Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39583—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials not provided for elsewhere, e.g. haptens, coenzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/14—Peptides, e.g. proteins
  • A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/54—F(ab')2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
  • G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins

Definitions

• the present disclosure relates to human antibodies and binding fragments thereof to carbohydrate antigens, as well as nucleic acids encoding such antibodies, complementary nucleic acids, polypeptides, vectors, host cells and methods of making and using thereof, including pharmaceutical compositions comprising said antibody and/or binding fragments. Further, methods are provided for administering antibodies to a subject in an amount effective to inhibit cancer cells. Specifically, antibodies that bind to stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4) and Globo H are disclosed herein, as well as related compositions and methods of use. Methods of use include, without limitation, cancer therapies and diagnostics.
• SSEA-3 stage-specific embryonic antigen 3
• SSEA-4 stage-specific embryonic antigen 4
• Globo H are disclosed herein, as well as related compositions and methods of use. Methods of use include, without limitation, cancer therapies and diagnostics.
• SSEA-4 stage-specific embryonic antigen-4 (Neu5Aca2 ⁇ 3Gai i ⁇ 3GalNAc i ⁇ 3Galal ⁇ 4Gai i ⁇ has been commonly used as a cell surface marker for pluripotent human embryonic stem cells and has been used to isolate mesenchymal stem cells and enrich neural progenitor cells (Kannagi R et al. (1983) EMBO J, 2:2355-2361).
• SSEA-4 stage-specific embryonic antigen-4
• Neurogen-4 stage-specific embryonic antigen-4
• the present disclosure is based on the discovery that Globo series antigens (Globo H, SSEA-3 and SSEA-4) are aberrantly expressed in a broad spectrum of cancers, but not on normal cells.
• human monoclonal antibodies to Globo series antigens can address the unmet need for effective treatment and/or prevention for cancer.
• Cancer cells expressing Globo series antigens can include, but are not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, stomach cancer, liver cancer, bile duct cancer, pancreatic cancer, colon cancer, kidney cancer, cervical cancer, ovarian cancer and prostate cancer.
• the present disclosure is directed to antibodies or binding fragments thereof specific to Globo series antigens.
• human B cells are isolated from peripheral blood of vaccinated subjects, plated at a density of one cell per well and cultured for secreted IgG production.
• the secreted IgGs are assayed for Globo H, SSEA-3 or SSEA-4 binding specificities.
• Genes encoding Ig VH, Ig VK or Ig ⁇ from positive wells are recovered using RT-PCR and cloned into expression vectors for generating anti-Globo H, SSEA-3 or SSEA-4 human monoclonal antibody.
• the light chains of the antibody is kappa type. In one embodiment, the light chain of the antibody is lamda type.
• the present disclosure provides an antibody, and/or an antigen- binding fragment thereof, comprising: a heavy chain variable domain (VH) comprising respective CDRs as disclosed herein and an amino acid sequence of at least about 80%, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 98, or 99 % sequence homology to the amino acid sequences as disclosed herein and/or a light chain variable domain (VL) comprising respective CDRs as disclosed herein and an amino acid sequence of at least about 80% , 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 98, or 99 % sequence homology to the amino acid sequences as disclosed herein respectively.
• VH heavy chain variable domain
• VL light chain variable domain
• the preent disclosure provides an antibody, or an antigen-binding fragment thereof capable of targeting the Globo-Series carbohydrate antigen, comprising: three heavy chain CDRs and corresponding three light chain CDRs of each respective clones as disclosed herein such as the ones disclosed in Tables 1-28.
• the present disclosure provides the antibody or antigen- binding fragment thereof comprising: the heavy chain variable domain comprising an amino acid sequence having at least about 80% identity to the full length heavy chain sequences of each clone as disclosed herein in Tables 1-28 further comprising the three corresponding heavy chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 sequences of the said corresponding clone; and the light chain variable domain comprising an amino acid sequence having at least about 80% identity to the full length light chain sequences of said clone as disclosed herein in Tables 1-28 further comprising the three corresponding light chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 sequences of said corresponding clone.
• CDRs heavy chain complementarity determining regions
• the present disclosure provides an antibody, or an antigen-binding fragment thereof capable of targeting the Globo-Series carbohydrate antigen, comprising: a.three heavy chain CDRs of SEQ ID NOs: 257, 258 and 259 or conservatively modified amino acid substitutions; and/or b. three light chain CDRs of SEQ ID NOs: 260, 261, and
• the antibody or antigen-binding fragment thereof of the above comprising: the light chain variable domain comprising an amino acid sequence having at least about 80% identity to
• SEQ ID NO: 3 further comprising the three heavy chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 (SEQ ID Nos 257, 258, 259); and/or the light chain variable domain comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 4 further comprising the three light chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 (SEQ ID Nos: 260, 261, 262).
• CDRs three heavy chain complementarity determining regions
• CDR1, CDR2 and CDR3 SEQ ID Nos 257, 258, 259
• the light chain variable domain comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 4 further comprising the three light chain complementarity determining regions (CDRs), CDR1, CDR2 and CDR3 (SEQ ID Nos: 260, 261, 262).
• the antibody or antigen-binding fragment thereof is selected from: (a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab') 2 ; or (e) a disulfide linked Fv.
• the antibody is an IgG or IgM.
• the present disclosure provides a pharmaceutical composition, comprising: an antibody or an antigen-binding fragment thereof; and at least one pharmaceutically acceptable carrier.
• the pharmaceutical composition further comprising at least one additional therapeutic agent.
• the present disclosure provides a method for inhibiting the proliferation of cancer cells, comprising the administering of an effective amount of an exemplary pharmaceutical composition to a subject in need thereof, wherein the proliferation of cancer cells is inhibited and/or decreased.
• the present disclosure provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the exemplary human antibody described herein.
• the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophageal cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.
• the present disclosure provides a method for staging cancer in a subject, comprising:
• the present disclosure provides a method for inhibiting the proliferation of cancer cells, comprising the administering of an effective amount of an pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof targeting Globo-series carbohydrate antigens to a subject in need thereof, wherein the proliferation of cancer cells is inhibited.
• the subject is human.
• the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject in need thereof an effective amount of the antibody or an antigen-binding fragment thereof targeting Globo-series carbohydrate antigens.
• the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.
• the subject is human.
• the present disclosure provides a method for cancer diagnosis in a subject, comprising:
• the markers consisting of Globo-H, SSEA-3 or SSEA-4.
• the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.
• the cell is cancer stem cell.
• the sample consists serum, blood, plasma, cells, cell medium, saliva, urine, lymph node fluid, tumor biopsy or tissue culture.
• the subject is human.
• the present disclosure provides a method of imaging a subject comprising:
• the imaging agent is a fluorophore, a dye, an MRI contrast agent or a radionuclide.
• the subject has a cancer, the method further defined as a method of detecting a cancer metastasis. In one embodiment, the subject is human. [0031] In one aspect, the present disclosure provides a method of isolating an antibody, or an antigen-binding fragment in a subject, comprising:
• the Globo series antigens comprising Globo-H, SSEA-3 or SSEA-4.
• the subject is human.
• the sample consists serum, blood, plasma, cells, cell medium, lymph node fluid, tumor biopsy or tissue culture.
• the present disclosure provides an antibody-drug conjugate (ADC) comprising a drug conjugated to an antibody or an antigen-binding fragment that binds Globo series antigens, wherein VH selected from SEQ ID No: 3, SEQ ID No: 7, SEQ ID No: 11, SEQ ID No: 15, SEQ ID No: 19, SEQ ID No: 23, SEQ ID No: 27, SEQ ID No: 31, SEQ ID No: 35, SEQ ID No: 39, SEQ ID No: 43, SEQ ID No: 47, SEQ ID No: 51, SEQ ID No: 55, , SEQ ID No: 59, SEQ ID No: 63, SEQ ID No: 67, SEQ ID No: 71, SEQ ID No: 75, SEQ ID No: 79, SEQ ID No: 83, SEQ ID No: 87, SEQ ID No: 91, SEQ ID No: 95, SEQ ID No: 99, SEQ ID No: 103, or SEQ ID No: 107 and VL
• the Globo series antigens comprising Globo-H, SSEA-3 or SSEA-4.
• the linker comprising a /?-nitrophenyl linker, a 4-(4-N- maleimidomethyl) cyclohexane-l-carboxyl hydrazide (MMCCH) linker, a maleimidocaproyl (MC) linker or a maleimidomethyl cyclohexane-l-carboxylate (MCC) linker.
• MMCCH 4-(4-N- maleimidomethyl) cyclohexane-l-carboxyl hydrazide
• MC maleimidocaproyl
• MCC maleimidomethyl cyclohexane-l-carboxylate
• the drug is a chemical compound or a biological agent. In one embodiment, the drug is an anti -proliferative agent.
• the anti -proliferative agent is selected from
• cyclophosphamide opiate, granulocyte colony-stimulating factor (GCSF), estrogen inhibitors (tamoxifen or Fareston), aromatase inhibitors (Arimidex, Aromasin or Femara), pituitary downregulators (Zoladex or Lupron), Novaldex (tamoxifen selective estrogen-receptor modulator), Evista (rolaxifene), Faslodex (estrogen receptor down-regulator), anticoagulant (Refludan), enzyme (Elitek), Hematopoietic growth factor, anti-neoplastic Agent
• HER1/EGFR tyrosine kinase inhibitor (Tarceva), VEGF protein inhibitor (Avastin), HER- 2/ErbB2 inhibitor (Tyverb/Tykerb), Interferon, Interleukin, Monoclonal antibody, or
• the anti -proliferative agent is selected from erlotinib (TARCEVA); docetaxel (TAXOTERE); gemcitabine (GEMZAR); cisplatin; carboplatin; paclitaxel (TAXOL); trastuzumab (HERCEPTIN); temozolomide (TEMODAL); tamoxifen (NOLVADEX, ISTUBAL, VALODEX); doxorubicin (ADRIAMYCIN); oxaliplatin
• ELOXATIN bortezomib
• VELCADE bortezomib
• sutent SUNITINIB
• letrozole FEMARA
• GLEEVEC imatinib mesylate
• MEK inhibitor Exelixis
• FASLODEX fulvestrant
• leucovorin folinic acid
• rapamycin RAPAMUNE
• lapatinib TYKERB
• lonafarnib SARASAR
• sorafenib NEXAVAR
• gefitinib IRESSA
• irinotecan CAMPTOSAR
• tipifarnib ZARNESTRA
• ABRAXANE Cremophor-free
• paclitaxel vandetanib
• ZACTFMA chloranmbucil
• TORISEL temsirolimus
• pazopanib canfosfamide
• TELCYTA thiotepa
• cyclosphosphamide CYTOXAN, NEOSAR
• 5-fluorouracil 5-FU
• vinorelbine NAVELBINE
• novantrone teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA); ibandronate; topoisomerase inhibitor RFS 2000; - difluoromethylornithine (DMFO); tamoxifen (NOLVADEX); raloxifene; droloxifene, 4- hydroxytamoxifen; tnoxifene; keoxifene; onapnstone; FARESTON (toremifine citrate); 4(5)- imidazoles; aminoglutethimide; MEGASE (megestrol acetate); AROMASIN (exemestane); formestanie; fadrozole; RIVISOR® (vorozole
• anastrozole flutamide; nilutamide; bicalutamide; leuprolide; goserelin; troxacitabine (a- 1,3-dioxolane nucleoside cytosine analog); lipid kinase inhibitor; oblimersen
• bevacizumab A VASTEST; cetuximab (ERBITUX); panitumumab (VECTIBIX); rituximab (RITUXAN); pertuzumab (OMNITARG); trastuzumab (HERCEPTIN); tositumomab (Bexxar, Corixia); gemtuzumab; or ozogamicin (MYLOTARG).
• the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject in need thereof an effective amount of the ADC as disclosed herein.
• the cancer is selected from the group consisting of sarcoma, skin cancer, leukemia, lymphoma, brain cancer, glioblastoma, lung cancer, breast cancer, oral cancer, head-and-neck cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, pancreatic cancer, intestinal cancer, colorectal cancer, kidney cancer, cervix cancer, endometrial cancer, ovarian cancer, testical cancer, buccal cancer, oropharyngeal cancer, laryngeal cancer and prostate cancer.
• the subject is human.
• Figure 1 shows the binding efficacy characterization between different human antibody clones by titration ELISA.
• Figure 1A uses Globo H-ceramide and
• Figure IB uses Globo H-lipid as the coating antigens.
• Figure 2 shows the binding efficacy characterization between different human antibody clones by titration ELISA.
• Figure 2A uses SSEA-3-ceramide and
• Figure 2B uses SSEA-3-lipid as the coating antigens.
• Figure 3 shows the binding efficacy characterization between different human antibody clones by titration ELISA.
• Figure 3A uses SSEA-4-ceramide and
• Figure 3B uses SSEA-4-lipid as the coating antigens.
• Anti-Globo series antigens human antibodies were developed and disclosed herein. Methods of use include, without limitation, cancer therapies and diagnostics.
• the antibodies described herein can bind to a broad spectrum of Globo series antigens-expressing cancer cells, thereby facilitating cancer diagnosis and treatment.
• Cells that can be targeted by the antibodies include carcinomas, such as those in skin, blood, lymph node, brain, lung, breast, mouse, esophagus, stomach, liver, bile duct, pancreas, colon, kidney, cervix, ovary, prostate cancer, etc.
• glycocan refers to a polysaccharide, or oligosaccharide. Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages between
• cellulose is a glycan (or more specifically a glucan) composed of B-l,4-linked D-glucose
• chitin is a glycan composed of B-l,4-linked N- acetyl-D-glucosamine.
• Glycans can be homo or heteropolymers of monosaccharide residues, and can be linear or branched. Glycans can be found attached to proteins as in glycoproteins and proteoglycans. They are generally found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes.
• N-Linked glycans are found attached to the R-group nitrogen (N) of asparagine in the sequon.
• the sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except praline.
• the term "antigen" is defined as any substance capable of eliciting an immune response.
• immunoogenicity refers to the ability of an
• immunogen antigen, or vaccine to stimulate an immune response.
• epitope is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.
• vaccine refers to a preparation that contains an antigen, consisting of whole disease-causing organisms (killed or weakened) or components of such organisms, such as proteins, peptides, or polysaccharides, that is used to confer immunity against the disease that the organisms cause.
• Vaccine preparations can be natural, synthetic or derived by recombinant DNA technology.
• the term "antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.
• the term “specifically binding,” refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10 "6 moles/liter, about 10 "7 moles/liter, or about 10 "8 moles/liter, or less.
• the phrase "substantially similar,” “substantially the same”, “equivalent”, or “substantially equivalent”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values, anti-viral effects, etc.).
• the difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the value for the reference/comparator molecule.
• the phrase "substantially reduced,” or “substantially different”, as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values).
• the difference between said two values is, for example, greater than about 10%), greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.
• Binding affinity generally refers to the strength of the sum total of
• binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
• the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer.
• vector 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.
• phage vector a type of vector
• viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
• 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
• 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, “recombinant 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.
• Polynucleotide or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
• the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction.
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer.
• the sequence of nucleotides may be interrupted by non-nucleotide components.
• a polynucleotide may be further modified after synthesis, such as by conjugation with a label.
• Other types of modifications include, for example, "caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals
• any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports.
• the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
• Other hydroxyls may also be derivatized to standard protecting groups.
• Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-,
• One or more phosphodiester linkages may be replaced by alternative linking groups.
• linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate”), P(S)S ("dithioate”), (0) R 2 ("amidate”), P(0)R, P(0)OR', CO or CH 2 ("formacetal”), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) optionally containing an ether (— O— ) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
• Oligonucleotide generally refers to short, generally single- stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length.
• oligonucleotide and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
• 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 generally 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.
• antibody and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments (as described in greater detail herein).
• An antibody can be chimeric, human and/or affinity matured.
• variable region or “variable domain” of an antibody refers to the amino- terminal domains of heavy or light chain of the antibody. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
• variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
• CDRs complementarity-determining regions
• FR framework
• the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
• the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen -binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
• the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
• Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
• Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
• Fv is the minimum antibody fragment which contains a complete antigen- recognition and -binding site.
• this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
• 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.
• the six CDRs confer antigen-binding specificity to the antibody.
• a single variable domain or half of an Fv comprising only three CDRs specific for an antigen
• the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
• Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
• Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
• F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
• the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
• antibodies can be assigned to different classes.
• immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi, and IgA 2 .
• the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
• An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
• full length antibody intact antibody
• whole antibody are used herein interchangeably, to refer to an antibody in its substantially intact form, not antibody fragments as defined below.
• Antibody fragments comprise only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody.
• an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.
• an antibody fragment for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding.
• an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody.
• such an antibody fragment may comprise an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
• Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
• the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones.
• a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones.
• each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
• the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al, Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
• the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to
• Antibodies of the present invention can also include chimerized monoclonal antibodies generated from antibodies of the present invention.
• the antibodies can be full-length or can comprise a fragment (or fragments) of the antibody having an antigen-binding portion, including, but not limited to, Fab, F(ab') 2 , Fab', F(ab)', Fv, single chain Fv (scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAb fragment (e.g., Ward et al, Nature, 341 :544-546 (1989)), an CDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
• Single chain antibodies produced by joining antibody fragments using recombinant methods, or a synthetic linker are also encompassed by the present invention.
• the antibodies or antigen-binding portions thereof of the present invention may be monospecific, bi-specific or multispecific.
• All antibody isotypes are encompassed by the present invention, including IgG (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 ), IgM, IgA (IgAi, IgA 2 ), IgD or IgE (all classes and subclasses are encompassed by the present invention).
• the antibodies or antigen-binding portions thereof may be mammalian (e.g., mouse, human) antibodies or antigen-binding portions thereof.
• the light chains of the antibody may be of kappa or lambda type.
• anti-cancer antibodies of the present invention include in combination with a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, of non-murine origin, preferably of human origin, which can be incorporated into an antibody of the present invention.
• Antibodies with a variable heavy chain region and a variable light chain region that are at least about 70%, at least about 75%, at least about 80%>, at least about 81%>, at least about 82%), at least about 83%>, at least about 84%>, at least about 85%>, at least about 86%>o, at least about 87%>>, at least about 88%>>, at least about 89%>>, at least about 90%>, at least about 91 >, at least about 92%>>, at least about 93%>>, at least about 94%>>, at least about 95%), at least about 96%>, at least about 97%>, at least about 98%>, at least about 99%> or about 100%) homologous to the variable heavy chain region and variable light chain region of the antibody produced by the reference antibody, and can also bind to a carbohydrate antigen (e.g. Globo H, SSEA-3 or SSEA-4). Homology can be present at either the amino acid or nucleotide sequence level
• substantially "homology" and/or "homologous sequences" of proteins of the invention include, without limitation, conservative amino acid substitutions, or for example alterations that do not effect the VH, VL or CDR domains of the antibodies, e.g., include scFv antibodies where a different linker sequence is used or antibodies where tag sequences or other components are added that do not contribute to the binding of antigen, or alterations to convert one type or format of antibody molecule or fragment to another type or format of antibody molecule or fragment (e.g., conversion from Fab to scFv or vice versa), or the conversion of an antibody molecule to a particular class or subclass of antibody molecule (e.g., the conversion of an antibody molecule to IgG or a subclass thereof, e.g., IgGl or IgG3).
• conservative amino acid substitutions or for example alterations that do not effect the VH, VL or CDR domains of the antibodies
• scFv antibodies where a different linker sequence is used
• a "conservative amino acid substitution”, as used herein, is one in which the amino acid residue is replaced with another amino acid residue having a similar side chain.
• Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, try
• Homology may be assessed by any convenient method. However, for determining the degree of homology between sequences, computer programs that make multiple alignments of sequences are useful, for instance Clustal W (Thompson et al., 1994). If desired, the Clustal W algorithm can be used together with BLOSUM 62 scoring matrix (Henikoff and Henikoff, 1992) and a gap opening penalty of 10 and gap extension penalty of 0.1, so that the highest order match is obtained between two sequences wherein at least 50% of the total length of one of the sequences is involved in the alignment.
• the antibodies or antigen-binding portions may be peptides.
• Such peptides can include variants, analogs, orthologs, homologs and derivatives of peptides, that exhibit a biological activity, e.g., binding of a carbohydrate antigen.
• the peptides may contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), peptides with substituted linkages, as well as other modifications known in the art.
• antibodies or antigen-binding portions thereof in which specific amino acids have been substituted, deleted or added. In an exemplary embodiment, these alternations do not have a substantial effect on the peptide's biological properties such as binding affinity.
• antibodies may have amino acid substitutions in the framework region, such as to improve binding affinity of the antibody to the antigen.
• a selected, small number of acceptor framework residues can be replaced by the corresponding donor amino acids.
• the donor framework can be a mature or germline human antibody framework sequence or a consensus sequence. Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science, 247: 1306-1310 (1990).
• the antibody, or antigen-binding portion thereof can be derivatized or linked to another functional molecule.
• an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent interaction, etc.) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag), amino acid linkers, signal sequences, immunogenic carriers, or ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, and staphylococcal protein A.
• One type of derivatized protein is produced by crosslinking two or more proteins (of the same type or of different types).
• Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
• spacer e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester
• homobifunctional e.g., disuccinimidyl suberate
• linkers are available from Pierce Chemical Company, Rockford, 111.
• Useful detectable agents with which a protein can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials.
• Non-limiting, exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, and, phycoerythrin.
• a protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase and the like.
• a protein can also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin).
• Nucleic acids encoding a functionally active variant of the present antibody or antigen-binding portion thereof are also encompassed by the present invention. These nucleic acid molecules may hybridize with a nucleic acid encoding any of the present antibody or antigen-binding portion thereof under medium stringency, high stringency, or very high stringency conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which is incorporated herein by reference.
• Specific hybridization conditions referred to herein are as follows: 1) medium stringency hybridization conditions: 6 X SSC at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 60°C; 2) high stringency hybridization conditions: 6 X SSC at about 45°C, followed by one or more washes in 0.2XSSC, 0.1% SDS at 65°C; and 3) very high stringency hybridization conditions: 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2XSSC, 1% SDS at 65°C.
• a nucleic acid encoding the present antibody or antigen-binding portion thereof may be introduced into an expression vector that can be expressed in a suitable expression system, followed by isolation or purification of the expressed antibody or antigen-binding portion thereof.
• a nucleic acid encoding the present antibody or antigen-binding portion thereof can be translated in a cell-free translation system.
• the present antibodies or antigen-binding portions thereof can be produced by host cells transformed with DNA encoding light and heavy chains (or portions thereof) of a desired antibody.
• Antibodies can be isolated and purified from these culture supernatants and/or cells using standard techniques. For example, a host cell may be transformed with DNA encoding the light chain, the heavy chain, or both, of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding, e.g., the constant region.
• substantially purified refers to a molecule (e.g. a compound) in a state that it is separated from substantially all other molecules normally associated with it in its native state.
• a substantially purified molecule is the predominant species present in a preparation.
• a substantially purified molecule may be greater than 60% free, preferably 75% free, or 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% free, or any range between any two recited percentages free from the other molecules (exclusive of solvent) present in the natural mixture.
• the present nuceic acids can be expressed in various suitable cells, including prokaryotic and eukaryotic cells, e.g., bacterial cells, (e.g., E. coli), yeast cells, plant cells, insect cells, and mammalian cells.
• prokaryotic and eukaryotic cells e.g., bacterial cells, (e.g., E. coli), yeast cells, plant cells, insect cells, and mammalian cells.
• bacterial cells e.g., E. coli
• yeast cells e.g., plant cells, insect cells, and mammalian cells.
• a number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC).
• ATCC American Type Culture Collection
• Non-limiting examples of the cells include all cell lines of mammalian origin or mammalian-like characteristics, including but not limited to, parental cells, derivatives and/or engineered variants of monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells.
• the engineered variants include, e.g., glycan profile modified and/or site-specific integration site derivatives.
• the present invention also provides for cells comprising the nucleic acids described herein.
• the cells may be a hybridoma or transfectant.
• the present antibody or antigen-binding portion thereof can be synthesized by solid phase procedures well known in the art.
• Solid Phase Peptide Synthesis A Practical Approach by E. Atherton and R. C. Sheppard, published by IRL at Oxford University Press (1989). Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed. M. W.Pennington and B. M. Dunn), chapter 7. Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984).
• G. Barany and R. B. Merrifield The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 1 and Vol. 2,
• hypervariable region when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops.
• antibodies comprise six hypervariable regions; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
• a number of hypervariable region delineations are in use and are encompassed herein.
• CDRs Determining Regions
• “Framework” or “FW” residues are those variable domain residues other than the hypervariable region residues as herein defined.
• variable domain residue numbering as in Kabat or "amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain.
• a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.
• the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
• Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
• the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
• diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL).
• VH heavy-chain variable domain
• VL light- chain variable domain
• VH-VL variable domain
• linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites.
• Diabodies are described more fully in, for example, EP 404,097; W093/1161; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
• a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein.
• An "affinity matured" antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).
• an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen.
• Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci. USA 91 :3809-3813 (1994); Schier et al. Gene 169: 147-155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al, J. Immunol.
• a “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
• An "agonist antibody”, as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.
• a “disorder” is any condition that would benefit from treatment with an antibody of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non- limiting examples of disorders to be treated herein include cancer.
• cell proliferative disorder and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation.
• the cell proliferative disorder is cancer.
• Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer cancer
• cancer cancer
• cancer cancer
• cancer cancer
• cancer cancer
• cancer cancer
• cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.
• Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.
• cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
• treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
• Treating or “treating” is referred to herein as administration of a therapeutic composition to a subject with the purpose to cure, alleviate, relieve, remedy, prevent, or ameliorate a disorder, symptoms of the disorder, a disease state secondary to the disorder, or predisposition toward the disorder.
• Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and/or tissue/organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
• antibodies of the invention are used to delay development of a disease or disorder.
• an "individual” or a “subject” is a vertebrate.
• the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats.
• the vertebrate is a human.
• mammal 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, etc. In certain embodiments, the mammal is human.
• an “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
• a "therapeutically effective amount" of a substance/molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, to elicit a desired response in the individual.
• a therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule are outweighed by the therapeutically beneficial effects.
• prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.
• One aspect of the present disclosure features the new antibody targeting the Globo series antigens (Globo H, SSEA-3, SSEA-4).
• SSEA-4 Globo series antigens
• SSEA-4 moiety is defined herein to be a glycan (i.e., a molecule containing a sugar moiety) that is SSEA-4 or a fragment or analog thereof.
• SSEA-4 is a glycan containing the hexasaccharide epitope (Neu5Aca2 ⁇ 3Gaipi ⁇ 3GalNAcpi ⁇ 3Galal ⁇ 4Gaipi ⁇ 4Glcpi), and optionally, a non-sugar moiety.
• Its fragment is a glycan containing a fragment of the hexasaccharide epitope and, if applicable, the non-sugar moiety.
• Globo H moiety is defined herein to be a glycan (i.e., a molecule containing a sugar moiety) that is Globo H or a fragment or analog thereof.
• Globo H is a glycan containing the hexasaccharide epitope (Fucal ⁇ 2 Gaipi ⁇ 3 GalNAcpi ⁇ 3 Galal ⁇ 4 Gaipi ⁇ 4 Glc), and optionally, a non-sugar moiety.
• Its fragment is a glycan containing a fragment of the hexasaccharide epitope and, if applicable, the non-sugar moiety.
• SSEA-3 moiety is defined herein to be a glycan (i.e., a molecule containing a sugar moiety) that is SSEA-3 or a fragment or analog thereof.
• SSEA-3 is a glycan containing the pentasaccharide epitope (Gaipi ⁇ 3GalNAcpi ⁇ 3Galal ⁇ 4Gaipi ⁇ 4Glcpi), and optionally, a non-sugar moiety.
• Its fragment is a glycan containing a fragment of the hexasaccharide epitope and, if applicable, the non-sugar moiety.
• CDRL2 KSDSDKYQGS
• CDRL3 QTWHTTTV
• CDRH2 VVWHDGRETHYGD S V sequence (CDRH)
• CDRL2 KSDSDKYQGS
• CDRL3 QTWHTSTV
• CDRH2 VVWHDGRETHYGD SV sequence (CDRH)
• CDRL2 KSDSENYRGS
• CDRL3 QTWHTSTV
• CDRH2 VVWHDGRETHYGD SV sequence (CDRH)
• CDRH3 DRGSDEPIDY
• CDRL2 KSDSNNYHGS
• CDRL3 QTWHTTTV
• CDRH2 VVWHNGRET YYED S V sequence (CDRH)
• CDRL2 KSDSEKYQGS
• CDRL3 QTWHANTV
• CDRH2 VVWHDGRETHYGD S V sequence (CDRH)
• CDRL2 KSDSEEYRAS
• CDRL3 MTWHTNKV
• CDRL2 KSDSDKHQGS
• CDRL3 MIWHVSGV
• CDRL2 RSDSDEEQGS
• CDRH2 VIWHNGRDRE YAD S V sequence (CDRH)
• CDRL2 KSDSEERRSS
• CDRL3 AIWHSNVV
• CDRL2 KSDSEKRRSS
• CDRL3 AIWHSNAV
• CDRL2 KSDSDIQHGS
• CDRL3 MIWYSTAV
• CDRH2 RIKSHSDGGTADYAAPV sequence (CDRH)
• CDRL3 MQGTYWPGT
• CDRL2 NADGSHIKGA
• CDRL3 QTWAPGW
• CDRH2 RIKSKNEAETTDYAAPV sequence (CDRH)
• CDRL3 MQRTHWPQT
• CDRL2 TDNQRPS
• CDRL3 AARDGSLDVW
• CDRH2 WMNSNSGDADSAQKF sequence (CDRH)
• CDRL2 MNSQRPS
• CDRL3 ATWDDNLRGW
• CDRH2 WMNSNSGDADSAQKF sequence (CDRH)
• CDRL2 MNSQRPS
• CDRL sequence (CDRL) CDRL3 : ATWDDNLRGW
• CDRH2 WMNSNSGNADSAQKF sequence (CDRH)
• CDRL2 MNNNRPS
• CDRL3 ATWDDNLRGW
• CDRH2 WMNSNTGDADSAQKF sequence (CDRH)
• CDRL3 MQGTYWPGT
• CDRH2 WMNSKYGNADSAHKF sequence (CDRH)
• CDRL2 MNNQRPS
• CDRL3 AVWDDNLRGW
• CDRH2 WMNSNTGDADSVQKF sequence (CDRH)
• CDRL2 MSNQRPS
• CDRL3 AVWDDNLRGW
• CDRH2 WMNSNTGDADSAQKF sequence (CDRH)
• CDRH3 MNYWGSKWDVNWFDP
• CDRL2 MSNKRPS
• CDRL3 AVWDDNLRGW
• CDRH2 WMNTNSGDTGYAQKF sequence (CDRH)
• CDRL2 KNSQRPS
• CDRL3 AAWDDSLSGSW
• CDRL2 DNDKRPS
• CDRL3 ATWDNRLDAV
• CDRL2 NGDGSHTKGDG
• CDRL3 QTWATGW
• CDRH2 WMNSRTGDADSAQNF sequence (CDRH)
• CDRL2 MNSQRPAG
• CDRL ATWDDNLRGW
• CDRL2 MSNRPRSG
• CDRL3 A VWDD SLRGW
• One aspect of the present disclosure features the new antibodies specific to SSEA-4.
• the anti-SSEA-4 antibody binds to Neu5Aca2 ⁇ 3Gai i ⁇ 3GalNAc i ⁇ 3Gala
• One aspect of the present disclosure features the new antibodies specific to SSEA-3.
• the anti-SSEA-3 antibody binds to 2Gai i ⁇ 3GalNAc i ⁇ 3Galal ⁇ 4Gai i ⁇
• One aspect of the present disclosure features the new antibodies specific to Globo H.
• the anti-Globo H antibody binds to Fucal ⁇ 2 Gai i ⁇ 3 GalNAc i ⁇ 3 Galal ⁇ 4 (3 ⁇ 41 ⁇ 1
• any of the antibodies described herein can be a full length antibody or an antigen- binding fragment thereof.
• the antigen binding fragment is a Fab fragment, a F(ab') 2 fragment, or a single-chain Fv fragment.
• the antigen binding fragment is a Fab fragment, a F(ab') 2 fragment, or a single-chain Fv fragment.
• the antibody is a human antibody, a chimeric antibody, or a single-chain antibody.
• any of the antibodies described herein has one or more characteristics of: (a) is a recombinant antibody, a monoclonal antibody, a chimeric antibody, a human antibody, an antibody fragment, a bispecific antibody, a monospecific antibody, a monovalent antibody, an IgGi antibody, an IgG 2 antibody, or derivative of an antibody; (b) is a human, murine, or chimeric antibody, antigen-binding fragment, or derivative of an antibody; (c) is a single- chain antibody fragment, a multibody, a Fab fragment, and/or an immunoglobulin of the IgG, IgM, IgA, IgE, IgD isotypes and/or subclasses thereof; (d) has one or more of the following characteristics: (i) mediates ADCC and/or CDC of cancer cells; (ii) induces and/or promotes apoptosis of cancer cells; (iii) inhibits proliferation of target cells of cancer cells; (iv
• the binding of the antibodies to their respective antigens is specific.
• the term "specific” is generally used to refer to the situation in which one member of a binding pair will not show any significant binding to molecules other than its specific binding partner (s) and e.g. has less than about 30%, preferably 20%, 10%, or 1 % cross-reactivity with any other molecule other than those specified herein.
• the present invention provides for a method for making a hybridoma that expresses an antibody that specifically binds to a carbohydrate antigen (e.g., Globo H).
• the method contains the following steps: immunizing an animal with a carbohydrate antigen (e.g., Globo H).
• composition that includes a carbohydrate antigen (e.g., Globo H); isolating splenocytes from the animal; generating hybridomas from the splenocytes; and selecting a hybridoma that produces an antibody that specifically binds to Globo H.
• a carbohydrate antigen e.g., Globo H
• isolating splenocytes from the animal generating hybridomas from the splenocytes
• selecting a hybridoma that produces an antibody that specifically binds to Globo H.
• Globo H carbohydrate antigen
• carbohydrate antigen is used to immunize mice
• the titers of the antibodies in the plasma can be monitored by, e.g., ELISA (enzyme-linked immunosorbent assay) or flow cytometry. Mice with sufficient titers of anti-carbohydrate antigen antibodies are used for fusions. Mice may or may not be boosted with antigen 3 days before sacrifice and removal of the spleen. The mouse splenocytes are isolated and fused with PEG to a mouse myeloma cell line. The resulting hybridomas are then screened for the production of antigen-specific antibodies. Cells are plated, and then incubated in selective medium.
• ELISA enzyme-linked immunosorbent assay
• Supematants from individual wells are then screened by ELISA for human anti-carbohydrate antigen monoclonal antibodies.
• the antibody secreting hybridomas are replated, screened again, and if still positive for anti-carbohydrate antigen antibodies, can be subcloned by limiting dilution.
• Exemplary Polyclonal antibodies against the anti-Globo series antigens antibodies may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method.
• Polyclonal antibodies include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies may be isolated from the serum.
• Polyclonal antibodies are generally raised in host animals (e.g., rabbit, mouse, horse, or goat) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCb, etc.
• a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
• Any mammalian animal may be immunized with the antigen for producing the desired antibodies.
• animals of Rodentia, Lagomorpha, or Primates can be used.
• Animals of Rodentia include, for example, mouse, rat, and hamster.
• Animals of Lagomorpha include, for example, rabbit.
• Animals of Primates include, for example, a monkey of
• Catarrhini old world monkey
• Catarrhini old world monkey
• Macaca fascicularis rhesus monkey
• baboon baboon
• chimpanzees
• antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc.
• PBS phosphate buffered saline
• the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion, and then administered to mammalian animals.
• Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining 1 mg or 1 ⁇ g of the peptide or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's incomplete adjuvant.
• Animals can be boosted until the titer plateaus by several administrations of antigen mixed with an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days. Animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. An appropriate carrier may also be used for immunization. After immunization as above, serum is examined by a standard method for an increase in the amount of desired antibodies. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
• antibodies can be made by the conventional hybridoma technology. Kohler et al., Nature, 256:495 (1975). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or rabbit, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that can specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. [00132] To prepare monoclonal antibodies, immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion.
• the immune cells used for cell fusion are preferably obtained from spleen.
• Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
• Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
• preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA.
• Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133 :3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
• the above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al. (Galfre et al., Methods Enzymol. 73 :3- 46, 1981). Lymphocytes are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).
• a suitable fusing agent such as polyethylene glycol
• Resulting hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine containing medium).
• HAT medium hyperxanthine, aminopterin, and thymidine containing medium.
• the cell culture is typically continued in the HAT medium for several days to several weeks, the time being sufficient to allow all the other cells, with the exception of the desired hybridoma (non-fused cells), to die. Then, the standard limiting dilution is performed to screen and clone a hybridoma cell producing the desired antibody.
• the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• the culture medium for the hybridomas typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
• Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
• the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay. Measurement of absorbance in enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/or
• immunofluorescence may be used to measure the antigen binding activity of the antibody of the invention.
• the antibody of the present invention is immobilized on a plate, protein of the invention is applied to the plate, and then a sample containing a desired antibody, such as culture supernatant of antibody producing cells or purified antibodies, is applied. Then, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme, such as alkaline phosphatase, is applied, and the plate is incubated. Next, after washing, an enzyme substrate, such as p-nitrophenyl phosphate, is added to the plate, and the absorbance is measured to evaluate the antigen binding activity of the sample.
• a fragment of the protein, such as a C-terminal or N-terminal fragment may be used in this method.
• hybridoma cells producing antibodies that bind to epitopes described herein can be identified and selected for further characterization.
• the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)).
• Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
• the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• the hybridoma cells may be grown in vivo as ascites tumors in an animal.
• the obtained hybridomas can be subsequently transplanted into the abdominal cavity of a mouse and the ascites are harvested.
• the obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the protein of the present invention is coupled.
• the antibody of the present invention can be used not only for purification and detection of the protein of the present invention, but also as a candidate for agonists and antagonists of the protein of the present invention.
• this antibody can be applied to the antibody treatment for diseases related to the protein of the present invention.
• Antibodies of the invention can be characterized for their physical/chemical properties and biological functions by various assays known in the art.
• Antibodies, or antigen-binding fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen.
• the affinity of an antibody for a carbohydrate antigen can be determined
• the present antibodies or antigen-binding portions thereof have in vitro and in vivo therapeutic, prophylactic, and/or diagnostic utilities.
• these antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, inhibit, prevent relapse, and/or diagnose cancer.
• Purified antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (UPLC), mass spectrometry, ion exchange
• antibodies are analyzed for their biological activity.
• antibodies of the invention are tested for their antigen binding activity.
• the antigen binding assays that are known in the art and can be used herein include without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
• immunoassays immunoprecipitation assays, fluorescent immunoassays, chemiluminescent immunoassays, nanoparticle immunoassays, aptamer immunoassays, and protein A immunoassays.
• An antibody of the invention may be used in, for example, in vitro, ex vivo and in vivo therapeutic methods.
• Antibodies of the invention can be used as an antagonist to partially or fully block the specific antigen activity in vitro, ex vivo and/or in vivo.
• at least some of the antibodies of the invention can neutralize antigen activity from other species.
• antibodies of the invention can be used to inhibit a specific antigen activity, e.g., in a cell culture containing the antigen, in human subjects or in other mammalian subjects having the antigen with which an antibody of the invention cross-reacts (e.g.
• an antibody of the invention can be used for inhibiting antigen activities by contacting the antibody with the antigen such that antigen activity is inhibited.
• the antigen is a human protein molecule.
• composition administration is referred to herein as providing a therapeutic composition of the invention to a patient.
• composition administration e.g., injection
• i.v. intravenous
• s.c. sub- cutaneous
• i.d. intradermal
• i.p. intraperitoneal
• intramuscular injection intramuscular injection.
• Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time.
• administration may be by the oral route or nasal route.
• administration may also be by surgical deposition of a bolus or positioning of a medical device.
• an antibody of the invention can be used in a method for inhibiting an antigen in a subject suffering from a disorder in which the antigen activity is detrimental, comprising administering to the subject an antibody of the invention such that the antigen activity in the subject is inhibited.
• the antigen is a human protein molecule and the subject is a human subject.
• the subject can be a mammal expressing the antigen with which an antibody of the invention binds.
• the subject can be a mammal into which the antigen has been introduced (e.g., by
• an antibody of the invention can be administered to a human subject for therapeutic purposes.
• an antibody of the invention can be administered to a non-human mammal expressing an antigen with which the antibody cross-reacts (e.g., a primate, pig or mouse) for veterinary purposes or as an animal model of human disease.
• a primate, pig or mouse e.g., a primate, pig or mouse
• animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration).
• Antibodies of the invention can be used to treat, inhibit, delay progression of, prevent/delay recurrence of, ameliorate, or prevent diseases, disorders or conditions associated with abnormal expression and/or activity of Globo series antigens (Globo H, SSEA-3, SSEA-4), including but not limited to cancer, muscular disorders, ubiquitin-pathway-related genetic disorders, immune/inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders.
• Globo series antigens Globo H, SSEA-3, SSEA-4
• an immunoconjugate comprising an antibody of the invention conjugated with a cytotoxic agent is administered to the patient.
• the immunoconjugate and/or antigen to which it is bound is/are internalized by cells expressing one or more proteins on their cell surface which are associated with Globo series antigens, resulting in increased therapeutic efficacy of the immunoconjugate in killing the target cell with which it is associated.
• the cytotoxic agent targets or interferes with nucleic acid in the target cell.
• cytotoxic agents include any of the chemotherapeutic agents noted herein (such as a maytansinoid or a calicheamicin), a radioactive isotope, or a ribonuclease or a DNA endonuclease.
• Antibodies of the invention can be used either alone or in combination with other compositions in a therapy.
• an antibody of the invention may be co-administered with another antibody, and/or adjuvant/therapeutic agents (e.g., steroids).
• an antibody of the invention may be combined with an anti-inflammatory and/or antiseptic in a treatment scheme, e.g. in treating any of the diseases described herein, including cancer, muscular disorders, ubiquitin-pathway-related genetic disorders, immune/inflammatory disorders, neurological disorders, and other ubiquitin pathway-related disorders.
• Such combined therapies noted above include combined administration (where the two or more agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, and/or following, administration of the adjunct therapy or therapies.
• An antibody of the invention can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
• Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
• the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
• Described herein are therapeutic methods that include administering to a subject in need of such treatment a therapeutically effective amount of a composition that includes one or more antibodies described herein.
• the subject in need of the treatment is diagnosed with, suspected of having, or at risk for cancer.
• the cancer include, but are not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer, kidney cancer, cervix cancer, ovary cancer and prostate cancer.
• the cancer is sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, or pancreas cancer.
• the cancer is brain cancer or glioblastoma multiforme (GBM) cancer.
• GBM glioblastoma multiforme
• the antibody is capable of targeting Globo series antigens-expressing cancer cells. In some embodiments, the antibody is capable of targeting Globo series antigens on cancer cells. In some embodiments, the antibody is capable of targeting Globo series antigens in cancers. [00155]
• the treatment results in reduction of tumor size, elimination of malignant cells, prevention of metastasis, prevention of relapse, reduction or killing of disseminated cancer, prolongation of survival and/or prolongation of time to tumor cancer progression.
• the treatment further comprises administering an additional therapy to said subject prior to, during or subsequent to said administering of the antibodies.
• the additional therapy is treatment with a chemotherapeutic agent.
• the additional therapy is radiation therapy.
• the methods of the invention are particularly advantageous in treating and preventing early stage tumors, thereby preventing progression to the more advanced stages resulting in a reduction in the morbidity and mortality associated with advanced cancer.
• the methods of the invention are also advantageous in preventing the recurrence of a tumor or the regrowth of a tumor, for example, a dormant tumor that persists after removal of the primary tumor, or in reducing or preventing the occurrence of a tumor.
• the methods as disclosed herein are useful for the treatment or prevention of a cancer, for example where a cancer is characterized by increased Globo H, SSEA-3 and/or SSEA-4 expression.
• the cancer comprises a cancer stem cell.
• the cancer is a pre-cancer, and/or a malignant cancer and/or a therapy resistant cancer.
• the cancer is a brain cancer.
• the subject to be treated by the methods described herein can be a mammal, more preferably a human.
• Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.
• a human subject who needs the treatment may be a human patient having, at risk for, or suspected of having cancer, which include, but not limited to, sarcoma, skin cancer, leukemia, lymphoma, brain cancer, lung cancer, breast cancer, oral cancer, esophagus cancer, stomach cancer, liver cancer, bile duct cancer, pancreas cancer, colon cancer, kidney cancer, cervix cancer, ovary cancer and prostate cancer.
• a subject having cancer can be identified by routine medical examination.
• an effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
• treating refers to the application or administration of a composition including one or more active agents to a subject, who has cancer, a symptom of cancer, or a predisposition toward cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect cancer, the symptom of cancer, or the predisposition toward cancer.
• “Development” or “progression” of cancer means initial manifestations and/or ensuing progression of cancer. Development of cancer can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein "onset” or “occurrence” of cancer includes initial onset and/or recurrence.
• compositions can be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
• inhalation spray topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
• parenteral as used herein includes subcutaneous, intracutaneous, intravenous,
• injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
• water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipients is infused.
• Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
• Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the antibody
• a pharmaceutical excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.
• the exemplary therapeutic compositions generally include a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
• a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intramuscular, intra-arterial, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, phosphate buffered saline, tris-buffered saline, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the pH value can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
• compositions suitable for an injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS).
• the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Exemplary Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• methods of preparation include vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
• Exemplary oral compositions generally include an inert diluent or an edible carrier.
• the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
• Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
• compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate or sterotes
• a glidant such as colloidal silicon dioxide
• a sweetening agent such as sucrose or saccharin
• the exemplary formulations of the invention can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
• binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
• fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
• lubricants e.g., magnesium stearate, talc or silica
• disintegrants e.g.
• compositions of the invention can be also introduced in microspheres or microcapsules, e.g., fabricated from poly-glycolic acid/lactic acid (PGLA) (see, U.S. Pat. Nos. 5,814,344; 5,100,669 and 4,849,222; PCT Publication Nos. WO 95/11010 and WO 93/07861).
• PGLA poly-glycolic acid/lactic acid
• Liquid preparations for oral administration can take the form of, for example, solutions, syrups, emulsions or suspensions, or they can be presented as a dry product for reconstitution with water or other suitable vehicle before use.
• Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid).
• suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
• emulsifying agents e.g., lecithin or acacia
• non-aqueous vehicles e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils
• preservatives e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid
• the preparations can also contain buffer
• compounds/formulations can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• Systemic administration can also be transmucosal or transdermal.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration may be accomplished through the use of nasal sprays or suppositories.
• the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
• the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
• the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, which is incorporated by reference herein.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the pharmaceutical formulations of the invention can be delivered parenterally, i.e., by intravenous (i.v.), subcutaneous (s.c), intraperitoneal (i.p.), intramuscular (i.m.), subdermal (s.d.), or intradermal (i.d.) administration, by direct injection, via, for example, bolus injection, continuous infusion, or gene gun (e.g., to administer a vector vaccine to a subject, such as naked DNA or RNA).
• Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• compositions can take such forms as excipients, suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the active ingredient can be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• a suitable vehicle e.g., sterile pyrogen-free water
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
• Therapeutic compositions which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected location to minimize potential damage to uninfected cells and, thereby, reduce side effects.

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• 2017
  • 2017-07-31 CN CN201780058430.0A patent/CN110062767B/zh active Active
  • 2017-07-31 KR KR1020197005486A patent/KR102528998B1/ko active Active
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