to the process described in this application by immunizing mice with a glyco- epitope that corresponds to that of the species of interest.
[00253] Also described herein is the use of an isolated antibody, e.g., monoclonal antibodies or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof or the pharmaceutical composition of the invention, for the manufacture of a medicament for use in veterinary applications in any mammal or vertebrate species. The medicament can be utilized in utero (for mammals) or in ovo (for birds, reptiles, egg-layer fishes, etc...), or in young animals, or in adult animals. The treatment can be used to modulate the development of cells or tissues of ectodermal origin, such as hair, feathers, scales, horns, claws, beaks, teeth, skin, sweat glands, sebaceous glands, larynx and trachea mucus-producing cells, Meibomian glands, preputial glands, mammary glands and salivary glands, In particular, the treatment can be used to temporarily or permanently modify hair, scale or feathers color and/or morphology.
[00254] In some embodiments, the use of the anti-glyco-epitope antibodies or antibody mimetic in a medicament for the treatment of a cancer in a subject is contemplated. The medicament can contain a therapeutically effective amount of the antibody. In some embodiments, the amount of a anti-glyco-epitope antibodies or antibody mimetic in the medicament is sufficient so that at least one beneficial result is observed, e.g., a lessening of a symptom. In some embodiments, the amount that is administered removes all of the symptoms of the cancer. In some embodiments, the amount is sufficient so that the level of a biomarker of cancer decreases in a subject after the medicament has been administered. In some embodiments, the amount of the antibody administered is about 0.001 to 1000, 0.1 to 100, 0.5 to 50, 1 to 10, 1, 3, or 10 mg of antibody/kg of subject. As will be appreciated by one of skill in the art, the actual amount of the antibody can depend upon the particular disorder (e.g., type of cancer, grade of cancer, if it is malignant or non-malignant etc.), the method of administration, the frequency of administration, the desired result, the characteristics of the patient, and the characteristics of the antibody. As will be appreciated by one of skill in the art, the use of the antibody in the preparation or manufacture of a medicament can involve any of the disclosed antibodies in any amount, sufficient to treat the particular condition it is directed to. In some embodiments, a medicament is prepared with one of the single chain anti-glyco-epitope antibodies selected from the group consisting of sc-Fv#l antibody, sc-Fv#8 antibody, sc-Fv#A7 antibody, sc- Fv#C2 antibody, sc-Fv#G2 antibody, and sc-Fv#H2 antibody.
[00255] As will be appreciated by one of skill in the art, the nature of the disorder can play a role in the amount, frequency, and method of administration. For example, in chronic disorders, relatively larger amounts, more potent antibodies, and/or more frequently administered doses of the antibody can be required. Similarly, in acute disorders, the amount of antibody required for treatment, including prophylaxis, can be relatively less. In subjects in which sensitization is initially required prior to the challenge, lower amounts of the antibody can be beneficial compared to the amount required for subjects that are naturally allergic. In such chronic systems, increased amounts of the antibody, as well as increased frequency of administration can be advantageous. The exact amount can readily be determined by one of skill in the art, in light of the present disclosure. One of skill in the art will further appreciate other factors and how to adjust the administration of the antibody accordingly.
[00256] If desired, the isotype of an anti-glyco-epitope antibody can be switched, for example to take advantage of a biological property of a different isotype. There are a number of isotypes of antibodies that are capable of the same, including, without limitation, the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgGl, and human IgG3. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather, the antibody as generated can possess any isotype and the antibody can be isotype switched thereafter using conventional techniques that are well known in the art. Such techniques include the use of direct recombinant techniques (see e.g., U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418), among others.
[00257] In some embodiments, an anti-glyco-epitope antibody as discussed herein is a mouse antibody. In some embodiments, the anti-glyco-epitope antibodies discussed herein can be human antibodies. If an antibody possessed desired binding to the glyco-epitope, it could be readily isotype switched to generate a human IgM, human IgGl, or human IgG3 isotype, while still possessing the same variable region (which defines the antibody's specificity and some of its affinity). Such molecule would then be capable of fixing complement and participating in CDC.
[00258] In the cell-cell fusion technique, a myeloma or other cell line is prepared that possesses a heavy chain with any desired isotype and another myeloma or other cell line is prepared that possesses the light chain. Such cells can, thereafter, be fused and a cell line expressing an intact antibody can be isolated.
[00259] Accordingly, as antibody candidates are generated that meet desired "structural"
attributes as discussed above, they can generally be provided with at least certain of the desired "functional" attributes through isotype switching.
[00260] Biologically active antibodies that bind glyco-epitopes are preferably used in a sterile pharmaceutical preparation or formulation to reduce the activity of glycomolecule. Anti-glyco- epitope antibodies preferably possess adequate affinity to potently interfere with the binding of glycoprotein to another entity, e.g., protein or glycoprotein. [00261] When used for in vivo administration, the antibody formulation is preferably sterile. This is readily accomplished by any method know in the art, for example by filtration through sterile filtration membranes. The modality of antibody administration is in accord with known methods.
Antibody mimetics
[00262] Antibody mimetics are organic or inorganic compounds that, like antibodies, can
specifically bind epitopes or antigens, but that are not structurally related to antibodies.
Accordingly, antibody mimetics encompass, but are not limited to nucleic acids, peptides or proteins, and in some embodiments, have a molar mass of about 3 to 20 kDa. Some antibody mimetics have an antibody-like beta-sheet structure. Antibody mimetics have several common advantages over antibodies, which include, but is not limited to, increased solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. Antibody mimetics are being developed as therapeutic and diagnostic agents (see Gebauer et al., "Engineered protein scaffolds as next-generation antibody therapeutics". 2009, Curr Opin Chem Biol.). In some embodiments, the antibody for use in the methods and compositions as disclosed herein can be any antibiotic mimetic as disclosed in Table 3.
[00263] Table 3 lists a number of different types of antibiotic mimetics
Figure imgf000059_0001
Kunitz domains of
Kunitz domain peptides various protease 6 kDa Ecallantide
inhibitors
10th type III domain of
Monobodies lO kDa Angiocept
fibronectin
[00264] (i) Affyboies and affibody molecules. In some embodiments, a glycomoleeule b d g entity is an AFFIBODY®. Affibody® Molecules mimic monoclonal antibodies, Compared to antibodies, the most striking dissimilarity of Affibody® Molecules is the small size,
Affibody® Molecules have a molecular weight of 6kDa, compared to the molecular weight of antibodies, which is 150kDa. In spite of its small size, the binding site of Affibody® Molecules is similar to that of an antibody.
[00265] The advantages of Affibody® Molecules over antibodies are: (i) their small size, (ii) he simple structure of the molecules (iii) its robust physical properties; able to withstand a broad range of analytical conditions, including extreme pH and elevated temperature (iv) its ability to fold correctly intracellularly (iv) Conjugation or directed coupling to matrices are facilitated by the unique C-terminal cysteine.
[00266] Affibody® Molecules have highly competitive properties for applications within affinity purification, sample preparation and protein detection. Affibodies are available commercially, for example from the sweedish company Affibody AB,
[00267] (ii) Affilins are a type of antibody mimetic that are genetically engineered proteins with the ability to selectively bind antigens. They are structurally derived from one of two proteins, gamma-B crystallin or ubiquitin, both occurring in humans. Affilins are constructed by modification of near- surface amino acids of these proteins and isolated by display techniques such as phage display. They resemble antibodies in their affinity to antigens but not in structure, which makes them a type of antibody mimetic. Affilins are being developed as potential new biopharmaceutical drugs.
[00268] (iii) Artificial binding proteins (Affitins) are a type of antibody mimetic and are
artificial proteins with the ability to selectively bind antigens. They are structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, a microorganism belonging to the archaea domain. By randomizing the amino acids on the binding surface of Sac7d and subjecting the resulting protein library to rounds of ribosome display, the affinity can be directed towards various proteins. Affitins are antibody mimetics and are being developed as an alternative to antibodies as tools in biotechnology. They have also been used as specific inhibitors for various enzymes, (see Krehenbrink, et al., (2008). "Artificial binding proteins (Affitins) as probes for conformational changes in secretin PulD". J. Mol Biol., 383 (5): 1058-68.
[00269] (iv) Anticalins are a type of antibody mimetic and are artificial proteins that are able to bind to antigens, either to proteins or to small molecules (Skerra A "Alternative binding proteins: anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities". FEBS J.2008: 275(11): 2677-83. They are not structurally related to antibodies, which makes them a type of antibody mimetic. Instead, they are derived from human lipocalins which are a family of naturally binding proteins. Anticalins are being used in lieu of monoclonal antibodies, but are about eight times smaller with a size of about 180 amino acids and a mass of about 20 kDa.
[00270] Anticalins have better tissue penetration than antibodies and are stable at temperatures up to 70 °C. Unlike antibodies, they can be produced in bacterial cells like E. coli in large amounts. While antibodies and most other antibody mimetics can only be directed at macromolecules like proteins, anticalins are able to selectively bind to small molecules as well. Characteristic for anticalins is their barrel structure formed by eight antiparallel β-strands pairwise connected by loops and an attached a-helix. The main structure of anticalins is identical to wild type lipocalins. Conformational deviations are primarily located in the four loops reaching in the ligand binding site. Mutagenesis of amino acids at the binding site allows for changing the affinity and selectivity.
[00271] (v) Avimers (short for avidity multimers) are a type of antibody mimetic and are
artificial proteins that are able to specifically bind to certain antigens via multiple binding sites. Since they are not structurally related to antibodies, they are classified as a type of antibody mimetic. Avimers have been developed by the biotechnology company Avidia, now part of Amgen, as potential new pharmaceutical drugs (Jeong, et al., (2005). "Avimers hold their own". Nature Biotech 23 (12): 1493-4).
[00272] Avimers typically consist of two or more peptide sequences of 30 to 35 amino acids each, connected by linker peptides. The individual sequences are derived from A domains of various membrane receptors and have a rigid structure, stabilised by disulfide bonds and calcium. Each A domain can bind to a certain epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity (hence the name).
[00273] Alternatively, the domains of avimers useful herein can be directed against glyco- epitopes on different target glycomolecules. This approach is similar to the one taken in the development of bispecific monoclonal antibodies. In a study, the plasma half-life of an anti- interleukin 6 avimer could be increased by extending it with an anti-immunoglobulin G domain. (Silverman, et al., (2005). "Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains". Nature Biotech 23 (12): 1556.
[00274] Avimers with two or three domains can bind to their targets glycomolecules in sub- nanomolar concentrations. They have improved heat stability compared with antibodies, but limited plasma half-life because of their smaller size. Half-life can be increased by binding them to antibodies.
[00275] Avimers can be produced in a library. A library theoretically containing up to 1023
different A domains serves as a starting point for the development of avimers. Domains targeting the desired glycolmolecule or glycol-epitope are selected with display techniques such as phage display. The most promising species are linked to a second A domain via a short linker peptide, forming a new library. This process can be repeated several times, yielding avimers with an increasing number of domains.
[00276] (vi) DARPins (an acronym for designed ankynn repeat proteins) are genetically
engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding (Binz HK et al., (2004). "High-affinity binders selected from designed ankyrin repeat protein libraries." Nature Biotech 22 (5): 575-582). DARPins are derived from natural ankyrin proteins and consist of at least three, usually four or five repeat motifs of these proteins. Their molecular mass is about 14 or 18 kDa (kilodaltons) for four- or five-repeat DARPins, respectively. DARPins are derived from naturally occurring ankyrin proteins, a protein class that is mediating high-affinity protein-protein interactions in nature. Sequence alignments of several thousand natural ankyrin repeat motifs (of about 33 amino acids each) combined with structure based design and recombinant DNA methods is used for generation of these proteins. DARPins are composed of repetitive structural units forming a stable protein domain with a large potential target interaction surface. Typically, DARPins are composed of four or five repeats, corresponding to the average size of natural ankyrin repeat protein domains. [00277] (vii) Monobodies, also known as adnectins, are a type of antibody mimetic and are genetically engineered proteins that are able to bind to antigens. Despite their name, monobodies are not structurally related to antibodies. Monobodies consist of 94 amino acids and have a molecular mass of about 10 kDa, fifteen times smaller than an IgG type antibody and comparable to the size of a single variable domain of an antibody. They are based on the structure of human fibronectin, more specifically on its tenth extracellular type III domain. This domain has a structure similar to antibody variable domains, with seven beta sheets forming a barrel and three exposed loops on each side corresponding to the three
complementarity determining regions (Koide A et al., "Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain". Methods Mol. Biol. 2007; 352: 95-109). Monobodies lack binding sites for metal ions and the central disulfide bond. Monobodies with specificity for different proteins can be tailored by modifying the loops BC (between the second and third beta sheets) and FG (between the sixth and seventh sheets) (Koide A et al., "The fibronectin type III domain as a scaffold for novel binding proteins". J. Mol. Biol.
1998;284 (4): 1141-51).
[00278] DETECTION OR TREATMENT OF VARIOUS TYPES OF CANCER
[00279] Cancers that originate in epithelial tissue (cellular tissue that lines cavities such as the stomach or lung) are called carcinomas. Epithelial tumors have a common biological profile and mechanism, such that antibodies or fragments thereof which are suitable for detection or treatment of one type of epithelial cancer, such as lung cancer, may also be suitable for other types of cancers.
[00280] Molecular classification of tumors based on their gene expression profiles promises to significantly refine diagnosis and management of cancer patients. Thomas et al. (American Journal of Pathology. 2001;159:1231-1238) have generated comprehensive gene expression profiles from 154 primary lung, colon, and ovarian adenocarcinomas to identify common profile and differences that allow discrimination of tumors in an organ- specific manner.
[00281] Kaufmann et al. (Histopathology, 2007: 29 ; 233 - 240) have demonstrated some molecular markers, excluding ovarian carcinomas, this combination had a sensitivity, specificity and predictive accuracy for mammary carcinomas of 0.83, 0.98 and 0.98, respectively. Carcinoembryonic antigen and/or cytokeratin 20 identified bronchogenic, gastric, pancreatic and colorectal carcinomas. [00282] Tumor necrosis factor receptor (TNFR) associated factor 4 (TRAF4) was initially identified as a gene amplified and overexpressed in breast carcinomas. Camilleri-Broet et al,( Oncogene (2007) 26, 142-147 ) evaluated whether TRAF4 protein overexpression exists in other cancer types. Immunohistochemistry analysis of tumor samples from 623 patients with 20 different tumor types showed that TRAF4 was overexpressed in 268 tumors (43%), including 82 of 137 lung adenocarcinomas (60%). TRAF4 protein overexpression was limited to cancer cells and the subcellular localization was consistently cytoplasmic in a large majority of cases.
[00283] Christopher et al. (Cancer Res 2006; 66: (6). 2953-2961) studied cancer
biomarkers related to the oncogenic process that are ubiquitously expressed by most malignancies with high sensitivity and specificity for conventional histopathologic evaluation by targeting genes whose expression is critical for invasion, metastatization, and cell survival.
[00284] Previously reports have identified common genetic traits associated with
aggressiveness, uncontrolled proliferation, and metastatic potential, which could, in turn, be exploited as ubiquitous identifiers of malignancy. They searched for genes overexpressed by cancer tissues in 373 archival cDNA microarray samples encompassing a variety of malignant and benign samples. All samples were prepared and processed identically and cohybridized consistently with a differentially labeled reference onto a 17.5K custom-made cDNA array. Novel candidate biomarkers were identified that could define malignancy with high levels of accuracy.
[00285] These data demonstrate that there is a common profile of tumors that originate from the same cell type as for epithelial cells. Therefore, the use of the scFv fragments and lectin- displacing antibodies as disclosed herein that were selected for adenocarcinoma of the lung are useful for the treatment and diagnosis of other cancers of epithelial cell origin.
[00286] Non-limiting examples of such other types of cancers include colorectal
adenocarcinomas, epithelial ovarian cancer, pancreatic cancer (including endocrine and exocrine pancreatic cancers, the latter including pancreatic ductal adenocarcinomas) and prostate cancer.
[00287] The efficacy of the antibodies and fragments according to at least some
embodiments of the present invention may optionally be tested according to a suitable assay as is known in the art for the above cancers. [00288] Diagnosis and analysis
[00289] Beside the therapeutic use of antibodies, for example for the treatment of diseases, such as cancer, these antibodies also constitute invaluable analytical tools. Most of these antibodies show cross-reactivity with mouse and human cancer cells. Monoclonal antibodies, according to at least some embodiments of the invention are also able to detect human, rat, rabbit and dog cancer cells. These antibodies were successfully tested in flow cytometry and Western blotting applications. The use of these reagents in immunohistochemistry is of real importance as a diagnostic tool, both for in vitro and in vivo diagnostic methods.
[00290] In some embodiments, the antibodies as disclosed herein for use in diagnosis, e.g., diagnosis of cancer, may optionally feature a ligand or tag. The ligand or tag may optionally comprise polyethylene glycol (PEG), a nanoparticle or particles, or a label. In some
embodiments, the label can be detected in vitro and/or in vivo. In some embodiments, detecting the binding of an antibody as disclosed herein to a cell in a biological sample, e.g., a serum or tissue sample is useful for in vitro diagnostic assays. In alternative embodiments, the detection of the binding of an antibody as disclosed herein to a cell present in a subject is useful for in vivo diagnostic assays.
[00291] Radio-imaging methods may optionally be used for in vivo diagnosis according to at least some embodiments of the present invention. These methods include but are not limited to, computer tomography (CT), magnetic resonance imaging (MRI) and spectroscopy (MRS), single photon emission computed tomography (SPECT), and positron emission tomography (PET). All of these techniques are non-invasive, and can be used to detect and/or measure a wide variety of tissue events and/or functions, such as detecting cancerous cells for example. Unlike PET, SPECT can optionally be used with two labels simultaneously. SPECT has some other advantages as well, for example with regard to cost and the types of labels that can be used. For example, US Patent No. 6,696,686, which is incorporated herein in its entirety by reference describes the use of SPECT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein. Briefly, an isolated antibody or fragment thereof against a biomarker associated with a particular disease, such as cancer for example, is administered to the subject, typically by injection. The isolated antibody or fragment thereof is preferably labeled, in this example with a radioisotope. The label is then used to determine the location of the isolated antibody or fragment thereof through one of the above technologies. [00292] Fluorophore-conjugated antibody for light and fluorescence imaging may also optionally be used, whether for in vitro tests or in-vivo imaging of cancer and cancer metastasis. In vivo fluorescence imaging resembles fluorescence microscopy in that both use a low-light camera and appropriate filters to collect fluorescence emission light from samples. The objects for imaging are typically the whole-body of small animals instead of cells in culture dishes or on slides. This extension into the in vivo setting allows visualization of biology in its intact and native physiological state.
[00293] Different fluorescent technologies (small-molecule fluorophores and quantum dot (QD) nanoparticles) or any molecules that absorb in the region of 400-1000 nm, including molecules that absorb in the near-infrared (NIR) region may optionally be used. This type of molecule can be efficiently used to visualize and investigate in vivo molecular targets because most tissues generate little NIR fluorescence. The most common organic NIR fluorophores are polymethines. Among them, pentamethine and heptamethine cyanines comprising
benzoxazole, benzothaizole, indolyl, 2-quinoline or 4-quinoline have been found to be the most useful [Rao et al. Current Opinion in Biotechnology 2007, 18:17-25]. A general, and simple, approach to improving the accumulation of contrast agents at the target site is to conjugate the fluorochrome to an antibody or an antibody fragment that binds to a specific molecular target. This approach is useful for tumor imaging, as cancers often overexpress certain surface receptors.
[00294] According to other embodiments of the present invention, the nanoparticle or
particles (attached to the isolated protein as a ligand or tag) may optionally comprise one or more of solid nanospheres microencapsulated with different dye compounds and/or magnetic properties, which may optionally be prepared from a variety of functional polymeric materials, e.g. polyacrolein, polyglutaraldehyde, polymethyl a-(hydroxymethyl) acrylate,
polychloromethylstyrene, cellulose and silica; uniform magnetic nanoparticles which may optionally comprise one or more of organic-inorganic hybrid particles composed of cores from micron-sized uniform polystyrene particles and shells from magnetite- silica nanospheres of approximately 30 nm diameter; and/or biodegradable, non-toxic, magnetic metal oxide (i.e. Fe304) nanoparticles of very narrow size distribution in sizes ranging from approximately 20 nm up to 0.5 microns; or non-magnetic and magnetic silica hollow micron-sized particles. [00295] Such nanoparticles may optionally be prepared as described for example with regard to US Patent No. 6,103,379, which is hereby incorporated by reference as if fully set forth herein.
[00296] COMBINATION DIAGNOSTIC PANELS
[00297] Any of the above described antibodies and fragments thereof may optionally be used in combination with an antibody, fragment thereof or other agent capable of detecting the presence of a known diagnostic marker to form a diagnostic panel. Non-limiting examples of such diagnostic markers which may optionally be combined in the diagnostic panel are given below.
[00298] Human chorionic gonadotropin (HCG) - HCG is another substance that appears normally in pregnancy and is produced by the placenta. If pregnancy is ruled out, HCG may indicate cancer in the testis, ovary, liver, stomach, pancreas, and lung. Marijuana use can also raise HCG levels.
[00299] CA 19-9 - This marker is associated with cancers in the colon, stomach, and bile duct. Elevated levels of CA 19-9 may indicate advanced cancer in the pancreas, but it is also associated with noncancerous conditions, including gallstones, pancreatitis, cirrhosis of the liver, and cholecystitis.
[00300] CA 15-3 - This marker is most useful in evaluating the effect of treatment for
women with advanced breast cancer. Elevated levels of CA 15-3 are also associated with cancers of the ovary, lung, and prostate, as well as noncancerous conditions such as benign breast or ovarian disease, endometriosis, pelvic inflammatory disease, and hepatitis. Pregnancy and lactation also can raise CA 15-3 levels.
[00301] CA 27-29 - This marker, like CA 15-3, is used to follow the course of treatment in women with advanced breast cancer. Cancers of the colon, stomach, kidney, lung, ovary, pancreas, uterus, and liver may also raise CA 27-29 levels. Noncancerous conditions associated with this substance are first trimester pregnancy, endometriosis, ovarian cysts, benign breast disease, kidney disease, and liver disease.
[00302] Neuron- specific enolase (NSE) - NSE is associated with several cancers, but it is used most often to monitor treatment in patients with neuroblastoma or small cell lung cancer.
[00303] Screening for complementary variable domain regions [00304] Given a specific variable domain region sequence, one of ordinary skill can easily screen for complementary variable domain region sequences using methods well known in the art. See, for example, Klimka et al., British Journal of Cancer (2000) 93: 252-260; Beboer et al., J. Mol. Biol. (2000) 296: 833-849; Radar et al., PNAS (1998) 95:8910-8915; Portolano et al, J. Immuno. (193) 150: 880-887; and Clarkson et al., Nature (1991) 352: 624-628, contents of all of which is herein incorporated by reference. For example, a heavy chain variable domain sequence comprising 1, 2, or 3 of the heavy chain CDR amino acid sequences described herein can be screened against a library of light chain variable domain sequences to obtain antibodies that bind human and/or mouse glyco-epitopes such as glycoproteins which are the binding sites for lectins. Alternatively, a light chain variable domain sequence comprising 1, 2, or 3 of the light chain CDRs described herein can be screened against a library of heavy chain variable domain sequences to obtain antibodies that bind human and/or mouse glyco-epitopes such as glycoproteins which are the binding sites for lectins. Without wishing to be bound by theory, this methodology can be used to humanize any known antibody. For example, a non-human variable domain sequence can be screened against human variable domain sequences and then the identified human variable domain sequences screened against a second set of human variable domain sequences.
[00305] Conjugation of antibodies with ligand/tags and/or fusion proteins
[00306] The term "fusion protein" as used herein refers to a polypeptide which comprises protein domains from at least two different proteins. For example, a fusion protein may comprise an antigen-binding portion or fragment of an antibody and a non-antibody protein.
[00307] A wide variety of ligands or tags can be coupled (i.e. linked) with the antibodies described herein. Without wishing to be bound by theory, the antibodies of the invention can be conjugated to either other peptides or other molecules to tailor, for example, the
bioavailability, serum half-life or shelf-life of the antibodies, immunogenicity, tolerance by human body, or to affect the solubility of the antibodies in pharmaceutically acceptable carriers. In some embodiments, the antibodies as disclosed herein can be coupled to form a fusion protein with a molecule or peptide to provide an additional functionality to the antibody, for example, enhanced therapeutic function such as coupling to a toxin. Although, conjugation with ligands and tags is discussed in reference to antibodies herein, it is to be understood that antibody fragments and antigen binding portions and fragments are also amenable to conjugation with ligands and tags.
[00308] In some embodiments, an antibody as disclosed herein can be fused with another molecule, for example, an antibody can be fused to at least one or more additional molecules, for example for therapeutic use and/or diagnostic use of antibodies. In some embodiments, an isolated antibody, e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof can be fused or conjugated to different proteins or molecules, for example to produce, but not limited to, bi-specific antibodies, antibody-drug conjugates (ADCs), antibody-radioisotope conjugates (e.g., radioimmunoconjugates), antibody-toxin fusion proteins (e.g., immunotoxins), antibody-enzyme fusion proteins for prodrug activation in Antibody Directed Enzyme Prodrug Therapy (ADEPT), and antibodies used for targeting gene delivery or drug-delivery systems.
[00309] In some embodiments, an isolated antibody, e.g., monoclonal antibody or isolated monoclonal antibody fragments or antigen binding portions or fragments thereof can be fused or conjugated to different ligands, including but not limited to, naturally occurring molecules, in some embodiments, a ligand can be a recombinant or synthetic molecules. Exemplary ligands include, but are not limited to, polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2- ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, aptamer, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g, steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., an alpha helical peptide, amphipathic peptide, RGD peptide, cell permeation peptide, endosomolytic/fusogenic peptide), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactors, lipopolysaccharide, an activator of p38 MAP kinase, an activator of NF-κΒ, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, gamma interferon, natural or recombinant low density lipoprotein (LDL), natural or recombinant high- density lipoprotein (HDL), bovine serum albumin (BSA), ovalbumin, keyhole limpet hemocyanin (KLH), and a cell-permeation agent (e.g., a.helical cell-permeation agent).
[00310] Ligands can be used for any number of reasons including, but no limited to,
targeting, PK modulation, and labeling/tagging. A targeting ligand can provide enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment. A PK modulating ligand can modulate pharmacokinetics of an antibody in vivo.
[00311] In some embodiments, the antibody of the invention is conjugated with a label/tag, such as a fluorescent label or a biotin label. Without wishing to be bound by theory, such labeling allows one to easily track the antibody, if necessary or to assist in purification of the antibody.
[00312] One can also design the ligand in such a way that is can be removed after
purification of the antibody is complete. For example, the ligand can be attached to the antibody via a linker that can be is easily cleavable under the appropriate conditions. Such conditions can include acid or basic pH, heating, sonication, enzymatic cleavage, and the like.
[00313] As used herein, the term "label" refers to a composition capable of producing a detectable signal indicative of the presence of a target. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods and devices described herein. For example, the antibody can be labeled with a detectable tag which can then be detected using an antibody specific to the label.
[00314] Exemplary fluorescent labels include, but are not limited to, Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester, Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red,
Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 or Cy7.
[00315] The ligands can be conjugated, either directly or through a linker, to the N-terminal, C-terminal, or the amino acid side chains of the heavy and/or light chain of the antibody. A ligand can be present on an amino acid when said amino acid is incorporated into the antibody heavy and/or light during synthesis. In some embodiments, the ligand can be incorporated via coupling to a "precursor" amino acid after said "precursor" amino acid has been incorporated into the antibody heavy and/or light chain. For example, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can be conjugated to the N-terminal of heavy and/or light chain of the antibody.
[00316] In another example, a monomer having a chemical group suitable for taking part in Click Chemistry reaction can be incorporated, e.g., an azide or alkyne group. In a subsequent operation, i.e., after incorporation of the precursor monomer antibody heavy and/or light chain, a ligand having complementary chemical group, e.g., an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together. [00317] In some embodiments, the covalent linkages between the antibody and a ligand are mediated by a linker. This linker can be cleavable linker or non-cleavable linker, depending on the application. As used herein, a "cleavable linker" refers to linkers that are capable of cleavage under various conditions. Conditions suitable for cleavage can include, but are not limited to, pH, UV irradiation, enzymatic activity, temperature, hydrolysis, elimination and substitution reactions, redox reactions, and thermodynamic properties of the linkage. In some embodiments, a cleavable linker can be used to release the antibody after transport to the desired target. The intended nature of the conjugation or coupling interaction, or the desired biological effect, will determine the choice of linker group.
[00318] As used herein, the term "non-peptide linker" means an organic moiety that
connects two parts of the peptide and such a moiety is not a peptide. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR1, C(O),
C(0)NH, SO, SO2, S02NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,
cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,
alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl,
alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,
alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,
alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S(O), S02, N(R1)2, C(O), cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R1 is hydrogen, acyl, aliphatic or substituted aliphatic. The two parts of the compound can be linked together by providing on each part of the molecule complementary chemical functionalities that undergo a coupling reaction.
[00319] In some embodiments, linkers can be non-covalent coupling of two parts of a
compound or two different molecules. Such non-covalent coupling can be achieved through, for example, ionic interactions, H-bonding, van der Waals interactions and affinity of one molecule for another. When non-covalent coupling is used, each part of the compound can be conjugated with a moiety that has complementary to another moiety that is conjugated to the second part of the compound. One example of such complementary coupling is the
biotin/avidin coupling. Other examples include but are not limited to affinity of an
oligonucleotide for its complementary strand, receptor/ligand binding, aptamer/ligand binding and antibody/antigen binding.
[00320] Many strategies are known in the art for conjugating peptides to peptides and other molecules. For example, Hermanson, G.T., Bioconjugate Techniques, 2nd Ed., Academic Press (2008) and Niemeyr, C. M., Bioconjugation Protocols: Strategies and Methods (Methods in Molecular Biology), Humana Press (2004) provide a number of methods and techniques for conjugating peptides to other molecules. Contents of both of these are herein incorporated by reference in their entirety for all purposes. For a review of site-specific introduction of non- natural amino acids into peptides for conjugation see A.J. de Graaf, et al., Biocojugate
Chemistry (2009) 20(7): 1281-1295, contents of which are herein incorporated in its entirety. Int. Pat. App. Pub. No.: WO92/13095, contents of which are herein incorporated in its entirety, describes methods for PEGylation of peptides.
[00321] One conjugation strategy is the biotin- sandwich method (Davis, et al., Proc. Natl.
Acad. Sci. USA 103:8155-8160 (2006)) in which a peptide is biotinylated and bound to biotinylated ligand using tetravalent streptavidin as a linker. To accomplish this, the peptide may be coupled to the 15 amino acid sequence of an acceptor peptide for biotinylation
(referred to as AP; Chen, et al., Nat. Methods 2:99-104 (2005)). The fusion proteins can be made by incorporating the extra sequences at the N- or the C-terminus of the peptide. The acceptor peptide sequence allows site- specific biotinylation by the E. coli enzyme biotin ligase (BirA; Chen, et al., Nat. Methods 2:99-104 (2005)). A ligand peptide can be similarly biotinylated for conjugation with a peptide described herein. Many commercial kits are available for biotinylating proteins. Non-peptidyl ligands agents can also be conjugated with biotin using methods well known in the art for conjugating biotin to non-peptide molecules, e.g. small organic molecules. In order to prevent steric interference between the biotin/avidin groups and the peptides or the ligands, a spacer may be included between them. [00322] The linkers and linking methods described herein can also be used for linking together heavy chain and light chain of an antibody, two or more Fv domains, and fragments thereof.
[00323] For simplicity, chemical moieties are defined and referred to throughout can be univalent chemical moieties (e.g., alkyl, aryl, etc.) or multivalent moieties under the appropriate structural circumstances clear to those skilled in the art.
[00324] The term "alkyl" refers to saturated non-aromatic hydrocarbon chains that may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include without limitation methyl, ethyl, propyl, allyl, or propargyl), which may be optionally inserted with N, O, S, SS, S02,C(0), C(0)0, OC(O), C(0)N or NC(O). For example, C C6 indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it.
[00325] The term "alkenyl" refers to an alkyl that comprises at least one double bond.
Exemplary alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl and the like.
[00326] The term "aryl" refers to monocyclic, bicyclic, or tricyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examplary aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
[00327] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12
membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examplary heteroaryl groups include, but are not limited to, pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, and the like.
[00328] The term "cyclyl", "cyclic" or "cycloalkyl" refers to saturated and partially
unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and the like. [00329] The term "heterocyclyl", "heterocycle" or "heterocyclic" refers to a nonaromatic 5- 8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Examplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
tetrahydrofuranyl, and the like.
[00330] The term "optionally substituted" means that the specified group or moiety, such as an alkyl, aryl group, heteroaryl group and the like, is unsubstituted or is substituted with one or more (typically 1-4 substituents) independently selected from the group of substituents listed below in the definition for "substituents" or otherwise specified.
[00331] The term "substituents" refers to a group "substituted" on an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, acyl, amino group at any atom of that group.
Suitable substituents include, without limitation, halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkylthio, CF3, N- morphilino, phenylthio, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido. In some embodiments, substituent can itself be optionally substituted. In some cases, two substituents, together with the carbons to which they are attached to can form a ring.
[00332] The term "halo" refers to any radical of fluorine, chlorine, bromine or iodine.
[00333] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,
heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents. Exemplary acyl groups include, but are not limited to, (Cp C6)alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t- butylacetyl, etc.), (C3- C6)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, pyrrolid-2-one-5 - carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl (e.g., thiophenyl-2-carbonyl, thiophenyl-3 -carbonyl, furanyl-2- carbonyl, furanyl-3 -carbonyl, lH-pyrroyl-2-carbonyl, lH-pyrroyl-3 -carbonyl,
benzo[b]thiophenyl-2-carbonyl, etc.). In addition, the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portion of the acyl group may be any one of the groups described in the respective definitions.
[00334] The term "alkoxy" refers to an -O-alkyl radical.
[00335] The term "aminoalkyl" refers to an alkyl substituted with an amino.
[00336] The term "mercapto" refers to an -SH radical.
[00337] The term "thioalkoxy" refers to an -S-alkyl radical.
[00338] The term "haloalkyl" refers to an alkyl group having one, two, three or more
halogen atoms attached thereto. Exemplary haloalkyl groups incude, but are not limited to chloromethyl, bromoethyl, trifluoromethyl, and the like.
[00339] Those skilled in the art will appreciate that the invention described herein is
susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification,
individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.
[00340] Various references are cited throughout this Specification, each of which is
incorporated herein by reference in its entirety.
[00341] The foregoing description will be more fully understood with reference to the
following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention.
EXAMPLES EXAMPLE 1
[00342] Materials and Methods
[00343] The scFvs described in the following examples were expressed in Escherichia coli bacteria in the form of maltose-binding protein-scFv fusion proteins (MBP-scFv as described in Bach, H., Mazor, Y., Shaky, S., Shoham-Lev, A., Berdichevsky, Y., Gutnik, D.L. and
Benhar, I. (2001). E. coli maltose-binding protein as a molecular chaperone for intracellular antibodies. J. Mol. Biol. 312:79-93). To observe the binding of soluble scFv fragments on live cells, staining of A549 cell by immunostaining and competition assays with the different lectins were performed on ice to prevent uptake of scFv fragments by the cells. The scFV fragments were further analyzed by competition with the specific lectin and the corresponding sugar ligand on the binding of Human A549 cancer cells that were fixed with or without specific lectins. The lectins used in methods include plant lectins such as Peanut Agglutinin (PNA), Ulex europaeus agglutinin (UEA), Soybean agglutinin (SBA), Dolichos biflorus (DBA), lotus tetragonolobus (LTL), Vicia villosa lectin (VVA), Maackia amurensis lectin (MAA) and Phaseolus vulgaris agglutinin (PHA); and animal lectins including Helix aspersa agglutinin (HA A) and Helix pomatia agglutinin (HP A).
[00344] Immunohistochemistry was performed as follows. Normal human fibroblasts and for A549 NSCLC cells, 3xl06 cells per sample, were seeded on glass cover slip in 6 wells plate. 24 hours later, immunohistochemistry protocol was applied as describe in methods, using scFv fragments. The staining was detected via peroxidase protocol.
[00345] For staining of human paraffin embedded lung cancer specimens with the different fragments the following protocol was used:
[00346] Antigen retrieval:
[00347] 1. Tissue sections were deparaffinized and rehydrated to water.
[00348] 2. Endogenous peroxidase was blocked with 3% H202 for 5 min.
[00349] 3. Slides were washed with distilled water for 10 min.
[00350] 4. Slides were then placed in citrate buffer at 100°C for 15min.
[00351] 7. Slides were then rinsed in distilled water twice and in PBS for 5 min.
[00352] 8. Treated slides were immunostained as described below.
[00353] Immuno staining :
[00354] All incubations were performed at room temperature and between incubation
periods, slides were washed with PBS. In the process, (a) slides were incubated for 30min incubation with 1% BSA; (b)slides were then followed by lh incubation with MBP-scFv (maltose binding protein); (c) slides underwent 30min incubation with secondary antibody mouse anti MBP; (d) the slides were further incubated for 30min with goat anti mouse peroxidase conjugated; and (e) peroxidase substrate was incubated for 5 min. Following completion of the process, slides were counterstained with hematoxylin and coverslipped with an aqueous mounting medium. [00355] Binding of scFv fragments to NSCLC A549 cells - ELISA assay - was performed as follows. A549 cells (15,000) were plated in each well of a 96 well plate.
[00356] 24h later, cells were washed with DMEM serum free medium. Each scFv fragment was first incubated at 37°C with mouse anti MBP antibodies to form bivalent complexes, than added to cells and allow for binding for lh at 37°C. Cells were washed three times with DMEM serum free medium and incubated with goat anti mouse peroxidase conjugated antibodies for additional 30 minutes at 37°C. Cells washed again and incubated with the substrate TMB. The reactions were stopped with 1M HC1 and analyzed by ELISA reader at OD460nm wavelength.
[00357] Cell Viability Assay was performed on NSCLC A549 cell line. Cells were seeded in 96 well plates (2x103 cells/well) in 10% FCS growing DMEM media. Following 24 hours, cells were treated with scFv # 1 Fragment complexed with mouse anti MBP to form bivalent complexes in serum free medium for 6 hours. The growth medium was adjusted to 10% FCS. Cell viability was determined by methylene blue assay following 95 hours treatment.
[00358] The methylene blue (MB) assay was used to assay cell growth and viability
including cell proliferation and doubling time, cell inhibition and apoptosis and to calibrate numbers of cells seeded on plate for the ELISA test. Cells seeded in 96 well plates were washed 3 times with PBS after which the cells are fixed with 200 μΐ 4% formaldehyde solution for 2 hrs at room temperature. Plates were washed with PBS and then incubated with 150 μΐ RNAse A (3 ug/ml) for 30 min at room temperature. Plates were equilibrated with 200 μΐ 0.1M sodium borate buffer pH 8.5, stained with 200ul 0.5% methylene blue (0.1 M sodium borate buffer ph 8.5) and then incubated for 10 min at room temperature. The cells were washed with tap water and cell-bound dye was eluted with 200 μΐ (microliter) 0.1M HC1. Eluted methylene blue was read in an ELISA plate reader at 595 nm. Methylene blue binds DNA in basic solution, and it is extracted in acidic condition. Staining of DNA by Methylene Blue correlates with cell survival.
[00359] Immunohistochemisty of Paraffin embedded tissue samples- scFvs binding
[00360] Antigen retrieval:
[00361] 1. Tissue sections were deparaffinized and rehydrated to water.
[00362] 2. Endogenous peroxidase was blocked with 3% H2O2 for 5 min.
[00363] 3. Slides were washed with distilled water for 10 min.
[00364] 4. Slides were then placed in citrate buffer at 100°C for 15min. [00365] 7. Slides were then rinsed in distilled water twice and in PBS for 5 min.
[00366] 8. Treated slides were immunostained as described below.
[00367] Immuno staining:
[00368] all incubations were performed at room temperature
[00369] as follows: (a) 30min incubation with 1% BSA. (b)lh incubation with MBP-scFvs (maltose binding protein-scFv fusion proteins) (c) 30min incubation with mouse antiMBP (d) 30min incubation with goat anti mouse peroxidase conjugated (e) peroxidase substrate was incubated for 5 min.
[00370] After immunostaining, slides were counterstained with hematoxylin and
coverslipped with an aqueous mounting medium.
[00371] Immunohistochemisty of A549 and Normal Human Fibroblast- scfvs binding.
[00372] 3xl06 A549 or lxlO6 Normal Human Fibroblast was plated on 2x2 cm sterile glass in 6 wells plate. 24h later, cells were incubated with 4% formaldehyde washed twice with lxPBS and placed for immunostaining.
[00373] Immunostaining:
[00374] All incubations were performed at room temperature as follows: (a) 30min
incubation with 1% BSA. (b)lh incubation with MBP-scFv (c) 30min incubation with mouse antiMBP (d) 30min incubation with goat anti mouse peroxidase conjugated (e) peroxidase substrate (invitrogen, #SKU 00-2014) was incubated for 5 min.
[00375] After immunostaining, slides were counterstained with hematoxylin and
coverslipped with an aqueous mounting medium.
[00376] scFVs binding to live cells - ELISA assay.
[00377] 15,000 A549 cells were plated in each well of 96 wells plate. 24h later, cells were washed with DMEM serum free, all incubation were performed at 37 °C in serum free medium. MBP-scFv was added to cells and incubated for lh. Cells were washed three times with DMEM serum free and incubated 30min with mouse antiMBP than incubated with goat anti mouse peroxidase conjugated. Cells washed again and incubated with TMB. Reaction was stopped with 1M HC1 and analyzed on ELISA plate reader at OD460nm.
[00378] Results
[00379] Table 4 represents the isolated scFv fragments as having binding properties to A549 cells as the indicated lectin. Each fragment was analyzed for its competition binding with the specified lectin and corresponding competing sugar molecule: [00380] Table 4: example of the isolated scFv fragments according to their lectin selection
Figure imgf000080_0001
[00381] Figure 1 shows micrographs of immunohistochemical staining of cells and specimens with scFv fragments as follows. Figure 1A shows staining of normal human fibroblasts with the different scFv fragments. Figure IB shows staining of NSCLC A549 cells with scFv fragments. Figure 1C shows staining of NSCLC human tumor specimens with scFv fragments.
[00382] Figure 2 shows quantitative binding of scFv fragments to NSCLC A549 cells.
[00383] The results described in Figures 1 and 2 suggest that the different scFv fragments bind and stain specifically and selectively with high specificity NSCLC A549 cells and human tumor cell specimens. The scFv fragments do not bind to normal fibroblast cells nor bind to variety of human normal tissue.
[00384] These results demonstrate that these human scFv fragments or their F(ab), F(ab)2, full human derived antibody or any molecule that is based on these scFv fragments can be used for cancer diagnosis, using cancer tissue specimens, circulating cancer cells in blood, blood serum and / or plasma, urine, saliva and feces, or any other biological sample as described herein, for binding to scFv fragments or their F(ab), F(ab)2, full human derived antibody or any molecule that is based on these scFv fragments.
[00385] In addition, these results suggest that these human scFv fragments or their F(ab), F(ab)2, full human derived antibody or any molecule that is based on these scFv fragments can be use to classify the grade and the stage of the tumor tissue.
[00386] Figure 3A shows survival of NSCLC A549 cells after treating the cells with MBP- scFvl fragments and 0.08uM antiMBP, while Figure 3B represents the survival of the cells after treatment with scFvland three different concentration of antiMBP (0.08uM, 0.165uM and
0.42uM) according to at least some embodiments of the present invention. As shown, the scFv fragments demonstrate specific and strong cell growth inhibition of these cancer cells by actually reducing cell survival. These results suggest that these human scFv fragments or their F(ab), F(ab)2, full human derived antibody or any molecule that is based on these scFv fragments can be use for therapeutics to treat human cancer diseases.
EXAMPLE 2
[00387] This Example relates to specific binding of scFv fragments according to at least some embodiments of the present invention to cancerous tissue samples as opposed to normal (non-cancerous) tissue samples.
[00388] Materials and Methods
[00389] scFv fragments were prepared as described with regard to Example 1.
[00390] Immunohistochemical staining of tissue sections with scFv fragments was performed as follows.
[00391] Paraffin embedded and fixed tissue samples were provided on slides in sections of about 5-10 μπι, including samples of both tumors and normal tissue from affected individuals, were obtained from the pathology department at a local hospital, under Helsinki regulations and approval.
[00392] The mounted sections of tissue first underwent deparaffinizion and hydration as follows:
[00393] The mounted sections were washed gradually with the following solutions:
[00394] 100% xylene (15 min): 100% xylene (15 min): 100% alcohol (5min): 100% alcohol (5min): 95% alcohol (5 min): 75% alcohol (5 min). The slides were further washed with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic at pH of 7.4 for 5 min, twice: PBS was added to cover the slide, and the slide was then incubated for an additional 5 minutes.
[00395]
[00396] The mounted sections of tissue were then incubated for 20 minutes with quenching solution containing 3% hydrogen peroxide in PBS buffer and then washed in PBS buffer for 5min, twice.
[00397]
[00398] The mounted sections of tissue then underwent blocking and staining using the ACUITY® advanced biotin free HRP polymer detection system (SIG-32904) kit from CO VANCE Inc (USA), according to the manufacturer's instructions. [00399] Briefly, the tissue samples were blocked with ACUITY advanced reagent 1 (serum blocker) for 30 min. Next 7.5ug/ml of a selected scfv fragment, coupled to MBP (Maltose Binding Protein), was used to detect the expression of carbohydrate moieties in tissue samples of NSCLC (non small cell lung cancer) and colorectal cancer from diagnosed individuals. The staining of normal tissue within the tissue sections on each slide was monitored to determine the binding specificity. After removal of the blocking solution, the scFv fragment-containing solution was added to each slide to cover the tissue sections. Slides were incubated for 1 hour at RT (Room Temp), followed by three consecutive washes of 10 min each with PBS.
[00400] Mouse anti MBP antibodies (1:1000 dilution, previously described) were added to slides, followed by incubation for 30 min at RT, followed by three washes with PBS. The slides were than incubated with ACUITY advanced reagent 2 (boost) 20min at RT followed by three washes with PBS. For specific binding detection, the slides were further incubated for 15min with ACUrfY advanced reagent 3 (HRP polymer). Slides were incubated with DAB - (3,3' Diaminobenzidine) reagent, (substrate chromogen for peroxidase activity detection) then counter stained with hematoxylin, followed by a series of graded alcohol washes (100% 5min, 95% 5min, 70% 5min), incubated for 5 min in xylem and covered with glass.
[00401] ELISA binding assay. Cells (A549 or LS-174-T) were plated in each well of 96 wells plate. The next day, cells were fixed with 4% formaldehyde lh and washed twice with PBS, cells were incubated with 3% H202 30min then blocked with 1% BSA, follow by incubation with scFvs or scFvs-IO-NIR, lh at room temperature. Cells were washed with PBSxl, then incubated with mouse anti MBP (1:1000), washed and incubated with goat anti mouse -HRP (1:1000). After washing the cells. TMB substrate was added. Reaction was stopped with 0.5M H2S04 read at OD 450nm.
[00402] Results
[00403] Table 5 presents the binding intensity of all 5 scFvs to NSCLC specimens and normal adjacent lung specimens as evaluated after immunohistochemical staining and to normal adjacent lung tissue. Intensity of staining was described as weak (+), moderate (++), strong (+++) or non- stained (-). Each of the tested specimens were positively stained with at list one of the scFvs. The staining pattern with all five scFvs was different from patient to patient. The binding of the scFvs to the normal lung specimens was very weak as shown.
[00404] Table 5: scFvs binding to NSCLC tissue samples. Limy Tumor patient sdv'l sdv8 scfv 2 ecFvG2 scFvH2 adenocarcinoma 5765 ÷÷- ÷÷÷ *÷- adenocar cinoma 17464 -÷ - - ÷÷ ÷ adenocar inoma 4710 ÷+÷ ÷÷÷ ÷÷- adenocar inoma 16320 - - - - - adenocar cinoma 16320 - - +÷ - adenocarcinoma 2420 ÷÷+ ÷÷+ ÷÷+ ÷+- - adenocarcinoma 2372 ÷-« ÷÷÷÷ adenocarcinoma 6804.200 I ÷÷- +÷ ÷÷÷
Adenocarcinoma gr de 1 123% ÷t- ÷ + - -
Adenocar inoma rade 1 3112 + ÷ - +
Adenocarcinoma de 1 1 1 02 ÷ - -
Adenocarcinoma grade 1 236.2001 ÷ - * - -
Adenocarcinoma grade 2 13856/99 it
Adenocarcinoma grade 2 4756 - - - -
Adenocarcinoma grade 2 11863 - - ÷ - -
Adenocarcinoma gr ade 1 5798 02 -÷ -÷ - ÷+- -
Adenocar cinoma gr ade 17697 2001 - -
Adenocar chroma gr ade 3 16968 ÷÷- - +÷ -
Adenocar inoma gr de 3 16317 ÷t- - -
Adenocarcinoma gr de 3 18414 ÷÷÷ ÷÷÷ - ÷÷÷ ÷
Adenocarcinoma grade 3 11891 - - - ÷~÷ -
Adenocarcinoma grade 3 10669 ÷÷- -
Adenocarcinoma grade 3 11891/00 - - - ÷÷- -
Adenocarcinoma grade 3 18074/2001 ÷÷ + ÷÷ + ÷÷+ ÷÷÷ ÷÷÷
Bronchelioalveolo carcinoma 13854 1 99 -÷ ÷÷ + ÷÷+ + ÷
Bronchelioalveolo carcinoma 51302000 ÷÷÷ ÷÷÷ t÷ ~
Lar ge Cell Carcinoma 7433
Large Cell Carcinoma 8165 00
poorly differ entiated squ mus grade 3 31842 02 ÷÷÷
SquamaCar cinoma 9438 - - - - -
Well Differ entiated Adenocarcinoma 22602001 ÷ ÷÷ - scFv V1 V8 C2 G2 H2
% of ¾|?e im n¾ &iaind 70% 60% 73% 73¾ 53% number of specimens stained 21 30 18-'30 22/30 22/30 16 30
Normal Aiijnceiit Lung tissireTissne patient # scFvl scFv8 seFvC2 scFvG2 scFvH2 aderrocar cinoma 6804_2 -/V - - -
Adenocar cinoma gr ade 1 14383 02 - -
Adenocar cinoma gr ade 1 236/2001 - - - - -
Adenocarcinoma grade 2 13356 10 -/~ - - - -
Adenocarcinoma grade 2 I7s.< 7 ?0 1 +÷ - ÷+ -
Adenocarcinoma grade 1 1Si's 1 00 +÷ -/- -÷ -
Adenocarcinoma grade 3 18074 2001 ÷÷ ÷÷÷ ÷÷÷
Br one helioalveol car cinoma 1 854 4 - - - - -
Br one helioalveolo car cinoma 8165Ό0 -
Br onchelioarVeoJo car cirroma 51302000 -÷ ÷ ÷
pootl differentiated sqiramirs gi ade 3 31842:02 - - - - -
Well Differentiated Adenocarcinoma 2260/2001 - - - -/V - [00405] As can be seen from Table 5, most of the above tumors were detected by at least one antibody. In terms of the graded cancers, typically more fragments reacted more strongly with the higher grade tumors. However, for the higher graded tumors, such as adenocarcinoma grade 3, some reactivity was also seen with the surrounding "normal" tissue. Without wishing to be limited by a single hypothesis, this reactivity could be due to the "normal" tissue including malignant cells.
[00406] As can be seen from Table 5, each of the immune molecules scFvl, scFv8, scFvG2, scFvC2 and scFvH2, bound to at least certain non- small cell lung cancer samples, with little to no binding to the adjacent "normal" tissues. scFvG2 alone bound to nearly all cancer samples, while those samples to which it did not bind were generally bound by scFvl. Therefore according to at least some embodiments of the present invention, there is provided use of each of the immune molecules scFvl, scFv8, scFvG2, scFvC2 and scFvH2 for diagnosis and optionally also treatment of non- small cell lung cancer, as well as combinations thereof. A non-limiting optional example of such a combination is the combination of scFvl and scFvG2.
[00407] Figure 4 shows example for immunohistochemical staining tumor array (TMA) contain samples from 32 colorectal patients and breast cancer with scFvs. A-M Coloreactal specimens - positive staining, N-0 Colorectal cancer specimens - negative staining, P-T breast cancer specimens - positive staining. Percentage of staining of colorectal cancer specimens with each one of the scFvs are summarized in Table 6 below.
[00408] Table 6
Figure imgf000084_0001
[00409] Figure 5 shows immunostaining of paraffin embedded colorectal specimens from two human patients with scFv8, demonstrating that the cancerous tissue is specifically bound while normal tissue is not bound.
[00410] Figure 6 shows that normal apparent tissue found close to the tumor is stained while normal tissue far from the tumor is not stained indicating that the scfvs may detect cancer progression in a very early stage. Staining was performed on paraffin embedded tumor specimens, immunostained with scFv8. The photographs are identified as follows: photograph no. 4 is of tissue taken from the tumor area, photograph no. 3 is of tissue taken from the peripheral area of the tumor, photograph no. 2 is of tissue taken near the tumor area and photograph no. 1 is of tissue taken far from the tumor.
[00411] Table 7 shows binding of four scFv fragments, scFvV8, scFvG2, scFvC2 and scFvH2 (described below as V8, G2, C2 and H2, respectively) to human colorectal and adjacent normal control specimens through immunohistochemistry staining. The results show that 91% (out of 12 tested) of colorectal cancer specimens were stained with scFv8, while 75% of the normal tissues were not stained.
[00412] Table 7: binding to human colorectal and adjacent normal control specimens.
Figure imgf000085_0001
[00413] As can be seen from Table 7, each of the immune molecules scFv8, scFvG2, scFvC2 and scFvH2, bound to at least certain colorectal cancer samples, with little to no binding to the adjacent "normal" tissues. scFvG2 alone bound to nearly all cancer samples, while those samples to which it did not bind were generally bound by scFv8. Therefore according to at least some embodiments of the present invention, there is provided use of each of the immune molecules scFv8, scFvG2, scFvC2 and scFvH2 for diagnosis and optionally also treatment of colorectal cancer, as well as combinations thereof. A non-limiting optional example of such a combination is the combination of scFv8 and scFvG2.
[00414] Figure 7 shows binding of scFv fragments to LS-174-T cells. Such binding is necessary in order to use scFv fragments as a bio-probe in animal model. All scFv fragments bound to LS-174-T cell line as demonstrated through the ELISA assay (the assay was performed as described in Example 1). scFv8 binding intensity is two fold higher.
[00415] Figure 8 shows immunohistochemistry binding to such cells as for Figure 7. Again, the results confirm that all fragments can be used in a mouse experimental model.
[00416] Conjugation of scFv8 to particles of iron oxide tagged with near infrared fluorescence (NIR) results in an actively binding molecule which fluoresces at OD 720/780nm, as indicated by the binding to A549 cell line. ELISA binding assay of the conjugant to A549 cell line is detailed in the Material and Methods section. scFv8-Iron Oxide NIR (scFv8-IO-NIR) conjugant also binds specifically to colon adenocarcinoma, as indicated by the specific binding to colon adenocarcinoma specimen in compare to adjacent normal control. Figure 9 shows the binding intensity of scFv8-IO-NIR to A549 cell line. Identification of binding done by mouse anti maltose binding protein follow by goat anti mouse peroxidase conjugated. The resultant peroxidase activity was measured at OD 450nm. The graph presents the relative values of scFv8 at concentration of lOug/ml and lug/ml and scFv8-IO-NIR in dilution of 1/2 1/4 and 1/8 to control secondary antibody.
[00417] Figure 10 shows the binding of scFv8 to human colon cancer specimen and adjacent normal control as indicated by immunohistochemistry (C- normal adjacent, D- colon adenocarcinoma), and binding of scFv8-IO-NIR to the same patient specimen (A- light scanning of normal adjacent and adenocarcinoma specimen B- 720/780nm scanning of normal adjacent and adenocarcinoma specimen. Table 8 below shows the extent to which staining of normal tissue occurred with scFv8. staining of normal tissue with scFv8
Figure imgf000087_0001
smooth
muscle ++
tonsil - uterus -
[00419] As can be seen from Table 8, while some tissues showed staining of smooth muscle and/or connective tissue and/or fibrin and/or endothelium (for example breast, duodenum, esophagus, gallbladder and ileum), most tissues showed little or no staining. Furthermore, the staining of the smooth muscle and/or connective tissue and/or fibrin and/or endothelium could be readily differentiated from staining of a tumor, given the shape and regularity of such staining.
EXAMPLE 3
[00420] This Example relates to in vivo experiments performed with the antibody fragments according to at least some embodiments of the present invention.
[00421] In-vivo/ ex-vivo protocol
[00422] CDl-Nude mice were implanted with 2*10Λ6 cells/30 micro-liter LS-174T cells in the colon. These cells induced expression of orthotropic human colon cancer tumors. 14-17 days post transplantation, mice were used for the in- vivo/ex- vivo experiments described herein, with the immunoreactive fragment scFv8, conjugated to albumin and to a dye fluorescing in the near infrared spectrum.
[00423] 200ul HAS(human serum albumin )-NIR (near infrared)- scFv8 in a concentration of lOOug/ml was inserted into the lumen of anesthetized mice and was incubated for 20 minutes. The lumen was washed with 5ml lxPBS, and sacrificed 3h after the final wash. The colon was then removed and imaged by using a fluorescence scanner (Odyssey).
[00424] The results are shown in Figure 11. Figure 11 shows images of colons taken from mice with colonic tumors after administration of scFv8-HAS-NIR. A. fluorescent imaging of the removed colon; the green dots are the tumors area stained with the probe. B. the removed colon photo in white light. C. translation of fluorescent intensity into red and blue color whereas red - the most intense fluorescence, and blue - the less intense fluorescence. Results are shown from two mice, designated as animals 62 and 63. Clearly, the immunoreactive molecules, such as scFv8, according to at least some embodiments of the present invention are suitable for imaging, for example of tumors, whether performed in vivo or of ex vivo tissue. EXAMPLE 4
[00425] Glycan targets ofscFvs
[00426] To demonstrate that the binding of the scFvs to their target protein involves a glycan epitope, competition experiments with PNA, UEA and SBA lectins were conducted. Each scFvs in a concentration of 20ug/ml in PBS buffer was mixed with PNA, UEA or SBA lectins at a concentration of 50ug/ml in PBS buffer and with BSA protein as a negative control. The scFvs/lectins mix or scFv/BSA mix was incubated with A549 cells plated in 96 well plates for lh at room temperature. The binding of each scFv was detected with anti MBP and then with goat anti mouse - HRP as previously described. Enzymatic reaction was evaluated with TMB reagent at OD 450nm. Each competition reaction was compared to the scFvs binding without the competitor lectin (Figure 12; Figure 12A shows scFvl competition with lectins; Figure 12B shows scFv8 competition with lectins; and Figure 12C shows scFvH2 competition with lectins).
[00427] For reliability of the competition assay, each lectin (PNA, SBA and UEA) were used for a self competition (data not shown). Indeed each lectin competed with itself but not with other lectins or BSA, except SBA which showed competition with itself and with PNA.
[00428] As appeared from the competition assays, the following results were obtained (also summarized in Figure 13). The target glycans of scFvl are N-acetylglucosamine, Galactose and Fucose. The target glycans of scFv8 are N-acetylglucosamine and Fucose. The target glycans of scFvH2 are Fucose and N-acetylglucosamine.
[00429] These data were shown to correspond to antibody fragment binding to cancer cells and the presence (or absence) of various glycans on these cells, as shown in Figure 14. Bold face type for results for scFvs binding indicate a positive correlation between lectin and scFvs binding to cancer cells, while regular type for results for scFvs binding indicate a lack of correlation between lectin and scFvs binding to cancer cells. For example, IHC staining of patient#14393 was negative for UEA, therefore negative for scFvH2 which compete with UEA. IHC staining of patient #5765 was positive for UEA therefore positive for scFvH2. IHC staining of lung cancer specimens with scFvs and the different lectins and their binding correlation is shown therefore to correlate.
[00430] Optionally, according to at least some embodiments of the present invention, there is provided a method of treatment of cancer in a subject, comprising determining if the cancer expresses a glyco-molecule which binds to N-acetylglucosamine, in which case the cancer may optionally be treated with one or more of scFvl, scFv8 or scFvH2; binds to fucose, in which case the cancer may optionally be treated with one or more of scFv8 or scFvH2; and/or binds to galactose in which case the cancer may optionally be treated with scFvl. Similarly, the above immune molecules may optionally be used for diagnosis of cancer according to the above binding.
31] While the invention has been described with respect to a limited number of embodiments, it will be appreciated that many variations, modifications and other applications of the invention may be made. In addition, any of the embodiments or portions thereof described herein may optionally be combined in any way.
SEQUENCE LISTING
SEQ ID NO:l - scFv # l:
AtggccCAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGCACTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCCATTAATAGTAATGGTGATAGCACCTACTAT CCAGACACTGTGAAGGACCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCT GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATGTCACCTCT CGAGACTCTATGATTTGGGGAGTCATCCTCCCTACTACTTTGACTTCTGGGGCCAGGGCACC CTGGTCACGGTCTCCTCAGGCTCAgcaggaggaggaggatccggtggtggtggttctggcgg cggcggctccGATATCGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA GAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCGGCTACTTAGCCTGGTACCAG CAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGCTGCATCCAGCAGGGCCACTGGCAT CCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGG AGCCTGAAGATTTTGCAGTGTATTACTGTCAACAATATTATGGATCTCCGTGGACGTTCGGC CAAGGGACCAAACTGGAAATCAAA
SEQ ID N0:2 - scFv # 8 - DNA sequence (5' to 3'):
AtggccGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCGCCTGGCCCTCTGTGGTCACGGAGGGGCTCGGTG GCCCAGGTGGTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTTTCACCATCTCCAG AGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGCCGT GTATTACTGTGCGAGGGACCCCGATCCCTATGGGTCGGAGAGTTTTCGCCTCTTCGGCACGT TTGACTACTGGGGCCAGGGCACCCTGGTCACAGTCTCCTCAGGCTCAgcaggaggaggagga tccggtggtggtggttctggcggcggcggctccGATATCGTGTTGACGCAGTCTCCAGGCAC CCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCA GCAGCTCCTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGT GCATCCGCCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATT ATGACTGGCCGCTCACTTTCGGCGGAGGGACCAAACTGGAAATCAAA
SEQ ID NO: 3 - scFv # A7 - DNA sequence (5' to 3'):
AtggCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCGAATATGGCATGAACTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTTTCAgGAATCAGTTGGAATAGTGATACCATAGACTAT GGAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCT GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCAAGACGTCAGTACT ACGCTATGGACGTCTGGGGCCCGGGCACCCTGGTCACAGTCTCTTCAggctcagcaggagga ggaggatccggtggtggtggttctggcggcggcggctccGATATCGTGCTGACTCAGCCACC CTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCCTGCTCCGGCAACAGTGGCAGCA TTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCTCCCAgGAAAAGCTCCCAAACTCCTCATC TATGACAATGATAAGCGACCCTCAgGGGTCCCTGACCGGTTCTCTGGCTCCAAGTCTGGCAC CTCAgCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAgGCTGATTATTACTGCAGCT CATATACAAGCAGCAGCACTCTCGTCGTATTCGGCGGAGGCACCAAGCTGACCGTCCTA SEQ ID NO: 4 - scFv # C2 - DNA sequence (5' to 3'):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCGTCATCAGCGGCGACATGGGCTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTTTCAGCTATTACTACTGGTGGTGGTAGCCCGAACTAT GCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCT GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCAAGAGACGAAGGAA TGGTGGGAGCCACATACTTCGACCACTGGGGCCAGGGCACCCTGGTCACAGTCTCTTCAGGC TCAGCAGGAGGAGGAGGATCCGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGATATCGTGCT GACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCCTGCTCTGGAA GCAACTCCAACATTGGCTACAGTCAAGTGTCCTGGTACCAGCAGTTCCCAGGAAAGGCCCCC AAACTCCTCATCTATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTGACCGGTTCTGTGGCTC CAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTGCGGTCCGAGGATGAGGCTGATT ATTACTGCCAGTCCTATGACAGCAGCCTAAATTCTTACATTTTCGGCGGAGGCACCAAGGTG ACCGTCCTC
SEQ ID NO: 5 - scFv # G2 - DNA sequence (5' to 3'):
ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAATTTTGTCATGAACTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTCTCACGTATTAATACTGATGGCACTAGCACAAACTAC GCGGACTCCGTGACGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCT GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGATCCCCCTATA CGATTTTTGGAGTGGTTCATTTTGACTACTGGGGCCAGGGCACCCTGGTCACGGTCTCTTCA
GgctcagcaggaggaggaggatccggtggtggtggatctgggggcggcggctccGATATCGT GCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGCTCTG GGAGCAGCTCCAACATCGATTCTAGTCCTGTAAACTGGTACCAGCAGCTCCCAGGAGCAGCT CCCAAACTCCTCATTTATCTGATTAATGAGCGACCCTCAGGGGTCCCTGACCGGTTCTCTGG CTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTGCGGTCCGAGGATGAGGCTG ATTATTACTGTCAGGTGTGGGATAGTATTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAG CTCACCGTCCTA
SEQ ID NO:6 - scFv # H2 - DNA sequence (5' to 3'):
atggccGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCGAATATGGCATGAACTGGGTCCGCCAGG CTCCAGGGAAGGGGCTGGAGTGGGTTTCcgGAATCAGTTGGAATAGTGATACCATAGACTAT GGAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCT GCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCAAGACGTCAGTACT ACGCTATGGACGTCTGGGGCCCGGGCACCCTGGTCACAGTCTCTTCAggctcagcaggagga ggaggatccggtggtggtggAtctgggGgcggcggctccGATATCGTGCTGACTCAGCCACC CTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCCTGCTCCGGCAACAGTGGCAGCA TTGCCAGCAACTCTGTGCAGTGGTACCAGCAGCTCCCAGGAAAAGCTCCCAAACTCCTCATC TATGACAATGATAAGCGACCCTCAGGGGTCCCTGACCGGTTCTCTGGCTCCAAGTCTGGCAC CTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGCAGCT CATATACAAGCAGCAGCACTCTCGTCGTATTCGGCGGAGGCACCAAGCTGACCGTCCTA
SEQ ID NO: 7 - scFv#l MAQVQLLESGGGLVQPGGSLRLSCAASGFTVSSNYMHWVRQAPGKGLEWVSAINSNGDSTYYPDTVK DRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARCHLSRLYDLGSHPPYYFDFWGQGTLVTVSSGS AGGGGSGGGGSGGGGSDIVLTQSPGTLSLSPGERATLSCRASQSVSSGYLAWYQQKPGQAPRLLI YAASSRATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQYYGSPWTFGQGTKLEIKAAADYK D
SEP ID NO 8 - scFv #8
MAEVQLLESGGGLVQPGGSLRLSCAASGFTAWPSVVTEGLGGPGGGSARLQARGWS GFHHLQRQFQEHAVSANEQPESRGHAVYYCARDPDPYGSESFRLFGTFDYWGQGTLVTVS SGSAGGGGSGGGGSGGGGSDIVLTQSPGTLSLSPGERATLSCRASQSVTSSSLAWYQQKP GQAPRLLIYGASARATGIPDRFSGSGSGTDFTLTI SRLEPEDFAVYYCQQYYDWPLTFGG GTKLEIKAAADYKD*
SEQ ID NO: 9 - scFv#A7
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSEYGMNWVRQAPGKGLEWVSGI SWNSDTIDYG DSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARRQYYAMDVWGPGTLVTVSSGSAGGGGSGGGGSG GGGSDIVLTQPPSASGTPGQRVTI SCSGNSGS IASNSVQWYQQLPGKAPKLLIYDNDKRPSGVPDRFSGS KSGTSASLAI SGLRSEDEADYYCSSYTSSSTLVVFGGGTKLTVL
SEQ ID NO: 10 - scFv#C2
MAEVQLLESGGGLVQPGGSLRLSCAASGFTVI SGDMGWVRQAPGKGLEWVSAITTGGGSPNYA DSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARDEGMVGATYFDHWGQGTLVTVSSGSAGGGGSGG GGSGGGGSDIVLTQPPSASGTPGQRVTI SCSGSNSNIGYSQVSWYQQFPGKAPKLLIYEVSKRPSGVPDR FCGSKSGTSASLAI SGLRSEDEADYYCQSYDSSLNSYIFGGGTKVTVL
SEQ ID NO: 11 - scFv#G2
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSNFVMNWVRQAPGKGLEWVSRINTDGTSTNYA DSVTGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARSPYTIFGVVHFDYWGQGTLVTVSSGSAGGGGSG GGGSGGGGSDIVLTQPPSASGTPGQRVTISCSGSSSNIDSSPVNWYQQLPGAAPKLLIYLINERPSGVPD RFSGSKSGTSASLAI SGLRSEDEADYYCQVWDS I SDHWVFGGGTKLTVL
SEQ ID NO: 12 - scFv#H2
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSEYGMNWVRQAPGKGLEWVSGI SWNSDTIDYG DSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARRQYYAMDVWGPGTLVTVSSGSAGGGGSGGGGSG GGGSDIVLTQPPSASGTPGQRVTI SCSGNSGS IASNSVQWYQQLPGKAPKLLIYDNDKRPSGVPDRFSGS KSGTSASLAI SGLRSEDEADYYCSSYTSSSTLVVFGGGTKLTVL

Claims

WHAT IS CLAIMED IS:
1. An isolated protein comprising an amino acid sequence selected from the group consisting of (a) SEQ ID NOs: 7-12; (b) amino acid sequences that differ from those sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitution; (c) amino acid sequences having at least 95% sequence identity to the sequences specified in (a) or (b); a polypeptide having an amino acid sequence encoded by a polynucleotide having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6; or an antigen binding portion or a functional fragment thereof.
2. The isolated protein of claim 1, wherein said isolated protein is an isolated monoclonal
antibody or isolated monoclonal antibody fragment or antigen binding portion or fragment thereof.
3. The isolated protein of claims 1 or 2, wherein said isolated protein is an immune molecule that interacts with a human and/or mouse glyco-epitope.
4. The isolated protein of claim 3, wherein said glyco-epitope comprises a tumor associated
carbohydrate antigen.
5. The isolated protein of claim 4, wherein said tumor associated carbohydrate antigen comprises a glycoprotein.
6. The isolated protein of any of claims 3-5, wherein said isolated protein interacts with said glyco-epitope by at least one of binding, neutralizing and/or displacing another molecule bound to said glyco-epitope.
7. The isolated protein of claim 6, wherein said glyco-epitope is associated with a cell and
wherein said neutralizing induces cell death, cell quiescence or cell apoptosis.
8. The isolated protein of any of claims 1-7, comprising an isolated monoclonal antibody or
isolated monoclonal antibody fragment or antigen binding portion or fragment thereof, that binds to a human and/or mouse tumor associated carbohydrate antigen, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region or the light chain variable region comprises an amino acid sequence selected from the group consisting of: (a) at least a portion of any of SEQ ID NOs: 7-12; (b) amino acid sequences that differ from those sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitution; (c) amino acid sequences having at least 95% sequence identity to the sequences specified in (a) or (b); and (d) amino acid sequences encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6.
9. The isolated protein of any of claims 1-7, comprising an isolated monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or fragment thereof, that binds to a human and/or mouse a tumor associated carbohydrate antigen, the antibody comprising a light chain variable region and a heavy chain variable region, wherein at least a portion of the combined light chain and heavy chain variable regions comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO 7-12; (b) amino acid sequences that differ from those sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions; and (c) amino acid sequences having at least 95% sequence identity to the sequences specified in (a) or (b).
10. The isolated protein of any of claims 1-7, comprising an isolated monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or fragment thereof, that binds a human and/or mouse tumor associated carbohydrate antigen, where the antibody, antibody fragment, or antigen portion or fragment thereof binds to the same antigenic determinant as does:
(a) an antibody comprising an amino acid sequence of any of : SEQ ID NOs: 7-12; and/or
(b) an scFv having an amino acid sequence of: any of SEQ ID NOs: 7-12;
wherein the isolated antibody, antibody fragment, or antigen binding portion or fragment thereof competes with the antibody of (a) or (b) for binding at that antigenic determinant.
11. A single-chain variable fragment having an amino acid sequence selected from the group consisting of any of SEQ ID NOs. 7-12 or an antigen binding portion or functional fragment thereof.
12. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of any of claims 1-11, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof displaces a lectin from the tumor associated carbohydrate antigen.
13. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof of claims 1-12, wherein the antibody, antibody fragment, or antigen binding portion or fragment thereof binds to a human and/or mouse tumor associated carbohydrate antigen with an affinity constant (KD) of at least 10~6M.
14. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of claims 1-13, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof is a humanized antibody, antibody fragment, or antigen binding portion or fragment thereof.
15. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of claims 1-14, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof is monovalent.
16. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of claims 1-14, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof is multivalent.
17. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of any of claims 1-16, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof is a single chain antibody, antibody fragment, or antigen binding portion or fragment thereof.
18. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of any of claims 1-17, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof is a Fab, F(ab)'2, Fv, Fab/c, Fv, single chain Fv (scFv), or Fd fragment.
19. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of any of claims 1-18, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof is a chimeric antibody, humanized antibody, antibody fragment, or antigen binding portion or fragment thereof.
20. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of any of claims 1-19, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof is a fusion protein.
21. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of any of claims 1-20, comprising a heavy chain and wherein said heavy chain is selected from the group consisting of heavy chain of IgG, IgM, IgA, IgE, single chain antibody, immunoglobulin-derived constructs, and non antibody binding proteins.
22. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of claim 21, wherein the IgG is selected from the group consisting of IgGl, IgG2, IgG3, IgG4, mutated IgGl that is no longer recognized by FcR, and mutated IgG4 that no longer undergoes heavy chain swapping.
23. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of claims 21 or 22, wherein said heavy chain of the antibody, antibody fragment, or antigen binding portion thereof is conjugated to a ligand and/or tag.
24. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of any of claims 1-23, wherein the protein, antibody, antibody fragment, or antigen binding portion or fragment thereof is conjugated to a ligand and/or a tag.
25. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of claim 24, comprising a light chain and wherein said light chain is conjugated to the ligand and/or tag.
26. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof of any of claims 23-25, wherein the ligand and/or tag is polyethylene glycol (PEG), a nanoparticle or particles, or a label.
27. The isolated protein, monoclonal antibody or isolated monoclonal antibody fragment or
antigen binding portion or functional fragment thereof of claim 26, wherein said label is a fluorescent label or a bioluminescent label.
28. An isolated nucleic acid molecule encoding an isolated protein, isolated monoclonal antibody or isolated monoclonal antibody functional fragment or antigen binding portion or fragment thereof of claims 1-27.
29. An expression vector comprising at least one copy of the nucleic acid molecule of claim 28.
30. A host cell comprising the expression vector of claim 29.
31. A transgenic non-human animal having a genome comprising the isolated nucleic acid
molecule of claim 28 and/or the expression vector of claim 29.
32. A hybridoma secreting isolated monoclonal antibody or isolated monoclonal antibody
fragment or antigen binding portion or fragment thereof of claims 1-27.
33. A pharmaceutical composition comprising an isolated protein, isolated monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof of claims 1-27, and a pharmaceutically acceptable carrier.
34. The pharmaceutical composition of claim 33, wherein said isolated protein or isolated
monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof is administered to a patient in need thereof at a dosage unit from 0.1 mg/kg of body weight to 100 mg/kg of body weight.
35. The pharmaceutical composition of claims 33 or 34, wherein the pharmaceutical composition is adapted to treat cancer.
36. The pharmaceutical composition of any of claims 33-35, wherein the cancer is related to the abnormal proliferative growth in of any one or more of the following organs and tissues: lung, bone, pancreatic, skin, head or neck, eye, uterus, ovary, rectum, anal region, stomach, colon, breast, fallopian tubes, endometrium, cervix, vagina, vulva, lymph including Hodgkin's and non-Hodgkin's and lymphocytic lymphomas, esophagus, small intestine, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, urethra, penis, prostate, blood including chronic or acute leukemia, bladder, kidney, the central nervous system (CNS) including spinal axis tumors, brain stem glioma; and pituitary.
37. The pharmaceutical composition of claim 36, wherein the cancer is lung cancer.
38. The pharmaceutical composition of claim 37, wherein said lung cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), carcinoids, cylindromas, sarcomas and bronchoalveolar lung cancer.
39. The pharmaceutical composition of claim 38, wherein said non-small cell lung cancer is
selected from the group consisting of squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma.
40. The pharmaceutical composition of any of claims 33-39, wherein the composition is
formulated for parenteral, intravenous, oral, subcutaneous, intradermal, intramuscular or topical, administration.
41. Use of an isolated protein or isolated monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof of any of claims 1-27 or the pharmaceutical composition of any of claims 33-39, for the preparation of a medicament for the treatment of cancer.
42. Use of an isolated protein or isolated monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof of any of claims 1-27 or the pharmaceutical composition of any of claims 33-39, for the preparation of a medicament for inducing apoptosis or cell death of cell expressing a tumor-associated carbohydrate antigen.
43. The use according to claim 42, for the treatment of cancer.
44. The use of claim 43, wherein the cancer is related to the abnormal proliferative growth in of any one or more of the following organs and tissues: lung, bone, pancreatic, skin, head or neck, eye, uterus, ovary, rectum, anal region, stomach, colon, breast, fallopian tubes, endometrium, cervix, vagina, vulva, lymph including Hodgkin's and non-Hodgkin's and lymphocytic lymphomas, esophagus, small intestine, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, urethra, penis, prostate, blood including chronic or acute leukemia, bladder, kidney, the central nervous system (CNS) including spinal axis tumors, brain stem glioma; and pituitary.
45. The use of claim 44, wherein the cancer is lung cancer.
46. The use of claim 45, wherein said lung cancer is selected from the group consisting of non- small cell lung cancer (NSCLC), small cell lung cancer (SCLC), carcinoids, cylindromas, sarcomas and broncho alveolar lung cancer.
47. The use of claim 46, wherein said non-small cell lung cancer is selected from the group
consisting of squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma.
48. Use of the isolated protein, isolated monoclonal antibody or isolated monoclonal antibody
fragment or antigen binding portion or functional fragment thereof of any of claims 1-27 or the pharmaceutical composition of any of claims 33-39, for the manufacture of a medicament for inducing apoptosis of cell expressing a tumor-associated carbohydrate antigen.
49. Use of the isolated protein, isolated monoclonal antibody or isolated monoclonal antibody
fragment or antigen binding portion or functional fragment thereof of any of claims 1-27 or the pharmaceutical composition of any of claims 33-39, for the diagnosis of cancer in a subject.
50. Use of the isolated protein, isolated monoclonal antibody or isolated monoclonal antibody
fragment or antigen binding portion or functional fragment thereof of any of claims 1-27 in an assay for the diagnosis of cancer in a subject.
51. An assay for determining the presence of a tumor associated carbohydrate antigen in a sample obtained from a subject, the assay comprising;
(a) an isolated protein or isolated monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof of any of claims 1-27;
(b) an agent to detect the binding of the isolated protein or isolated monoclonal
antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment of (a).
52. The use of the assay of claim 51 for determining the presence of a tumor associated
carbohydrate antigen in a sample obtained from a subject.
53. A method of increasing apoptosis of a cell expressing a tumor-associated carbohydrate antigen, comprising administering to a subject in need thereof, the isolated protein or the isolated monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof of any of claims 1-27 or the pharmaceutical composition of any of claims 33-39.
54. The method of claim 53, wherein the method comprises administering to said subject in need thereof an amount of the isolated protein or isolated monoclonal antibody or isolated monoclonal antibody fragment or antigen binding portion or functional fragment thereof of any of claims 1-27 or the pharmaceutical composition of any of claims 33-39 sufficient to promote cell death or apoptosis of a cell expressing a tumor-associated carbohydrate antigen in a tissue of said subject in need thereof.
55. The method of claims 53 or 54, wherein the subject is suffering from cancer.
56. The method of claim 55, wherein the cancer is adenocarcinoma.
57. The method of claim 55, wherein the cancer is related to the abnormal proliferative growth in of any one or more of the following organs and tissues: lung, bone, pancreatic, skin, head or neck, eye, uterus, ovary, rectum, anal region, stomach, colon, colorectal, breast, fallopian tubes, endometrium, cervix, vagina, vulva, lymph including Hodgkin's and non-Hodgkin's and lymphocytic lymphomas, esophagus, small intestine, endocrine system, thyroid gland, parathyroid gland, adrenal gland, soft tissue, urethra, penis, prostate, blood including chronic or acute leukemia, bladder, kidney, the central nervous system (CNS) including spinal axis tumors, brain stem glioma; and pituitary.
58. The method of claim 57, wherein the cancer is lung cancer.
59. The method of claim 58, wherein said lung cancer is selected from the group consisting of non- small cell lung cancer (NSCLC), small cell lung cancer (SCLC), carcinoids, cylindromas, sarcomas and bronchoalveolar lung cancer.
60. The method of claim 59, wherein said non-small cell lung cancer is selected from the group consisting of squamous cell carcinoma (also called epidermoid carcinoma), adenocarcinoma, and large cell carcinoma.
61. A polynucleotide having a nucleic acid sequence according to any of SEQ ID NOs 1-6.
62. An isolated molecule having at least one CDR having an amino acid sequence selected from one of the following sequence sets: SEQ ID NOs: 13-15; 16-18, 19; 20-22; 23-25; 26-28; 29- 31; 32-34; 35-37; and 38-40.
63. The isolated molecule of claim 62, comprising three CDRs having amino acid sequences
selected from one of the following sequence sets, given in the order of CDR1, CDR2 and CDR3: SEQ ID NOs: 13-15; 16-18, 20-22; 23-25; 26-28; 29-31; 32-34; 35-37; and 38-40.
64. Use of each of the proteins scFvl, scFv8, scFvG2, scFvC2 and scFvH2 for diagnosis and
optionally also treatment of non- small cell lung cancer, as well as combinations thereof.
65. Use of claim 64, comprising use of a combination of scFvl and scFvG2
66. Use of each of the proteins scFv8, scFvG2, scFvC2 and scFvH2 for diagnosis and optionally also treatment of colorectal cancer, as well as combinations thereof.
67. Use of claim 66, wherein a combination of scFv8 and scFvG2 are used.
68. A method of treatment of cancer in a subject, comprising determining if the cancer expresses a glyco-molecule which binds to N-acetylglucosamine, in which case the cancer may optionally be treated with one or more of scFvl, scFv8 or scFvH2; binds to fucose, in which case the cancer may optionally be treated with one or more of scFv8 or scFvH2; and/or binds to galactose in which case the cancer may optionally be treated with scFvl.
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