WO2021017892A1 - 针对抗cd73抗体和变体的方法和组合物 - Google Patents

针对抗cd73抗体和变体的方法和组合物 Download PDF

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WO2021017892A1
WO2021017892A1 PCT/CN2020/102678 CN2020102678W WO2021017892A1 WO 2021017892 A1 WO2021017892 A1 WO 2021017892A1 CN 2020102678 W CN2020102678 W CN 2020102678W WO 2021017892 A1 WO2021017892 A1 WO 2021017892A1
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seq
acid sequence
amino acid
cdr
antibody
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PCT/CN2020/102678
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French (fr)
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姜伟东
陈奕颖
曾琪铃
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上海复宏汉霖生物技术股份有限公司
上海复宏汉霖生物制药有限公司
上海复宏汉霖生物医药有限公司
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Priority to CA3145897A priority Critical patent/CA3145897A1/en
Priority to CN202080053981.XA priority patent/CN114206930A/zh
Priority to JP2022505389A priority patent/JP2022542165A/ja
Priority to BR112022001359A priority patent/BR112022001359A2/pt
Priority to EP20847134.2A priority patent/EP4006054A4/en
Publication of WO2021017892A1 publication Critical patent/WO2021017892A1/zh
Priority to US17/584,258 priority patent/US20220162334A1/en

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • 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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • 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]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/77—Internalization into the cell
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    • 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

Definitions

  • the present invention generally relates to anti-CD73 antibodies, their variants or mutants or antigen-binding fragments thereof, and methods of using them to treat human cancer.
  • CD73 (called exo-5'-nucleotidase (NT5E, EC 3.1.3.5)) is a glycosyl-phosphatidylinositol (GPI)-linked 70-kDa cell surface enzyme found in most tissues ( Zhang B., Cancer Res. [Cancer Research], 70(16):6407-6411(2010)).
  • CD73 (originally defined as lymphocyte differentiation antigen) is believed to act as a common signal transduction molecule on T lymphocytes and as an adhesion molecule, which is important for the binding of lymphocytes to endothelium.
  • CD73 can control a variety of physiological responses, including epithelial ion and fluid transport, ischemic preconditioning, tissue damage, platelet function, hypoxia, and vascular leakage (Zhang B., Cancer Res. [Cancer Research], 70 (16):6407-6411 (2010)).
  • adenosine The immunosuppressive effect of extracellular adenosine has been demonstrated (Stagg J. et al., Oncogene [oncogene], 29(39): 5346-5358 (2010)).
  • the interaction of adenosine and its receptors can inhibit most immune cell functions, including natural killer (NK) cell cytotoxicity, macrophage phagocytosis, T cell cytotoxicity and cytokine release (Goto T. et al., J. Immunol. [Journal of Immunology], 130(3): 1350-1355 (1983); Ohta A. et al., J. Immunol. [Journal of Immunology], 183(9): 5487-5493 (2009)).
  • NK natural killer
  • CD73 is a key molecule involved in adenosine signaling.
  • CD73 hydrolyzes adenosine monophosphate (AMP) (product from the adenosine triphosphate (ATP) hydrolysis pathway through CD39) to adenosine to affect the tumor microenvironment (Allard B. et al., Immunol. Rev. [Immunol Review], 276(1): 121-144 (2017); Resta R. et al., Immunol. Rev. [Immunology Review], 161: 95-109 (1998)).
  • AMP adenosine monophosphate
  • ATP adenosine triphosphate
  • CD73 is involved in cell-cell and cell-matrix interactions, and showed that CD73 is involved in drug resistance and tumor promotion (Spychala J.,Pharmacol.Ther.[pharmacology and therapeutics],87(2-3):161 -173 (2000)). It has been confirmed that CD73 is overexpressed in several types of cancer cells (Gao ZW. et al., Biomed. Res. Int. [International Biomedical Research], 2014: 460654 (2014)), and this expression is related to the poor prognosis of some cancers or patients Survival related (Loi S. et al., Proc. Natl. Acad. Sci.
  • anti-CD73 monoclonal antibody (mAb) treatment can inhibit metastasis and tumor angiogenesis (Allard B. et al., Int. J. Cancer[International Journal of Cancer], 134(6):1466-1473 (2014) ; Terp MG. et al., J. Immunol.
  • CD73 has produced favorable anti-tumor effects in preclinical models, and the combination therapy of CD73 blockade together with other immunomodulators is a particularly attractive treatment option (Antonioli L. et al., Trends Cancer [cancer] Trend], 2(2):95-109(2016)).
  • the combination with anti-CD73 mAb can significantly enhance the anti-tumor activity of anti-CTLA-4 (cytotoxic T lymphocyte antigen 4) mAb and anti-PD-1 (programmed cell death protein 1) mAb (Allard B. et al., Clin. Cancer) Res. [Clinical Cancer Research], 19(20): 5626-5635 (2013)).
  • the present invention in one aspect relates to anti-CD73 antibodies, variants, mutants, and/or antigen-binding fragments thereof, methods for preparing them, including them Pharmaceutical compositions, and methods of using them to treat human subjects.
  • the disclosed anti-CD73 antibodies and their affinity variants and/or mutants or antigen-binding fragments can be used in the treatment of diseases related to CD73 expression, CD73 overexpression, and/or abnormal CD73 function (such as cancer or fibrosis) Used alone or in combination with other agents.
  • the antibodies provided herein can also be used to detect by administering anti-CD73 antibodies, and/or their affinity variants/mutants or antigen-binding fragments thereof to a patient and in a sample from the patient (e.g., in vivo or ex vivo) Anti-CD73 antibody, and/or variant/mutant or antigen-binding fragment thereof that bind to CD73 protein, or by contacting the anti-CD73 antibody, and/or variant/mutant or antigen-binding fragment thereof with a sample from a patient And qualitatively or quantitatively detect the anti-CD73 antibody that binds to the CD73 protein, and/or the affinity variant/mutant or antigen-binding fragment thereof, and detect the CD73 protein in the patient or patient sample.
  • the present invention provides anti-CD73 antibodies and variants, and/or antigen-binding fragments thereof.
  • the present invention provides at least eighteen (18) anti-CD73 antibodies, variants and/or antigen-binding fragments thereof, namely anti-CD73 antibodies, and/or variant N1 and variant N1 variants ( N1#2, N1#2-P, N1#9 and N1#9-PH), N2, N4 and N4 variants (N4#1, N4#4, N4#4-3, N4#5, N4#6 , N4#6-2, N4#6-3, N4#6-3-P, N4#6-4, N4#6-4-P and N4#6-5).
  • the light chain and heavy chain nucleic acids and/or their encoded amino acid sequences of each of these (18) anti-CD73 antibodies and/or variants thereof are provided in the sequence listing below.
  • the following tables 1 and 2 respectively provide each light chain (CDR-L1, CDR-L2, and CDR-L3) and each heavy chain (CDR-H1, CDR-H1, and CDR-L3) of each anti-CD73 antibody and/or its variants.
  • Anti-CD73 antibodies, variants, and/or mutant heavy chain CDR sequences are listed in Table 2.
  • the anti-CD73 antibody of the present invention includes one or more N-glycosylation sites in one or more CDRs of its variable domains. mutation.
  • the resulting deglycosylated antibody retains the same functions as the parental non-deglycosylated antibody.
  • the anti-CD73 antibody of the present invention is a full-length IgG antibody, wherein the light chain is composed of the above-mentioned light chain variable region and the human antibody light chain constant region, wherein The heavy chain is composed of the variable region of the heavy chain and the constant region of the human antibody heavy chain.
  • the anti-CD73 antibody of the present invention is a full-length IgG antibody, wherein the light chain is composed of the above-mentioned light chain variable region and the human antibody light chain constant region, so The light chain constant region is shown in SEQ ID NO. 115, wherein the heavy chain is composed of the above heavy chain variable region and the human antibody heavy chain constant region, and the heavy chain constant region is shown in 112, 113 or 114.
  • the anti-CD73 antibody of the present invention is a full-length IgG antibody, wherein:
  • Antibody variant N1#2-P The light chain is shown in SEQ ID NO.93 and the heavy chain is shown in SEQ ID NO.116, or
  • Antibody variant N1#9-PH The light chain is shown in SEQ ID NO.94 and the heavy chain is shown in SEQ ID NO.117, or
  • Antibody variant N4#6-3-P The light chain is shown in SEQ ID NO.95 and the heavy chain is shown in SEQ ID NO.118, or
  • Antibody variant N4#6-4-P The light chain is shown in SEQ ID NO.96 and the heavy chain is shown in SEQ ID NO.119.
  • the anti-CD73 antibody comprises the Fc sequence of human IgG.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single-chain Fv (scFv) , Fv fragments, diabodies, and linear antibodies.
  • the antibody is a multispecific antibody.
  • the anti-CD73 antibody, variant or antigen-binding fragment thereof is conjugated to a therapeutic agent. In some embodiments, according to (or as applied to) any of the above embodiments, the anti-CD73 antibody, variant or antigen-binding fragment thereof is conjugated to a label. In some embodiments, according to (or as applied to) any of the above embodiments, the label is selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes.
  • the present invention provides an isolated nucleic acid molecule encoding an anti-CD73 antibody, variant, mutant or antigen-binding fragment thereof according to (or as applied to) any of the above-mentioned embodiments.
  • An expression vector encoding a nucleic acid molecule according to (or as applied to) any of the above embodiments is also provided.
  • a cell comprising an expression vector according to (or as applied to) any of the above embodiments.
  • the present invention also provides a method of producing an anti-CD73 antibody, variant or antigen-binding fragment thereof, the method comprising culturing a cell according to (or as applied to) any of the above-mentioned embodiments and recovering the antibody or antigen-binding fragment thereof from the cell culture.
  • the cell is a mammalian cell.
  • the mammalian cell is a CHO cell.
  • the cells are stable mammalian cell lines.
  • the stable mammalian cell line is a CHO cell line.
  • an isolated antibody that specifically binds to human CD73 on the cell surface and can neutralize the 5'-extracellular nuclease activity of soluble or membrane-bound human CD73 protein is provided.
  • Antibodies can induce intracellular internalization of CD73.
  • an antibody that binds to soluble or membrane-bound human CD73 protein and can inhibit its enzymatic activity, wherein the antibody is not internalized into cells expressing CD73.
  • the present invention provides a composition comprising an anti-CD73 antibody, variant, mutant or antigen-binding fragment thereof according to (or as applied to) any of the foregoing embodiments and a pharmaceutically acceptable carrier.
  • the present invention provides an anti-CD73 antibody, variant, mutant, or antigen-binding fragment thereof according to (or as applied to) any of the above-mentioned embodiments in contact with a sample and detecting the anti-CD73 antibody that binds to the CD73 protein.
  • Method for detecting CD73 protein in samples is used in an immunohistochemical assay (IHC) or in an ELISA assay.
  • a method of treating cancer in a subject comprising administering to the subject an effective amount of a composition according to (or as applied to) any of the foregoing embodiments.
  • a composition comprising an anti-CD73 antibody, variant, mutant or antigen-binding fragment thereof according to (or as applied to) any of the above-mentioned embodiments, for use in cancer treatment.
  • the use of the anti-CD73 antibody, variant, mutant or antigen-binding fragment thereof according to (or as applied to) any of the above-mentioned embodiments in the manufacture of a medicine for the treatment of cancer (such as cancer related to the expression of CD73) is provided.
  • the cancer is selected from melanoma, head and neck cancer, urothelial cancer, breast cancer (e.g., triple-negative breast cancer, TNBC), gastric cancer, classic Huo Chikin’s lymphoma (cHL), non-Hodgkin’s lymphoma, primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer (NSCLC) ), esophageal cancer, nasopharyngeal cancer (NPC), biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer and salivary gland cancer.
  • breast cancer e.g., triple-negative breast cancer, TNBC
  • gastric cancer e.g., classic Huo Chikin’s lymphoma (cHL), non-Hodgkin’s lymphoma, primary mediastinal B-cell lymphoma (NHL PMBCL), me
  • the subject is further administered a therapeutic agent selected from the group consisting of an antitumor agent, a chemotherapeutic agent, a growth inhibitor, and Cytotoxic agent.
  • a therapeutic agent selected from the group consisting of an antitumor agent, a chemotherapeutic agent, a growth inhibitor, and Cytotoxic agent.
  • the subject is further administered radiation therapy and/or surgery.
  • Figure 1A-1B Light chains of anti-CD73 lead clones N1, N2, and N4 selected from the initial phage panning (Figure 1A) ( Figure 1A discloses SEQ ID NO 79-81 in the order of appearance) and heavy chain The amino acid sequence alignment of the variable regions ( Figure 1B) ( Figure 1B discloses SEQ ID NO 97-99 in the order of appearance).
  • Figure 1A discloses SEQ ID NO 79-81 in the order of appearance
  • Figure 1B discloses SEQ ID NO 97-99 in the order of appearance.
  • Figures 2A-2C CD73 binding of selected antibodies. Test the selected antibodies with recombinant human CD73 protein (by ELISA (Figure 2A)), CD73-expressing human tumor cells MDA-MB-231 (human breast cancer) cells ( Figure 2B) and NCI-H292 (human mucoepidermoid lung cancer) Binding of cells ( Figure 2C) (by flow cytometry). Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive control and negative control, respectively.
  • Figure 3A-3B Effect of anti-CD73 lead clone on soluble CD73 enzymatic activity and cellular CD73 enzymatic activity.
  • the recombinant CD73 protein ( Figure 3A) and NCI-H292 cells ( Figure 3B) were incubated with anti-CD73 antibodies, ATP and AMP.
  • the CellTiter-Glo assay was used to determine the AMP concentration in the sample.
  • the luminescence reading indicates CD73 enzyme activity.
  • Anti-CD73 ref-1 antibody and APCP were used as positive controls, and HLX01 (anti-CD20) was used as negative controls.
  • Anti-CD73 lead clone antibody-mediated CD73 internalization. After anti-CD73 antibody treatment, the cell surface expression of CD73 was measured by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive control and negative control, respectively.
  • Figures 5A-5C CD73 binding of selected antibodies with different IgG Fc regions.
  • the variable sequences of N1, N2 and N4 were also cloned into the human wild-type or C219S mutant IgG2 Fc backbone.
  • the binding of different IgG isotypes of N1 ( Figure 5A), N2 ( Figure 5B), and N4 ( Figure 5C) to CD73-expressing human MDA-MB-231 cells was tested by flow cytometry.
  • Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive control and negative control, respectively.
  • Figure 6A-6C The effect of anti-CD73 lead clones with different IgG Fc regions on cell CD73 enzyme activity.
  • the anti-CD73 lead clones N1 ( Figure 6A), N2 ( Figure 6B), and N4 ( Figure 6C) with different IgG isotypes were tested for their ability to inhibit cell CD73 enzyme activity.
  • the MDA-MB-231 cells were incubated with anti-CD73 antibody. Add ATP, AMP and CellTiter-Glo reagents and record the luminescence. APCP was used as a positive control in the enzyme activity assay.
  • Figures 7A-7C Antibody-mediated CD73 internalization of anti-CD73 antibodies with different IgG Fc regions. After the cells were incubated with cells of different IgG isotypes with N1 ( Figure 7A), N2 ( Figure 7B) and N4 ( Figure 7C), the cell surface expression of CD73 was measured by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive control and negative control, respectively.
  • FIG 8A-8B Tumor growth inhibitory activity of the anti-CD73 lead clone in MDA-MB-231 (human triple-negative breast cancer) xenograft mouse model.
  • the tumor growth curve is shown in Figure 8A.
  • the individual tumor volume on day 45 is shown in Figure 8B. All data points are mean ⁇ SEM.
  • Figure 9 Amino acid sequence alignment of light chain variable regions derived from affinity matured N1 and N4 variants. N1 and N4 variants with better CD73 binding affinity and soluble CD73 enzyme blocking activity were identified from affinity maturation experiments based on in vitro phage display. The CDRs (complementarity determining regions) defined by Kabat are underlined and marked in bold. Figure 9 respectively discloses SEQ ID NO 82-85, 88, 86-87 and 89-92 in the order of appearance.
  • Figure 10 Amino acid sequence alignment of the heavy chain variable regions derived from affinity matured N1 and N4 variants. N1 and N4 variants with better CD73 binding affinity and soluble CD73 enzyme blocking activity were identified from affinity maturation experiments based on in vitro phage display. The CDRs (complementarity determining regions) defined by Kabat are underlined and marked in bold. These selected variable sequences are then cloned into the human IgG2 Fc backbone to become full-length antibodies.
  • Figure 10 respectively discloses SEQ ID NO 100-103, 106, 104-105 and 107-110 in the order of appearance.
  • Figures 11A-11B CD73 binding of N1 and N4 variants. The selected variants were tested by flow cytometry for binding to CD73-expressing MDA-MB-231 ( Figure 11A) and NCI-H292 cells ( Figure 11B). HLX01 (anti-CD20) was used as a negative control.
  • Figure 12 Effect of N1 and N4 variants on the enzymatic activity of immobilized CD73.
  • the N1 and N4 variants were tested for their ability to inhibit the enzymatic activity of immobilized CD73.
  • After incubating the anti-CD73 antibody and CD73 protein add ATP, AMP and CellTiter-Glo reagents and record the luminescence.
  • Anti-CD73 ref-1 antibody and anti-CD73 ref-2 antibody were used as positive controls, and HLX01 (anti-CD20) was used as negative control.
  • Figure 13 Antibody-mediated CD73 internalization of N1 and N4 variants. NCI-H292 cells were incubated with N1 and N4 variants, and the cell surface expression of CD73 was measured by flow cytometry. Anti-CD73 ref-1 antibody and anti-CD73 ref-2 antibody were used as positive controls, and HLX01 (anti-CD20) was used as negative control.
  • Figure 14 Amino acid sequence alignment of the light chain of N1 and N4 front variants.
  • the CDRs (complementarity determining regions) defined by Kabat are underlined and marked in bold.
  • Figure 14 respectively discloses SEQ ID NO 93-96 in the order of appearance.
  • Figure 15 Amino acid sequence alignment of the heavy chain variable regions of N1 and N4 front variants.
  • the CDRs complementarity determining regions defined by Kabat are underlined and marked in bold. These sequences were then cloned into the human IgG2 Fc backbone to become full-length antibodies.
  • Figure 15 respectively discloses SEQ ID NO 111, 101, and 108-109 in the order of appearance.
  • Figures 16A-16C CD73 binding capacity of N1 and N4 front variants.
  • the binding of anti-CD73 antibody to recombinant human CD73 protein was tested by ELISA ( Figure 16A), and the CD73 antibody was tested by flow cytometry with CD73-expressing human tumor cells MDA-MB-231 ( Figure 16B) and NCI-H292 ( Figure 16C) ) Combination.
  • Anti-CD73 ref-1 antibody and anti-CD73 ref-2 antibody were used as positive controls, and HLX01 (anti-CD20) was used as negative control.
  • Figures 17A-17C Effect of N1 and N4 front variants on soluble CD73 enzymatic activity (Figure 17A) and cell surface CD73 enzymatic activity ( Figure 17B, Figure 17C). Test the ability of anti-CD73 antibody to inhibit human recombinant CD73 protein and CD73 enzyme activity on the cell surface.
  • Recombinant CD73 protein Figure 17A
  • MDA-MB-231 Figure 17B
  • NCI-H292 Figure 17C
  • Anti-CD73 ref-1 antibody, anti-CD73 ref-2 antibody and APCP were used as positive controls.
  • HLX01 (anti-CD20) was used as a negative control.
  • Figures 18A-18B Antibody-mediated CD73 internalization of N1 and N4 front variants.
  • MDA-MB-231 ( Figure 18A) and NCI-H292 ( Figure 18B) cells were incubated with anti-CD73 N1 and N4 variants. After incubation, the cell surface expression of CD73 was measured by flow cytometry.
  • Anti-CD73 ref-1 and anti-CD73 ref-2 antibodies were used as positive controls, and HLX01 (anti-CD20) was used as negative control.
  • FIGS 19A-19B Reversal of the inhibitory effect of AMP on T cell activity by N1 and N4 front variants.
  • the isolated human T cells were cultured together with CD3/CD28 beads, AMP and serially diluted antibodies at 37°C for 4 days. Proliferation of CD3 + T cells was followed by CellTiter-Glo assay (Figure 19A), and IFN- ⁇ secretion was measured using human IFN- ⁇ ELISA MAX TM Deluxe kit ( Figure 19B).
  • Anti-CD73 ref-1 and anti-CD73 ref-2 antibodies were used as positive controls, and HLX04 (anti-VEGF) was used as negative control.
  • FIG. 20 Tumor growth inhibitory activity of N1 and N4 front variants in an MDA-MB-231 (human triple negative breast cancer) xenograft mouse model.
  • FIG. 21 Tumor growth inhibitory activity of N1 and N4 front variants in NCI-H292 (human mucoepidermoid lung cancer) xenograft mouse model.
  • the first dose of the test product was given 3 days after tumor inoculation.
  • Mice were treated intraperitoneally with antibodies at 50, 10 and 2 mg/kg twice a week for 3 weeks. All data points are mean ⁇ SEM.
  • Figure 22 Effect of N1 and N4 front variants on cell CD73 enzyme activity in NCI-H292 xenograft tumor.
  • the N1 and N4 front variants were tested for their ability to inhibit CD73 enzyme activity in the NCI-H292 xenograft model.
  • Antibody, APCP and placebo were injected intraperitoneally on day 0. Tumors were removed on days 1, 3, and 7 after antibody administration.
  • the CD73 enzyme activity in tumors was measured by CellTiter-Glo assay.
  • Figure 23A-23B Effect of N1 and N4 front variants on CD73 expression and CD73 enzyme activity in MDA-MB-231 xenograft tumors.
  • Test the ability of anti-CD73 antibody to down-regulate surface CD73 expression and inhibit CD73 enzyme activity in the MDA-MB-231 xenograft model. Seven days before antibody treatment, mice (n 4 mice/group) were subcutaneously transplanted with MDA-MB-231 cells. Antibody and placebo were injected on day 0. Tumors were collected 1, 3 and 7 days after antibody administration.
  • CD73 expression was measured by the mean fluorescence intensity (MFI) of staining ( Figure 23A), and CD73 enzymatic activity in tumors was measured by the CellTiter-Glo assay ( Figure 23B).
  • MFI mean fluorescence intensity
  • Figures 24A-24B Cross binding of N1 and N4 front variants to CD73-expressing cells in mice and monkeys.
  • HLX01 was used as a negative control.
  • the present invention relates to anti-CD73 antibodies, variants, mutants, and/or antigen-binding fragments thereof, methods of preparing them, pharmaceutical compositions containing them, and methods of using them to treat human subjects.
  • the present invention further provides that the anti-CD73 antibody specifically binds to CD73 and can inhibit tumor growth by inducing macrophage-mediated phagocytosis.
  • the disclosed antibodies exhibit improved efficacy and/or anti-tumor activity compared to conventional anti-CD73 monoclonal antibodies used to treat clinical conditions such as cancer or fibrosis.
  • immunoconjugates nucleic acids encoding novel anti-CD73 antibodies, their affinity variants or antigen-binding fragments thereof (as described herein), and compositions (such as pharmaceutical compositions).
  • the present invention provides at least eighteen (18) anti-CD73 antibodies, variants and/or antigen-binding fragments thereof, namely the anti-CD73 antibodies, variants, and/or specified in Table 1 and Table 2. Or mutants.
  • the light chain and heavy chain nucleic acid and/or the encoded amino acid sequence of each of these anti-CD73 antibodies, variants and/or mutants thereof are provided in the sequence-sequence listing section disclosed below.
  • Tables 1 and 2 provide each light chain (CDR-L1, CDR-L2, and CDR-L3) and each heavy chain (CDR-H1) of each anti-CD73 antibody, its variants and/or mutants, respectively. , CDR-H2, and CDR-H3) CDR sequences.
  • the present invention also provides methods for using novel anti-CD73 antibodies, affinity variants and/or mutants or antigen-binding fragments thereof to detect CD73 in samples (such as in vivo or ex vivo samples), and/or comprising such antibodies, Compositions of variants and/or mutants or antigen-binding fragments thereof for the treatment of clinical conditions or diseases related to CD73 expression, CD73 overexpression and/or abnormal CD73 function (such as cancer or fibrosis).
  • the disclosed anti-CD73 antibodies and affinity variants and/or mutants or antigen-binding fragments thereof can be used in the treatment of diseases related to CD73 expression, CD73 overexpression, and/or abnormal CD73 function (such as cancer). Or fibrosis) in combination with other agents.
  • the present invention further provides the use of such antibodies, variants or antigen-binding fragments thereof in the manufacture of drugs for cancer treatment.
  • the antibodies provided herein can also be used to detect by administering anti-CD73 antibodies, and/or their affinity variants/mutants or antigen-binding fragments thereof to a patient and in a sample from the patient (e.g., in vivo or ex vivo) Anti-CD73 antibody, and/or variant/mutant or antigen-binding fragment thereof that bind to CD73 protein, or by contacting the anti-CD73 antibody, and/or variant/mutant or antigen-binding fragment thereof with a sample from a patient And qualitatively or quantitatively detect the anti-CD73 antibody that binds to the CD73 protein, and/or the affinity variant/mutant or antigen-binding fragment thereof, and detect the CD73 protein in the patient or patient sample.
  • ratios, concentrations, amounts, and other numerical data can be expressed in range format herein. It will be further understood that the endpoints of each range are important relative to and independent of the other endpoints. It should also be understood that many values are disclosed herein, and in addition to the value itself, each value is also disclosed herein as “about” that particular value. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one specific value, and/or to "about” other specific values. Similarly, when a value is expressed as an approximation by using the antecedent “about”, it will be understood that the particular value forms an additional aspect. For example, if the value "about 10" is disclosed, then “10” is also disclosed.
  • additional aspects include from one specific value and/or to other specific values.
  • a range excluding one or both of those included limitations is also included in the present invention, for example the phrase'x to y'includes from'x' to The range of'y', and the range greater than'x' and smaller than'y'.
  • the range can also be expressed as an upper limit, such as'about x, y, z or less', and should be interpreted as including a specific range of'about x','about y', and'about z', and'less than x' ,'Less than y'and'less than z'range.
  • the phrase'about x, y, z or greater' should be interpreted as including the specific ranges of'about x','about y'and'about z'as well as'greater than x','greater than y'and'greater than z' Range.
  • the terms “about”, “approximately”, “at or about” and “substantially” mean that the amount or value in question can be an exact value or provide equivalents as recited in the claims or teachings herein The value of the result or effect.
  • References to "about” values or parameters herein refer to the usual error ranges of the corresponding values that are easily known by those skilled in the art. That is, it should be understood that quantities, sizes, formulations, parameters and other quantities and properties are not and need not be precise, but can (as required) be approximately and/or larger or smaller, which reflects tolerances, conversion factors , Rounding, measurement error, etc., and other factors known to those skilled in the art to obtain equivalent results or effects.
  • the term “optional” or “optionally” means that an event or situation described later may or may not occur, and the description includes examples where the event or situation occurs and implementations that do not occur example.
  • CD73 and “CD-73” can be used interchangeably, and refer to a cell surface enzyme encoded by a human gene containing 8 exons whose cytogenetic position is 3q13.12 And the molecular position of the base pair is 85,449,584-85,495,791 on chromosome 6 (Homo sapiens Annotation Release 109, GRCh38.p12).
  • CD73 protein is an enzyme with 5'-nucleotidase (using Zn +2 as a cofactor) (EC classification: 3.1.3.5).
  • the protein can hydrolyze extracellular nucleotides into membrane permeable nucleosides, and participate in the regulation of nucleobases, nucleosides, nucleotides and nucleic acid metabolism. It is related to the intracellular location mainly on the plasma membrane via the GPI anchor.
  • the human CD73 protein has two isotypes produced by alternative splicing.
  • the canonical isoform (UniProtKB identifier P21589) contains 574 amino acids and has a molecular weight of approximately 63 kDa.
  • CD73 is also called exo-5'-nucleotidase and 5'-nucleotidase. Mutations in the CD73 gene are associated with calcification of joints and arteries, that is, hereditary arterial and joint multiple calcification syndrome.
  • relapse refers to the recovery of cancer or disease after the disappearance of the disease on clinical assessment.
  • diagnosis of distant metastasis or local recurrence can be regarded as recurrence.
  • refractory or “resistant” refers to a cancer or disease that does not respond to treatment.
  • maintenance therapy refers to a predetermined retreatment that is used to help maintain the effect of the previous treatment. Maintenance therapy is usually given to help maintain cancer remission or prolong the response to a particular therapy regardless of disease progression.
  • invasive cancer refers to cancer that has spread beyond the tissue layer (where the cancer begins to enter normal surrounding tissues). Invasive cancer may or may not be metastatic.
  • non-invasive cancer refers to very early cancer or cancer that has not spread beyond the tissue of origin.
  • progression-free survival in oncology refers to the length of time during and after treatment that cancer does not grow. Progression-free survival includes the amount of time that the patient experiences a complete or partial response, and the amount of time that the patient experiences stable disease.
  • progressive disease in oncology can refer to the increase in the mass of the mass or the spread of the tumor since the beginning of the treatment and the tumor growth has exceeded 20%.
  • a “disorder” is any clinical condition associated with CD73 expression, CD73 overexpression, and/or abnormal CD73 function in a subject (eg, a mammal, such as a human), such that the subject will benefit from using the disclosed anti-CD73 Treatment or prevention of antibodies, variants, mutants and/or fragments thereof, and/or pharmaceutical compositions provided herein.
  • mammals suffering from or in need of prevention of CD73 overexpression This includes chronic and acute disorders or diseases, including those pathological conditions that predispose mammals to the disorder in question.
  • disorders to be treated herein include the disclosed cancers (such as head and neck cancer, throat cancer, colorectal cancer, lung cancer, etc.) or fibrosis (including idiopathic pulmonary fibrosis).
  • tumor refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues.
  • antibody is used in the broadest sense, and specifically covers, for example, single monoclonal antibodies (including agonists, antagonists, and neutralizing antibodies), antibody compositions with multiple epitope specificities, polyclonal antibodies, single-chain antibodies And antibody fragments (see below), as long as they specifically bind to natural polypeptides and/or exhibit the biological or immunological activity of the present invention.
  • the antibody binds to an oligomeric form of the target protein, such as a trimer form.
  • the antibody specifically binds to a protein, and the binding can be inhibited by the monoclonal antibody of the present invention (for example, the deposited antibody of the present invention, etc.).
  • the phrase "functional fragment or analog" of an antibody is a compound that has the same qualitative biological activity as the antibody to which it refers.
  • the functional fragment or analog of the antibody of the present invention may be an antibody capable of specifically binding to CD73.
  • the antibody can induce macrophage-mediated phagocytosis of CD73-expressing cancer cells.
  • an “isolated antibody” is an antibody that has been identified and separated and/or recovered from a component of its natural environment.
  • the pollutant components of its natural environment are substances that interfere with the diagnostic or therapeutic use of antibodies, and may include enzymes, hormones, and other protein or non-protein solutes.
  • the antibody will be purified to (1) higher than 95% by weight of the antibody as determined by the Lowry method, and most preferably higher than 99% by weight, (2) by using spin Cup sequencer, sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) Under reducing or non-reducing conditions using Coomassie blue or preferably silver staining, as determined by SDS-PAGE The uniformity.
  • Isolated antibodies include antibodies in situ within recombinant cells because at least one component of the antibody's natural environment will not be present. However, generally, isolated antibodies are prepared by at least one purification step.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the ⁇ and ⁇ chains, and four CH domains of the ⁇ and ⁇ isotypes.
  • Each L chain has a variable domain (VL) at the N-terminus and a constant domain (CL) at the other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of VH and VL together forms a single antigen binding site.
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively. Based on relatively small differences in CH sequence and function, the gamma and alpha classes are further divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • variable refers to the fact that certain segments of variable domains differ greatly in sequence between antibodies.
  • the V domain mediates antigen binding and defines the specificity of a specific antibody to its specific antigen.
  • variability is not evenly distributed across the 110 amino acid span of the variable domain.
  • the V region is composed of relatively constant segments called framework regions (FR) of 15-30 amino acids. These segments consist of shorter regions called “hypervariable regions” (each region is 9- 12 amino acids) separated.
  • FR framework regions
  • hypervariable regions each region is 9- 12 amino acids
  • CDR complementarity determining region
  • CDR complementarity determining region
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, that is, the antibodies of the individuals comprising the population are the same, except for possible naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be interpreted as requiring the production of antibodies by any specific method.
  • monoclonal antibodies that can be used in the present invention can be prepared by the hybridoma method first described in Kohler G. et al. Nature [Nature], 256(5517):495-497 (1975), or can be prepared in bacteria, eukaryotic Recombinant DNA methods are used for preparation in animal or plant cells (see, for example, US Patent No. 4,816,567). You can also use Clackson T. et al., Nature[Nature], 352(6336):624-628(1991), Marks JD. et al., J.Mol. Biol. [Molecular Biology Journal], 222(3): 581-597 (1991), and techniques such as those described in the Examples below to isolate "monoclonal antibodies” from phage antibody libraries.
  • Monoclonal antibodies herein include “chimeric” antibodies, in which a portion of the heavy chain and/or light chain is the same or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and this or these The remaining part of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, as long as they exhibit the biological activity of the present invention (see US Patent No. 4,816,567; and Morrison SL. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 81(21):6851-6855 (1984)).
  • Chimeric antibodies of interest herein include "primatized” antibodies, which comprise variable domain antigen binding sequences derived from non-human primates (eg, Old World monkeys, apes, etc.), and human constant region sequences.
  • “Intact” antibodies are antibodies that contain an antigen binding site and CL and at least the heavy chain constant domains CH1, CH2, and CH3.
  • the constant domain may be a natural sequence constant domain (for example, a human natural sequence constant domain) or an amino acid sequence variant thereof.
  • the intact antibody has one or more effector functions.
  • the constant domain (constant region) is preferably a human antibody constant region, more preferably a human IgG1, human IgG2, human IgG3 or human IgG4 antibody constant region.
  • the optional heavy chain constant region sequence is shown in SEQ ID NO: 112, 113 or 114 As shown, the specific sequence of the optional light chain constant region is shown in SEQ ID NO: 115.
  • Antibody fragments comprise a part of a complete antibody, preferably the antigen binding region or variable region of the complete antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata G. et al., Protein Eng. Chem.], 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • the expression “linear antibody” generally refers to the antibody described in Zapata G. et al., Protein Eng. [protein engineering] 8(10): 1057-1062 (1995). In short, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
  • Fab antigen-binding fragments
  • Fc fragment
  • the Fab fragment consists of the entire L chain together with the variable domain (VH) of the H chain and the first constant domain of a heavy chain (CH1).
  • VH variable domain
  • CH1 first constant domain of a heavy chain
  • Each Fab fragment is monovalent in terms of antigen binding, that is, it has a single antigen binding site.
  • the treatment of the antibody by pepsin produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen binding activity, and is still capable of cross-linking the antigen.
  • Fab'-SH is the designation for Fab' herein, in which one or more cysteine residues of the constant domain carry free sulfhydryl groups.
  • F(ab')2 antibody fragments were originally produced as a pair of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.
  • the Fc fragment contains the carboxy terminal portions of the two H chains held together by disulfide bonds.
  • the effector function of an antibody is determined by the sequence in the Fc region, which is also the part recognized by the Fc receptor (FcR) found on certain types of cells.
  • the “variant Fc region” includes an amino acid sequence that differs from the natural sequence Fc region due to at least one "amino acid modification" as defined herein.
  • the variant Fc region has at least one amino acid substitution, for example, from about one to about ten amino acid substitutions in the Fc region of the native sequence or the Fc region of the parent polypeptide, And preferably from about one to about five amino acid substitutions.
  • the variant Fc region herein will have at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology or a native sequence Fc region. At least about 99% homology.
  • the variant Fc region herein will have at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology with the Fc region of the parent polypeptide. Or at least about 99% homology.
  • polypeptide comprising an Fc region refers to a polypeptide comprising an Fc region, such as an antibody or immunoadhesion protein (see definition elsewhere herein).
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the purification of the polypeptide or by recombinant engineering of the nucleic acid encoding the polypeptide.
  • composition comprising a polypeptide (including an antibody) having an Fc region may comprise a polypeptide population with all K447 residues removed, a polypeptide population without K447 residues removed, or a mixture of polypeptides with and without K447 residues The peptide population.
  • Antibody effector functions refer to those biological activities attributable to the Fc region of the antibody (natural sequence Fc region or amino acid sequence variant Fc region), and vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors; and B cell activation .
  • the "native sequence Fc region” includes an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. Examples of Fc sequence are described in, for example, but not limited to: Sequences of Proteins of Immunological Interest [protein sequence of immunological interest], 5th edition, Elvin A. Kabat et al., Public Health Service [Public Health Service], National Institutes of Health [National Institutes of Health], Bethesda [Bethesda], MD. [Maryland], 1991.
  • Fv is the smallest antibody fragment, which contains a complete antigen recognition site and an antigen binding site.
  • the fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain that are tightly non-covalently associated. From the folding of these two domains, six hypervariable loops (each from the 3 loops of the H chain and the L chain) are emitted. These hypervariable loops contribute amino acid residues for antigen binding and give the antibody and antigen binding specificity .
  • variable domain or a half Fv containing only three CDRs against the original specificity
  • diabodies refers to small antibody fragments prepared by constructing sFv fragments (see the preceding paragraph) with a short linker (about 5-10 residues) between the VH and VL domains, so that the chain of the V domain is realized Pairing between, rather than within the chain, produces a bivalent fragment, that is, a fragment with two antigen binding sites.
  • Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are more fully described in, for example, EP 404,097; WO 93/11161; and Hollinger P. et al., Proc. Natl. Acad. Sci. USA, [Proceedings of the National Academy of Sciences] 90(14): 6444-6448 (1993).
  • Humanized forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequences derived from non-human antibodies.
  • humanized antibodies are human immunoglobulins (receptor antibodies), in which residues from the hypervariable region of the receptor are derived from non-human species (e.g., mouse, rat, rabbit, or non-human primate). ) With the desired antigen specificity, affinity and capacity of the hypervariable region (donor antibody) residue replacement.
  • the framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues.
  • humanized antibodies can include residues not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance.
  • a humanized antibody will include at least one, and typically substantially all of the two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all All the FRs above are those of the human immunoglobulin sequence.
  • the humanized antibody will also optionally include at least a portion of an immunoglobulin constant region, typically at least a portion of a human immunoglobulin.
  • Jones PT. et al. Nature[Nature], 321(6069):522-525 (1986); Riechmann L. et al., Nature[Nature], 332(6162): 323-329 (1988) ); and Presta LG, Curr. Opin. Biotechnol. [Biotechnology when the previous review], 3(4):394-398(1992).
  • sequence comparison computer program ALIGN-2 is used to generate% amino acid sequence identity values.
  • the ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with the user documentation in the US Copyright Office (Washington DC, 20559). Registered under the US copyright registration number TXU510087.
  • the ALIGN-2 program is publicly available through Genentech, Inc. of South San Francisco, California.
  • the ALIGN-2 program should be compiled for use on the UNIX operating system (preferably digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.
  • Fc receptor or "FcR” is used to describe a receptor that binds to the Fc region of an antibody.
  • the FcR of the present invention is an FcR ( ⁇ receptor) that binds to an IgG antibody, and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants of these receptors and alternatively spliced form.
  • FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("inhibitory receptor”), and FcyRIIA and FcyRIIB have similar amino acid sequences (these sequences differ mainly in their cytoplasmic domains).
  • the activated receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M, Annu. Rev. Immunol. [Annual Review of Immunology], 15:203-234 (1997)).
  • the term includes allotypes, such as FcyRIIIA allotypes: FcyRIIIA-Phe158, FcyRIIIA-Val158, FcyRIIA-R131 and/or FcyRIIA-H131.
  • FcRs are reviewed in Ravetch JV. et al., Annu. Rev.
  • FcR encompasses other FcRs, including FcRs identified in the future.
  • the term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer RL. et al., J. Immunol. [Journal of Immunology], 117(2):587-593 (1976) and Kim JK. Et al., Eur. J. Immunol. [European Journal of Immunology], 24(10): 2429-2434 (1994)).
  • FcRn refers to the neonatal Fc receptor (FcRn).
  • FcRn is similar in structure to the major histocompatibility complex (MHC) and consists of an ⁇ chain that is non-covalently bound to ⁇ 2-microglobulin.
  • MHC major histocompatibility complex
  • FcRn plays a role in passively delivering immunoglobulin IgG from mother to offspring and regulating serum IgG levels.
  • FcRn can act as a salvage receptor, binding and transporting (both intracellular or transcellular) pinocytosis IgG in an intact form, and rescues them from the default degradation pathway.
  • CH1 domain (also referred to as “C1" of "H1") of the human IgG Fc region generally extends from about amino acid 118 to about amino acid 215 (EU numbering system).
  • the "hinge region” is generally defined as extending from Glu216 of human IgG1 to Pro230 (Burton DR., Mol. Immunol. [Molecular Immunology], 22(3):161-206 (1985)).
  • the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine residues forming the S-S bond between the heavy chains in the same position.
  • the “CH2 domain” (also referred to as “C2” of “H2”) of the human IgG Fc region generally extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique because it is not closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the complete natural IgG molecule. It is speculated that carbohydrates can provide a substitute for domain-domain pairing and help stabilize the CH2 domain (Burton DR., Mol. Immunol. [Molecular Immunology], 22(3):161-206 (1985) ).
  • the “CH3 domain” (also known as “C2” or “H3” domain) contains the region of residues near the C-terminus of the CH2 domain in the Fc region (that is, from about amino acid residue 341 to the C-terminus of the antibody sequence (typically At amino acid residue 446 or 447 of IgG)).
  • the “functional Fc region” has the "effector function” of the native sequence Fc region.
  • effector functions include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors; BCR ) And so on.
  • Such effector functions generally require the combination of an Fc region and a binding domain (e.g., antibody variable domain), and can be assessed using, for example, various assays disclosed herein.
  • C1q is a polypeptide containing the binding site of the Fc region of an immunoglobulin. C1q and the two serine proteases C1r and C1s form the complex C1 (the first component of the complement-dependent cytotoxicity (CDC) pathway). Human C1q can be purchased commercially from, for example, Quidel, San Diego, California (CA).
  • binding domain refers to the region of a polypeptide that binds to another molecule.
  • the binding domain may comprise the portion of its polypeptide chain responsible for binding to the Fc region (for example, its alpha chain).
  • a useful binding domain is the extracellular domain of the FcR ⁇ chain.
  • an antibody with a variant IgG Fc (the variant has "altered” FcR binding affinity or ADCC activity) has enhanced or reduced FcR binding activity (e.g., Fc ⁇ R or Fc ⁇ R) compared to the parent polypeptide or a polypeptide comprising a native sequence Fc region. FcRn) and/or ADCC active antibodies.
  • FcR binding activity e.g., Fc ⁇ R or Fc ⁇ R
  • FcRn native sequence Fc region.
  • a variant Fc that "shows increased binding" to an FcR binds at least one FcR that has a higher affinity (for example, a lower apparent Kd or IC50 value) than the parent polypeptide or native sequence IgG Fc.
  • the improvement in binding compared to the parent polypeptide is about 3-fold, preferably about 5-fold, 10-fold, 25-fold, 50-fold, 60-fold, 100-fold, 150-fold, 200-fold, up to 500-fold, Or about 25% to 1000% improvement in binding.
  • a polypeptide variant that "shows reduced binding" to an FcR binds at least one FcR that has a lower affinity (e.g., a higher apparent Kd or a higher IC50 value) than the parent polypeptide.
  • the reduction in binding compared to the parent polypeptide can be a reduction in binding of about 40% or more.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells such as natural killer (NK) cells, neutrophils, and macrophages.
  • FcR Fc receptor
  • Antibodies “arm” cytotoxic cells and are absolutely necessary for this killing.
  • the polypeptide of is a polypeptide that mediates ADCC more effectively in vitro or in vivo when the amounts of the polypeptide with the variant Fc region and the polypeptide with the wild-type Fc region (or parent polypeptide) are substantially the same in the assay.
  • any in vitro ADCC assay known in the art is used to identify such variants, such as an assay or method for determining ADCC activity (e.g., in animal models, etc.).
  • the preferred variant is about 5-fold to about 100-fold (e.g., from about 25 to about 50-fold) more effective in mediating ADCC than the wild-type Fc (or parent polypeptide).
  • “Complement-dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement.
  • the activation of the classical complement pathway is triggered by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen.
  • C1q the first component of the complement system
  • an antibody of the appropriate subclass
  • Polypeptide variants with altered Fc region amino acid sequence and increased or decreased Clq binding capacity are described in US Patent No. 6,194,551 B1 and WO99/51642. The contents of these patent publications are expressly incorporated herein by reference (see also Idusogie EE. et al., J. Immunol. [Journal of Immunology], 164(8): 4178-4184 (2000)).
  • an “effective amount” of an anti-CD73 antibody (or fragment thereof) or composition as disclosed herein is an amount sufficient to achieve a specific stated purpose.
  • the “effective amount” can be determined empirically and by known methods related to the stated purpose.
  • the term “therapeutically effective amount” refers to the amount of the anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition as disclosed herein that is effective to "treat” a disease or disorder in a mammal (also known as a patient).
  • a therapeutically effective amount of the anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition as disclosed herein can reduce the number of cancer cells; reduce tumor size or weight; inhibit (ie, in a certain Slow down to a certain extent and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (ie slow down and preferably stop to a certain extent) tumor metastasis; inhibit tumor growth to a certain extent; and/or to a certain extent Relieve one or more symptoms associated with cancer.
  • the anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition as disclosed herein can prevent the growth of existing cancer cells and/or kill existing cancer cells.
  • the antibody (or its variant or The antigen-binding fragment) or composition may be cytostatic and/or cytotoxic.
  • the therapeutically effective amount is a growth inhibitory amount.
  • the therapeutically effective amount is an amount that prolongs the survival of the patient.
  • the therapeutically effective amount is an amount that improves the patient's progression-free survival.
  • the “growth inhibitory amount” of the anti-CD73 antibody (or variant or antigen-binding fragment thereof) of the present invention or the composition as disclosed herein is an amount capable of inhibiting the growth of cells (especially tumors, such as cancer cells) in vitro or in vivo.
  • the "growth inhibitory amount" of the polypeptide, antibody, antagonist or composition of the present disclosure for the purpose of inhibiting the growth of neoplastic cells can be determined empirically and by known methods or by the examples provided herein.
  • the "cytotoxic amount” of the anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition of the present invention is an amount capable of destroying cells (especially tumors, such as cancer cells) in vitro or in vivo.
  • the "cytotoxic amount” of the anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition of the present invention for the purpose of inhibiting the growth of neoplastic cells can be determined empirically and by methods known in the art.
  • the “growth inhibitory amount” of the anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition of the present invention is an amount capable of inhibiting the growth of cells (especially tumors, such as cancer cells) in vitro or in vivo.
  • the "growth inhibitory amount" of the anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition of the present disclosure for the purpose of inhibiting the growth of neoplastic cells can be implemented empirically and by known methods or by the implementation provided herein The case is determined.
  • pharmaceutically acceptable or “pharmacologically compatible” refers to materials that are not biologically or otherwise undesirable.
  • the material may be incorporated into a pharmaceutical composition administered to a patient, Without causing any significant undesirable biological effects or interactions in a harmful manner with any other components of the composition containing the material.
  • the pharmaceutically acceptable carrier or excipient preferably meets the required standards for toxicology and manufacturing testing and/or is included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration (U.S. Food and Drug Administration).
  • detection is intended to include determining the presence or absence of a substance, or quantifying the amount of a substance (such as CD73). Therefore, the term refers to the use of materials, compositions and methods of the present invention for qualitative and quantitative determination. Generally, the specific technique used for detection is not important to the practice of the present invention.
  • the "detection" may include: observing the presence or absence of CD73 gene products, mRNA molecules, or CD73 polypeptides; changes in the level of CD73 polypeptides or the amount bound to the target; and the biological function/activity of CD73 polypeptides Variety.
  • "detecting” may include detecting wild-type CD73 levels (eg, mRNA or polypeptide levels).
  • the detection may include quantifying any value between 10% and 90%, or any value change (increase or decrease) between 30% and 60% or above 100% compared to the control.
  • the detection may include quantifying any value (including end values) or more (for example, 100 times) changes between 2 times and 10 times.
  • label refers to a detectable compound or composition that can be directly or indirectly conjugated to an antibody.
  • the label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze a chemical change of a detectable substrate compound or composition.
  • the present invention relates to antibodies that bind to the CD73 receptor as disclosed herein.
  • the disclosed anti-CD73 antibodies, and their affinity variants and/or mutants, or antigen-binding fragments thereof can be used in various therapeutic and diagnostic methods.
  • Anti-CD73 antibodies are antibodies that have sufficient affinity and specificity to bind to CD73.
  • the disclosed anti-CD73 antibodies (or variants or mutants or antigen-binding fragments thereof) can be used as therapeutic agents in targeting and interfering with diseases or disorders related to the expression of CD73.
  • the disclosed anti-CD73 antibodies (or variants/mutants or antigen-binding fragments thereof) show minimal binding to other proteins.
  • it is preferred that the disclosed anti-CD73 antibody (or variant/mutant or antigen-binding fragment thereof) is a human or recombinant humanized anti-CD73 monoclonal antibody.
  • the disclosed anti-CD73 antibodies, and their affinity variants and/or mutants, or antigen-binding fragments thereof can increase the internalization of CD73 in cells expressing CD73, thereby resulting in a decrease
  • the disclosed anti-CD73 antibodies, and their affinity variants and/or mutants or antigen-binding fragments are considered to be involved in other clinical conditions related to CD73 expression (such as fibrosis, including idiopathic lung fibrosis). Chemical) has a beneficial effect.
  • the disclosed anti-CD73 antibodies are due to a decrease in CD73 protein (and correspondingly, a decrease in 5'-nucleotidase activity) of appropriate cells expressing CD73
  • their affinity variants and/or mutants or antigen-binding fragments can have positive clinical benefits for patients such as fibrosis (including idiopathic pulmonary fibrosis).
  • the anti-CD73 antibody comprises the CDR, variable heavy chain region, and/or variable light chain region of any of the antibodies disclosed herein.
  • the present invention provides anti-CD73 antibodies, their affinity variants/mutants, and/or antigen-binding fragments thereof.
  • the specific anti-CD73 antibody of the present invention, its one or more affinity variants/one or more mutants, and/or one or more antigen-binding fragments thereof comprise a light chain (LC) variable domain sequence (comprising the specific CDR-L1, CDR-L2 and CDR-L3 sequence listed in Table 1 above) and heavy chain (HC) variable domain sequence (comprising the specific sequence listed in Table 2 above) Specific CDR-H1, CDR-H2 and CDR-H3 sequences).
  • LC light chain
  • HC heavy chain
  • each light chain and heavy chain of each specific anti-CD73 antibody of the present invention, its one or more affinity variants/one or more mutants, and/or one or more antigen-binding fragments thereof The nucleic acid and its amino acid sequence are also provided in the sequence listing below.
  • the present invention provides heavy chain and light chain variable domains, and the CDRs mentioned herein are combined in all possible pairs to generate a large number of anti-CD73 antibodies and one or more affinity variants/a One or more mutants, and/or one or more antigen-binding fragments thereof.
  • the one or more amino acid substitutions are one or more conservative amino acid substitutions.
  • amino acid substitutions do not substantially reduce the ability of the antibody to bind to the antigen.
  • conservative changes eg, conservative substitutions as provided herein
  • the methods described in the Examples below can be used to assess the binding affinity of anti-CD73 antibody variants.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp; Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val
  • Non-conservative substitutions will require the exchange of members of one of these categories for another category.
  • exemplary substitution variants are affinity matured antibodies, which can be conveniently produced, for example, using phage display-based affinity maturation techniques (such as those disclosed herein).
  • one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (such as binding affinity).
  • Changes e.g., substitutions
  • HVR can be made in the HVR, for example to improve antibody affinity.
  • HVR "hot spots” ie residues encoded by codons that undergo mutations at high frequency during the somatic cell maturation process
  • affinity maturation diversity is introduced into the variable selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis) In the genes. Then a secondary library is generated. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method of introducing diversity involves HVR targeted methods, in which several HVR residues (eg 4-6 residues at a time) are randomized. Alanine scanning mutagenesis or modeling can be used, for example, to specifically identify HVR residues involved in antigen binding.
  • anti-CD73 antibodies, variants or mutants or antigen-binding fragments thereof may lack N-glycosylation motifs in the variable regions of the heavy or light chains, which may result in differences within a batch of antibodies, Causes changes in function, immunogenicity or stability.
  • Methods for analyzing antibody glycosylation include, but are not limited to, for example, chromatography (e.g., cation exchange chromatography (CEX) or liquid chromatography), mass spectrometry (e.g., electrospray ionization mass spectrometry), and capillary electrophoresis-sodium lauryl sulfate .
  • chromatography e.g., cation exchange chromatography (CEX) or liquid chromatography
  • mass spectrometry e.g., electrospray ionization mass spectrometry
  • capillary electrophoresis-sodium lauryl sulfate capillary electrophoresis-sodium lauryl s
  • the mutant of a specific anti-CD73 antibody mutant contains one or more mutations at the N-glycosylation site in one or more CDR regions, such as the sequon N-X-S/T.
  • Anti-CD73 antibody mutants with one or more mutants at the N glycosylation site eliminate the N glycosylation site but still retain the equivalent function.
  • the binding affinity of anti-CD73 antibodies, variants and/or mutants or antigen-binding fragments thereof to CD73 is higher than the binding affinity to CD73 homologs.
  • anti-CD73 antibodies, and/or variants or antigen-binding fragments thereof "specifically bind" CD73 (ie, have no more than about 1x 10 -7 M, preferably no more than about 1x 10 -8 and most preferably no more than A binding affinity (Kd) value of about 1 x 10 -9 M), but has a binding affinity for non-CD73 that is at least about 50 times weaker than its binding affinity for CD73, or at least about 500 times, or at least about 1000 times.
  • the anti-CD73 antibody that specifically binds to CD73 may be any of various types of antibodies as defined above, but is preferably a humanized or human antibody.
  • the degree of binding of the anti-CD73 antibody to the non-target protein is less than About 10% of the binding of the antibody to CD73.
  • Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule (usually a molecule of similar structure that does not have binding activity). For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., excess unlabeled target).
  • the term “specifically binds” to a specific polypeptide or epitope on a specific polypeptide target or “specifically binds to a specific polypeptide or epitope on a specific polypeptide target” or to a specific polypeptide or epitope on a specific polypeptide target Having “specificity” can be manifested by, for example, having at least about 10 -4 M, alternatively at least about 10 -5 M, alternatively at least about 10 -6 M, alternatively at least about 10 -7 for the target M, alternatively at least about 10 -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 M, or larger Kd molecules.
  • the term “specific binding” refers to binding in which a molecule binds
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCC is the mechanism of action of therapeutic antibodies against tumor cells.
  • ADCC is a cell-mediated immune defense by which effector cells of the immune system actively lyse target cells (e.g., cancer cells), and the membrane surface antigens of these target cells are covered by specific antibodies (e.g., anti-CD73 antibodies and/or as described herein). Or affinity variants) combined.
  • target cells e.g., cancer cells
  • specific antibodies e.g., anti-CD73 antibodies and/or as described herein.
  • affinity variants e.g., anti-CD73 antibodies and/or as described herein.
  • the anti-CD73 antibody and/or affinity variant exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) effector functions similar to the reference anti-CD73 monoclonal antibody, as described in the Examples, for example Proved by the measurement.
  • the ADCC effector function activity of the anti-CD73 antibody and/or its affinity variant or mutant described herein is at least about 80%, at least about 80%, of the ADCC effector function activity of the reference anti-CD73 antibody About 85%, 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%, at least about 100%, or more than 100% (e.g., about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125% , Or about 130%), including any range between these values.
  • the anti-CD73 antibody, and/or affinity variants thereof, or mutants thereof are between about 0.1 pM to 200 pM (0.2 nM) (e.g., about 0.1 pM, about 0.25 pM, about 0.5 pM, About 0.75pM, about 1pM, about 5pM, about 10pM, about 20pM, about 30pM, about 40pM, about 50pM, about 60pM, about 70pM, about 80pM, about 90pM, about 100pM, about 110pM, about 120pM, about 130pM, about A Kd of 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, or more than about 190 pM, including any range between these values) binds human CD73.
  • 0.2 nM e.g., about 0.1 pM, about 0.25 pM, about 0.5 pM, About 0.75pM, about 1
  • the binding affinity of the anti-CD73 antibody, and/or its affinity variant or mutant thereof to CD73 is about 1%, about 5%, or about 10% higher than the binding affinity of the reference anti-CD73 antibody to CD73 , About 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or more than about 100% (e.g., about 105%, about 110%, about 120%, or about 130%).
  • the binding affinity of anti-CD73, and/or variants or mutants thereof, to CD73 is about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times higher than the binding affinity of the reference anti-CD73 antibody to CD73. Times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.25 times, about 2.5 times, about 2.75 times, about 3 times, about 3.25 times, about 3.5 times, About 3.75 times, about 4 times, about 4.25 times, about 4.5 times, about 4.75 times, or more than about 4.75 times, including any range between these values.
  • the anti-CD73 antibodies provided herein, and/or variants or mutants thereof have an extended half-life in vivo compared to a reference anti-CD73 antibody.
  • the in vivo half-life of the anti-CD73 antibodies described herein, and/or variants or mutants thereof is not shorter than the in vivo half-life of the reference anti-CD73 antibody.
  • the anti-CD73 antibodies provided herein, and/or variants or mutants thereof exhibit similar pharmacokinetic properties to the reference anti-CD73 antibody.
  • the anti-CD73 antibodies provided herein, and/or variants or mutations thereof exhibit about 50%, about 55%, about 60%, about 65 percent of the serum concentration-time curve of the reference anti-CD73 antibody. %, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than 95% (such as about 96%, about 97%, about 98%, about 99%, or more AUC (area under the curve) of about 99%) (including any range between these values).
  • the anti-CD73 antibodies of the present invention comprise the Fc sequence of human IgG (for example, human IgG1 or human IgG4).
  • the Fc sequence has been altered or otherwise altered so that it lacks antibody-dependent cytotoxicity (ADCC) effector function, which is usually related to its binding to Fc receptors (FcR).
  • ADCC antibody-dependent cytotoxicity
  • FcR Fc receptors
  • the anti-CD73 antibodies of the present disclosure, and/or variants or mutants thereof may be in the following forms: Fab, Fab', F(ab')2, single chain Fv (scFv), Fv fragment; diabody and linear antibody.
  • the anti-CD73 antibodies and/or variants or mutants thereof of the present disclosure may be multispecific antibodies that bind to CD73 but also bind to one or more other mutants thereof and inhibit one or more of its functions. , And/or variants or mutants thereof.
  • anti-CD73 antibodies of the present disclosure, and/or variants or mutants thereof can be combined with therapeutic agents (for example, cytotoxic agents, radioisotopes, and chemotherapeutic agents) or markers for detecting CD73 in patient samples or in vivo by imaging. (E.g., radioisotopes, fluorescent dyes, and enzymes) for conjugation.
  • therapeutic agents for example, cytotoxic agents, radioisotopes, and chemotherapeutic agents
  • markers for detecting CD73 in patient samples or in vivo by imaging E.g., radioisotopes, fluorescent dyes, and enzymes
  • Other modifications are also provided herein, including conjugation of toxins with anti-CD73 antibodies, and/or variants or mutants thereof.
  • the anti-CD73 antibodies of the present invention, and/or variants or mutants thereof can be used in the therapies described herein and can be used in patient samples (e.g., via FACS, immunohistochemistry (IHC), ELISA assay) or patients CD73 protein in the detection.
  • Monoclonal antibodies can be prepared, for example, using the hybridoma method (as described in Kohler G. et al., Nature [Nature], 174(5):2453-2455 (2005)) or can be prepared by recombinant DNA methods (U.S. Patent No. 4,816,567) Or it can be produced by the method described herein in the examples below.
  • hamsters, mice, or other suitable host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or can produce antibodies that specifically bind to the immunizing agent.
  • lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include a polypeptide or a fusion protein of a protein of interest or a protein-containing composition.
  • a polypeptide or a fusion protein of a protein of interest or a protein-containing composition Generally, if human-derived cells are desired, peripheral blood lymphocytes ("PBL") are used, or if non-human mammalian origin is desired, spleen cells or lymph node cells are used. Then use a suitable fusion agent (such as polyethylene glycol) to fuse lymphocytes with immortalized cell lines to form hybridoma cells (Monoclonal Antibodies: principles and practice [Monoclonal Antibodies: Principles and Practice], James W. Goding, New York : Academic Press [Academic Press Company], 1986, pages 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Generally, rat or mouse myeloma cell lines are used.
  • Hybridoma cells can be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cell lacks the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium of the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), these substances prevent HGPRT The growth of defective cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred immortalized cell lines are those that fuse efficiently, support the stable high-level expression of antibodies by selected antibody-producing cells, and are sensitive to culture media, such as HAT media.
  • a more preferred immortalized cell line is a murine myeloma cell line, which can be obtained from: for example, Salk Institute [Salk Institute] Cell Distribution Center [cellular distribution center], San Diego [San Diego], California [California] and American Type Culture Collection[American Type Culture Collection], Manassas[Manassas], Virginia[Virginia]. It also describes the use of human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies (Kozbor D. et al., J. Immunol.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can be determined by, for example, the Scatchard analysis of Munson PJ. et al., Anal. Biochem. [Analytical Biochemistry], 107(1):220-239 (1980).
  • the clone can be subcloned by a limiting dilution procedure and grown by standard methods (Monoclonal Antibodies: principles and practice [Monoclonal Antibodies: Principles and Practice], James W. Goding, New York: Academic Press [Academic Publishing Company], 1986, pages 59-103).
  • Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
  • hybridoma cells can grow as ascites in a mammal.
  • the monoclonal antibodies secreted by subclones can be separated from the culture medium or ascites fluid by conventional immunoglobulin purification procedures (such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography) Or purification.
  • immunoglobulin purification procedures such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography
  • Monoclonal antibodies can also be prepared by recombinant DNA methods known in the art and/or later developed. Using conventional procedures (for example, by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies), the DNA encoding the monoclonal antibodies provided herein can be easily isolated And sequencing. The hybridoma cells provided herein are used as a preferred source of such DNA.
  • the DNA is placed in expression vectors, and these vectors are then transfected into host cells that do not otherwise produce immunoglobulins (such as ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) in order to The synthesis of monoclonal antibodies is obtained in recombinant host cells. It is also possible, for example, by replacing the coding sequences of human heavy and light chain constant domains with homologous murine sequences (for example, Morrison et al., supra), or by covalently binding all or part of the coding sequence of a non-immunoglobulin polypeptide to Immunoglobulin coding sequences to modify DNA. This non-immunoglobulin polypeptide can replace the constant domain of the antibody provided herein, or can replace the variable domain of an antigen binding site of the antibody provided herein to produce a chimeric bivalent antibody.
  • immunoglobulins such as ape COS cells, Chinese hamster ovary (CHO)
  • the antibody is a monovalent antibody.
  • Methods for preparing monovalent antibodies are known in the art. For example, one method involves the recombinant expression of immunoglobulin light chains and modified heavy chains.
  • the heavy chain is usually truncated at any point in the Fc region to prevent heavy chain cross-linking.
  • the related cysteine residue is substituted with another amino acid residue or deleted to prevent cross-linking.
  • the anti-CD73 antibody of the present invention and/or a variant or mutant thereof is a human antibody. They can also be humanized antibodies.
  • the humanized form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin, an immunoglobulin chain, or a fragment (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding sub-sequences of antibodies).
  • Humanized antibodies include human immunoglobulins (acceptor antibodies), in which the residues from the acceptor CDR are derived from non-human species (donor antibodies) (such as mice, rats with the desired specificity, affinity, and ability). Mouse or rabbit) residues in the CDR.
  • the Fv framework residues of the human immunoglobulin can be replaced by corresponding non-human residues.
  • Humanized antibodies can also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulin, and all or substantially All FR regions are those of the consensus sequence of human immunoglobulins.
  • the humanized antibody also preferably comprises at least a part of an immunoglobulin constant region (Fc), typically at least a part of a human immunoglobulin constant region (for example, Jones PT.
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced from a source that is non-human. These non-human amino acid residues are often referred to as "import” residues, and they are typically taken from an “import” variable domain. According to one embodiment, basically following the method of Winter and colleagues, humanization is carried out by substituting rodent CDR or CDR sequences for the corresponding sequences of human antibodies (Jones PT. et al., Nature [Nature], 321 (6069) :522-525(1986); Riechmann L. et al., Nature[Natural],332(6162):323-327(1988); Verhoeyen M.
  • humanized antibody refers to an antibody in which a region substantially smaller than a complete human variable domain is replaced with a corresponding sequence from a non-human species.
  • humanized antibodies are typically antibodies in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.
  • human antibodies can be generated.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • JH antibody heavy chain joining region
  • chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
  • the transfer of human germline immunoglobulin gene arrangement to such germline mutant mice will result in the production of human antibodies after antigen challenge. See, for example, Jakobovits A. et al., Proc. Natl. Acad. Sci.
  • human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals, such as mice that have partially or completely inactivated endogenous immunoglobulin genes. After challenge, the production of human antibodies was observed, which was closely similar to that seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire.
  • This method is known in the art, for example, see Marks JD. et al., Biotechnology [Biotechnology], 10(7): 779-783 (1992); Lonberg N. et al., Nature [Natural], 368( 6474):856-859(1994); Morrison SL.,Nature[Nature],368(6474):812-813(1994); Fishwild DM. et al., Nat.
  • phage display technology can be used to obtain variable (V) structures from immunoglobulins from unimmunized donors.
  • the domain gene bank produces human antibodies and antibody fragments in vitro.
  • the antibody V domain sequence is cloned in frame into the primary or secondary coat protein gene of filamentous bacteriophage (such as M13 or fd), and displayed as a functional antibody on the surface of the phage particle Fragment.
  • Phage display can be performed in a variety of formats, for example, as described in the Examples section below or as, for example, Johnson KS.
  • V gene segments can be used for phage display.
  • Clackson T. et al. Nature[Nature], 352(6336):624-628 (1991) isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice .
  • a library of V genes from unimmunized human donors can be constructed, and antibodies against multiple antigens (including autoantigens) can be isolated basically according to the technique described below: Marks JD. et al., J. Mol. Biol.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries. Hoogenboom HR. et al., J.Mol.Biol.[Molecular Biology Journal],227(2):381-388(1991); Marks JD. et al.,J.Mol.Biol.[Molecular Biology Journal], 222(3):581-597 (1991).
  • the technology of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Monoclonal Antibodies and Cancer Therapy [monoclonal antibodies and cancer therapy], Cole et al., Alan Liss, Inc. [Alan Liss Co., Ltd.], 1985, p. 77, and Boerner P. et al., J. Immunol. [Journal of Immunology], 147(1): 86-95 (1991)).
  • Multispecific antibodies are monoclonal antibodies with binding specificities for two or more different antigens, preferably human antibodies or humanized antibodies (for example, bispecific antibodies with binding specificities for at least two antigens).
  • one of the binding specificities may be for the a5-1 protein, and the other may be for any other antigen.
  • the other antigens are cell surface proteins or receptors or receptor subunits.
  • the cell surface protein may be a natural killer (NK) cell receptor. Therefore, according to one embodiment, the bispecific antibody of the present invention can bind to both CD73 and, for example, a second cell surface receptor.
  • bispecific antibodies Suitable methods for preparing bispecific antibodies are known in the art.
  • the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities (Milstein C et al., Nature [Natural], 305 ( 5934):537-540 (1983)). Due to the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished through an affinity chromatography step. Similar methods are disclosed in the art, for example in WO93/08829 and Traunecker A. et al., EMBO J. [Journal of the European Society of Molecular Biology], 10(12): 3655-3659 (1991).
  • An antibody variable domain (antibody-antigen binding site) with the desired binding specificity can be fused to an immunoglobulin constant domain sequence.
  • the fusion is preferably with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. It is preferred that the first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one of these fusions.
  • the DNA encoding the immunoglobulin heavy chain fusion and (if necessary) the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism.
  • a leucine zipper is used to generate bispecific antibodies (Kostelny SA. et al., J. Immunol. [Journal of Immunology], 148(5): 1547-1553 (1992)).
  • the leucine zipper peptides from Fos and Jun proteins were linked to the Fab' parts of two different antibodies by gene fusion.
  • the antibody homodimer is reduced in the hinge region to form a monomer, and then oxidized to form an antibody heterodimer. This method can also be used to produce antibody homodimers.
  • trispecific antibodies can be prepared (Tutt A. et al., J. Immunol. [Journal of Immunology], 147 (1) 60-69 (1991)).
  • Heteroconjugate antibodies consist of two covalently linked antibodies.
  • such antibodies have been proposed to target immune system cells to unwanted cells and used to treat HIV infection. It is contemplated that these antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents.
  • an immunotoxin can be constructed using a disulfide bond exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those known in the art.
  • the antibodies provided herein in terms of effector function to enhance, for example, the effectiveness of the antibodies in the treatment of cancer.
  • one or more cysteine residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region.
  • the homodimeric antibody thus generated may have improved internalization capabilities and/or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). See Caron PC. et al., J.Exp.Med.[Journal of Experimental Medicine],176(4):1191-1195(1992) and Shopes B.,J.Immunol.[Journal of Immunology],148(9): 2918-2922 (1992).
  • heterobifunctional crosslinkers known in the art to prepare homodimeric antibodies with enhanced anti-tumor activity.
  • the antibody can be engineered to have dual Fc regions so that it can have enhanced complement lysis and ADCC capabilities. See, for example, Stevenson GT. et al., Anticancer Drug Des. [Anticancer Drug Design], 3(4):219-230 (1989).
  • the antibody of the invention has at least one altered effector function selected from the group consisting of ADCC, CDC, and improved FcRn binding compared to natural IgG or parent antibodies. Examples of several useful specific mutations are described in, for example, Shields RL. et al., J. Biol. Chem. [Journal of Biological Chemistry], 276(6): 6591-6604 (2001); Presta, LG., etc. Human, Biochem. Soc. Trans. [Report by the Society of Biochemistry] 30(4): 487-490 (2002); and WO00/42072.
  • the Fc receptor mutation is a substitution at at least one position selected from the group consisting of: 238, 239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439, wherein the numbering of residues in the Fc region is based on the EU numbering system.
  • the Fc receptor mutation is a D265A substitution.
  • the Fc receptor mutation is a D
  • the present invention also relates to a compound conjugated with a cytotoxic agent (such as a chemotherapeutic agent), a toxin (for example, an enzymatically active toxin or fragment thereof of bacterial, fungal, plant or animal origin), or a radioisotope (ie, a radioconjugate) Immunoconjugates of antibodies, or variants or mutants thereof.
  • a cytotoxic agent such as a chemotherapeutic agent
  • a toxin for example, an enzymatically active toxin or fragment thereof of bacterial, fungal, plant or animal origin
  • a radioisotope ie, a radioconjugate
  • Enzyme-active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, acacia somatin A Chain, Capsule Lotus Root Toxin A Chain, Alpha-Cytotoxin, Tung Oil Protein, Carnation Toxin, Phytolacaamericana Protein (PAPI, PAPII and PAP-S), Momordicacharantia Inhibitor, Jatropha toxin, crotonin, sapaonaria officinalis inhibitor, multiflora toxin, mitogellin, restrictocin, phenomycin, sapaonaria officinalis Enomycin and tricothecenes.
  • diphtheria A chain non-binding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas a
  • radionuclides can be used to produce radioconjugated antibodies. Examples include 212 Bi, 131 I, 131 In, 90 Y and 186 Re. Useful Exemplary chemotherapeutic agents that generate such immunoconjugates include those described elsewhere herein.
  • the anti-CD73 antibodies of the present invention, and/or variants or mutants thereof are conjugated to the maytansinoid DM1.
  • bifunctional protein coupling agents for example, N-succinimidyl-3-(2-pyridinedimercapto)propionate (SPDP), iminosulfane (IT), imidate double Functional derivatives (such as dimethyl adipimidate HCl), active esters (such as suberic acid disuccinimidyl ester), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis( Azide benzoyl) hexamethylene diamine), double nitrogen derivatives (such as bis-(p-diazobenzoyl)-ethylene diamine), diisocyanate (such as tolylene 2,6-diisocyanate), And bi-reactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) to prepare antibody and cytotoxic agent conjugates.
  • SPDP N-succinimidyl-3-(2-pyridinedimercapto)propionate
  • I iminosulfane
  • the ricin immunotoxin can be prepared as described in Vitetta ES. et al., Science [Science], 238(4830): 1098-1104 (1987).
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine-pentaacetic acid (MX-DTPA) is an exemplary chelate for conjugating radionucleotides to antibodies. mixture. See, WO 94/11026.
  • the anti-CD73 antibodies of the present invention, and/or variants or mutants thereof can be conjugated with a "receptor” (such as streptavidin) for tumor pre-targeting, wherein The patient is administered an antibody-receptor conjugate, followed by a scavenger to remove unbound conjugate from the circulation, and then a "ligand” conjugated to a cytotoxic agent (e.g., radionucleotide) (e.g., Avidin).
  • a cytotoxic agent e.g., radionucleotide
  • Avidin e.g., Avidin
  • Covalent modifications of anti-CD73 antibodies, variants, mutants, and fragments thereof are included within the scope of the present disclosure.
  • One type of covalent modification involves reacting the target amino acid residue of the polypeptide with an organic derivatizing agent capable of reacting with a selected side chain or N-terminal or C-terminal residue of the polypeptide.
  • Derivatization with bifunctional agents is useful, for example, as a method for cross-linking a polypeptide with a water-insoluble support matrix or surface for purification of antibodies, and vice versa.
  • crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide ester (for example, with 4-azidosalicylic acid) Acid esters), homobifunctional imidates (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl-propionate)), bifunctional maleimides (Such as bis-N-maleimido-1,8-octane) and agents (such as methyl-3-[(p-azidophenyl)-dithio]propionimidate).
  • Another type of covalent modification of the polypeptide includes linking the polypeptide to a variety of non-toxic substances in a manner known in the art (e.g., listed in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337)
  • protein polymers such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene.
  • a chimeric molecule comprising a polypeptide fused with another heterologous polypeptide or amino acid sequence (for example, an immunoadhesion protein or a peptibody), the anti-CD73 antibody of the present invention, and/or a variant or a mutant thereof Can also be modified.
  • another heterologous polypeptide or amino acid sequence for example, an immunoadhesion protein or a peptibody
  • this chimeric molecule comprises a fusion of a polypeptide and a protein transduction domain that targets the polypeptide for delivery to various tissues, and more specifically crosses the cerebral blood barrier, where
  • the protein transduction domain of the human immunodeficiency virus TAT protein is used (Schwarze SR. et al., Science 285(5433):1569-1572 (1999)).
  • such chimeric molecules comprise a fusion of a polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind.
  • Epitope tags are usually located at the amino or carboxyl end of the polypeptide. The presence of such polypeptides in the form of epitope tags can be detected using antibodies against the tag polypeptides.
  • the provision of an epitope tag makes it easy for polypeptides to be purified by affinity purification using anti-tag antibodies or another type of affinity matrix that binds the epitope tag.
  • a variety of tag polypeptides and their respective antibodies are known in the art.
  • Examples include poly-His or poly-His-gly tags; influenza HA tag polypeptide and its antibody 12CA5 (Field J. et al., Mol. Cell. Biol. [ Journal of Molecular Cell Biology], 8(5): 2159-2165 (1988)); c-myc tag and its 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies (Evan GI. et al., Mol. Cell. Biol [Journal of Molecular Cell Biology], 5(12):3610-3616(1985)); and herpes simplex virus glycoprotein D (gD) label and its antibody (Paborsky LR. et al., Protein Eng. [protein engineering] , 3(6):547-553(1990)).
  • influenza HA tag polypeptide and its antibody 12CA5 Field J. et al., Mol. Cell. Biol. [ Journal of Molecular Cell Biology], 8(5): 2159-2165 (1988)
  • tag peptides include Flag peptides (Hopp TP. et al., Bio/Technology [Biotechnology], 6:1204-1210 (1988)); KT3 epitope peptides (Martin GA. et al., Science [Science], 255 (5041) ):192-194(1992)); ⁇ -tubulin epitope peptide (Skinner MP. et al., J.Biol.Chem.[Journal of Biological Chemistry],266(9):15163-15166(1991)); And T7 gene 10 protein peptide tag (Lutz-Freyermuth C. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 87(16): 6393-6397 (1990)).
  • Flag peptides Hopp TP. et al., Bio/Technology [Biotechnology], 6:1204-1210 (1988)
  • KT3 epitope peptides
  • the chimeric molecule may comprise a fusion of a polypeptide with an immunoglobulin or a specific region of an immunoglobulin.
  • a bivalent form such as "immunoadhesion proteins”
  • fusions may be fusions to the Fc region of IgG molecules.
  • the Ig fusion of the present invention includes a polypeptide that contains approximately or only human residues 94-243, residues 33-53, or residues 33-52 to replace at least one variable region in an Ig molecule.
  • the immunoglobulin fusion comprises the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of an IgG1 molecule.
  • the production of immunoglobulin fusions is known in the art, see, for example, U.S. Patent No. 5,428,130.
  • the anti-CD73 antibodies of the present invention, and/or variants or mutants thereof can also be formulated as immunoliposomes.
  • the antibody-containing liposomes are prepared by methods known in the art, as described below: Epstein DA. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 82(11): 3688- 3692 (1985); and Hwang KJ. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 77(7): 4030-4034 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. US Patent No. 5,013,556 discloses liposomes with enhanced circulation time.
  • Particularly useful liposomes can be produced from a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE) by a reverse phase evaporation method.
  • the liposomes are extruded through a filter with a defined pore size to produce liposomes with the desired diameter.
  • the Fab' fragment of the antibody of the present disclosure can be conjugated with liposomes via a disulfide bond exchange reaction, such as Martin FJ. et al., J. Biol. Chem. [Journal of Biological Chemistry], 257(1): 286-288 ( 1982).
  • the liposomes also optionally contain anti-tumor agents, growth inhibitors or chemotherapeutic agents (such as doxorubicin). See, Gabizon A. et al., J. Natl. Cancer Inst. [Journal of the National Cancer Institute], 81(19): 1484-1488 (1989).
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical compositions provided herein can be administered to subjects (eg, mammals such as humans) to treat CD73 expression, CD73 over Diseases, disorders, or clinical conditions related to the expression or abnormal function of CD73.
  • cancers related to the expression of CD73 may be, but are not limited to, such as lymphoma, kidney cancer, lung cancer, colon cancer, liver cancer, gastric cancer, primary exudative lymphoma, osteosarcoma, and non-Hodgkin's lymphoma.
  • Additional examples of diseases, disorders or clinical conditions associated with CD73 expression may be fibrosis, including idiopathic pulmonary fibrosis.
  • the present invention provides the anti-CD73 antibodies and/or variants/mutants (or fragments thereof) described herein for the manufacture of drugs for the treatment of cancer or fibrosis in a subject.
  • the subject to be treated is a mammal (eg, human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.).
  • the subject is a human.
  • the subject is a clinical patient, clinical trial volunteer, experimental animal, and the like. In certain embodiments, it is suspected that the subject has cancer or fibrosis or is at risk.
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal such as a human) to treat Cancer recurrence or recurrence of cancers associated with CD73 expression.
  • a subject e.g., a mammal such as a human
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal such as a human) to treat Refractory or resistant cancers associated with CD73 expression.
  • a subject e.g., a mammal such as a human
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal such as a human) to provide For adjuvant therapy of cancers related to CD73 expression.
  • a subject e.g., a mammal such as a human
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal such as a human) to provide For maintenance therapy of cancers related to CD73 expression.
  • a subject e.g., a mammal such as a human
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal such as a human) to treat An aggressive cancer associated with CD73 expression.
  • a subject e.g., a mammal such as a human
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal such as a human) to treat Non-invasive cancer associated with CD73 expression.
  • a subject e.g., a mammal such as a human
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal such as a human) to increase Progression-free survival of subjects diagnosed with cancer associated with CD73 expression.
  • a subject e.g., a mammal such as a human
  • the cancers associated with CD73 expression that can be treated using the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof can be multidrug resistant (MDR) or drug sensitive.
  • MDR multidrug resistant
  • cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, hematological cancer, and leukemia.
  • cancers associated with CD73 expression that can be treated with the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof include, but are not limited to: colorectal cancer, ovarian cancer, gastric cancer, gallbladder cancer, Leukemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), prostate cancer, melanoma, breast cancer, triple negative breast cancer (TNBC), bladder cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, glue Hematoma, head and neck cancer, thyroid cancer, B-cell acute lymphoblastic leukemia, medulloblastoma, atypical teratomoid/rhaboid tumor, and oral squamous cell carcinoma.
  • colorectal cancer ovarian cancer
  • gastric cancer gallbladder cancer
  • Leukemia chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), prostate cancer, melanoma, breast cancer,
  • the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof can be used to treat cancers associated with CD73 expression, including but not limited to: blood (or hematopoietic tissue) cancer, bladder cancer, Brain cancer, breast cancer (for example, triple negative breast cancer, TNBC), central and peripheral nervous system cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer (including small cell lung cancer), esophageal cancer, gallbladder cancer , Gastrointestinal cancer, cancer of the urogenital tract, lymphoid lineage lymphoid system hematopoietic tumor, bone marrow lineage hematopoietic tumor, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer, prostate cancer, skin cancer (including squamous cell carcinoma) and thyroid cancer .
  • blood (or hematopoietic tissue) cancer including but not limited to: blood (or hematopoietic tissue) cancer, bladder cancer, Brain cancer, breast cancer (for
  • the cancers associated with CD73 expression that can be treated using the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof can be hematological cancers.
  • the hematological cancer is selected from leukemia, myeloma, or lymphoma, including but not limited to: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML ), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), Hodgkin's lymphoma, classic Hodgkin's lymphoma (cHL), non-Hodgkin's lymphoma, primary mediastinal B-cell lymphoma (NHL PMBCL), non-Hodgkin's lymphoma, multiple myeloma, single mye
  • AML acute mye
  • Exemplary hematopoietic tumors of the lymphoid lineage include, but are not limited to: leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hair cell Lymphoma and Burkitt's lymphoma.
  • Hematopoietic tumors of the myeloid lineage include but are not limited to: acute and chronic myeloid leukemia, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma.
  • the cancers associated with CD73 expression that can be treated using the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof can be cancers of the brain or central nervous system.
  • Tumors of the central and peripheral nervous system include but are not limited to: astrocytoma, neuroblastoma, glioma, and schwannoma.
  • Additional exemplary brain or central nervous system cancers include: glioma, medulloblastoma, primitive neuroectodermal tumor (PNET), acoustic neuroma, glioma, meningioma, pituitary adenoma, schwannoma, CNS Lymphoma, primitive neuroectodermal tumor, craniopharyngioma, chordoma, medulloblastoma, central neuroblastoma, central neurocytoma, pineoblastoma, pineoblastoma, SARS Type teratomoid rhabdoid tumor, chondrosarcoma, chondroma, choroid plexus carcinoma, choroid plexus papilloma, craniopharyngioma, embryonic dysplastic neuroepithelioma, gangliocytoma, germ cell tumor , Hemangioblastoma, Hemangiopericytoma and metastatic brain tumors.
  • PNET
  • the glioma is selected from the group consisting of ependymoma, astrocytoma, oligodendroglioma, and oligoastrocytoma.
  • the glioma is selected from juvenile pilocytic astrocytoma, subependymal giant cell astrocytoma, ganglion glioma, subependymoma, xanthoma multiforme Pleomorphic xanthoastrocytoma, anaplastic astrocytoma, glioblastoma multiforme, brainstem glioma, oligodendroglioma, ependymoma, oligodendrocyte Tumor, cerebellar astrocytoma, connective tissue hyperplastic pediatric astrocytoma, subependymal giant cell astrocytoma, diffuse astrocytoma, mixed glioma, optic
  • exemplary tumors associated with CD73 expression that can be treated with the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof include, but are not limited to: squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, Sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial lung cancer, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms tumor, cervical Cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, osteosarcoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma , Acoustic neuroma, oligodendroglioma, meningioma,
  • Targeting cancer such as melanoma, non-small cell lung cancer (NSCLC), head and neck cancer, urothelial cancer, breast cancer (such as triple negative breast cancer, TNBC), gastric cancer, classic Hodgkin’s lymphoma (cHL), non- Hodgkin’s lymphoma Primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small-cell lung cancer), esophageal cancer, nasopharyngeal cancer (NPC), biliary tract cancer, colorectal cancer Cancer, cervical cancer, thyroid cancer) or any other diseases that exhibit abnormal CD73 function and the clinical description of these diseases are known in the art. Such methods include, but are not limited to, for example, immunohistochemistry, PCR, fluorescence in situ hybridization (FISH). Additional details regarding diagnostic methods for abnormal CD activity or expression are described in the art.
  • FISH fluorescence in situ hybridization
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be administered by any suitable route, including, for example, intravenous, intramuscular, or subcutaneous.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be administered by parenteral administration.
  • Parenteral refers to intravenous, subcutaneous and intramuscular administration.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be combined with at least one agent (such as a second, third, or fourth Medicament) administered in combination.
  • the at least one agent may be any suitable agent known to treat cancer or fibrosis (including idiopathic pulmonary fibrosis).
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be administered in combination with at least one agent for cancer treatment.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be combined with at least one agent for cancer treatment (such as oxa Liplatin, leucovorin, fluorouracil (5-FU), gemcitabine, paclitaxel (including protein-bound paclitaxel (such as but not limited to nanoparticle albumin-bound paclitaxel, which is sometimes referred to as "albumin-bound paclitaxel ( nab-paclitaxel)”), or a combination thereof) administered in combination.
  • at least one agent for cancer treatment such as oxa Liplatin, leucovorin, fluorouracil (5-FU), gemcitabine, paclitaxel (including protein-bound paclitaxel (such as but not limited to nanop
  • At least one agent for cancer treatment include, but are not limited to: chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, anti-angiogenic agents, cytokines, hormone therapy, gene therapy, biological therapy, and radiation therapy.
  • at least one agent known to treat cancer may be a known cytostatic or cytotoxic or anticancer agent (such as an antitumor agent), growth inhibitor, cytotoxic agent, or chemotherapeutic agent (such as docetaxel).
  • anti-CD73 antibodies and/or variants/mutants are conjugated with additional agents.
  • the at least one medicament includes uracil mustard, nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, piperobromide ( pipobroman), triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozotocin (streptozotocin), dacarbazine, temozolomide, thiotepa, altretamine, methotrexate, 5-fluorouracil, fluorouridine, arabinosporin, 6 -Mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, bortezomib, vinblastine, vincristine, vinorelbine, vindesine , Bleomycin (bleomycin), actinomycin D, daunorubic
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be administered in combination with at least one agent, the at least one agent being
  • alkylating agents such as Thiotepa and CYTOXAN(R) cyclophosphamide
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines aziridines, such as benzodopa, carboquone, meturedopa and uredopa
  • ethyleneimine and methylamelamines including hexamethylmelamine , Tratamide, Trietylenephosphoramide, Triethiylenethiophosphoramide and Trimethylolomelamine
  • Polyacetyls acetogenins) (especially bullatacin (bullatacin) ) And bullatacinone (bullatacinone); ⁇ -9-tetrahydrocannabinol
  • dynemicin including dynemicin A; esperamicin; and neocarzinostatin chromophore and related colors Protein enediyne antibiotic chromophore), aclacinomysins, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, Carabicin, carmine, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazonium -5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN(R), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrololino- Doxorubicin, doxorubicin HC
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be administered in combination with at least one agent, the at least one agent being Angiogenesis inhibitors, such as but not limited to: angiostatin (plasminogen fragment); anti-angiogenic antithrombin III; Angiozyme; ABT-627; Bay 12-9566; flumioxamide (Benefin); Bevar Monoclonal antibody; BMS-275291; Cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; Comprestin (combretastatin) A-4; Endothelial inhibitor (collagen XVIII fragment); Fibronectin fragment; gro- ⁇ ; halofuginone; heparinases; heparin polyhexose fragment; HMV833; human chorionic gonadotropin (hCG); IM-862; interferon ⁇ / ⁇ /
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be administered in combination with at least one agent, which is selected from From DNA interacting agents, such as cisplatin or doxorubicin; topoisomerase II inhibitors, such as etoposide; topoisomerase I inhibitors, such as CPT-11 or topotecan; tubulin interactions Agents, such as naturally occurring or synthetic paclitaxel, docetaxel, or epothilone (e.g.
  • hormone agents such as tamoxifen; thymidilate synthase inhibitors, such as 5- Fluorouracil; and antimetabolites, such as methotrexate, other tyrosine kinase inhibitors, such as Iressa and OSI-774; angiogenesis inhibitors; BTK inhibitors, SYK inhibitors, ITK inhibitors, PI3 -Kinase inhibitors, FLT3 inhibitors, EGF inhibitors; PAK inhibitors, VEGF inhibitors; CDK inhibitors; SRC inhibitors; c-Kit inhibitors; Her1/2 inhibitors and monoclonal antibodies against growth factor receptors , Such as Erbitux (EGF) and Herceptin (Her2) and other protein kinase modulators.
  • differentiation agents such as ATRA, EZH2 inhibitors, DNMT inhibitors, corticosteroids, IDH1 inhibitors, IDH2 inhibitors and vitamin C.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or pharmaceutical compositions provided herein can be combined with small molecules that enhance DNA damage killing in cancer cells (including PARP inhibitor, MDM2 inhibitor, NAMPT inhibitor, and HSP90 inhibitor) are administered in combination.
  • small molecules that enhance DNA damage killing in cancer cells including PARP inhibitor, MDM2 inhibitor, NAMPT inhibitor, and HSP90 inhibitor
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or pharmaceutical compositions provided herein can be combined with one or more immunotherapeutic agents (including antagonists, Antibodies and immunomodulators, including but not limited to: HERCEPTINS, RITUXANS, OVAREX TM , PANOREX@, BEC2, IMC-C225, VITAMIN, CAMPATH@I/H, Smart MI95, LYMPHOCIDE TM , Smart ID10 and ONCOLYM TM , Ritux Xiximab, gemtuzumab (gemtuzumab) or trastuzumab (trastuzumab) were given in combination.
  • immunotherapeutic agents including antagonists, Antibodies and immunomodulators, including but not limited to: HERCEPTINS, RITUXANS, OVAREX TM , PANOREX@, BEC2, IMC-C225, VITAMIN, CAMPATH@I/H, Smart MI95, LYMPHO
  • the immunotherapeutic agent may target PD-1, PD-L1, PD-L2, CTLA4, LAG3, TIGIT, TIM3, CEACAM-1, Galectin-1, Galectin-9 , BLTA, CD69, CD113, GPR56, 2B4, CD48, PD1H, LAIR1, TIM-1, TIM-4, VISTA, GARP, CD73, CD39, A2AR, B7-1, B7-2, 4-1BBL, CD137, CD28 , CD28H, GITR, GITRL, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3, or a combination of antibodies against such targets.
  • the immunotherapeutic agent may be targeted to PD-1, PD-L1, PD-L2, CTLA4, LAG3, TIGIT, TIM3, CEACAM-1, galectin-1, galectin-9 , BLTA, CD69, CD113, GPR56, 2B4, CD48, PD1H, LAIR1, TIM-1, TIM-4, VISTA, GARP, CD73, CD39, A2AR, or a combination of antagonists; targeting B7-1, B7- 2.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or pharmaceutical compositions provided herein can be combined with cell surface molecules on targeted immune cells or cancer cells ( Including but not limited to: EGFR, HER2, CD38, mesothelin, CD33, CD37, CD19, CD20, CD3, CD123, CD70, BAFFR, CD4, CD8, CD56, CD38) antibodies or combinations of such antibodies for combined administration .
  • targeted immune cells or cancer cells Including but not limited to: EGFR, HER2, CD38, mesothelin, CD33, CD37, CD19, CD20, CD3, CD123, CD70, BAFFR, CD4, CD8, CD56, CD38
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be combined with targeted immune cells or cell surface molecules on cancer cells (including But not limited to: EGFR, HER2) antibodies or a combination of such antibodies for combined administration.
  • targeted immune cells or cell surface molecules on cancer cells including But not limited to: EGFR, HER2
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be combined with at least one chemotherapeutic agent (e.g., alkylating agent, anti- Metabolites, platinate agents, paclitaxel, antihormones, topoisomerase inhibitors, tubulin agents, signaling inhibitors (e.g., kinase inhibitors) and other chemotherapeutic agents are administered in combination.
  • chemotherapeutic agents are chemical compounds useful in cancer treatment.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide Alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone, Meturedopa and uredopa; ethyleneimine and methylamelamines, including hexamethylmelamine, tritamide, trietylenephosphoramide, triethylene sulfide Triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); ⁇ -9-tetrahydrocannabinol (Dronnabinol, ); ⁇ -Lappaquinone; Lapamol; colchicine; betulinic acid; camptothecin (including synthetic analog topotecan CPT-11 (irinotecan, ), acetylcamptothecin (ace
  • dynemicin including dynemicin A; esperamicin; and new cancer Neocarzinostatin chromophore (neocarzinostatin chromophore) and related chromophore enediyne antibiotic chromophore), aclacinomysins, actinomycin, authramycin, azaserine ), bleomycin, actinomycin C, carabicin (carabicin), carcinomycin, carzinop hilin), chromomycin (chromomycinis), actinomycin D, daunorubicin, detorubicin (detorubicin), 6-diazo-5-oxo-L-norleucine, doxorubicin (include Morpholino-doxorubicin, cyanomorpholino-doxorubicin (include Morpholino-doxorubicin, cyanomorpholino-doxorubicin,
  • chemotherapeutic agents include “antihormonal agents” or “endocrine therapeutic agents” used to modulate, reduce, block or inhibit the action of hormones that can promote cancer growth. They may be hormones themselves, including but not limited to: anti-estrogens with mixed agonist/antagonist spectrum, including tamoxifen 4-hydroxy tamoxifen, toremifene Idoxifene, droloxifene, raloxifene Travoxifene, keoxifene and selective estrogen receptor modulators (SERM) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant And EM800 (such agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover and/or inhibit ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestin ( formestane) and exemestane And non-steroidal aromatase inhibitors such as anastrozole Letrozole And amino
  • taxane is a chemotherapeutic agent that has the function of inhibiting the depolymerization of microtubules.
  • examples include paclitaxel Paclitaxel (ABRAXANE TM ) albumin engineered nanoparticle formulation, and docetaxel
  • the preferred taxane is paclitaxel.
  • EGFR inhibitor refers to a compound that binds to or otherwise directly interacts with EGFR and prevents or reduces its signaling activity, and may alternatively be referred to as "EGFR antagonist".
  • EGFR antagonist examples include antibodies and small molecules that bind to EGFR.
  • antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Patent No.
  • EMD7200 humanized EGFR antibody (EMD/Merck) that targets EGFR and competes with both EGF and TGF- ⁇ for EGFR binding
  • Human EGFR antibody HuMax-EGFR (GenMab company); known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and is in the US patent number
  • the anti-EGFR antibody can be conjugated with a cytotoxic agent, thereby generating an immunoconjugate (see, for example, EP659,439A2, Merck Patent GmbH).
  • EGFR antagonists include small molecules, such as the compounds described in the following: U.S.
  • EGFR antagonists include OSI-774 (CP-358774, Erlotinib, Genentech/OSI Pharmaceuticals); PD 183805 (CI 1033, 2-acrylamide, N-[4-[(3-chloro-4--fluorophenyl)amino]-7- [3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (Iressa ( IRESSA) J) 4-(3′-chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca) ; ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382(N8-(3-chloro-4-fluoro-benzene Yl)-N2-(1
  • Tyrosine kinase inhibitors are molecules that inhibit the tyrosine kinase activity of tyrosine kinases such as HER receptors.
  • examples of such inhibitors include the EGFR-targeted drugs mentioned in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 (available from Takeda); CP-724,714 (ErbB2 receptor Oral selective inhibitors of human tyrosine kinases (Pfizer and OSI); dual HER inhibitors, such as EKB-569 that preferentially binds to EGFR but inhibits cells overexpressing both HER2 and EGFR (available from Wyeth); Lapatinib (GW572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis Company (Novartis); pan-HER inhibitors, such as canertinib (CI
  • tyrosine Acid phosphorylation inhibitors tryphostins
  • US Patent No. 5,804,396 tyrosine Acid phosphorylation inhibitors
  • ZD6474 Astra Zeneca
  • PTK-787 Novartis/Schering AG
  • pan-HER inhibitor Such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate (Gleevac); Novartis (Novartis)); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone) ); or as described in any of the following patent publications: U.S.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be combined with actinomycin D, capecitabine, carboplatin , Cisplatin, Colchicine, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide, 5-Fluorouracil, Gemcitabine, Melphalan, Methotrexate, Silk Schitomycin C, mitoxantrone, paclitaxel, thalidomide, topotecan, vinblastine, and vincristine are given in combination.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or pharmaceutical compositions provided herein can be combined with one or more additional therapies or clinical interventions (such as Radiation therapy, surgery, chemotherapy and/or targeted therapy) are given in combination.
  • additional therapies or clinical interventions such as Radiation therapy, surgery, chemotherapy and/or targeted therapy
  • the disclosed anti-CD73 antibodies and/or variants (or fragments thereof) and/or the pharmaceutical compositions provided herein are administered in combination with radiation therapy for the disclosed cancers.
  • the anti-CD73 antibodies, variants, mutants or fragments thereof provided herein will be administered in an effective dose, for example, in minimizing toxicity and side effects
  • the dosage is effective to treat the indication. It is generally preferred to use the maximum dose of the pharmacological agent of the present disclosure (alone or in combination with other therapeutic agents), that is, the highest safe dose based on sound medical judgment. However, those of ordinary skill in the art will understand that for medical reasons, psychological reasons, or almost any other reason, patients may adhere to lower doses or tolerable doses.
  • the effective dose may be such that the desired response is to inhibit the progression of the disease or disorder, such as the progression of cancer.
  • the desired response to the treatment of a disease or condition can also be to delay the onset or even prevent the onset of the disease or condition.
  • the toxicity and therapeutic efficacy of the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein can be determined by standard drug procedures in cell cultures or experimental animals, for example to determine LD 50 (50% of the dose lethal population) and the ED 50 (the dose therapeutically effective in 50% of the patient population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50.
  • Compounds that exhibit a large therapeutic index can be desired.
  • anti-CD73 antibodies and/or variants or mutants (or fragments thereof) that exhibit toxic side effects and/or the pharmaceutical compositions provided herein can be used, careful design should be made to target such compounds to the affected The delivery system to the site of the tissue, thereby minimizing potential damage to uninfected cells, and thereby reducing side effects.
  • Dose of anti-CD73 antibodies disclosed and / or variant or mutant thereof (or fragment thereof) and / or pharmaceutical compositions provided herein preferably comprise and ED 50 with little or no toxicity within a range of circulating concentrations.
  • the dosage can vary within this range according to the dosage form used and the route of administration used.
  • the therapeutically effective amount can be assessed initially from cell culture assays. It can be formulated in animal models to achieve a circulating plasma concentration dose range comprises determining the cell culture experiments the IC 50. This information can be used to more accurately determine the useful dose in humans.
  • the level in plasma can be measured, for example, by high performance liquid chromatography.
  • the lower level of proliferation or survival of the contacted cells may indicate that the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) and/or the pharmaceutical compositions provided herein may be effective treatment options in the subject Obstacles.
  • tumor cell lines or malignant cell lines can be used to screen protocols.
  • Many assays known in the art can be used to assess this survival and/or growth; for example, by measuring 3H-thymidine incorporation, by direct cell counting, by detecting known genes such as proto-oncogenes or cell cycle markers
  • Cell proliferation can be measured by changes in transcriptional activity; cell viability can be assessed by trypan blue staining, and differentiation can be visually assessed based on changes in morphology.
  • the disclosed anti-CD73 antibodies and/or variants for therapy can be tested in a suitable animal model system (including but not limited to rats, mice, chickens, cows, monkeys, rabbits, etc.) Or mutants (or fragments thereof) and/or pharmaceutical compositions provided herein.
  • a suitable animal model system including but not limited to rats, mice, chickens, cows, monkeys, rabbits, etc.
  • mutants (or fragments thereof) and/or pharmaceutical compositions provided herein include mice, such as Hann B. et al., Curr. Opin. Cell Biol. [New Perspectives in Cell Biology] 13(6):778-784 (2001) Described, its entire content is incorporated herein by reference.
  • the daily dose can be about 0.1 ⁇ g/kg total body weight to about 100 mg/kg total body weight (e.g., about 5 ⁇ g/kg, about 10 ⁇ g/kg, about 100 ⁇ g/kg, about 500 ⁇ g/kg, about 1 mg/kg, about 50 mg/kg , Or a range defined by any two of the foregoing values), preferably from about 0.3 ⁇ g/kg total body weight to about 10 mg/kg total body weight (for example, about 0.5 ⁇ g/kg, about 1 ⁇ g/kg, about 50 ⁇ g/ kg, about 150 ⁇ g/kg, about 300 ⁇ g/kg, about 750 ⁇ g/kg, about 1.5 mg/kg, about 5 mg/kg, or a range defined by any two of the foregoing values), more preferably from about 1 ⁇ g/kg kg total weight to 1 mg/kg total weight (for example, about 3 ⁇ g/kg, about 15 ⁇ g/kg, about 75 ⁇ g/kg, about 300 ⁇ g/kg, about 900 ⁇ g/
  • compositions containing anti-CD73 antibodies, variants, mutants or fragments thereof can be administered once, twice, three or four times a day.
  • the composition can also be administered less frequently than daily, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, Once every three weeks, once a month, once every two months, once every three months, or once every six months.
  • the composition can also be administered in a sustained-release formulation, for example, in an implant that gradually releases the composition for use over a period of time, and allows the composition to be administered at a lower frequency, such as once a month, every time Give once every 2-6 months, once a year, or even a single dose.
  • Sustained release devices (such as pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) can be administered by injection.
  • the antibody (or fragment thereof) can also be administered, for example, in a sustained release formulation in an implant that gradually releases the composition for use over a period of time, and allows the composition to be administered at a lower frequency, such as every Give once a month, once every 2-6 months, once a year, or even a single dose.
  • Sustained release devices (such as pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) can be administered by injection or surgical implantation in various locations.
  • Cancer treatment can be assessed by, for example, but not limited to, tumor regression, tumor weight or size reduction, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression, and/or activity.
  • Methods to determine the efficacy of therapy can be used, including, for example, measuring the response by radiography.
  • the treatment efficacy is measured as the percentage of tumor growth inhibition (%TGI), calculated using the equation 100-(T/C x 100), where T is the average relative tumor volume of the tumor being treated, and C is The average relative tumor volume of untreated tumors.
  • %TGI is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, About 92%, about 93%, about 94%, about 95%, or more than 95%.
  • the %TGI of the anti-CD73 and/or variants or mutants thereof is the same as or higher than the %TGI of the reference anti-CD73 antibody, for example about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times , About 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 2.1 times, about 2.2 times, about 2.3 times, about 2.4 times, about 2.5 times, about 2.6 times, about 2.7 times (including any range between these values), or about 2.7 times higher than the %TGI of the reference anti-CD73 antibody.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) of the present invention can be prepared into pharmaceutical compositions containing suitable carriers or excipients to make them suitable for administration.
  • suitable carriers or excipients By combining the antibody (or fragment thereof) with the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences) 16th edition, Osol, A. Edit (1980)) mix and obtain a suitable pharmaceutical composition of the antibody in the form of a lyophilized formulation or an aqueous solution.
  • Acceptable carriers, excipients or stabilizers are non-toxic to the receptor at the dose and concentration used, and include buffers (such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; Preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens , Such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine,
  • Exemplary antibody formulations The preparation is described in WO 98/56418, which is expressly incorporated herein by reference.
  • Lyophilized pharmaceutical compositions suitable for subcutaneous administration are described in WO 97/04801. Such lyophilized pharmaceutical compositions can be suitably diluted
  • the dose weight constitutes a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the mammal to be treated herein.
  • the disclosed pharmaceutical composition is suitable for parenteral or intravenous administration.
  • the disclosed pharmaceutical compositions suitable for parenteral administration may be aqueous and non-aqueous formulations that are isotonic with the blood of the intended subject; aqueous and non-aqueous sterile suspensions may include suspension systems, It is designed to target compounds to the blood components of one or more organs.
  • the formulation may be presented in unit-dose or multi-dose sealed containers (e.g., ampoules or vials). Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type previously described.
  • Parenteral and intravenous formulations may include minerals and other materials to make them compatible with the type of injection or delivery system selected.
  • Commonly used pharmaceutically acceptable carriers for parenteral administration include water, suitable oils, saline, aqueous dextrose (glucose) solutions or related sugar solutions, and glycols such as propylene glycol or polyethylene glycol.
  • the solution for parenteral administration preferably contains a water-soluble salt of the active ingredient, a suitable stabilizer and (if necessary) a buffer substance, and antioxidants (such as sodium bisulfite, sodium sulfite or ascorbic acid) alone or in combination are suitable stabilizers Agent. Citrate and sodium EDTA can also be used as carriers.
  • parenteral solutions may contain preservatives such as benzalkonium chloride, methyl paraben or propyl paraben, or chlorobutanol. Suitable pharmaceutical carriers are described in Remington [Remington] cited above.
  • Injectable formulations can be prepared in a conventional form, either as a liquid solution or suspension; as a solid form suitable for dissolution or suspension in a liquid before injection; or as an emulsion.
  • a sterile injectable suspension is formulated using a suitable pharmaceutically acceptable carrier and other optional components as described above according to techniques known in the art.
  • the formulation can also be injected locally to the target site, for example, injection into a specific part of the body to treat cancer or fibrosis is desirable.
  • the advantage of local administration via injection is that it limits or avoids exposure of the entire body to one or more active agents (e.g., inhibitors and/or other therapeutic agents).
  • the term local administration includes regional administration, for example, administration of a formulation directed to a part of the body by delivery to a blood vessel serving the body area.
  • Local delivery can be direct, for example, intratumoral delivery.
  • Local delivery can also be almost direct, that is, intralesional or intraperitoneal, that is, delivered to an area sufficiently close to the tumor or infection site so that the inhibitor exhibits the desired pharmacological activity. Therefore, when local delivery is desired, the pharmaceutical formulation is preferably delivered intralesional, intratumoral or intraperitoneal.
  • the pharmaceutical composition is in unit dosage form.
  • the composition is divided into unit doses containing appropriate amounts of active ingredients.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as lyophilized powder in vials or ampoules.
  • the pharmaceutical composition herein may also contain more than one active compound necessary for the specific indication being treated, preferably those active compounds that have complementary activities without adversely affecting each other.
  • active compounds that have complementary activities without adversely affecting each other.
  • Such molecules are suitably present in combination in amounts effective for the intended purpose.
  • the effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are usually used at the same dosage and route of administration as described herein, or at from about 1% to 99% of the dosages used so far.
  • the active ingredient can also be embedded in microcapsules prepared, for example, by coacervation technology or by interfacial polymerization, for example, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) Or hydroxymethyl cellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in a coarse emulsion.
  • colloidal drug delivery systems e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules
  • hydroxymethyl cellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in a coarse emulsion Such technologies are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Editor (1980).
  • sustained release formulations can be prepared.
  • suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, such as films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide (US Patent No.
  • L- Copolymer of glutamic acid and ethyl-L-glutamic acid non-degradable ethylene-vinyl, degradable lactic acid-glycolic acid copolymer (such as LUPRON DEPOT TM (comprised of lactic acid-glycolic acid copolymer and leucoacetate ) Injectable microspheres composed of Ruilin)) and poly-D-(-)-3-hydroxybutyric acid.
  • polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins in a shorter period of time.
  • the encapsulated antibodies When the encapsulated antibodies remain in the body for a long time, they will denature or aggregate due to exposure to humidity at 37°C, leading to loss of biological activity and possible changes in immunogenicity.
  • a reasonable strategy can be designed according to the involved mechanism to achieve stability. For example, if it is found that the aggregation mechanism is the formation of intermolecular SS bonds through thio-disulfide bond exchange, one can modify sulfhydryl residues, freeze-dry from acidic solutions, control moisture content, use appropriate additives, and develop specific polymers Matrix composition to achieve stabilization.
  • Lipofectins or liposomes can be used to deliver the polypeptides and antibodies (or fragments thereof) or compositions of the present invention into cells.
  • antibody fragments the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • peptide molecules can be designed to retain the ability to bind the target protein sequence.
  • Such peptides can be chemically synthesized and/or produced by recombinant DNA technology. See, for example, Marasco WA. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90(16): 7889-7893 (1993).
  • the active ingredient can also be embedded in microcapsules prepared, for example, by coacervation technology or by interfacial polymerization, for example, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) Or hydroxymethyl cellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in coarse emulsion.
  • colloidal drug delivery systems e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules
  • hydroxymethyl cellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in coarse emulsion e.g., hydroxymethyl cellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in coarse emulsion.
  • sustained release formulations can be prepared.
  • suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody (or fragments thereof) in the form of shaped articles, such as films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide (US Patent No.
  • L- Copolymer of glutamic acid and ethyl-L-glutamic acid non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer (such as LUPRON DEPOT TM (from lactic acid-glycolic acid copolymer and acetic acid) Injectable microspheres composed of Prorelin)) and poly-D-(-)-3-hydroxybutyric acid.
  • polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins in a shorter period of time.
  • the encapsulated antibodies When the encapsulated antibodies remain in the body for a long time, they will denature or aggregate due to exposure to humidity at 37°C, leading to loss of biological activity and possible changes in immunogenicity.
  • a reasonable strategy can be designed according to the involved mechanism to achieve stability. For example, if it is found that the aggregation mechanism is the formation of intermolecular SS bonds through thio-disulfide bond exchange, one can modify sulfhydryl residues, freeze-dry from acidic solutions, control moisture content, use appropriate additives, and develop specific polymers Matrix composition to achieve stabilization.
  • the formulation contains greater than about 0.5 mg/mL, greater than about 1 mg/mL, greater than about 2 mg/mL, greater than about 3 mg/mL, greater than about 4 mg/mL, greater than about 5 mg /mL, higher than about 6mg/mL, higher than about 7mg/mL, higher than about 8mg/mL, higher than about 9mg/mL, higher than about 10mg/mL, higher than about 11mg/mL, higher than about 12mg/mL mL, higher than about 13mg/mL, higher than about 14mg/mL, higher than about 15mg/mL, higher than about 16mg/mL, higher than about 17mg/mL, higher than about 18mg/mL, higher than about 19mg/mL , Higher than about 20 mg/mL, higher than about 21 mg/mL, higher than about 22 mg/mL, higher than about 23 mg/mL, higher than about 24 mg/mL, higher than about
  • the formulation containing the disclosed antibody should have a pH (in the final formulation) that is not similar to the isoelectric point of the antibody or fragment, for example, if the pH of the formulation is 7, then the pI is from 8 to 9 or Higher may be appropriate. Although not wishing to be bound by theory, it is thought that this may ultimately provide a final formulation with improved stability, for example, the antibody or fragment remains in solution.
  • the disclosed pharmaceutical formulations having a pH in the range of 4.0 to 7.0 may include: 1 mg/mL to 200 mg/mL of the antibody according to the present disclosure and 1 to 100 mM buffer, and any Choose one or more of the following: (a) 0.001% to 1% surfactant, (b) 10mM to 500mM stabilizer, (c) 10mM to 500mM stabilizer and 5mM to 500mM tonicity Agent, and/or (d) 5mM to 500mM tonicity agent.
  • Suitable buffers include, but are not limited to, citrate, glycine, phosphate, acetate, succinate, and bicarbonate.
  • a formulation of about pH 6 may contain 1 to 50 mg/mL of antibody, 20 mM L-histidine HCl, 240 mM trehalose, and 0.02% polysorbate 20.
  • a formulation of approximately pH 5.5 may contain 1 to 50 mg/mL of antibody, 20 mM citrate buffer, 240 mM sucrose, 20 mM arginine, and 0.02% polysorbate 20.
  • the anti-CD73 antibody and/or variants or mutants thereof are formulated to include citrate, NaCl, acetate, amber Acid salt, glycine, polysorbate 80 (Tween 80) or any combination of the foregoing buffers.
  • anti-CD73 antibodies and/or variants thereof e.g., at higher than about 0.5 mg/mL, higher than about 1 mg/mL, higher than about 5 mg/mL, higher than about 10 mg/mL, higher It is formulated at a concentration of about 15 mg/mL, higher than about 20 mg/mL, or higher than about 25 mg/mL (including any range between these values) in a buffer containing about 100 mM to about 150 mM glycine.
  • the anti-CD73 antibody and/or variants thereof are formulated in a buffer containing about 50 mM to about 100 mM NaCl.
  • the anti-CD73 antibody and/or variants or mutants thereof (e.g., at more than about 0.5 mg/mL, greater than about 1 mg/mL, greater than about 5 mg/mL, greater than about 10 mg /mL, greater than about 15mg/mL, greater than about 20mg/mL, or greater than about 25mg/mL (including any range between these values) is formulated in a buffer containing about 10mM to about 50mM acetate In the liquid. In certain embodiments, the anti-CD73 antibody and/or variants thereof are formulated in a buffer containing about 10 mM to about 50 mM succinate.
  • the anti-CD73 antibody and/or variants or mutants thereof (e.g., at more than about 0.5 mg/mL, greater than about 1 mg/mL, greater than about 5 mg/mL, greater than about 10 mg /mL, greater than about 15mg/mL, greater than about 20mg/mL, or greater than about 25mg/mL (including any range between these values) is formulated to contain about 0.005% to about 0.02% polysorbate Ester 80 in the buffer.
  • the anti-CD73 antibody and/or variants or mutants thereof are formulated in a suitable buffer to provide a buffered pH between about 5.1 and 5.6.
  • a buffer contains a formulation of 10 mM citrate, 100 mM NaCl, 100 mM g
  • a buffer contains a formulation of 10 mM citrate, 100 mM NaCl, 100 mM
  • Size exclusion chromatography is a well-known and widely used method for protein stability studies to detect potential fragmentation and aggregation corresponding to physical and chemical instabilities.
  • the% relative to the initial high molecular weight species includes 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL.
  • mL of the anti-CD73 antibody and/or its variants or mutant formulations described herein show less than about 1.6%, 1.4%, 1.2%, 1.0%, 0.8%, 0.6%, 0.4%, 0.2%, or 0.1%
  • the increase in high molecular weight species (HMWS) (including any range between these values).
  • the% relative to the initial high molecular weight species includes 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL.
  • mL of the anti-CD73 antibody formulation described herein shows less than about 2.0%, 1.8%, 1.6%, 1.4%, 1.2%, 1.0%, 0.8%, 0.6%, 0.4%, 0.2%, or 0.1% (including these values Any range between) the increase in high molecular weight species.
  • the% relative to the initial high molecular weight species includes 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL mL of the anti-CD73 antibody and/or its variants or mutant formulations described herein show less than about 3.3%, 3.2%, 3.1%, 3.0%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.2%, 2.0%, 1.8%, 1.6%, 1.4%, 1.2%, 1.0%, 0.8%, 0.6%, 0.4%, 0.2%, or 0.1% (including any range between these values) Increase in high molecular weight species.
  • the% relative to the initial low molecular weight species includes 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL.
  • mL of the anti-CD73 antibody and/or its variants or mutant formulations described herein show less than about 1.6%, 1.4%, 1.2%, 1.0%, 0.8%, 0.6%, 0.4%, 0.2%, or 0.1% (Including any range between these values) increase in low molecular weight species (LMWS).
  • the% relative to the initial low molecular weight species includes 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL.
  • mL of the anti-CD73 antibody and/or its variants or mutant formulations described herein show less than about 2.0%, 1.8%, 1.6%, 1.4%, 1.2%, 1.0%, 0.8%, 0.6%, 0.4%, An increase of 0.2%, or 0.1% (including any range between these values) of low molecular weight species.
  • the% relative to the initial monomer includes 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL
  • the anti-CD73 antibody and/or its variants or its mutant formulations described herein show no more than about 0.2%, 0.4%, 0.6%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3% , 1.4%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1 %, 3.2%, 3.3%, 3.4%, or 3.5% (including any range between these values) monomer reduction.
  • the% relative to the initial monomer includes 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL
  • the anti-CD73 antibody and/or its variants or its mutant formulations described herein show no more than about 0.2%, 0.4%, 0.6%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3% , 1.4%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1 %, 3.2%, 3.3%, 3.4%, or 3.5% (including any range between these values) monomer reduction.
  • the% relative to the initial monomer includes 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL
  • the anti-CD73 antibody and/or its variants or its mutant formulations described herein show no more than about 0.2%, 0.4%, 0.6%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3% , 1.4%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1 %, 3.2%, 3.3%, 3.4%, or 3.5% (including any range between these values) monomer reduction.
  • Cation exchange chromatography is a well-known and widely used tool to detect protein degradation events such as deamidation or oxidation (Moorhouse KG. et al., J. Pharm. Biomed. Anal. [Journal of Pharmaceutical and Biomedical Analysis] 16 (4):593-603 (1997)).
  • the degradation products are typically called acidic or alkaline substances. Acidic species are variants that elute earlier than the main peak of CEX, while basic species are variants that elute later than the main peak of CEX.
  • the anti-CD73 antibody described herein contains 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL and / Or its variants or the acidic peak part of the preparation of its mutants does not exceed about 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the total protein ( Include any range between these values).
  • the anti-CD73 antibodies described herein and / Or its variants or the acidic peak part of the preparation of its mutants does not exceed about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17 of the total protein %, or 18% (including any range between these values).
  • the preparation of the acidic peak part does not exceed about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16 of the total protein %, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, or 27% (including any range between these values).
  • the anti-CD73 antibody described herein contains 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL and / Or its variants or mutants (or fragments) of the formulation of the basic peak part does not exceed about 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46% of the total protein (Including any range between these values).
  • the formulation of the basic peak part does not exceed about 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46% of the total protein (Including any range between these values).
  • the anti-CD73 antibody described herein contains 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL and / Or its variants or mutants (or fragments) of the formulation of the basic peak part does not exceed about 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46% of the total protein (Including any range between these values).
  • the formulation of the basic peak part is not less than about 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46% (including any range between these values).
  • the anti-CD73 antibody described herein contains 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, or 25 mg/mL and / Or its variants or mutants (or fragments) of the formulation of the basic peak part is not less than about 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46% (including any range between these values).
  • the formulations used for in vivo administration must be sterile. This can be easily achieved, for example, by filtering through a sterile filter membrane.
  • the labeled anti-CD73 antibodies, variants, mutants, fragments, and derivatives and analogs thereof that specifically bind to CD73 can be used for diagnostic purposes to detect, diagnose, or monitor CD73 expression, abnormal expression and/or Activity-related diseases and/or disorders.
  • the disclosed anti-CD73 antibodies and/or variants or mutants (or fragments thereof) provided herein can be used in in situ, in vivo, ex vivo, and in vitro diagnostic assays or imaging assays.
  • the method for detecting the expression of CD73 includes (a) using one or more antibodies of the present disclosure to determine the expression of the polypeptide in the cells (such as tissue) or body fluid of an individual, and (b) comparing the expression level of the gene with a standard gene The expression level, where an increase or decrease in the measured gene expression level compared to the standard expression level is indicative of abnormal expression.
  • the diagnosis includes: (a) administering to the mammal an effective amount of labeled anti-CD73 antibody and/or variants or mutants (or fragments) thereof; (b) waiting for a period of time after administration , To allow the labeled anti-CD73 antibody and/or its variants or mutants (or fragments) to be preferentially concentrated in the subject's CD73-expressing site (and to eliminate unbound labeled molecules to background levels); ( c) determining the background level; and (d) detecting the labeled molecule in the subject, so that the detection of the labeled molecule above the background level indicates that the subject has a specific disease or disorder related to CD73 expression or abnormal expression .
  • the background level can be determined by different methods, which include comparing the amount of labeled molecules detected with a standard value previously determined
  • anti-CD73 antibodies and/or variants or mutants (or fragments) thereof provided herein can be used to determine protein levels in biological samples using classical immunohistological methods known to those skilled in the art (for example, see Jalkanen et al., J. Cell. Biol. [Journal of Cell Biology] 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. [Journal of Cell Biology] 105:3087-3096 (1987)).
  • Other antibody-based methods that are useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels include enzyme labels (e.g., glucose oxidase); radioisotopes, such as iodine (131 I, 125 I, 123 I, 121 I), carbon (14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In, 112 In, 111 In), technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), Palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), samarium ( 153 Sm), lutetium ( 177 Lu), gadolinium ( 159 Gd), promethium ( 149 Pm), lanthanum ( 140 La), ytterbium ( 175 Yb), holmium ( 166 Ho), yttrium ( 90 Y), scandium ( 47 Sc), rhenium
  • enzyme labels
  • the level of nucleic acid or mRNA encoding CD73 in a cell can be measured, for example, via fluorescence in situ hybridization using a nucleic acid-based probe corresponding to the nucleic acid encoding CD73 or its complement; (FISH; see WO 98/45479 published in October 1998), Southern blotting, Northern blotting or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR).
  • FISH fluorescence in situ hybridization using a nucleic acid-based probe corresponding to the nucleic acid encoding CD73 or its complement
  • FISH fluorescence in situ hybridization
  • Southern blotting Southern blotting
  • Northern blotting or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR).
  • RT-PCR real-time quantitative PCR
  • cells in a mammal can be exposed to an antibody, which is optionally labeled with a detectable label (e.g., a radioisotope), and can be taken, for example, by external scanning radioactivity or by analysis from a mammal previously exposed to the antibody To evaluate the binding of antibodies to cells (for example, biopsy or other biological samples).
  • a detectable label e.g., a radioisotope
  • the present invention relates to a kit comprising the disclosed anti-CD73 antibody, variant, mutant and/or fragments thereof, and/or the pharmaceutical composition provided herein, and the following One or more: (a) at least one agent known to treat cancer; or (b) instructions for treating cancer.
  • the disclosed anti-CD73 antibodies, variants, mutants and/or fragments thereof, and/or the pharmaceutical compositions provided herein, and at least one agent known to treat cancer are co-formulated.
  • the disclosed anti-CD73 antibody, its variants, mutants and/or fragments, and/or the pharmaceutical composition provided herein, and at least one agent known to treat cancer are packaged together .
  • one agent known to treat cancer is a hormone therapy agent.
  • the hormone therapeutic agent is selected from one or more of the following group consisting of leuprolide, tamoxifen, raloxifene, megestrol, fulvestrant, Triptorelin, medroxyprogesterone, letrozole, anastrozole, exemestane, bicalutamide, goserelin, histrelin, fluoxyrelin , Estramustine, flutamide, toremifene, degarelix (degarelix), nilutamide, abarelix (abarelix) and testosterone, or a pharmaceutically acceptable salt, hydrate Compounds, solvates or polymorphs.
  • the antitumor antibiotic agent is selected from one or more of the following group consisting of doxorubicin, mitoxantrone, bleomycin, daunorubicin, Nematoxin D, epirubicin, idarubicin, pracamycin, mitomycin, pentostatin and valrubicin, or a pharmaceutically acceptable salt, hydrate, Solvates or polymorphs.
  • the anti-metabolite agent is selected from one or more of the following group consisting of gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed Trixet, fludarabine, nelarabine, cladribine, clofarabine, arabinoside, decitabine, praltrexate, fluorouridine, methamine Pterin and thioguanine, or pharmaceutically acceptable salts, hydrates, solvates, or polymorphs thereof.
  • the alkylating agent is selected from one or more of the following group consisting of carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmos Stine, busulfan, nitrosourea, dacarbazine, oxaliplatin, ifosfamide, nitrogen mustard, temozolomide, thiatepa, bendamustine and streptozotocin, Or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.
  • the mitosis inhibitor is selected from one or more of the following group consisting of irinotecan, topotecan, rubitecan, cabazitaxel , Docetaxel, paclitaxel, etoposide (etopside), vincristine, ixabepilone, vinorelbine, vinblastine and teniposide, or a pharmaceutically acceptable salt, hydrate, or solvent Compounds or polymorphs.
  • the mTOR inhibitor is selected from one or more of the group consisting of everolimus, siroliumus and temsirolimus, Or a pharmaceutically acceptable salt, hydrate, solvate or polymorph thereof.
  • the kit further includes instructions for providing the disclosed anti-CD73 antibody, variants, mutants, and/or fragments thereof, and/or the pharmaceutical composition provided herein in combination with surgery.
  • the kit further comprises instructions for providing the disclosed anti-CD73 antibodies, variants, mutants and/or fragments thereof, and/or the pharmaceutical composition provided herein in combination with surgery, wherein these instructions provide To perform surgery before administering at least one compound.
  • the kit further comprises instructions for providing the disclosed anti-CD73 antibody, its variants, mutants and/or fragments, and/or the pharmaceutical composition provided herein in combination with surgery, wherein these instructions provide Surgery is performed after administration of the disclosed anti-CD73 antibodies, variants, mutants and/or fragments thereof, and/or the pharmaceutical compositions provided herein.
  • the kit further comprises instructions that provide the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or the pharmaceutical composition provided herein in combination with surgery, wherein these instructions provide Surgery is performed after administration of the disclosed anti-CD73 antibodies, variants, mutants and/or fragments thereof, and/or the pharmaceutical compositions provided herein, and wherein these instructions provide for administration of the disclosed anti-CD73 antibodies, variants thereof Variants, mutants and/or fragments, and/or pharmaceutical compositions provided herein are used to achieve preoperative tumor reduction.
  • the kit further comprises instructions for providing the compound in combination with surgery, wherein these instructions provide for the administration of the disclosed anti-CD73 antibodies, variants, mutants and/or fragments thereof, and/or provided herein
  • the pharmaceutical composition is followed by surgery, and wherein these instructions provide for the surgery to be performed almost simultaneously with the administration of the disclosed anti-CD73 antibodies, variants, mutants and/or fragments thereof, and/or the pharmaceutical compositions provided herein.
  • the kit further includes instructions for providing the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or pharmaceutical compositions in combination with radiotherapy.
  • the kit further comprises instructions for providing the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or pharmaceutical compositions in combination with radiotherapy, wherein these instructions provide Radiotherapy is performed prior to administration of the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or pharmaceutical compositions.
  • the kit further comprises instructions for providing the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or pharmaceutical compositions in combination with radiotherapy, wherein these instructions provide The step of administering the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or pharmaceutical compositions is followed by radiotherapy.
  • the kit further comprises instructions for providing the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or pharmaceutical compositions in combination with radiotherapy, wherein these instructions provide The steps of administering the disclosed anti-CD73 antibodies, variants, mutants, and/or fragments thereof, and/or pharmaceutical compositions are performed almost simultaneously with radiotherapy.
  • the kit further comprises a plurality of dosage forms, the plurality comprising one or more dosages; wherein each dosage comprises a therapeutically effective amount of the disclosed anti-CD73 antibody, variant, mutant and/or Fragment, and/or pharmaceutical composition and at least one medicament.
  • the kit further comprises a plurality of dosage forms, the plurality comprising one or more dosages; wherein each dosage comprises a therapeutically effective amount of the disclosed anti-CD73 antibodies, variants, mutants and/or Fragments thereof, and/or pharmaceutical compositions and at least one medicament, and each dose of the disclosed anti-CD73 antibodies, variants, mutants and/or fragments thereof, and/or pharmaceutical compositions and known treatments At least one agent for cancer is co-formulated.
  • the kit further comprises a plurality of dosage forms, the plurality comprising one or more dosages; wherein each dosage comprises a therapeutically effective amount of the disclosed anti-CD73 antibody, variant, mutant and/or Fragments thereof, and/or pharmaceutical compositions and at least one medicament, and each dose of the disclosed anti-CD73 antibodies, variants, mutants and/or fragments thereof, and/or pharmaceutical compositions and known treatments At least one medicament for cancer is packaged together.
  • the kit further comprises a plurality of dosage forms, the plurality comprising one or more dosages; wherein each dosage comprises a therapeutically effective amount of the disclosed anti-CD73 antibody, variant, mutant and/or Fragments, and/or pharmaceutical compositions (formulated for intravenous administration), and at least one medicament (formulated for oral administration and/or intravenous administration).
  • the kit further comprises a plurality of dosage forms, the plurality comprising one or more dosages; wherein each dosage comprises a therapeutically effective amount of the disclosed anti-CD73 antibodies, variants, mutants and/or Fragments thereof, and/or pharmaceutical compositions (formulated for intravenous administration), and at least one medicament (formulated for oral administration).
  • the kit further comprises a plurality of dosage forms, the plurality comprising one or more dosages; wherein each dosage comprises a therapeutically effective amount of the disclosed anti-CD73 antibody, variant, mutant and/or Fragments thereof, and/or pharmaceutical compositions (formulated for intravenous administration), and at least one medicament (formulated for intravenous administration).
  • kits which provide two or more components (which can be active or inactive). Ingredients, carriers, diluents, etc.) and instructions for preparing the actual dosage form for the patient or the person administering the drug to the patient.
  • Such kits may have all necessary materials and ingredients contained therein, or these kits may contain instructions for using or manufacturing materials or components that must be independently obtained by the patient or the person administering the drug to the patient.
  • the kit may include optional components that facilitate the administration of a unit dose to the patient, such as vials for reconstitution in powder form, syringes for injections, customized IV delivery systems, inhalers Wait.
  • the kit may include instructions for preparing and administering the composition.
  • the kit can be manufactured as a single-use unit dose for one subject, for multiple uses for a specific subject (at a constant dose or in which the efficacy of a single compound can vary as the therapy progresses); or the kit It may contain multiple doses suitable for administration to multiple patients ("bulk packages").
  • the kit components can be packed in cartons, blister packs, bottles, tubes, etc.
  • kits can be packaged in a daily dosing regimen (eg, packaged on cards, packaged with dosing cards), blister packs, or blow-molded plastic packaging Wait).
  • a daily dosing regimen eg, packaged on cards, packaged with dosing cards
  • blister packs e.g., blister packs, or blow-molded plastic packaging Wait.
  • blow-molded plastic packaging Wait e.g., blow-molded plastic packaging Wait.
  • the present invention also provides a pharmaceutical package or kit, which comprises one or more containers filled with one or more components of the pharmaceutical composition of the present invention.
  • a pharmaceutical package or kit which comprises one or more containers filled with one or more components of the pharmaceutical composition of the present invention.
  • Related to such a container or such a container may be an identification in a form that is regulated by a government agency that regulates the manufacture, use, or sale of drugs or biological products, and the identification reflects the approval of the manufacturing, use, or sale agency for human administration.
  • kits may also include compounds and/or products that are co-packaged, co-formulated, and/or co-delivered with other components.
  • a drug manufacturer, drug distributor, physician, compounding shop, or pharmacist may provide a kit containing the disclosed compound and/or product, and another component for delivery to patient.
  • kits can be used with the disclosed preparation method, the disclosed use or treatment method, and/or the disclosed composition.
  • kits that can be used for various purposes, such as kits for isolating or detecting CD73 in patients, optionally combined with preparations.
  • the kit may contain the anti-CD73 antibodies provided herein and/or variants or mutants (or fragments) thereof coupled to beads (eg, SEPHAROSE TM beads).
  • a kit containing antibodies (or fragments thereof) can be provided for in vitro detection and quantification of CD73 in, for example, ELISA or Western blotting.
  • the kit contains a container and a marking or packaging insert on or associated with the container.
  • the container contains a composition comprising at least one anti-CD73 antibody provided herein and/or a variant or mutant (or fragment) thereof.
  • Additional containers that can be included contain, for example, diluents and buffers, control antibodies.
  • the label or package insert can provide a description of the composition and instructions for the intended in vitro or diagnostic use.
  • the article of manufacture may include a container and a marking or packaging insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes and the like.
  • the container can be made of a variety of materials, such as glass or plastic.
  • the container contains a composition effective to treat the condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is the anti-CD73 antibody provided herein and/or a variant or mutant (or fragment) thereof.
  • the label or package insert indicates that the composition is used to treat a particular condition.
  • the labeling or packaging insert will further include instructions for administering the antibody composition to the patient.
  • Articles of manufacture and kits containing the combination therapies described herein are also contemplated.
  • Package inserts refer to instructions usually included in the commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings for the use of such therapeutic products.
  • the package insert indicates that the composition is used to treat cancer (such as head and neck cancer, lung cancer, or colorectal cancer).
  • the preparation may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • the human Fab initial phage display library was panned with human CD73-ECD/His. Magnetic coating with streptavidin M-280 (Thermo Fisher Scientific #11205D) conjugated biotinylated huCD73-ECD/His after four rounds of panning, the CD73 enzyme with CD73 binding activity and immobilization was selected Fifty-two possible clones of blocking activity and four sequences (N1 to N4) were determined. The selected variable sequence was then cloned into the L234F, L235E, and P331S mutants of the human IgG1 Fc backbone to be used for in vitro functional analysis (CD73 binding, CD73 enzymatic activity blocking, antibody-mediated CD73 internalization) Full-length antibody.
  • N1, N2 and N4 were also lead cloned into human wild-type or C219S mutant IgG2 Fc backbone. According to in vitro efficacy and in vivo efficacy tests (MDA-MB-231 xenograft model), N1 and N4 were selected for the following affinity maturation and determination of the wtIgG2 backbone.
  • the antibody lead clone N1 and lead clone N4 were used in affinity maturation experiments based on in vitro phage display to generate additional clones with improved CD73 binding activity performance and CD73 enzyme blocking activity performance.
  • the CDR-L1/CDR-L3/CDR-H3 (concentrated in 3 CDRs) nucleic acid library of the N1 lead clone was generated by PCR, cloned into a phage display vector and transformed into E. coli TG1 cells to generate a phage library.
  • N4#1, N4#4, N4#5, and N4#6 cloned CDR-L1/CDR-L2/CDR-H2 nucleic acid libraries were generated to generate phage libraries.
  • select clones N4#4-3, N4#6-2, N4#6-3, N4#6-4, N4#6-5 with better binding activity and enzyme blocking activity were generated.
  • N1#2-P, N1#9-PH, N4#6-3-P, N4#6-4-P has better soluble CD73 enzyme blocking effect, and N4#6-3-P, N4#6-4-P can induce surface CD73 internalization.
  • N1#9-PH, N4#6-3-P, N4#6-4-P can alleviate AMP-mediated T cell suppression and have anti-tumor activity against MDA-MB-231 xenografts in vivo.
  • N4#6-4-P can induce CD73 down-regulation and block CD73 enzyme activity in xenograft tumors, and has better anti-tumor activity than other anti-CD73 antibodies in the NCI-H292 xenograft model.
  • the amino acid sequence alignment of the light chain and heavy chain variable regions of the anti-CD73 lead clones N1, N2, and N4 selected from the initial phage panning are shown in Figure 1A and Figure 1B, respectively.
  • the amino acid sequence alignment of the light chain variable region derived from the affinity matured N1 and N4 variants is shown in Figure 9, where the CDR (complementarity determining region) defined by Kabat is underlined and marked in bold, and will be derived from the affinity
  • the amino acid sequence alignment of the heavy chain variable regions of mature N1 and N4 variants is shown in Figure 10, in which the CDRs (complementarity determining regions) defined by Kabat are underlined and marked in bold.
  • variable sequences are then cloned into the human IgG2 Fc backbone to become full-length antibodies.
  • the amino acids of the constant region of the light chain and the constant region of the human antibody can be selected as follows (not limited to the amino acid sequence of the variable region of the light and heavy chains herein):
  • wtIgG2 isotype heavy chain constant region (SEQ ID NO: 112)
  • mtIgG2 isotype heavy chain constant region (SEQ ID NO: 113)
  • tmtIgG1 isotype heavy chain constant region (SEQ ID NO: 114)
  • N1#2-P heavy chain (SEQ ID NO: 116)
  • N1#9-PH heavy chain (SEQ ID NO: 117)
  • N4#6-4-P heavy chain (SEQ ID NO: 119)
  • Figure 14 shows the amino acid sequence alignment of the light chain of the N1 and N4 front variants, in which the Kabat-defined CDR (complementarity determining region) is underlined and marked in bold
  • Figure 15 shows the N1 and N4 front variants Alignment of the amino acid sequence of the variable region of the heavy chain, in which the CDR (complementarity determining region) defined by Kabat is underlined and marked in bold.
  • An ELISA assay was performed to evaluate the binding of the selected antibody to recombinant human CD73-ECD/His protein.
  • Goat anti-human IgG Fd (or Fc) antibody was coated on a 96-well EIA microtiter plate overnight at 4°C at 30 ng per well. After blocking with 5% skim milk (in PBS), serially diluted antibodies were added and incubated for 1 hour at room temperature. Unbound antibody was removed, and the wells were washed three times with 1 ⁇ PBST containing 0.05% Tween20. The biotin-labeled CD73-ECD/His protein was added to the wells at 30 nanograms per well, and incubated for another hour at room temperature.
  • the unbound biotin-labeled CD73-ECD/His was washed three times with 1 ⁇ PBST containing 0.05% Tween20. Then avidin-HRP was added to the wells and incubated at room temperature for 30 minutes. The excess secondary antibody was removed by washing three times with 1 ⁇ PBST containing 0.05% Tween20. After washing, the plate was developed using TMB. Read the absorbance at a wavelength of 450nm by a microplate reader.
  • the whole cell binding ability of anti-CD73 antibodies was tested by incubating MDA-MB-231 or NCI-H292 cells with serially diluted antibodies in FACS buffer (PBS containing 2% FBS) for 30 minutes at 4°C. The cells were washed with FACS buffer, and FITC-labeled goat anti-human IgG (H+L) antibody (or PE-labeled goat anti-human IgG Fc ⁇ ) was used to detect the binding for another 30 minutes at 4°C. The binding of the antibody to the cell surface is measured by the mean fluorescence intensity (MFI) of the stain. Cytomics FC500 or CytoFLEX flow cytometer (Beckman Coulter Inc.) was used for flow cytometry analysis.
  • FACS buffer PBS containing 2% FBS
  • Figures 2B and 2C show that the selected anti-CD73 antibodies bind to CD73-expressing human tumor cells MDA-MB-231 (human breast cancer) cells ( Figure 2B) and NCI-H292 (human mucoepidermoid lung cancer) cells ( Figure 2C).
  • anti-CD73 ref-1 antibody and HLX01 were used as positive and negative controls, respectively. These data indicate that all selected anti-CD73 antibodies can bind to human CD73 recombinant protein and human tumor cells expressing CD73.
  • Figures 11A-11B also show the CD73 binding of N1 and N4 variants.
  • the selected variants were tested by flow cytometry for binding to CD73-expressing MDA-MB-231 ( Figure 11A) and NCI-H292 ( Figure 11B) cells.
  • HLX01 anti-CD20
  • the N4 variant has better CD73 binding capacity than the N1 variant.
  • the order of whole cell binding capacity is: N4#5, N4#6-4, N4#6-5>N4#6-2>N4#4-3, N4#6, N4#6-3, N1#9> N1#2.
  • Figures 16A-16C show the CD73 binding capacity of N1 and N4 front variants.
  • the binding of anti-CD73 antibody to recombinant human CD73 protein was tested by ELISA ( Figure 16A), and the anti-CD73 antibody was tested by flow cytometry and CD73-expressing human tumor cells MDA-MB-231 ( Figure 16B) and NCI-H292 ( Figure 16C) ) Combination.
  • Anti-CD73 ref-1 antibody and anti-CD73 ref-2 antibody were used as positive controls, and HLX01 (anti-CD20) was used as negative control.
  • HLX01 anti-CD20
  • Example 3 The effect of anti-CD73 lead clone on soluble CD73 enzyme activity and cellular CD73 enzyme activity.
  • the CD73-expressing cells (5E3-1E4 cells/well) were resuspended in the culture medium and placed in a 96-well microplate (or pre-seeded for 24 hours). Three-fold (or five-fold) serial dilutions of antibody were added to each culture. After 15 minutes of incubation at 37°C, AMP (final concentration 225 ⁇ M) was added and incubated for 4-20 hours. After centrifugation, the culture supernatant was collected and placed in a 96-well microtiter plate, and ATP was added to a final concentration of 100 ⁇ M for incubation for 2 min. Add the same volume of CellTiter-Glo reagent to each well and mix for 2 minutes. After incubation for 10 min, luminescence was measured.
  • AMP final concentration 225 ⁇ M
  • the anti-CD73 lead clones N1, N2, and N4 with different IgG isotypes were tested for their ability to inhibit cell CD73 enzyme activity.
  • the MDA-MB-231 cells were incubated with anti-CD73 antibody.
  • APCP was used as a positive control in the enzyme activity assay.
  • the effects of these anti-CD73 lead clones with different IgG Fc regions on cell CD73 enzymatic activity are shown in Figure 6A (lead clone N1), 6B (lead clone N2) and 6C (lead clone N4).
  • the wtIgG2 isotype of the N1 clone has better blocking activity than the tmtIgG1 and mtIgG2 isotypes.
  • the tmtIgG1 isotype of N2 clone has better blocking activity than wtIgG2 and mtIgG2 isotypes.
  • N4 with wtIgG2 isotype showed the best blocking activity than tmtIgG1 and mtIgG2 isotypes.
  • the tmtIgG1 isotype has the following Fc site-specific changes: L234F, L235E, and P331S.
  • Figures 17A-17C also show the effect of N1 and N4 front variants on soluble CD73 enzymatic activity (Figure 17A) and cell surface CD73 enzymatic activity ( Figure 17B and Figure 17C).
  • Recombinant CD73 protein ( Figure 17A), MDA-MB-231 ( Figure 17B) and NCI-H292 ( Figure 17C) cells were incubated with anti-CD73 antibody. Add ATP, AMP and CellTiter-Glo and record luminescence.
  • Anti-CD73 ref-1 antibody, anti-CD73 ref-2 antibody and APCP were used as positive controls.
  • HLX01 (anti-CD20) was used as a negative control.
  • N1#9-PH is better than N4#6-3-P and N4#6-4P in inhibiting soluble CD73 enzyme activity.
  • N1#9-PH and N4#6-4-P showed comparable blocking activity on cell surface CD73 enzyme activity.
  • Tumor cells (5E4 to 1E5 cells/well) were suspended in the culture medium and incubated with different concentrations of anti-CD73 antibodies for 4 hours. After removing the medium, the cells were suspended in FACS buffer (PBS containing 2% FBS) and incubated with mouse anti-human CD73 antibody (4G4) at 4°C for 30 minutes. The cells were washed with FACS buffer and incubated with FITC-labeled goat anti-mouse IgG (H+L) antibody or Alexa488-labeled goat anti-mouse IgG Fc ⁇ antibody. Cytomics FC500 or CytoFLEX flow cytometer (Beckman Coulter Inc.) was used for flow cytometry analysis. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive control and negative control, respectively.
  • N4-tmtIgG1 can induce CD73 internalization.
  • N1-tmtIgG1 and N2-tmtIgG1 cannot induce CD73 internalization.
  • Figure 7 also shows the antibody-mediated CD73 internalization of anti-CD73 antibodies with different IgG Fc regions.
  • the cells were incubated with the different IgG isotypes of N1 ( Figure 7A), N2 ( Figure 7B) and N4 ( Figure 7C)
  • the cell surface expression of CD73 was measured by flow cytometry.
  • Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive control and negative control, respectively.
  • the data shows that N4 (instead of N1 and N2) can induce CD73 internalization. There is no significant difference between different IgG isotypes.
  • Figure 13 also shows antibody-mediated CD73 internalization of N1 and N4 variants.
  • NCI-H292 cells were incubated with N1 and N4 variants, and the cell surface expression of CD73 was measured by flow cytometry.
  • Anti-CD73 ref-1 antibody and anti-CD73 ref-2 antibody were used as positive controls, and HLX01 (anti-CD20) was used as negative control.
  • the data indicates that after affinity maturation, the N4 variant can induce CD73 internalization, and the N1 variant still cannot induce CD73 internalization.
  • Figures 18A and 18B also show antibody-mediated CD73 internalization of the N1 and N4 front variants.
  • MDA-MB-231 ( Figure 18A) and NCI-H292 ( Figure 18B) cells were incubated with anti-CD73 N1 and N4 variants. After incubation, the cell surface expression of CD73 was measured by flow cytometry.
  • Anti-CD73 ref-1 and anti-CD73 ref-2 antibodies were used as positive controls, and HLX01 (anti-CD20) was used as negative control.
  • Example 5 CD73 binding of selected antibodies with different IgG Fc regions.
  • variable sequences of N1, N2 and N4 were cloned into human wild-type or C219S mutant IgG2 Fc backbone.
  • the binding of different IgG isotypes of N1 ( Figure 5A), N2 ( Figure 5B), and N4 ( Figure 5C) to CD73-expressing human MDA-MB-231 cells was tested by flow cytometry.
  • Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive control and negative control, respectively.
  • the data shown in Figures 5A-5C indicate that there is no difference between the different IgG isotypes of N1 in terms of CD73 binding ability.
  • the data also shows that the N2 clone (tmtIgG1 isotype) has better CD73 binding activity than wtIgG2 and mtIgG2 isotypes, and the N4 clone (tmtIgG1 isotype) has better CD73 binding activity than wtIgG2 and mtIgG2 isotypes.
  • Example 6 Anti-CD73 lead in MDA-MB-231 (human triple-negative breast cancer) xenograft mouse model The cloned tumor growth inhibitory activity.
  • tumor volume (length x width 2 )/2.
  • the tumor growth curve is shown in Figure 8A.
  • the individual tumor volume on day 45 is shown in Figure 8B. All data points are mean ⁇ SEM.
  • the data proves that, except for the tmtIgG1 isotypes of clones N1, N2 and N4, all anti-CD73 antibodies have anti-tumor activity in the MDA-MB-231 xenograft model (TGI ⁇ 50%).
  • N1-wtIgG2, N1-mtIgG2, N2-wtIgG2 and N2-mtIgG2 almost completely inhibited tumor growth.
  • Figure 20 also shows the tumor growth inhibitory activity of the N1 and N4 front variants in the MDA-MB-231 (human triple negative breast cancer) xenograft mouse model.
  • Example 7 Effect of N1 and N4 variants on the enzymatic activity of immobilized CD73.
  • the N1 and N4 variants were tested for their ability to inhibit the enzymatic activity of immobilized CD73.
  • Goat anti-human IgG Fd antibody was coated on a white high-affinity 96-well microtiter plate overnight at 4°C at 300 ng per well. After blocking with 5% skim milk (in PBS), serially diluted antibodies were added and incubated for 1 hour at room temperature. Unbound antibodies were removed, and the wells were washed three times with 1 ⁇ PBST containing 0.05% Tween 20.
  • N1 and N4 variants The effect of N1 and N4 variants on the enzymatic activity of immobilized CD73 is shown in FIG. 12. These data indicate that after affinity maturation, all N1 and N4 variants can inhibit CD73 enzyme activity.
  • the CD73 enzyme blocking activity of the N1 variant is better than the N4 variant and the anti-CD73 ref-2 antibody, but it is comparable to the anti-CD73 ref-1 antibody. Block the enzyme activity of immobilized CD73: N1#2, N1#9>N4#6-4, N4#6-5>N4#4-3, N4#6-2>N4#6-3, N4#5> N4#6.
  • Example 8 Reversal of the inhibitory effect of AMP on T cell activity by N1 and N4 front variants.
  • CD73 catalyzes the extracellular production of adenosine by adenosine monophosphate (AMP).
  • Adenosine suppresses immune response (including immune response of T cells, NK cells and dendritic cells) by activating A2aR and A2bR receptors.
  • the functional effects of CD73 inhibition by anti-CD73 antibodies on T cell proliferation were also tested.
  • Human T cells were isolated from peripheral blood mononuclear cells (PBMC) using MagniSort human T cell enrichment kit (eBioscience (eBioscience, Inc.)), and suspended in recombinant human IL-2 (eBioscience, Inc.) containing 50IU/mL (eBioscience, Inc.)) in complete medium (RPMI-1640 containing 10% FBS).
  • PBMC peripheral blood mononuclear cells
  • eBioscience eBioscience, Inc.
  • recombinant human IL-2 eBioscience, Inc.
  • RPMI-1640 complete medium
  • a volume of 100 ⁇ L T cells (5E4 cells / reaction) with Dynabeads TM T- activating factor human CD3 / CD28 (Thermo Fisher Scientific Company (Thermo Fisher Scientific)) at 1: cell ratio of 0.5 were mixed ratio of beads .
  • AMP (1 mM) and serially diluted antibodies were added to each reaction in a volume of 100 ⁇ L.
  • Reactions containing no activated beads (unstimulated group), only CD3/CD28 beads (stimulated group), or CD3/CD28 beads and AMP (AMP-mediated inhibition group) were used as assay controls.
  • Anti-CD73 ref-1 antibody, anti-CD73 ref-2 antibody and APCP were used as positive controls.
  • HLX04 (anti-VEGF) was used as a negative control.
  • the cells were cultured at 37°C for 4 days. On day 4, cells were collected, washed, and resuspended in fresh RPMI-1640 medium. T cell proliferation was analyzed by CellTiter-Glo assay. The culture supernatant was collected for cytokine measurement.
  • Human IFN- ⁇ ELISA MAX TM Deluxe Kit Biolegend Co., Ltd. (BioLegend, Inc.) was used to measure the level of IFN- ⁇ .
  • FIGS 19A and 19B The reversal of the inhibitory effect of AMP on T cell activity by the N1 and N4 front variants is shown in Figures 19A and 19B.
  • Proliferation of CD3 + T cells was followed by CellTiter-Glo assay (Figure 19A), and IFN- ⁇ secretion was measured using human IFN- ⁇ ELISA MAX TM Deluxe kit ( Figure 19B).
  • Anti-CD73 ref-1 antibody, anti-CD73 ref-2 antibody and APCP were used as positive controls.
  • HLX04 (anti-VEGF) was used as a negative control.
  • the data shown in Figures 19A and 19B indicate that the N1 and N4 front variants can enhance T cell proliferation and IFN- ⁇ secretion in a dose-dependent manner.
  • the data further indicate that N4#6-3-P, N4#6-4-P and N1#9-PH can alleviate AMP-mediated T cell suppression.
  • N1 and N4 depend on NCI-H292 (human mucoepidermoid lung cancer) xenograft mouse model Tumor growth inhibitory activity of the pre-variant.
  • Figure 21 shows the tumor growth inhibitory activity of N1 and N4 front variants in the NCI-H292 (human mucoepidermoid lung cancer) xenograft mouse model.
  • the first dose of the test product was given 3 days after tumor inoculation.
  • Mice were treated intraperitoneally with antibodies at 50, 10 and 2 mg/kg twice a week for 3 weeks. All data points are mean ⁇ SEM.
  • the data proves that N4#6-4-P has better anti-tumor activity than other anti-CD73 antibodies in the NCI-H292 xenograft model.
  • Example 10 N1 and N4 front variants in NCI-H292 xenograft tumor on CD73 enzyme activity of cells The role of.
  • a total of 5E6NCI-H292 cells in 100 ⁇ L of PBS were mixed with 100 ⁇ L of artificial basement membrane (Matrigel) (Corning, California (CA), USA) (at a ratio of 1:1), and before the antibody was administered
  • Matrigel artificial basement membrane
  • CA California
  • mice Male BALB/c nude mice (Biolasco, Taipei, Taiwan) were subcutaneously implanted into the flanks of both sides. Mice were treated intraperitoneally with 30 mg/kg antibody, APCP, and placebo on day 0, and tumors were collected on days 1, 3, and 7.
  • Tumor cells (2E4) was resuspended in assay buffer (25mM Tris, 5mM MgCl 2, pH 7.46) and treated with ATP (final concentration 100 M) and AMP (300 uM final concentration) treated at 37 °C 45 minutes. The same volume of CellTiter-Glo reagent was added to the reaction mixture and the contents (in a white 96-well microplate) were mixed for 2 min. After incubation for 10 min, luminescence was measured.
  • assay buffer 25mM Tris, 5mM MgCl 2, pH 7.46
  • ATP final concentration 100 M
  • AMP 300 uM final concentration
  • Figure 22 shows the effect of N1 and N4 front variants on cell CD73 enzyme activity in NCI-H292 xenograft tumors.
  • the N1 and N4 front variants were tested for their ability to inhibit CD73 enzyme activity in the NCI-H292 xenograft model.
  • Antibody, APCP and placebo were injected intraperitoneally on day 0. Tumors were removed on days 1, 3, and 7 after antibody administration.
  • the CD73 enzyme activity in tumors was measured by CellTiter-Glo assay.
  • N1 and N4 front variants can inhibit the CD73 enzyme activity in the NCI-H292 xenograft model.
  • N4#6-3-P and N4#6-4-P block CD73 enzyme activity in tumors better than N1#9-PH and anti-CD73 reference antibody.
  • Example 11 MDA-MB-231 xenograft tumors N1 and N4 front variants on CD73 expression and fineness The role of cell CD73 enzyme activity.
  • a total of 1E7MDA-MB-231 cells in 100 ⁇ L of PBS were mixed with 100 ⁇ L of artificial basement membrane (Matrigel) (Corning, California (CA), USA) (at a ratio of 1:1), and then administered Seven days before the antibody, female NOD/SCID mice (Biolasco, Taipei, Taiwan) were subcutaneously implanted into the flanks of both sides. Mice were treated intraperitoneally with 2 mg/kg antibody and 10 mL/kg placebo on day 0. Tumors were collected on days 1, 3 and 7.
  • 5E4 tumor cells were resuspended in FACS buffer (PBS containing 2% FBS) and treated with anti-human and anti-mouse FcR blocking reagents at 4°C for 30 minutes. After centrifugation, the cells were then incubated with mouse anti-human CD73 antibody (4G4) at 4°C for 30 minutes. The cells were washed with FACS buffer and incubated with Alexa 488 labeled goat anti-mouse IgG Fc ⁇ antibody at 4°C for another 30 minutes. The flow cytometry analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter Inc.).
  • a total 2E4 tumor cells were resuspended in assay buffer (25mM Tris, 5mM MgCl 2, pH 7.46) for measuring CD73 activity.
  • the cell suspension was treated with ATP (final concentration 100 ⁇ M) and AMP (final concentration 300 ⁇ M) at 37°C for 45 minutes.
  • the same volume of CellTiter-Glo reagent was added to the reaction mixture and mixed for 2 min (in a white 96-well microplate). After incubation for 10 min, luminescence was measured.
  • Figures 23A and 23B show the effect of N1 and N4 front variants on CD73 expression and cellular CD73 enzyme activity in MDA-MB-231 xenograft tumors.
  • CD73 expression was measured by the mean fluorescence intensity (MFI) of staining ( Figure 23A), and CD73 enzymatic activity in tumors was measured by the CellTiter-Glo assay ( Figure 23B).
  • MFI mean fluorescence intensity
  • Figure 23B The data proves that compared with the placebo group, N4#6-4-P can down-regulate the surface CD73 expression and inhibit the CD73 enzyme activity in the MDA-MB-231 xenograft mouse model.
  • Anti-CD73 antibody binding was assessed by incubating 4T1 or LLC-MK2 cells (1E5 cells/test) with serially diluted antibodies in FACS buffer (PBS containing 2% FBS) for 30 minutes at 4°C. The cells were washed with FACS buffer, and the binding was detected with FITC-labeled goat anti-human IgG (H+L) antibody pair at 4°C for another 30 minutes. The binding of the antibody to the cell surface is measured by the mean fluorescence intensity (MFI) of the stain. Cytomics FC500 or CytoFLEX flow cytometer (Beckman Coulter Inc.) was used for flow cytometry analysis.
  • FACS buffer PBS containing 2% FBS
  • Figures 24A and 24B show the cross binding of N1 and N4 front variants to CD73 expressing cells in mice and monkeys.
  • the binding of anti-CD73 antibodies to CD73-expressing mouse breast cancer 4T1 cells ( Figure 24A) and monkey kidney epithelial LLC-MK2 cells ( Figure 24B) was tested by flow cytometry. HLX01 was used as a negative control. These data indicate that the N1 and N4 front variants are cross-reactive with monkey CD73-expressing cells. The front variants of N1 and N4 cannot cross-bind with mouse CD73.

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Abstract

提供了抗CD73抗体、变体、和其抗原结合片段，这些抗体、变体、和其抗原结合片段以高亲和力与人CD73结合并抑制CD73的酶活性，并且任选地诱导CD73内化。还提供了编码该抗CD73抗体、变体、和其抗原结合片段的分离的核酸分子，和相关的表达载体，和宿主细胞。提供了制备抗CD73抗体、变体、和其抗原结合片段的方法。还提供了相关的药物组合物和使用这些药物组合物治疗受试者的方法。

Description

针对抗CD73抗体和变体的方法和组合物 技术领域
本发明总体上涉及抗CD73抗体、它们的变体或突变体或其抗原结合片段，以及使用其治疗人癌症的方法。
背景技术
CD73(称为外-5’-核苷酸酶(NT5E，EC 3.1.3.5))是在大多数组织中发现的糖基-磷脂酰肌醇(GPI)-连接的70-kDa细胞表面酶(Zhang B.,Cancer Res.[癌症研究],70(16):6407-6411(2010))。CD73(最初被定义为淋巴细胞分化抗原)被认为在T淋巴细胞上起共同信号传导分子的作用，并且起黏附分子的作用，这对淋巴细胞与内皮结合是重要的。最近的研究表明CD73可以控制多种生理应答，包括上皮离子和液体转运、缺血预处理、组织损伤、血小板功能、缺氧和血管渗漏(Zhang B.,Cancer Res.[癌症研究],70(16):6407-6411(2010))。
已经证明了细胞外腺苷的免疫抑制作用(Stagg J.等人,Oncogene[癌基因],29(39):5346–5358(2010))。腺苷与其受体相互作用可以抑制大多数免疫细胞功能，包括自然杀伤(NK)细胞的细胞毒性、巨噬细胞吞噬作用、T细胞的细胞毒性和细胞因子释放(Goto T.等人,J.Immunol.[免疫学杂志],130(3):1350-1355(1983)；Ohta A.等人,J.Immunol.[免疫学杂志],183(9):5487-5493(2009))。阻断腺苷信号传导可以改善免疫疗法。CD73是参与腺苷信号传导的关键分子。CD73将腺苷一磷酸(AMP)(来自通过CD39进行的三磷酸腺苷(ATP)水解途径的产物)水解为腺苷以影响肿瘤微环境(Allard B.等人,Immunol.Rev.[免疫学综述],276(1):121-144(2017)；Resta R.等人,Immunol.Rev.[免疫学综述],161:95-109(1998))。CD73腺苷轴构成免疫肿瘤学中最有希望的途径之一。
研究报道了CD73参与细胞-细胞和细胞-基质相互作用，并表明CD73参与药物抗性和肿瘤促进(Spychala J.,Pharmacol.Ther.[药理学与治疗学],87(2-3):161–173(2000))。已证实CD73在若干种癌细胞中过表达(Gao ZW.等人,Biomed.Res.Int.[国际生物医学研究],2014:460654(2014))，并且该表达与一些癌症的预后不良或患者存活相关(Loi S.等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],110(27):11091-11096(2013)；Turcotte M.等人,Cancer Res.[癌症研究],75(21):4494-4503(2015)；Xiong L.等人,Cell Tissue Res.[细胞组织研究],355(2):365-374(2014))。在一些例子中，抗CD73单克隆抗体(mAb)治疗可以抑制转移和肿瘤血管发生(Allard B.等人,Int. J.Cancer[国际癌症杂志],134(6):1466-1473(2014)；Terp MG.等人,J.Immunol.[免疫学杂志],191(8):4165-4173(2013))。此外，研究人员已经证明了CD73-腺苷在癌症中的免疫抑制作用，并且认为靶向阻断CD73或腺苷受体可以有效地促进抗肿瘤免疫并增强第一代免疫检查点阻断剂的活性(Allard D.等人、Immunotherapy[免疫疗法]、8(2):145-163(2016))。
近年来，靶向CD73在临床前模型中产生了有利的抗肿瘤作用，并且CD73阻断连同其他免疫调节剂的组合治疗是特别有吸引力的治疗选择(Antonioli L.等人,Trends Cancer[癌症趋势],2(2):95-109(2016))。与抗CD73mAb的组合可以显著增强抗CTLA-4(细胞毒性T淋巴细胞抗原4)mAb和抗PD-1(程序性细胞死亡蛋白1)mAb的抗肿瘤活性(Allard B.等人,Clin.Cancer Res.[临床癌症研究],19(20):5626-5635(2013))。
然而，尽管针对CD73的研究取得进展并且利用该蛋白质得到临床益处，仍然需要开发针对CD73的有效、安全且强有力的抗体药剂。本发明满足了这些需求和其他需求。
发明内容
根据本发明的一个或多个目的，如本文所体现和广泛描述的，本发明在一个方面涉及抗CD73抗体、变体、突变体、和/或其抗原结合片段，制备它们的方法，包含它们的药物组合物，和使用它们治疗人受试者的方法。例如，所披露的抗CD73抗体及其亲和变体和/或突变体或抗原结合片段可以在治疗与CD73表达、CD73过表达、和/或异常CD73功能相关的疾病(如癌症或纤维化)中单独使用或与其他药剂组合使用。本文提供的抗体还可以用于通过向患者给予抗CD73抗体、和/或它们的亲和变体/突变体或其抗原结合片段并且在来自该患者的样品中(例如，体内或离体)检测与CD73蛋白结合的抗CD73抗体、和/或变体/突变体或其抗原结合片段，或通过将抗CD73抗体、和/或变体/突变体或其抗原结合片段与来自患者的样品进行接触并且定性地或定量地检测与CD73蛋白结合的抗CD73抗体、和/或亲和变体/突变体或其抗原结合片段，而在患者或患者样品中检测CD73蛋白。
本发明提供了抗CD73抗体和变体、和/或其抗原结合片段。在某些实施例中，本发明提供了至少十八(18)种抗CD73抗体、变体和/或其抗原结合片段，即抗CD73抗体、和/或变体N1及变体N1变体(N1#2、N1#2-P、N1#9和N1#9-PH)，N2，N4及N4变体(N4#1、N4#4、N4#4-3、N4#5、N4#6、N4#6-2、N4#6-3、N4#6-3-P、N4#6-4、N4#6-4-P和N4#6-5)。在下文序列表中提供了这些(18种)抗CD73抗体和/或其变体中每一种的轻链和重链的核酸和/或其编码氨基酸序列。下表1和2中分别提供了每种抗CD73抗体、和/或其变体的每条轻链(CDR-L1、CDR-L2、 和CDR-L3)和每条重链(CDR-H1、CDR-H2、和CDR-H3)的CDR序列。
表1.抗CD73抗体、变体、和/或突变体轻链CDR序列
Figure PCTCN2020102678-appb-000001
表2.抗CD73抗体、变体、和/或突变体重链CDR序列
Figure PCTCN2020102678-appb-000002
在一些实施例中，根据(或如应用于)任何上述实施例，本发明的抗CD73抗体在其可变结构域的一个或多个CDR的N-糖基化位点处包含一个或多个突变。 所得去糖基化的抗体保持与亲本非去糖基化抗体相同的功能。
在一些实施例中，根据(或如应用于)任何上述实施例，本发明的抗CD73抗体为全长IgG抗体，其中轻链为上述轻链可变区和人抗体轻链恒定区组成，其中重链为上述重链可变区和人抗体重链恒定区组成。
在一些实施例中，根据(或如应用于)任何上述实施例，本发明的抗CD73抗体为全长IgG抗体，其中轻链由上述轻链可变区和人抗体轻链恒定区组成，所述轻链恒定区如SEQ ID NO.115所示，其中重链由上述重链可变区和人抗体重链恒定区组成，其中重链恒定区如112、113或114所示。
在一些实施例中，根据(或如应用于)任何上述实施例，本发明的抗CD73抗体为全长IgG抗体，其中：
(抗体变体N1#2-P)轻链为SEQ ID NO.93所示和重链为SEQ ID NO.116所示，或
(抗体变体N1#9-PH)轻链为SEQ ID NO.94所示和重链为SEQ ID NO.117所示，或
(抗体变体N4#6-3-P)轻链为SEQ ID NO.95所示和重链为SEQ ID NO.118所示，或
(抗体变体N4#6-4-P)轻链为SEQ ID NO.96所示和重链为SEQ ID NO.119所示。
表3抗体的CDR、VH、VL、LV和HC序列号对应表
Figure PCTCN2020102678-appb-000003
在一些实施例中，根据(或如应用于)任何上述实施例，抗CD73抗体包含人IgG的Fc序列。在一些实施例中，根据(或如应用于)任何上述实施例，抗原结合片段选自下组，该组由以下组成：Fab、Fab’、F(ab')2、单链Fv(scFv)、Fv片段、双抗体、和线性抗体。在一些实施例中，根据(或如应用于)任何上述实施例，抗体是多特异性抗体。
在一些实施例中，根据(或如应用于)任何上述实施例，抗CD73抗体、变体或其抗原结合片段与治疗剂缀合。在一些实施例中，根据(或如应用于)任何上述实施例，抗CD73抗体、变体或其抗原结合片段与标记缀合。在一些实施例中，根据(或如应用于)任何上述实施例，标记选自下组，该组由以下组成：放射性同位素、荧光染料、和酶。
本发明提供了编码根据(或如应用于)任何上述实施例的抗CD73抗体、变体、突变体或其抗原结合片段的分离的核酸分子。还提供了编码根据(或如应用于)任何上述实施例的核酸分子的表达载体。还提供了包含根据(或如应用于)任何上述实施例的表达载体的细胞。本发明还提供了产生抗CD73抗体、变体或其抗原结合片段的方法，该方法包括培养根据(或如应用于)任何上述实施例的细胞和从细胞培养物回收抗体或其抗原结合片段。在一些实施例中，根据(或如应用于)任何上述实施例，细胞是哺乳动物细胞。在一些实施例中，根据(或如应用于)任何上述实施例，哺乳动物细胞是CHO细胞。在一些实施例中，根据(或如应用于)任何上述实施例，细胞是稳定的哺乳动物细胞系。在一些实施例中，根据(或如应用于)任何上述实施例，稳定的哺乳动物细胞系是CHO细胞系。
在某些实施例中，提供了在细胞表面与人CD73特异性结合并且能够中和可溶性或膜结合的人CD73蛋白的5'-胞外核苷酸酶活性的分离抗体。抗体可以诱导CD73的细胞内内化。
在某些实施例中，提供了一种与可溶性或膜结合的人CD73蛋白结合并能够抑制其酶活性的抗体，其中所述抗体不被内化到表达CD73的细胞中。
本发明提供了包含根据(或如应用于)任何上述实施例的抗CD73抗体、变体、突变体或其抗原结合片段和药学上可接受的载体的组合物。
本发明提供了通过使根据(或如应用于)任何上述实施例的抗CD73抗体、变体、突变体或其抗原结合片段与样品接触并且检测与CD73蛋白结合的抗CD73抗体而在来自患者的样品中检测CD73蛋白的方法。在一些实施例中，根据(或如应用于)任何上述实施例，抗CD73抗体、变体或其抗原结合片段在免疫组织化学测定(IHC)或在ELISA测定中使用。
还提供了在受试者中治疗癌症的方法，该方法包括向受试者给予有效量的 根据(或如应用于)任何上述实施例的组合物。还提供了包含根据(或如应用于)任何上述实施例的抗CD73抗体、变体、突变体或其抗原结合片段的组合物，用于癌症治疗。提供了根据(或如应用于)任何上述实施例的抗CD73抗体、变体、突变体或其抗原结合片段在制造用于治疗癌症(如与CD73的表达相关的癌症)的药物中的用途。
在一些实施例中，根据(或如应用于)任何上述实施例，癌症选自黑色素瘤、头颈癌、尿路上皮癌、乳腺癌(例如，三阴性乳腺癌，TNBC)、胃癌、经典型霍奇金淋巴瘤(cHL)、非霍奇金淋巴瘤原发性纵隔B-细胞淋巴瘤(NHL PMBCL)、间皮瘤、卵巢癌、肺癌(例如，小细胞肺癌和非小细胞肺癌(NSCLC))、食管癌、鼻咽癌(NPC)、胆道癌、结肠直肠癌、宫颈癌、甲状腺癌和唾液腺癌。在一些实施例中，根据(或如应用于)任何上述实施例，向受试者进一步给予选自下组的治疗剂，该组由以下组成：抗肿瘤剂、化学治疗剂、生长抑制剂和细胞毒性剂。在一些实施例中，根据(或如应用于)任何上述实施例，向受试者进一步给予放射疗法和/或手术。
通过研究以下附图和详细描述，本发明的其他系统、方法、特征、和优点对于本领域技术人员将是显而易见的或将变得显而易见。旨在将所有这些另外的系统、方法、特征和优点包括在本说明书内、本发明的范围内，并且由所附权利要求保护。此外，所描述的实施例的所有任选和优选特征和修改都可用于本文所教导的本发明的所有方面。此外，所附权利要求书的个体特征连同描述的实施例的所有任选的和优选的特征和修改都是彼此可组合的并且可互换的。
附图说明
参考以下附图可以更好地理解本发明的许多方面。
图1A-1B.从初始噬菌体淘选而选择的抗CD73先导克隆N1、N2和N4的轻链(图1A)(图1A分别按出现的顺序披露了SEQ ID NO 79-81)和重链可变区(图1B)(图1B分别按出现的顺序披露了SEQ ID NO 97-99)的氨基酸序列比对。通过用人CD73-ECD/His筛选人Fab初始噬菌体展示文库，鉴定了具有CD73酶结合和阻断活性的三个选择的抗体先导克隆。然后将这些选择的可变序列克隆到人IgG1 Fc主链的L234F、L235E、P331S突变体中，以变成全长抗体。本文列出了抗体先导克隆的序列比对，并且将Kabat定义的CDR(互补决定区)加下划线并用粗体标记。
图2A-2C.选择的抗体的CD73结合。测试选择的抗体与重组人CD73蛋白(通过ELISA(图2A))、表达CD73的人肿瘤细胞MDA-MB-231(人乳腺癌)细胞(图2B)和NCI-H292(人黏液表皮样肺癌)细胞(图2C)(通过流式细胞术)的结合。将抗CD73 ref-1抗体和HLX01(抗CD20)分别用作阳性对照和阴性对照。
图3A-3B.抗CD73先导克隆对可溶性CD73酶活性和细胞CD73酶活性的作用。测试抗CD73抗体抑制人重组CD73蛋白和细胞表面CD73的酶活性的能力。将重组CD73蛋白(图3A)和NCI-H292细胞(图3B)与抗CD73抗体、ATP和AMP一起孵育。使用CellTiter-Glo测定来确定样品中的AMP浓度。冷光读数值指示CD73酶活性。将抗CD73 ref-1抗体和APCP用作阳性对照，而将HLX01(抗CD20)用作阴性对照。
图4.抗CD73先导克隆的抗体介导的CD73内化。在抗CD73抗体处理后，通过流式细胞术测量CD73的细胞表面表达。将抗CD73 ref-1抗体和HLX01(抗CD20)分别用作阳性对照和阴性对照。
图5A-5C.具有不同IgG Fc区的选择的抗体的CD73结合。还将N1、N2和N4的可变序列克隆到人野生型或C219S突变体IgG2 Fc主链中。通过流式细胞术测试N1(图5A)、N2(图5B)、N4(图5C)的不同IgG同种型与表达CD73的人MDA-MB-231细胞的结合。将抗CD73 ref-1抗体和HLX01(抗CD20)分别用作阳性对照和阴性对照。
图6A-6C.具有不同的IgG Fc区的抗CD73先导克隆对细胞CD73酶活性的作用。测试具有不同IgG同种型的抗CD73先导克隆N1(图6A)、N2(图6B)、N4(图6C)抑制细胞CD73酶活性的能力。将MDA-MB-231细胞与抗CD73抗体一起孵育。添加ATP、AMP和CellTiter-Glo试剂并记录冷光。在酶活性测定中将APCP用作阳性对照。
图7A-7C.具有不同IgG Fc区的抗CD73抗体的抗体介导的CD73内化。在将细胞与具有N1(图7A)、N2(图7B)和N4(图7C)的不同IgG同种型的细胞孵育后，通过流式细胞术测量CD73的细胞表面表达。将抗CD73 ref-1抗体和HLX01(抗CD20)分别用作阳性对照和阴性对照。
图8A-8B.MDA-MB-231(人三阴性乳腺癌)异种移植小鼠模型中抗CD73先导克隆的肿瘤生长抑制活性。将小鼠(n＝5只小鼠/组)皮下移植MDA-MB-231细胞。在肿瘤接种后14天，当移植的肿瘤大小达到150-300mm 3时，给予测试制品的第一剂量。在指定时间用5mg/kg抗体腹膜内处理小鼠。肿瘤生长曲线示于图8A中。第45天的个体肿瘤体积示于图8B中。所有数据点均为平均值±SEM。
图9.衍生自亲和力成熟的N1和N4变体的轻链可变区的氨基酸序列比对。从基于体外噬菌体展示的亲和力成熟实验鉴定具有更好CD73结合亲和力和可溶性CD73酶阻断活性的N1和N4变体。将Kabat定义的CDR(互补决定区)加下划线并用粗体标记。图9分别按出现的顺序披露了SEQ ID NO 82-85、88、86-87和89-92。
图10.衍生自亲和力成熟的N1和N4变体的重链可变区的氨基酸序列比对。从基于体外噬菌体展示的亲和力成熟实验鉴定具有更好CD73结合亲和力和可溶 性CD73酶阻断活性的N1和N4变体。将Kabat定义的CDR(互补决定区)加下划线并用粗体标记。然后将这些选择的可变序列克隆到人IgG2 Fc主链中，以变成全长抗体。图10分别按出现的顺序披露了SEQ ID NO 100-103、106、104-105和107-110。
图11A-11B.N1和N4变体的CD73结合。通过流式细胞术来测试选择的变体与表达CD73的MDA-MB-231(图11A)和NCI-H292细胞(图11B)的结合。将HLX01(抗CD20)用作阴性对照。
图12.N1和N4变体对固定化CD73酶活性的作用。测试N1和N4变体抑制固定化CD73酶活性的能力。孵育抗CD73抗体和CD73蛋白后，添加ATP、AMP和CellTiter-Glo试剂并记录冷光。将抗CD73 ref-1抗体、抗CD73 ref-2抗体用作阳性对照，而将HLX01(抗CD20)用作阴性对照。
图13.N1和N4变体的抗体介导的CD73内化。将NCI-H292细胞与N1和N4变体一起孵育，并通过流式细胞术测量CD73的细胞表面表达。将抗CD73 ref-1抗体和抗CD73 ref-2抗体用作阳性对照，而将HLX01(抗CD20)用作阴性对照。
图14.N1和N4靠前变体的轻链的氨基酸序列比对。将Kabat定义的CDR(互补决定区)加下划线并用粗体标记。图14分别按出现的顺序披露了SEQ ID NO 93-96。
图15.N1和N4靠前变体的重链可变区的氨基酸序列比对。将Kabat定义的CDR(互补决定区)加下划线并用粗体标记。然后将这些序列克隆到人IgG2 Fc主链中，以变成全长抗体。图15分别按出现的顺序披露了SEQ ID NO 111、101、和108-109。
图16A-16C.N1和N4靠前变体的CD73结合能力。通过ELISA测试抗CD73抗体与重组人CD73蛋白的结合(图16A)，以及通过流式细胞术测试CD73抗体与表达CD73的人肿瘤细胞MDA-MB-231(图16B)和NCI-H292(图16C)的结合。将抗CD73 ref-1抗体和抗CD73 ref-2抗体用作阳性对照，而将HLX01(抗CD20)用作阴性对照。
图17A-17C.N1和N4靠前变体对可溶性CD73酶活性(图17A)和细胞表面CD73酶活性(图17B，图17C)的作用。测试抗CD73抗体抑制人重组CD73蛋白和细胞表面CD73酶活性的能力。将重组CD73蛋白(图17A)、MDA-MB-231(图17B)和NCI-H292(图17C)细胞与抗CD73抗体一起孵育。添加ATP、AMP和CellTiter-Glo并记录冷光。将抗CD73 ref-1抗体、抗CD73 ref-2抗体和APCP用作阳性对照。将HLX01(抗CD20)用作阴性对照。
图18A-18B.N1和N4靠前变体的抗体介导的CD73内化。将MDA-MB-231(图18A)和NCI-H292(图18B)细胞与抗CD73 N1和N4变体一起孵育。孵育后，通过流式细胞术测量CD73的细胞表面表达。将抗CD73 ref-1和抗CD73 ref-2抗体用 作阳性对照，而将HLX01(抗CD20)用作阴性对照。
图19A-19B.通过N1和N4靠前变体逆转AMP对T细胞活性的抑制作用。将分离的人T细胞与CD3/CD28珠、AMP和连续稀释的抗体一起在37℃培养4天。CD3 +T细胞的增殖之后进行CellTiter-Glo测定(图19A)，并使用人IFN-γELISA MAX TM Deluxe试剂盒(图19B)测量IFN-γ分泌。将抗CD73 ref-1和抗CD73 ref-2抗体用作阳性对照，而将HLX04(抗VEGF)用作阴性对照。
图20.MDA-MB-231(人三阴性乳腺癌)异种移植小鼠模型中N1和N4靠前变体的肿瘤生长抑制活性。将小鼠(n＝5只小鼠/组)皮下移植MDA-MB-231细胞。在肿瘤接种后7天，当移植的肿瘤大小达到约100mm 3时，给予测试制品的第一剂量。每周两次用10mg/kg和2mg/kg的抗体腹膜内处理小鼠，持续5-6周。所有数据点均为平均值±SEM。
图21.NCI-H292(人黏液表皮样肺癌)异种移植小鼠模型中N1和N4靠前变体的肿瘤生长抑制活性。将小鼠(n＝5只小鼠/组)皮下移植NCI-H292细胞。肿瘤接种后3天给予测试制品的第一剂量。每周两次用50、10和2mg/kg的抗体腹膜内处理小鼠，持续3周。所有数据点均为平均值±SEM。
图22.NCI-H292异种移植肿瘤中N1和N4靠前变体对细胞CD73酶活性的作用。测试N1和N4靠前变体在NCI-H292异种移植模型中抑制CD73酶活性的能力。在抗体处理前4天，将小鼠(n＝4只小鼠/组)皮下移植NCI-H292细胞。在第0天腹膜内注射抗体、APCP和安慰剂。在抗体给予后第1、3和7天切除肿瘤。通过CellTiter-Glo测定来测量肿瘤中的CD73酶活性。
图23A-23B.MDA-MB-231异种移植肿瘤中N1和N4靠前变体对CD73表达和细胞CD73酶活性的作用。测试抗CD73抗体在MDA-MB-231异种移植模型中下调表面CD73表达和抑制CD73酶活性的能力。在抗体处理前7天，将小鼠(n＝4只小鼠/组)皮下移植MDA-MB-231细胞。在第0天注射抗体和安慰剂。在抗体给予后第1、3和7天收集肿瘤。通过染色的平均荧光强度(MFI)来测量CD73表达(图23A)，并通过CellTiter-Glo测定来测量肿瘤中的CD73酶活性(图23B)。
图24A-24B.N1和N4靠前变体与小鼠和猴的表达CD73的细胞的交叉结合。通过流式细胞术测试抗CD73抗体与表达CD73的小鼠乳腺癌4T1细胞(图24A)和猴肾上皮LLC-MK2细胞(图24B)的结合。将HLX01用作阴性对照。
本发明的另外的优点将部分地在下面的描述中列出，并且部分地将从该描述而显而易见，或者可以通过本发明的实践来了解。将借助于所附权利要求中具体指出的要素和组合来实现和获得本发明的优点。应当理解，前述一般描述和下面的详细描述都只是示例性和描述性的，并不是对要求保护的本发明的限制。
具体实施方式
在一个方面，本发明涉及抗CD73抗体、变体、突变体、和/或其抗原结合片段，制备它们的方法，包含它们的药物组合物，和使用它们治疗人受试者的方法。本发明进一步提供了抗CD73抗体与CD73特异性结合并且可以经由诱导巨噬细胞介导的吞噬作用而抑制肿瘤生长。所披露的抗体展示出与用于治疗临床病症(如癌症或纤维化)的常规的抗CD73单克隆抗体相比改善的功效和/或抗肿瘤活性。还提供了免疫缀合物，编码新颖的抗CD73抗体、它们的亲和变体或其抗原结合片段的核酸(如本文描述的)，和组合物(如药物组合物)。
在某些实施例中，本发明提供了至少十八(18)种抗CD73抗体、变体和/或其抗原结合片段，即表1和表2中指定的抗CD73抗体、变体、和/或突变体。在下文披露的序列-序列表部分中提供了这些抗CD73抗体、其变体和/或突变体中每一种的轻链和重链的核酸和/或其编码氨基酸序列。表1和2中分别提供了每种抗CD73抗体、其变体和/或突变体的每条轻链(CDR-L1、CDR-L2、和CDR-L3)和每条重链(CDR-H1、CDR-H2、和CDR-H3)的CDR序列。
本发明还提供了使用新颖的抗CD73抗体、其亲和变体和/或突变体或抗原结合片段以检测样品(如体内或离体样品)中CD73的方法，和/或包含此类抗体、变体和/或突变体或其抗原结合片段的组合物，所述组合物用于治疗与CD73表达、CD73过表达和/或异常CD73功能(如癌症或纤维化)相关的临床病症或疾病。在其他实施例中，所披露的抗CD73抗体及亲和变体和/或突变体或其抗原结合片段可以在治疗与CD73表达、CD73过表达、和/或异常CD73功能相关的疾病(如癌症或纤维化)中与其他药剂组合使用。本发明进一步提供了此类抗体、变体或其抗原结合片段在制造用于癌症治疗的药物中的用途。
本文提供的抗体还可以用于通过向患者给予抗CD73抗体、和/或它们的亲和变体/突变体或其抗原结合片段并且在来自该患者的样品中(例如，体内或离体)检测与CD73蛋白结合的抗CD73抗体、和/或变体/突变体或其抗原结合片段，或通过使抗CD73抗体、和/或变体/突变体或其抗原结合片段与来自患者的样品进行接触并且定性地或定量地检测与CD73蛋白结合的抗CD73抗体、和/或亲和变体/突变体或其抗原结合片段，而在患者或患者样品中检测CD73蛋白。
受益于前述描述和相关附图中呈现的教导，所披露的组合物和方法的所属领域的技术人员将想到本文披露的许多修改和其他实施例。因此，应当理解本发明不限于所披露的具体实施例，改进和其他实施例旨在包括于所附权利要求的范围内。技术人员将认识到本文描述的方面的许多变体和改编。这些变体和改编旨在包括在本发明的教导中并且涵盖于本文的权利要求中。
尽管本文采用了特定术语，但它们仅以一般性和描述性意义使用，而不是出于限制的目的。
如将对于本领域技术人员清楚的是，在阅读本发明说明书时，本文描述和展 示的单独实施例的每一个具有离散的组分和特征，这些组分和特征可以在不偏离本发明的范围或精神的情况下易于与任何其他若干个实施例的特征分离或组合。
可以按照所叙述的事件的顺序或按照逻辑上可行的任何其他顺序来进行任何叙述的方法。也就是说，除非另外明确说明，否则决不旨在将本文阐述的任何方法或方面解释为要求以特定顺序执行其步骤。因此，在方法权利要求没有在权利要求或描述中具体陈述这些步骤将限于特定顺序的情况下，在任何方面都不旨在推断顺序。这适用于任何可能的用于解释的非表达基础，包括关于步骤或操作流程的安排的逻辑问题，从语法组织或标点符号衍生的明确含义，或说明书中描述的方面的数量或类型。
本文引用的所有参考文献，包括专利申请和出版物，均通过引用以其整体并入本文。仅提供本文讨论的这些出版物在本申请的提交日期之前的披露。本文不应被解释为承认本发明无权凭借先前的本发明而早于此类出版物。此外，本文提供的出版日期可能与实际出版日期不同，这可能需要独立确认。
虽然本发明的方面可以在具体的法定类别(例如系统法定类别)中描述和要求保护，但这仅是出于方便的目的，并且本领域技术人员将理解可以在任何法定类别中描述和要求保护本发明的每个方面。
还应当理解，本文使用的术语仅是出于对具体的方面进行说明的目的，并不旨在进行限制。除非另外定义，否则本文使用的所有技术和科学术语具有与所披露的组合物和方法所属领域的普通技术人员通常理解的含义相同的含义。将进一步理解，术语(如在常用词典中定义的那些术语)应被解释为具有与其在说明书和相关领域的上下文中一致的含义，并且除非本文明确定义，否则不应被解释为理想化或过于正式的意义。
在描述本发明的各个方面之前，除非另外指示，否则提供以下定义并应当使用以下定义。另外的术语可以在本发明的其他地方定义。
定义
应当理解，本文描述的本发明的方面和实施例包括“包含”‘方面和实施例’、“由‘方面和实施例’组成”和“基本上由‘方面和实施例’组成”。
如本文使用的，“包含”应被解释为指定如提及的所声明的特征、整数、步骤或组件的存在，但不排除一个或多个特征、整数、步骤或组分或其组的存在或添加。此外，术语“通过”、“包含(comprising、comprises、comprised of)”、“包括(including、includes、included)”，“涉及(involving、involves、involved)”和“例如”中的每个以其开放的、非限制性的意义使用，并且可以互换使用。此外，术语“包含”旨在包括术语“基本上由……组成”和 “由……组成”涵盖的实施例和方面。类似地，术语“基本上由......组成”旨在包括术语“由……组成”涵盖的实施例。
应当注意，比率、浓度、量和其他数值数据可以在本文中以范围格式表示。将进一步理解，每个范围的端点相对于其他端点都是重要的，并且独立于其他端点。还应当理解，本文披露了许多值，并且除了值本身之外，每个值在本文中也被披露为“约”该特定值。例如，如果披露了值“10”，则也披露了“约10”。在本文中范围可以表达为从“约”一个具体值，和/或至“约”其他具体值。类似地，当通过使用先行词“约”将值表示为近似值时，将理解该特定值形成一个另外的方面。例如，如果披露了值“约10”，则也披露了“10”。
当表达范围时，另外的方面包括从一个特定值和/或至其他特定值。例如，在所声明的范围包括一个或两个限制的情况下，排除那些包括的限制中的一个或两个的范围也包括于本发明中，例如短语‘x至y’包括从‘x’至‘y’的范围，以及大于‘x’且小于‘y’的范围。该范围也可以表示为上限，例如‘约x、y、z或更小’，并且应该被解释为包括‘约x’、‘约y’和‘约z’的特定范围，以及‘小于x’、‘小于y’和‘小于z’的范围。同样，短语‘约x，y，z或更大’应解释为包括‘约x’、‘约y’和‘约z’的特定范围以及‘大于x’、‘大于y’和‘大于z’的范围。此外，短语“约‘x’至‘y’”，其中‘x’和‘y’是数值，包括“约‘x’至约‘y’”。
应当理解，此范围格式出于方便和简洁的目的而被使用，并且因此应当被灵活地解释为不仅包括被明确叙述为范围界限的数值，而且包括该范围内所涵盖的所有单独数值或子范围，就像每个数值和子范围被明确叙述。为了说明，“约0.1％至5％”的数值范围应该被解释为不仅包括明确叙述的值：约0.1％至约5％，还包括在指定范围内的单独的值(例如，约1％、约2％、约3％、和约4％)，以及子范围(例如，约0.5％至约1.1％、约0.5％至约2.4％、约0.5％至约3.2％、和约0.5％至约4.4％，以及其他可能的子范围)。
如本文使用的，术语“约”、“大约”、“在或约”和“基本上”意指所讨论的量或值可以是精确值或提供如在本文权利要求或教导中叙述的等效结果或作用的值。本文对“约”值或参数的引用是指本技术领域的技术人员容易知道的相应值的通常误差范围。也就是说，应当理解，量、大小、配方、参数和其他数量和性质不是也不必是精确的，但可以(根据需要)是大约和/或更大或更小的，这反映公差、转换因子、四舍五入、测量误差等，以及本领域技术人员已知的其他因素，从而获得等效结果或作用。在一些情况下，无法合理确定提供等效结果或作用的值。在这种例子中，通常理解，如本文使用的，“约”和“在或约”意指标称值表示±10％变化，除非另外指示或推断。通常，量、大小、配方、参数或其他数量或特征是“约”、“大约”或“在或约”，无论是否明确 地声明为此。应当理解，在定量值之前使用“约”、“大约”或“在或约”时，除非另外特别声明，否则参数还包括特定的定量值本身。也就是说，本文对“约”值或参数的引用包括(并描述)针对该值或参数本身的方面。例如，涉及“约X”的描述包括“X”的描述。
如本文使用的，术语“任选的”或“任选地”意指随后描述的事件或情况可以发生或可以不发生，并且该描述包括所述事件或情况发生的实施例和不发生的实施例。
如本文使用的，“CD73”和“CD-73”可以互换使用，并且是指由包含8个外显子的人基因编码的细胞表面酶，该人基因的细胞遗传学位置为3q13.12，并且碱基对的分子位置为在染色体6(智人注释发布(Homo sapiens Annotation Release)109，GRCh38.p12)上85,449,584-85,495,791。CD73蛋白是一种具有5’-核苷酸酶(利用Zn +2作为辅因子)的酶(EC分类：3.1.3.5)。该蛋白质能够将细胞外核苷酸水解成膜可渗透的核苷，并参与核碱基、核苷、核苷酸和核酸代谢的调节。其与经由GPI锚的主要在质膜上的细胞内位置相关。人CD73蛋白具有两种通过替代性剪接产生的同种型。典型同种型(UniProtKB标识符P21589)包含574个氨基酸并且具有约63kDa的分子量。CD73也被称为外-5’-核苷酸酶和5’-核苷酸酶。CD73基因的突变与关节和动脉的钙化相关，即遗传性动脉和关节多发性钙化综合征。
如本文使用的，“治疗(treatment或treating)”是用于获得有益的或所希望的结果(包括临床结果)的方法。出于本发明的目的，有益的或所希望的临床结果包括但不限于以下中的一种或多种：缓解由疾病引起的一种或多种症状、减少疾病的程度、稳定疾病(例如，预防或延迟疾病的恶化)、预防或延迟疾病的传播(例如，转移)、预防或延迟疾病的重现、延迟或减缓疾病的进展，改善疾病状态、提供疾病的缓解(部分或全部)、减少治疗疾病所需的一种或多种其他药物的剂量、延迟疾病的进展、增加或改善生活质量、增加体重增长和/或延长存活。“治疗”还涵盖减少癌症的病理后果(像例如，肿瘤体积)。本文提供的方法考虑了这些治疗方面中的任何一个或多个。
术语“重现”、“复发”或“复发的”是指在临床评估疾病消失后癌症或疾病的恢复。远处转移或局部重现的诊断可视为复发。
术语“难治性”或“抗性”是指对治疗没有应答的癌症或疾病。
术语“辅助疗法”是指在初次疗法后给予的疗法，通常是手术。针对癌症或疾病的辅助疗法可以包括免疫疗法、化学疗法、放射疗法、或激素疗法。
术语“维持疗法”是指预定的再治疗，其用于帮助维持先前治疗的效果。通常给予维持疗法以帮助保持癌症缓解或延长对特定疗法的应答，而不管疾病进展如何。
术语“侵袭性癌症”是指已经扩散到组织层(在其中癌症开始进入正常周围组织)之外的癌症。侵袭性癌症可能是也可能不是转移性的。
术语“非侵袭性癌症”是指非常早期的癌症或未扩散到起源组织之外的癌症。
肿瘤学中的术语“无进展存活”是指治疗期间和治疗之后癌症不生长的时间长度。无进展存活包括患者经历完全应答或部分应答的时间量，以及患者经历稳定疾病的时间量。
肿瘤学中的术语“进展性疾病”可以指自治疗开始以来由于肿块的质量增加或肿瘤扩散肿瘤生长超过20％。
“障碍”是受试者(例如，哺乳动物，如人)中与CD73表达、CD73过表达、和/或异常CD73功能相关的任何临床病症，使得受试者将受益于使用所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物的治疗或预防。例如，患有或需要预防CD73过表达的哺乳动物。这包括慢性和急性障碍或疾病,包括使哺乳动物易患所讨论的障碍的那些病理状况。本文中待治疗的障碍的非限制性实施例包括所披露的癌症(如头颈癌、咽喉癌、结肠直肠癌、肺癌等)或纤维化(包括特发性肺纤维化)。
如本文使用的，“肿瘤”是指无论恶性还是良性的所有赘生性细胞生长和增殖、以及所有癌前期与癌性细胞和组织。
术语“抗体”以最广泛的含义使用，并且具体涵盖例如单一单克隆抗体(包括激动剂、拮抗剂和中和抗体)，具有多表位特异性的抗体组合物，多克隆抗体，单链抗体和抗体片段(参见下文)，只要它们特异性结合天然多肽和/或表现出本发明的生物活性或免疫活性。根据一个实施例，抗体结合靶蛋白的寡聚形式，例如三聚体形式。根据另一个实施例，抗体特异性结合蛋白，该结合可以被本发明的单克隆抗体抑制(例如本发明的保藏抗体等)。短语抗体的“功能片段或类似物”是具有与其所参考的抗体相同的定性生物学活性的化合物。例如，本发明的抗体的功能片段或类似物可以是能够特异性结合CD73的抗体。在一个实施例中，抗体可以诱导对表达CD73的癌细胞的巨噬细胞介导的吞噬作用。
“分离的抗体”是已经从其天然环境的组分中鉴定和分离和/或回收的抗体。其天然环境的污染组分是会干扰抗体的诊断或治疗用途的物质，并且可包括酶、激素和其他蛋白质或非蛋白质溶质。在优选的实施例中，抗体将被纯化至(1)如通过Lowry法所确定的按抗体的重量计高于95％，并且最优选地按重量计高于99％，(2)通过使用旋转杯测序仪，足以获得N末端或内部氨基酸序列的至少15个残基的程度，或(3)在使用考马斯蓝或优选地银染色的还原或非还原条件下，通过SDS-PAGE所确定的均一性。分离的抗体包括重组细胞内的原位抗体，因为抗体的天然环境的至少一种组分将不存在。然而，通常，通过至少一个纯化步 骤制备分离的抗体。
基本的4链抗体单元是由两条相同的轻(L)链和两条相同的重(H)链组成的异四聚体糖蛋白(IgM抗体由5个基本的异四聚体单元连同另外的称为J链的多肽组成，并且因此含有10个抗原结合位点，而分泌的IgA抗体可以聚合以形成包含2-5个基本的4链单元与J链的多价组合)。在IgG的情况下，4链单元通常为约150,000道尔顿。每条轻(L)链与重(H)链通过一个共价二硫键连接，而两条H链通过一个或多个二硫键(取决于H链同种型)相互连接。每条H和L链还具有规则间隔的链内二硫桥。每条H链在N末端具有可变结构域(VH)，随后是α和γ链中每一条的三个恒定结构域(CH)，以及μ和ε同种型的四个CH结构域。每条L链在N末端具有可变结构域(VL)，在其另一端具有恒定结构域(CL)。VL与VH对齐，并且CL与重链的第一恒定结构域(CH1)对齐。特定的氨基酸残基被认为在轻链和重链可变结构域之间形成界面。VH和VL的配对一起形成单个抗原结合位点。关于不同类别抗体的结构和特性，参见例如，Basic and Clinical Immunology[基础和临床免疫学],第8版,Daniel P.Stites等人,Appleton&Lange[阿普尔顿和兰格出版社],Norwalk[诺沃克],CT[康涅狄格州],1994,第71页和第6章。
来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被指定为两种明显不同的类型之一，称为κ和λ。取决于其重链(CH)的恒定结构域的氨基酸序列，可以将免疫球蛋白指定为不同类别或同种型。存在五类免疫球蛋白：IgA、IgD、IgE、IgG、和IgM，具有分别被命名为α、δ、γ、ε、和μ的重链。基于CH序列和功能中的相对较小差异，γ和α类进一步被分为亚类，例如，人表达以下亚类：IgG1、IgG2、IgG3、IgG4、IgA1、和IgA2。
术语“可变”是指可变结构域的某些区段在抗体之间的序列上差异很大的事实。V结构域介导抗原结合并定义特定抗体对其特定抗原的特异性。然而，在可变结构域的110个氨基酸跨度上变异性不是均匀分布的。相反，V区由15-30个氨基酸的称为框架区(FR)的相对不变的区段组成，这些区段由被称为“高变区”的较短区域(每个区域长9-12个氨基酸)分隔。天然重链和轻链的可变结构域各自包含主要采用β片层构型的四个FR，其通过三个高变区连接，这三个高变区连接形成连接β片层结构的环，并且在一些情况下形成β-片层结构的一部分。每条链中的高变区由FR维持密切接近地在一起，并且在高变区来自其他链的情况下，对形成抗体的抗原结合部位作出贡献(参见Sequences of Proteins of Immunological Interest[免疫学关注的蛋白序列],第5版,Elvin A.Kabat等人,Public Health Service[公共卫生署],National Institutes of Health[国立卫生研究院],Bethesda[贝塞斯达],MD[马里兰州],1991)。恒定结构域不直接参与抗体与抗原的结合，但表现出各种效应 子功能，例如抗体参与抗体依赖性细胞毒性(ADCC)。
如本文使用的，术语“CDR”或“互补决定区”旨在意指在重链和轻链多肽的可变区内发现的非连续抗原结合位点。这些特定区已经描述于：Kabat EA.等人,J.Biol.Chem.[生物化学杂志],252(19):6609-6616(1977)；Sequences of Proteins of Immunological Interest[免疫学关注的蛋白序列],第5版,Elvin A.Kabat等人,Public Health Service[公共卫生署],National Institutes of Health[国立卫生研究院],Bethesda[贝塞斯达],MD[马里兰州],1991；Chothia C.等人,J.Mol.Biol.[分子生物学杂志],196(4):901-917(1987)；以及MacCallum RM.等人,J.Mol.Biol.[分子生物学杂志],262(5):732-745(1996)，其中定义包括当彼此比较时氨基酸残基的重叠或子集。然而，应用任一定义来指代抗体或移植的抗体或其变体的CDR旨在落入如本文定义和使用的术语的范围内。涵盖如上述每篇参考文献定义的CDR的氨基酸残基列于下表4中作为比较。
表4
Figure PCTCN2020102678-appb-000004
如本文使用的，术语“单克隆抗体”是指从基本上均一的抗体群体中获得的抗体，即包含群体的个体的抗体是相同的，除了可以少量存在的可能天然存在的突变。单克隆抗体是高度特异性的，其针对单个抗原性位点。此外，与多克隆抗体制剂(其包括针对不同决定簇(表位)的不同抗体)不同，每种单克隆抗体针对抗原上的单个决定簇。除了其特异性之外，单克隆抗体的优点还在于它们可以不被其他抗体污染地合成。修饰语“单克隆”不应解释为需要通过任何特定方法产生抗体。例如，可用于本发明的单克隆抗体可以通过首次描述于Kohler G.等人Nature[自然],256(5517):495-497(1975)的杂交瘤方法进行制备，或可以在细菌、真核动物或植物细胞中使用重组DNA方法进行制备(参见例如，美国专利号4,816,567)。还可以使用Clackson T.等人,Nature[自然],352(6336):624-628(1991)，Marks JD.等人,J.Mol.Biol.[分子生物学杂志],222(3):581-597(1991)，以及例如下文实施例中描述的技术从噬菌体抗体文库中分离“单克隆抗体”。
本文的单克隆抗体包括“嵌合”抗体，其中重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源，而这个或 这些链的剩余部分与衍生自另一物种或属于另一抗体类或亚类的抗体、以及此类抗体的片段中的相应序列相同或同源，只要它们表现出本发明的生物活性即可(参见美国专利号4,816,567；和Morrison SL.等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]81(21):6851-6855(1984))。本文的目的嵌合抗体包括“灵长类化”抗体，其包含衍生自非人灵长类(例如，旧世界猴、猿等)的可变结构域抗原结合序列，和人恒定区序列。
“完整”抗体是包含抗原结合位点以及CL和至少重链恒定结构域CH1、CH2和CH3的抗体。恒定结构域可以是天然序列恒定结构域(例如人天然序列恒定结构域)或其氨基酸序列变体。优选地，完整抗体具有一种或多种效应子功能。恒定结构域(恒定区)优选人抗体恒定区，更优选人IgG1、人IgG2、人IgG3或人IgG4抗体恒定区，可选的重链恒定区具体序列如SEQ ID NO：112、113或114所示，可选的轻链恒定区具体序列如SEQ ID NO：115所示。
“抗体片段”包含完整抗体的一部分，优选完整抗体的抗原结合区或可变区。抗体片段的实施例包括Fab、Fab'、F(ab')2、和Fv片段；双抗体；线性抗体(参见美国专利号5,641,870，实施例2；Zapata G.等人,Protein Eng.[蛋白质工程化],8(10):1057-1062(1995))；单链抗体分子；和由抗体片段形成的多特异性抗体。表达“线性抗体”通常是指Zapata G.等人,Protein Eng.[蛋白质工程化]8(10):1057-1062(1995)中描述的抗体。简而言之，这些抗体包含一对串联Fd区段(VH-CH1-VH-CH1)，这些区段与互补的轻链多肽一起形成一对抗原结合区。线性抗体可以是双特异性的或单特异性的。
木瓜蛋白酶对抗体的消化产生了两个相同的抗原结合片段(称为“Fab”片段)和残留的“Fc”片段(此名称反映了易于结晶的能力)。Fab片段由整个L链连同H链的可变区结构域(VH)以及一条重链(CH1)的第一恒定结构域组成。每个Fab片段就抗原结合而言是单价的，即它具有单个抗原结合位点。胃蛋白酶对抗体的处理产生了单个大的F(ab’)2片段，其大致对应于具有二价抗原结合活性的两个二硫键连接的Fab片段，并且仍然能够交联抗原。Fab’片段与Fab片段的区别在于在包括来自抗体铰链区的一或多个半胱氨酸的CH1结构域的羧基末端具有另外的残基。Fab'-SH是本文对于Fab'的命名，其中恒定结构域的一个或多个半胱氨酸残基携带游离巯基。F(ab’)2抗体片段最初是作为Fab'片段对(在其之间有铰链半胱氨酸)生产的。抗体片段的其他化学偶联也是已知的。
Fc片段包含通过二硫键维持在一起的两条H链的羧基末端部分。抗体的效应子功能由Fc区中的序列确定，该区也是在某些类型的细胞上发现的Fc受体(FcR)识别的部分。
“变体Fc区”包含由于如本文定义的至少一个“氨基酸修饰”而不同于天 然序列Fc区的氨基酸序列。优选地，与天然序列Fc区或亲本多肽的Fc区相比，变体Fc区具有至少一个氨基酸取代，例如在天然序列Fc区或亲本多肽的Fc区中从约一个至约十个氨基酸取代，并且优选从约一个至约五个氨基酸取代。在一个实施例中，本文变体Fc区将与天然序列Fc区具有至少约80％同源性、至少约85％同源性、至少约90％同源性、至少约95％同源性或至少约99％同源性。根据另一个实施例中，本文变体Fc区将与亲本多肽的Fc区具有至少约80％同源性、至少约85％同源性、至少约90％同源性、至少约95％同源性或至少约99％同源性。
术语“包含Fc区的多肽”是指一种包含Fc区的多肽，如抗体或免疫黏附蛋白(参见本文其他地方的定义)。可以例如在多肽的纯化期间或通过对编码多肽的核酸进行重组工程化来去除Fc区的C末端赖氨酸(根据EU编号系统的残基447)。因此，根据本披露的包含具有Fc区的多肽(包括抗体)的组合物可以包含去除所有K447残基的多肽群体、没有去除K447残基的多肽群体、或具有和不具有K447残基的多肽混合物的多肽群体。
抗体“效应子功能”是指可归因于抗体的Fc区(天然序列Fc区或氨基酸序列变体Fc区)的那些生物学活性，并且随抗体同种型而变化。抗体效应子功能的实施例包括：C1q结合和补体依赖的细胞毒性；Fc受体结合；抗体依赖的细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体的下调；和B细胞活化。“天然序列Fc区”包含与天然存在的Fc区的氨基酸序列相同的氨基酸序列。Fc序列的实施例描述于例如但不限于：Sequences of Proteins of Immunological Interest[免疫学关注的蛋白序列],第5版,Elvin A.Kabat等人,Public Health Service[公共卫生署],National Institutes of Health[国立卫生研究院],Bethesda[贝塞斯达],MD.[马里兰州],1991。
“Fv”是最小抗体片段，其含有完整抗原识别位点和抗原结合位点。该片段由紧密非共价缔合的一个重链可变区结构域和一个轻链可变区结构域的二聚体组成。从这两个结构域的折叠中发出六个高变环(各自来自H链和L链的3个环)，这些高变环贡献氨基酸残基以用于抗原结合并赋予抗体与抗原结合特异性。
然而，甚至单个可变结构域(或仅包含三个对抗原有特异性的CDR的半个Fv)具有识别并结合抗原的能力，虽然通常以比完整结合位点更低的亲和力进行。
“单链Fv”(也缩写为“sFv”或“scFv”)是包含连接成单个多肽链的VH和VL抗体结构域的抗体片段。优选地，sFv多肽进一步包含在VH与VL结构域之间的多肽接头，该多肽接头使得sFv能够形成用于抗原结合的所希望的结构。关于scFv的综述，参见A.Pluckthun,The Pharmacology of Monoclonal Antibodies[单克隆抗体的药理学]第113卷,Rosenburg和Moore编辑,Springer-Verlag[施普林格出版社],纽约,1994,第269-315页中的 “Antibodies from Escherichia coli[来自大肠杆菌的抗体]”；或Ahmad ZA.等人,Clin.Dev.Immunol.[临床与发育免疫学],2012:980250(2012)。
术语“双抗体”是指通过构建在VH和VL结构域之间具有短接头(约5-10个残基)的sFv片段(参见前述段落)制备的小抗体片段，这样实现V结构域的链间而不是链内配对，产生二价片段，即具有两个抗原结合位点的片段。双特异性双抗体是两个“交叉”sFv片段的异二聚体，其中两个抗体的VH和VL结构域存在于不同的多肽链上。双抗体更全面地描述于，例如，EP 404,097；WO 93/11161；和Hollinger P.等人,Proc.Natl.Acad.Sci.USA,[美国国家科学院院刊]90(14):6444-6448(1993)。
“人源化”形式的非人(例如啮齿动物)抗体是嵌合抗体，其含有衍生自非人抗体的最小序列。大多数情况下，人源化抗体是人免疫球蛋白(受体抗体)，其中来自受体高变区的残基被来自非人物种(例如小鼠、大鼠、兔或非人灵长类)的具有所希望的抗原特异性、亲和力和容量的高变区(供体抗体)的残基替换。在一些实施例中，人免疫球蛋白的框架区(FR)残基被相应的非人残基替换。
另外，人源化抗体可以包括未在受体抗体或供体抗体中发现的残基。进行这些修饰以进一步改善抗体性能。通常，人源化抗体将包括至少一个、并且典型地两个可变结构域中的基本上全部，其中全部或基本上全部的高变环对应于非人类免疫球蛋白的那些，并且全部或基本上全部的FR是人类免疫球蛋白序列的那些。人源化抗体还将任选地包括免疫球蛋白恒定区的至少一部分，典型地是人免疫球蛋白的至少一部分。关于另外的细节，参见Jones PT.等人,Nature[自然],321(6069):522-525(1986)；Riechmann L.等人,Nature[自然],332(6162):323-329(1988)；以及Presta LG,Curr.Opin.Biotechnol.[生物技术当前述评],3(4):394-398(1992)。
将“关于本文鉴定的多肽和抗体序列的氨基酸序列同一性百分比(％)”或“同源性”定义为在对齐序列(考虑任何保守取代作为序列同一性的一部分)后，候选序列中与所比较多肽中的氨基酸残基相同的氨基酸残基的百分比。出于确定氨基酸序列同一性百分比的目的，可以用本领域技术中的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。然而，出于本文的目的，使用序列比较计算机程序ALIGN-2生成氨基酸序列同一性％值。ALIGN-2序列比较计算机程序由基因泰克公司(Genentech,Inc.)编写，并且源代码已经与美国版权局(U.S.Copyright Office)(华盛顿特区(Washington D.C.)，20559)中的用户文档一起提交，其在美国版权登记号TXU510087下注册。ALIGN-2程序 可通过加利福尼亚州南旧金山的基因泰克公司(Genentech,Inc.)公开获得。应编译ALIGN-2程序以在UNIX操作系统(优选数字UNIX V4.0D)上使用。所有序列比较参数均由ALIGN-2程序设置并且不变。
术语“Fc受体”或“FcR”用于描述结合抗体Fc区的受体。在一个实施例中，本发明的FcR是结合IgG抗体的FcR(γ受体)，并且包括FcγRI、FcγRII和FcγRIII亚类的受体，包括这些受体的等位基因变体和可替代地剪接形式。FcγRII受体包括FcγRIIA(“活化受体”)和FcγRIIB(“抑制受体”)，FcγRIIA和FcγRIIB具有类似的氨基酸序列(这些序列主要在其细胞质结构域中不同)。活化受体FcγRIIA在其细胞质结构域中含有基于免疫受体酪氨酸的活化基序(ITAM)。抑制受体FcγRIIB在其细胞质结构域中含有基于免疫受体酪氨酸的抑制基序(ITIM)(参见
Figure PCTCN2020102678-appb-000005
M,Annu.Rev.Immunol.[免疫学年度评论],15:203-234(1997))。该术语包括同种异型，如FcγRIIIA同种异型：FcγRIIIA-Phe158、FcγRIIIA-Val158、FcγRIIA-R131和/或FcγRIIA-H131。FcRs综述于Ravetch JV.等人,Annu.Rev.Immunol.[免疫学年度评论],9:457-492(1991)；Capel PJ.等人,Immunomethods[免疫方法],4(1):25-34(1994)；和de Haas M.等人,J.Lab.Clin.Med.[实验与临床医学杂志],126(4):330-341(1995)中。本文的术语“FcR”涵盖其他FcR，包括将来鉴定的FcR。该术语还包括新生儿受体FcRn，其负责将母体IgG转移至胎儿(Guyer RL.等人,J.Immunol.[免疫学杂志],117(2):587-593(1976)和Kim JK.等人,Eur.J.Immunol.[欧洲免疫学杂志],24(10):2429-2434(1994))。
术语“FcRn”是指新生儿Fc受体(FcRn)。FcRn在结构上与主要组织相容性复合体(MHC)类似，并且由与β2-微球蛋白非共价结合的α链组成。新生儿Fc受体FcRn的多种功能综述于Ghetie V.等人,Annu.Rev.Immunol.[免疫学年度评论],18:739-766(2000)中。FcRn在从母亲到后代的被动递送免疫球蛋白IgG和调节血清IgG水平中起作用。FcRn可以作为补救受体，以完整形式结合和运输(两者都在细胞内或跨细胞)胞饮化的IgG，并从默认的降解途径中拯救它们。
人IgG Fc区的“CH1结构域”(也称为“H1”的“C1”)“结构域”通常从约氨基酸118延伸至约氨基酸215(EU编号系统)。
“铰链区”通常定义为从人IgG1的Glu216延伸至Pro230(Burton DR.,Mol.Immunol.[分子免疫学],22(3):161-206(1985))。其他IgG同种型的铰链区可以通过将第一个和最后一个形成重链间S-S键的半胱氨酸残基置于相同位置而与IgG1序列比对。
Fc区的“下铰链区”通常定义为紧邻铰链区C末端的残基区，即Fc区的残基233至239。在先前的报道中，FcR结合通常归因于IgG Fc区的下铰链区中 的氨基酸残基。
人IgG Fc区的“CH2结构域”(也称为“H2”的“C2”)“结构域”通常从约氨基酸231延伸至约氨基酸340。CH2结构域是独特的，因为其不与另一个结构域紧密配对。而是，两个N连接的支链碳水化合物链插入完整天然IgG分子的两个CH2结构域之间。据推测，碳水化合物可以提供结构域-结构域配对的替代物并有助于稳定CH2结构域(Burton DR.,Mol.Immunol.[分子免疫学],22(3):161-206(1985))。
“CH3结构域”(也称为“C2”或“H3”结构域)包含Fc区中靠CH2结构域C末端的残基区域(即从约氨基酸残基341至抗体序列的C末端(典型地在IgG的氨基酸残基446或447处))。
“功能Fc区”具有天然序列Fc区的“效应子功能”。示例性“效应子功能”包括C1q结合；补体依赖的细胞毒性；Fc受体结合；抗体依赖的细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体；BCR)的下调等。此类效应子功能通常需要Fc区与结合结构域(例如抗体可变结构域)组合，并且可以使用例如本文披露的各种测定法进行评估。
“C1q”是包含免疫球蛋白Fc区的结合位点的多肽。C1q与两种丝氨酸蛋白酶C1r和C1s一起形成复合物C1(补体依赖的细胞毒性(CDC)途径的第一组分)。人C1q可以从例如加利福尼亚州(CA)圣地亚哥(San Diego)的基代尔公司(Quidel)商业购买。
术语“结合结构域”是指与另一分子结合的多肽的区域。在FcR的情况下，结合结构域可以包含其负责结合Fc区的多肽链部分(例如，其α链)。一个有用的结合结构域是FcRα链的细胞外结构域。
具有变体IgG Fc(该变体具有“改变的”FcR结合亲和力或ADCC活性)的抗体是与亲本多肽或包含天然序列Fc区的多肽相比具有增强或减弱的FcR结合活性(例如，FcγR或FcRn)和/或ADCC活性的抗体。针对FcR“表现出增加的结合”的变体Fc结合至少一个比亲本多肽或天然序列IgG Fc具有更高的亲和力(例如，更低的表观Kd或IC50值)的FcR。根据一些实施例，与亲本多肽相比，结合的改善为约3倍，优选地约5倍、10倍、25倍、50倍、60倍、100倍、150倍、200倍、高达500倍、或约25％至1000％的结合的改善。针对FcR“表现出减少的结合”的多肽变体结合至少一个比亲本多肽具有更低的亲和力(例如，更高的表观Kd或更高的IC50值)的FcR。与亲本多肽相比结合的减少可以为约40％或更多的结合减少。
“抗体依赖的细胞介导的细胞毒性”或“ADCC”是指一种细胞毒性形式，其中分泌的Ig与某些细胞毒性细胞(例如自然杀伤(NK)细胞、中性粒细胞和巨噬细胞)上存在的Fc受体(FcR)结合使得这些细胞毒性效应细胞特异性结合 携带抗原的靶细胞，并随后用细胞毒素杀伤该靶细胞。抗体“武装”细胞毒性细胞并且对于这种杀伤是绝对必需的。用于介导ADCC的原代细胞NK细胞仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。造血细胞上的FcR表达总结于Ravetch JV.等人,Annu.Rev.Immunol.[免疫学年度评论]9:457-492(1991)的第464页的表3中。为了评估目的分子的ADCC活性，可以进行体外ADCC测定(如美国专利号5,500,362或5,821,337或下文实施例中描述的)。用于此类测定的有用的效应细胞包括外周血单核细胞(PBMC)和自然杀伤(NK)细胞。可替代地或另外地，可以在体内评估目的分子的ADCC活性，例如在动物模型中，如Clynes R.等人,Proc.Natl.Acad.Sci.USA,[美国国家科学院院刊]95(2):652-656(1998)中披露的。
包含变体Fc区(“表现出增加的ADCC”或在人效应细胞的存在下比具有野生型IgG Fc的多肽或亲本多肽更有效地介导抗体依赖的细胞介导的细胞毒性(ADCC))的多肽是当在测定中具有变体Fc区的多肽和具有野生型Fc区的多肽(或亲本多肽)的量基本上相同时，在体外或体内基本上更有效地介导ADCC的多肽。通常，使用本领域已知的任何体外ADCC测定鉴定此类变体，如用于确定ADCC活性的测定或方法(例如在动物模型中等)。在一个实施例中，优选的变体是比野生型Fc(或亲本多肽)从约5倍至约100倍(例如从约25至约50倍)更有效地介导ADCC。
“补体依赖的细胞毒性”或“CDC”是指在补体存在下靶细胞的裂解。经典补体途径的活化由补体系统的第一组分(C1q)与(合适的亚类的)抗体(该抗体与其同源抗原结合)的结合而引发。为了评估补体活化，可以进行CDC测定(例如，如Gazzano-Santoro H.等人,J.Immunol.Methods[免疫学方法杂志],202(2):163-171(1997)中描述的)。具有改变的Fc区氨基酸序列、和增加的或减少的C1q结合能力的多肽变体描述于美国专利号6,194,551B1和WO99/51642。这些专利公开物的内容通过引用明确地并入本文(还参见Idusogie EE.等人,J.Immunol.[免疫学杂志],164(8):4178-4184(2000))。
如本文披露的抗CD73抗体(或其片段)或组合物的“有效量”是足以实现特定声明的目的的量。“有效量”可以凭经验和通过与所声明的目的相关的已知方法确定。术语“治疗有效量”是指如本文披露的抗CD73抗体(或变体或其抗原结合片段)或组合物在哺乳动物(亦称患者)中有效“治疗”疾病或障碍的量。在癌症的情况下，如本文披露的抗CD73抗体(或变体或其抗原结合片段)或组合物的治疗有效量能减少癌细胞的数量；减小肿瘤大小或重量；抑制(即，在一定程度上减慢并且优选地停止)癌细胞浸润到外周器官中；抑制(即，在一定程度上减慢并且优选地停止)肿瘤转移；在一定程度上抑制肿瘤生长；和/或在一定程度上缓解与癌症相关的一种或多种症状。在一定程度上，如本文披 露的抗CD73抗体(或变体或其抗原结合片段)或组合物可以防止现有癌细胞生长和/或杀死现有癌细胞，该抗体(或其变体或抗原结合片段)或组合物可以是细胞抑制性的和/或细胞毒性的。在一个实施例中，治疗有效量是生长抑制量。在另一个实施例中，治疗有效量是延长患者存活的量。在另一个实施例中，治疗有效量是改善患者无进展存活的量。
本发明的抗CD73抗体(或变体或其抗原结合片段)或如本文披露的组合物的“生长抑制量”是能够在体外或体内抑制细胞(尤其是肿瘤，例如癌细胞)生长的量。出于抑制赘生性细胞生长的目的的本披露的多肽、抗体、拮抗剂或组合物的“生长抑制量”可以凭经验和通过已知的方法或通过本文提供的实施例确定。
本发明的抗CD73抗体(或变体或其抗原结合片段)或组合物的“细胞毒性量”是能够在体外或体内破坏细胞(尤其是肿瘤，例如癌细胞)的量。出于抑制赘生性细胞生长的目的的本发明的抗CD73抗体(或变体或其抗原结合片段)或组合物的“细胞毒性量”可以凭经验和通过本领域已知的方法确定。
本发明的抗CD73抗体(或变体或其抗原结合片段)或组合物的“生长抑制量”是能够在体外或体内抑制细胞(尤其是肿瘤，例如癌细胞)生长的量。出于抑制赘生性细胞生长的目的的本披露的抗CD73抗体(或变体或其抗原结合片段)或组合物的“生长抑制量”可以凭经验和通过已知的方法或通过本文提供的实施例确定。
如本文使用的，“药学上可接受的”或“药理学上相容的”是指不具有生物学或其他方面不希望的材料，例如，该材料可以掺入给予患者的药物组合物中，而不会与包含该材料的该组合物的任何其他组分以有害的方式引起任何显著的不希望的生物学作用或相互作用。药学上可接受的载体或赋形剂优选满足毒理学和制造测试的所需标准和/或包含在由美国食品和药物管理局(U.S.Food and Drug administration)编制的非活性成分指南中。
术语“检测”旨在包括确定物质的存在或不存在，或量化物质(如CD73)的量。因此，该术语是指本发明的材料、组合物和方法用于定性和定量确定的用途。通常，用于检测的特定技术对于本发明的实践并不重要。
例如，根据本发明的“检测”可以包括：观察CD73基因产物、mRNA分子、或CD73多肽的存在或不存在；CD73多肽水平或与靶标结合的量的变化；CD73多肽的生物学功能/活性的变化。在一些实施例中，“检测”可以包括检测野生型CD73水平(例如，mRNA或多肽水平)。检测可以包括与对照相比量化在10％和90％之间的任何值，或在30％和60％之间或100％以上的任何值的变化(增加或减少)。检测可以包括量化在2倍至10倍之间的任何值(包括端值)或更多(例如100倍)的变化。
当在本文中使用时，术语“标记”是指可直接或间接与抗体缀合的可检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)，或者在酶标记的情况下可以催化可检测的底物化合物或组合物的化学改变。
抗CD73抗体和亲和变体/突变体
本发明涉及结合如本文披露的CD73受体的抗体。所披露的抗CD73抗体、及它们的亲和变体和/或突变体、或其抗原结合片段可以用于各种治疗性和诊断性方法。抗CD73抗体是以具有足够的亲和力和特异性与CD73结合的抗体。在一些实施例中，所披露的抗CD73抗体(或其变体或突变体或抗原结合片段)可以在靶向和干扰与CD73的表达相关的疾病或病症中用作治疗剂。在各个方面，所披露的抗CD73抗体(或变体/突变体或其抗原结合片段)显示与其他蛋白质的最小结合。在一些实施例中，优选所披露的抗CD73抗体(或变体/突变体或其抗原结合片段)是人或重组人源化抗CD73单克隆抗体。
不希望受特定理论的束缚，据信所披露的抗CD73抗体、及它们的亲和变体和/或突变体、或其抗原结合片段可以增加表达CD73的细胞中CD73的内化，从而导致减少在所披露的抗CD73抗体、及它们的亲和变体和/或突变体、或其抗原结合片段结合的细胞上的质膜上CD73蛋白的量。因此，质膜上CD73蛋白量的减少与和CD73相关的5’-核苷酸酶活性的降低是相关的。在一些方面，再次不希望受特定理论的束缚，据信由于5’-核苷酸酶活性降低，与CD73相关的5'-核苷酸酶活性的降低(经由增加的内化)可以经由肿瘤位点处增强的免疫细胞功能来减少肿瘤细胞增殖。
如本文披露的，所披露的抗CD73抗体、及它们的亲和变体和/或突变体或抗原结合片段被认为在与CD73表达相关的其他临床病症(例如纤维化，包括特发性肺纤维化)中具有有益作用。不希望受特定理论的束缚，据信在其他此类临床病症中，由于表达CD73的适当细胞的CD73蛋白减少(和相应的，5’-核苷酸酶活性降低)，所披露的抗CD73抗体及它们的亲和变体和/或突变体或抗原结合片段可以对患者(例如纤维化(包括特发性肺纤维化))具有积极的临床益处。
根据某些实施例中，抗CD73抗体包含本文披露的抗体中的任一个的CDR、可变重链区、和/或可变轻链区。
本发明提供了抗CD73抗体、它们的亲和变体/突变体、和/或其抗原结合片段。在某些实施例中，本发明的特定抗CD73抗体、它的一种或多种亲和变体/一种或多种突变体，和/或其一种或多种抗原结合片段包含轻链(LC)可变结构域序列(包含如上表1中列出的特异性CDR-L1、CDR-L2和CDR-L3序列)和重链(HC)可变结构域序列(包含如上表2中列出的特异性CDR-H1、CDR-H2和CDR-H3序列)。本发明的每个特定抗CD73抗体、它的一种或多种亲和变体/一种或多种 突变体、和/或其一种或多种抗原结合片段的每条轻链和重链的核酸和它的氨基酸序列也在下文序列表中提供。本发明提供了重链和轻链可变结构域，并且本文提到的CDR以所有可能的成对方式组合，以生成大量抗CD73抗体、及它们的一种或多种亲和变体/一种或多种突变体、和/或其一种或多种抗原结合片段。
在某些实施例中，一个或多个氨基酸取代是一个或多个保守氨基酸取代。在某些实施例中，氨基酸取代基本上不降低抗体与抗原结合的能力。例如，可以进行基本上不降低所披露的抗体与靶CD73的结合亲和力的保守的改变(例如，如本文提供的保守取代)。可以使用下文实施例中描述的方法来评估抗CD73抗体变体的结合亲和力。
保守取代示于“保守取代”的标题下表5中。“示例性取代”的标题下的表4提供了更实质的变化，并且如下文参考氨基酸侧链类别进一步描述的。氨基酸取代可以引入目的抗体中，并针对所希望的活性(例如保留/改善的CD73结合、降低的免疫原性、或改善的ADCC或CDC)筛选产物。
表5：保守取代
原始残基 示例性取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；Norleucine Leu
Leu(L) Norleucine；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；Norleucine Leu
非保守取代将需要将这些类别中的一个的成员交换为另一个类别。示例性取代变体是亲和力成熟的抗体，该抗体可以方便地生成，例如使用基于噬菌体展示的亲和力成熟技术(例如本文披露的那些)。简而言之，对一个或多个CDR残基进行突变，并且将变体抗体在噬菌体上展示并针对特定生物学活性(例如结合亲和力)进行筛选。可以在HVR中进行改变(例如，取代)，例如以改善抗体亲和力。此类改变可以在HVR“热点”(即由在体细胞成熟过程期间以高频率经历突变的密码子编码的残基)中进行(参见例如，Chowdhury,Methods Mol.Biol.[分子生物学方法]207:179-196(2008))和/或在SDR(a-CDR)中进行，测试所得变体VH或VL的结合亲和力。通过构建二级文库和从二级文库重选进行的亲和力成熟已经描述于例如Hoogenboom HR,Methods in Mol.Biol.[分子生物学方法]178:1-37(2002)；Antibody Phage Display[抗体噬菌体展示],Philippa O'Brien,R.A.,Humana Press[哈门那出版社],Totowa[托托瓦],NJ[新泽西州],2001。
在亲和力成熟的一些实施例中，通过多种方法(例如，易错PCR，链改组，或寡核苷酸定向诱变)中的任一种，将多样性引入经选择用于成熟的可变基因中。然后产生二级文库。然后筛选文库以鉴别具有所希望的亲和力的任何抗体变体。引入多样性的另一种方法涉及HVR定向方法，其中使若干HVR残基(例如一次4-6个残基)随机化。可例如使用丙氨酸扫描诱变或模型化来特异性地鉴别抗原结合中涉及的HVR残基。
在某些实施例中，抗CD73抗体、变体或突变体或其抗原结合片段可能在重链或轻链可变区中缺乏N糖基化基序，这可能导致一批抗体内的差异，造成功能、免疫原性或稳定性改变。分析抗体糖基化的方法包括但不限于例如色谱法(例如阳离子交换色谱法(CEX)或液相色谱法)、质谱法(例如电喷雾电离质谱法)和毛细管电泳-十二烷基硫酸钠。此类方法描述于例如，Jung ST.等人,Curr.Opin.Biotechnol.[生物技术当前述评],22(6):858-67(2011)；Cummings RD,Etzler ME.Antibodies and Lectins in Glycan Analysis.[多糖分析中的抗体和凝集素].于:Varki A,Cummings RD,Esko JD等人编辑.Essentials of Glycobiology[糖生物学的本质].第2版.Cold Spring Harbor[冷泉港](纽约):Cold Spring Harbor Laboratory Press[冷泉港实验室出版社]；2009.第45章；Mulloy B,Hart GW,Stanley P.Structural Analysis of Glycans.[聚糖的结构分析]于:Varki A,Cummings RD,Esko JD等人编辑.Essentials of Glycobiology.[糖生物学的本质]第2版.Cold Spring Harbor[冷泉港](纽约):Cold Spring Harbor Laboratory Press[冷泉港实验室出版社]；2009.第47章；Leymarie N.等人,Anal.Chem.[分析化学], 84(7):3040-3048(2012)；Fernandez DD.,European Biopharmaceutical Review[欧洲生物制药评论]第106-110页(2005)；以及Raju TS,Methods Mol.Biol.[分子生物学方法],988:169-180(2013)。
在某些实施例中，特定抗CD73抗体突变体的突变体在一个或多个CDR区中的N糖基化位点处包含一个或多个突变，例如序列子N-X-S/T。在N糖基化位点具有一个或多个突变体的抗CD73抗体突变体消除了N糖基化位点但仍保留了等同的功能。
在某些实施例中，抗CD73抗体、变体和/或突变体或其抗原结合片段对CD73的结合亲和力高于对CD73同源物的结合亲和力。通常，抗CD73抗体、和/或变体或其抗原结合片段“特异性结合”CD73(即，具有不超过约1x 10 -7M，优选地不超过约1x 10 -8和最优选地不超过约1x 10 -9M的结合亲和力(Kd)值)，但对非CD73具有比其对CD73的结合亲和力弱至少约50倍、或至少约500倍、或至少约1000倍的结合亲和力。特异性结合CD73的抗CD73抗体可以是如上定义的任何各种类型的抗体，但优选是人源化或人抗体。
在一些实施例中，如通过本领域已知的方法(如ELISA、荧光活化细胞分选(FACS)分析、或放射免疫沉淀(RIA))确定的，抗CD73抗体与非靶蛋白的结合程度小于抗体与CD73的结合的约10％。可以例如通过确定与对照分子(通常是不具有结合活性的具有类似结构的分子)的结合相比的分子的结合来测量特异性结合。例如，可以通过与靶标(例如，过量的未标记靶标)类似的对照分子竞争来确定特异性结合。在这种情况下，如果标记的靶标与探针的结合被过量的未标记的靶标竞争性地抑制，则指示特异性结合。如本文使用的，术语“特异性结合”特定多肽或特定多肽靶上的表位或“与特定多肽或特定多肽靶上的表位特异性结合”或对特定多肽或特定多肽靶上的表位具有“特异性”可以通过以下表现：例如，对靶标具有至少约10 -4M、可替代地至少约10 -5M、可替代地至少约10 -6M、可替代地至少约10 -7M、可替代地至少约10 -8M、可替代地至少约10 -9M、可替代地至少约10 -10M、可替代地至少约10 -11M、可替代地至少约10 -12M、或更大的Kd的分子。在一个实施例中，术语“特异性结合”是指以下结合：其中分子与特定多肽或特定多肽上的表位结合而基本上不结合任何其他多肽或多肽表位。
抗体依赖的细胞介导的细胞毒性(ADCC)是针对肿瘤细胞的治疗性抗体的作用机制。ADCC是细胞介导的免疫防御，免疫系统的效应细胞借此主动裂解靶细胞(例如，癌细胞)，这些靶细胞的膜表面抗原被特异性抗体(例如，如本文描述的抗CD73抗体和/或亲和变体)结合。在一些实施例中，抗CD73抗体和/或亲和变体表现出与参照抗CD73单克隆抗体类似的抗体依赖的细胞介导的细胞毒性(ADCC)效应子功能，如例如通过实施例中描述的测定所证明的。
例如，在某些实施例中，本文描述的抗CD73抗体和/或其亲和变体或突变体的ADCC效应子功能活性是参照抗CD73抗体的ADCC效应子功能活性的至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％、至少约100％、或多于100％(例如，约105％、约106％、约107％、约108％、约109％、约110％、约111％、约112％、约113％、约114％、约115％、约116％、约117％、约118％、约119％、约120％、约121％、约122％、约123％、约124％、约125％、或约130％)，包括这些值之间的任何范围。
在某些实施例中，抗CD73抗体、和/或其亲和变体或其突变体表现出与参照抗CD73抗体类似的对CD73的结合亲和力。在某些实施例中，与CD73结合是通过ELISA证明的，如实施例中描述的。例如，抗CD73和/或其亲和变体或其突变体对CD73的结合亲和力比参照抗CD73抗体对CD73的结合亲和力高约1％、约5％、约10％、约15％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约95％、约96％、约97％、约98％、约99％、约100％、或多于100％(例如，约105％、约106％、约107％、约108％、约109％、约110％、约111％、约112％、约113％、约114％、约115％、约116％、约117％、约118％、约119％、约120％、约121％、约122％、约123％、约124％、约125％、或多于约125％)。
在某些实施例中，抗CD73抗体、和/或其亲和变体或其突变体以在约0.1pM至200pM(0.2nM)之间(例如约0.1pM、约0.25pM、约0.5pM、约0.75pM、约1pM、约5pM、约10pM、约20pM、约30pM、约40pM、约50pM、约60pM、约70pM、约80pM、约90pM、约100pM、约110pM、约120pM、约130pM、约140pM、约150pM、约160pM、约170pM、约180pM、约190pM、或多于约190pM，包括这些值之间的任何范围)的Kd结合人CD73。在某些实施例中，抗CD73抗体、和/或其亲和变体或其突变体与CD73的结合亲和力比参照抗CD73抗体与CD73的结合亲和力高约1％、约5％、约10％、约15％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约95％、约96％、约97％、约98％、约99％、约100％、或多于约100％(例如，约105％、约110％、约120％、或约130％)。在某些实施例中，抗CD73、和/或其变体或其突变体与CD73的结合亲和力比参照抗CD73抗体与CD73的结合亲和力高约1.1倍、约1.2倍、约1.3倍、约1.4倍、约1.5倍、约1.6倍、约1.7倍、约1.8倍、约1.9倍、约2倍、约2.25倍、约2.5倍、约2.75倍、约3倍、约3.25倍、约3.5倍、约3.75倍、约4倍、约4.25倍、约4.5倍、约4.75倍、或多于约4.75倍，包括这些值之间的任何范围。
在某些实施例中，与参照抗CD73抗体相比，本文提供的抗CD73抗体、和/或其变体或其突变体具有延长的体内半衰期。在某些实施例中，本文描述的抗 CD73抗体、和/或其变体或其突变体的体内半衰期不短于参照抗CD73抗体的体内半衰期。
在某些实施例中，本文提供的抗CD73抗体、和/或其变体或其突变体表现出与参照抗CD73抗体类似的药代动力学特性。在某些实施例中，本文提供的抗CD73抗体、和/或其变体或其突变表现出参照抗CD73抗体的血清浓度-时间曲线的约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％、或高于95％(如约96％、约97％、约98％、约99％、或多于约99％)(包括这些值之间的任何范围)的AUC(曲线下面积)。
在某些实施例中，本发明的抗CD73抗体、和/或其变体或其突变体包含人IgG(例如，人IgG1或人IgG4)的Fc序列。在某些实施例中，Fc序列已经被改变或以其他方式被改变，使得其缺乏抗体依赖的细胞毒性(ADCC)效应子功能，这通常和其与Fc受体(FcR)的结合相关。有许多可以改变效应子功能的Fc序列的变化或突变的实施例。例如，WO00/42072，以及Shields等人J Biol.Chem.[生物化学杂志]9(2):6591-6604(2001)描述了具有改善或减弱的与FcR的结合的抗体变体。这些出版物的内容通过引用明确地并入本文。本披露的抗CD73抗体、和/或其变体或其突变体可以处于以下形式：Fab、Fab'、F(ab')2、单链Fv(scFv)、Fv片段；双抗体和线性抗体。此外，本披露的抗CD73抗体、和/或其变体或其突变体可以是与CD73结合但也与一个或多个其他其突变体结合并抑制其一种或多种功能的多特异性抗体、和/或其变体或突变体。本披露的抗CD73抗体、和/或其变体或其突变体可以与治疗剂(例如，细胞毒性剂、放射性同位素和化学治疗剂)或用于通过成像在患者样品中或体内检测CD73的标记(例如，放射性同位素、荧光染料和酶)进行缀合。本文还提供了其他修饰，包括毒素与抗CD73抗体、和/或其变体或突变体的缀合。
本文还考虑了编码抗CD73抗体、和/或其变体和/或其突变体的核酸分子，和包含编码CDR和/或重链可变结构域和/或轻链可变结构域的核酸分子的表达载体，并且还考虑了包含这些核酸分子的细胞。本发明的抗CD73抗体、和/或其变体或其突变体可以在本文描述的疗法中使用并且可以用于在患者样品(例如，经由FACS、免疫组织化学(IHC)、ELISA测定)或患者中检测CD73蛋白。
单克隆抗体
单克隆抗体可以例如使用杂交瘤方法(如描述于Kohler G.等人,Nature[自然],174(5):2453-2455(2005))制备或可以通过重组DNA方法(美国专利号4,816,567)制作或可以通过下文实施例中本文描述的方法产生。在杂交瘤方法中，典型地用免疫剂使仓鼠、小鼠或其他适当的宿主动物免疫，以引发产生或能够产生与免疫剂特异性结合的抗体的淋巴细胞。可替代地，可使淋巴细胞体 外免疫。
免疫剂将典型地包括多肽或目的蛋白的融合蛋白或包含蛋白质的组合物。通常，如果希望是人源细胞，则使用外周血淋巴细胞(“PBL”)，或者如果希望是非人哺乳动物来源，则使用脾细胞或淋巴结细胞。然后使用合适的融合剂(例如聚乙二醇)将淋巴细胞与永生化细胞系融合以形成杂交瘤细胞(Monoclonal Antibodies:principles and practice[单克隆抗体：原理和实践],James W.Goding,纽约:Academic Press[学术出版社公司],1986,第59-103页)。永生化细胞系通常是转化的哺乳动物细胞，特别是啮齿动物、牛和人来源的骨髓瘤细胞。通常，采用大鼠或小鼠骨髓瘤细胞系。杂交瘤细胞可以在合适的培养基中培养，该培养基优选地含有一种或多种抑制未融合的永生化细胞的生长或存活的物质。例如，如果亲代细胞缺少酶次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT或HPRT)，杂交瘤的培养基典型地包含次黄嘌呤、氨蝶呤和胸苷(HAT培养基)，这些物质阻止HGPRT缺陷型细胞的生长。
优选的永生化细胞系是有效融合、支持经选择的产生抗体的细胞稳定高水平表达抗体、并且对培养基(如HAT培养基)敏感的那些。更优选的永生化细胞系是鼠骨髓瘤细胞系，其可以获得自：例如Salk Institute[索尔克研究所]Cell Distribution Center[细胞分布中心],San Diego[圣地亚哥],California[加利福尼亚州]和American Type Culture Collection[美国典型培养物保藏中心],Manassas[马纳萨斯],Virginia[弗尼吉亚州]。还描述了人骨髓瘤及小鼠-人杂合骨髓瘤细胞系用于产生人单克隆抗体(Kozbor D.等人,J.Immunol.[免疫学杂志],133(6):3001-3005(1984)；Monoclonal Antibody Production Techniques and Applications[单克隆抗体生产技术及应用],Brodeur等人,Marcel Dekker,Inc.[马塞尔德克尔公司]:纽约,1987,第51-63页)。
然后可以测定培养杂交瘤细胞的培养基中是否存在针对多肽的单克隆抗体。由杂交瘤细胞产生的单克隆抗体的结合特异性可以通过免疫沉淀或通过体外结合测定来确定，例如放射免疫测定(RIA)或酶联免疫吸附测定(ELISA)。此类技术和测定在本领域中是已知的。单克隆抗体的结合亲和力可以例如通过Munson PJ.等人,Anal.Biochem.[分析生物化学],107(1):220-239(1980)的斯卡查德(Scatchard)分析来确定。
在鉴定出所希望的杂交瘤细胞后，可以将克隆通过有限稀释程序进行亚克隆并通过标准方法生长(Monoclonal Antibodies:principles and practice[单克隆抗体：原理和实践],James W.Goding,纽约:Academic Press[学术出版社公司],1986,第59-103页)。用于此目的的合适培养基包括例如杜氏改良培养基(Dulbecco's Modified Eagle's Medium)和RPMI-1640培养基。可替代地，杂交瘤细胞可以在哺乳动物体内作为腹水生长。
亚克隆分泌的单克隆抗体可以通过常规的免疫球蛋白纯化程序(例如蛋白A-琼脂糖、羟基磷灰石色谱法、凝胶电泳、透析或亲和色谱法)从培养基或腹水液中分离或纯化。
单克隆抗体也可以通过本领域已知的和/或后来开发的重组DNA方法制备。使用常规的程序(例如，通过使用能够与编码鼠类抗体的重链和轻链的基因特异性结合的寡核苷酸探针)，可以容易地对编码本文提供的单克隆抗体的DNA进行分离和测序。本文提供的杂交瘤细胞用作这种DNA的优选来源。一旦被分离，DNA被置于表达载体中，然后将这些载体转染到不另外产生免疫球蛋白的宿主细胞(如猿COS细胞、中国仓鼠卵巢(CHO)细胞或骨髓瘤细胞)中，以便在重组宿主细胞中获得单克隆抗体的合成。还可以例如通过以同源鼠类序列取代人重链和轻链恒定域的编码序列(例如，Morrison等人，同上)，或通过将非免疫球蛋白多肽全部或部分的编码序列共价结合至免疫球蛋白编码序列来修饰DNA。这种非免疫球蛋白多肽可以取代本文提供的抗体的恒定结构域，或者可以取代本文提供的抗体的一个抗原结合位点的可变结构域以产生嵌合二价抗体。
在某些实施例中，本发明提供的抗CD73抗体和/或其变体或其突变体由稳定的哺乳动物细胞系表达。在某些实施例中，本发明提供的抗CD73抗体和/或其变体或其突变体以约2.0克/升、约2.5克/升、约3.0克/升、约3.5克/升、约4.0克/升、约4.5克/升、约5.0克/升、约5.5克/升、约6.0克/升、约6.5克/升、约7.0克/升、或多于约7.0克/升(包括这些值之间的任何范围)的滴度从稳定的哺乳动物细胞系表达。在某些实施例中，表达本发明提供的抗CD73抗体和/或其变体或其突变体的稳定的哺乳动物细胞系是CHO细胞系。
在某些实施例中，抗体是单价抗体。用于制备单价抗体的方法是本领域已知的。例如，一种方法涉及免疫球蛋白轻链和修饰的重链的重组表达。通常在Fc区中的任何点截短重链以防止重链交联。可替代地，相关的半胱氨酸残基被另一个氨基酸残基取代或被删除以防止交联。
体外方法也适于制备单价抗体。可以使用但不限于本领域已知的技术来完成消化抗体以产生其片段(特别是Fab片段)。
人抗体和人源化抗体
本发明的抗CD73抗体、和/或其变体或其突变体是人抗体。它们也可以是人源化抗体。非人(例如鼠类)抗体的人源化形式是嵌合免疫球蛋白、免疫球蛋白链或其典型地含有衍生自非人免疫球蛋白的最小序列的片段(例如Fv、Fab、Fab’、F(ab’)2或抗体的其他抗原结合子序列)。人源化抗体包括人免疫球蛋白(受体抗体)，其中来自受体CDR的残基被来自非人物种(供体抗体)(如具有所希望的特异性、亲和力和能力的小鼠、大鼠或兔)的CDR的残基替代。在 一些实施例中，人免疫球蛋白的Fv框架残基可以被相应的非人残基替代。人源化抗体还可以包含既不在受体抗体中也不在导入的CDR或框架序列中发现的残基。通常，人源化抗体可包含至少一个、典型地两个可变结构域中的基本上全部，其中全部或基本上全部的CDR区对应于非人免疫球蛋白的那些区，以及全部或基本上全部的FR区是人免疫球蛋白共有序列的那些区。人源化抗体还优选地包含免疫球蛋白恒定区(Fc)的至少一部分，典型地是人免疫球蛋白恒定区的至少一部分(例如，Jones PT.等人,Nature[自然],321(6069):522-525(1986)；Riechmann L.等人,Nature[自然],332(6162):323-327(1988)；Presta LG,Curr.Opin.Biotechnol.[生物技术当前述评],3(4):394-398(1992))。
通常，人源化抗体具有从非人来源引入的一个或多个氨基酸残基。这些非人氨基酸残基通常被称为“输入”残基，它们典型地取自一种“输入”可变结构域。根据一个实施例中，基本上按照Winter和同事的方法，通过用啮齿动物CDR或CDR序列取代人抗体的相应序列来进行人源化(Jones PT.等人,Nature[自然],321(6069):522-525(1986)；Riechmann L.等人,Nature[自然],332(6162):323-327(1988)；Verhoeyen M.等人.,Science[科学],239(4847):1534-1536(1988))。因此，在此所述的“人源化”抗体是指如下的抗体：其中基本上小于一个完整的人可变结构域的区域被来自非人物种的相应序列取代。在实践中，典型地，人源化抗体是如下抗体：其中一些CDR残基和可能的一些FR残基被来自啮齿动物抗体中类似位点的残基取代。
作为人源化的替代，可以生成人抗体。例如，现在有可能产生转基因动物(例如小鼠)，这些转基因动物能够在免疫后在不存在内源性免疫球蛋白产生的情况下产生完整的人抗体库。例如，已经描述了在嵌合和种系突变小鼠中的抗体重链连接区(JH)基因的纯合性缺失导致内源性抗体产生的完全抑制。将人种系免疫球蛋白基因排列转移到此类种系突变小鼠中将导致在抗原激发后人抗体的产生，参见例如Jakobovits A.等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],90(6):2551-2555(1993)；Jakobovits A.等人,Nature[自然],362(6417):255-258(1993)；Bruggemann M.等人,Year Immunol.[免疫学年报],7:33-40(1993)；美国专利号5,545,806、5,569,825、5,591,669；5,545,807；和WO 97/17852。
可替代地，可以通过将人免疫球蛋白基因座引入转基因动物中来制备人抗体，这些转基因动物例如是已经使内源性免疫球蛋白基因部分地或完全地失活的小鼠。在激发后，观察到人抗体的产生，这在所有方面都紧密地类似于在人中所见，包括基因重排、组装及抗体库。该方法在本领域中是已知的，例如参见Marks JD.等人,Biotechnology[生物技术],10(7):779-783(1992)；Lonberg N. 等人,Nature[自然],368(6474):856-859(1994)；Morrison SL.,Nature[自然],368(6474):812-813(1994)；Fishwild DM.等人,Nat.Biotechnol.[自然生物技术],14(7):845-851(1996)；Neuberger M.,Nat.Biotechnol.[自然生物技术],14(7):826(1996)；Lonberg N.等人,Int.Rev.Immunol.[国际免疫学综述],13(1):65-93(1995)。
可替代地，噬菌体展示技术(McCafferty J.等人,Nature[自然],348(6301):552-554(1990))可以用于从来自未免疫供体的免疫球蛋白可变(V)结构域基因库体外产生人抗体和抗体片段。根据该技术的一个实施例，将抗体V结构域序列框内克隆到丝状细菌噬菌体的主要或次要外壳蛋白基因中(例如M13或fd)，并在噬菌体颗粒的表面上展示为功能性抗体片段。噬菌体展示能以多种形式进行，例如，如下文实施例部分中描述的或如例如Johnson KS.等人,Curr.Opin.Struct.Biol.[结构生物学观点],3(4):564-571(1993)中综述的。若干个来源的V基因区段可用于噬菌体展示。Clackson T.等人,Nature[自然],352(6336):624-628(1991)从衍生自经免疫的小鼠的脾脏的V基因的小随机组合文库中分离出多种抗噁唑酮抗体。可以构建来自未免疫的人供体的V基因的库，并且可以基本上按照以下描述的技术分离针对多种抗原(包括自身抗原)的抗体：Marks JD.等人,J.Mol.Biol.[分子生物学杂志],222(3):581-597(1991)，或Griffith AD.等人,EMBO J.[欧洲分子生物学学会杂志]12(2):725-734(1993)。还参见，美国专利号5,565,332和5,573,905。
如上讨论的，还可以使用本领域已知的方法通过体外活化的B细胞生成人抗体。
还可以使用本领域已知的各种技术(包括噬菌体展示文库)产生人抗体。Hoogenboom HR.等人,J.Mol.Biol.[分子生物学杂志],227(2):381-388(1991)；Marks JD.等人,J.Mol.Biol.[分子生物学杂志],222(3):581-597(1991)。Cole等人和Boerner等人的技术也可用于制备人单克隆抗体(Monoclonal Antibodies and Cancer Therapy[单克隆抗体与癌症疗法],Cole等人,Alan Liss,Inc.[艾伦利斯有限公司],1985,第77页，以及Boerner P.等人,J.Immunol.[免疫学杂志],147(1):86-95(1991))。
多特异性抗体
多特异性抗体是对两种或更多种不同抗原具有结合特异性的单克隆抗体，优选人抗体或人源化抗体(例如，对至少两种抗原具有结合特异性的双特异性抗体)。例如，结合特异性中的一种可以是针对a5～1蛋白质，另一种可以是针对任何其他抗原。根据一个优选的实施例，其他抗原是细胞表面蛋白或受体或受体亚基。例如，细胞表面蛋白可以是自然杀伤(NK)细胞受体。因此，根据一 个实施例，本发明的双特异性抗体可以结合CD73和例如第二细胞表面受体两者。
用于制备双特异性抗体的合适的方法是本领域已知的。例如，双特异性抗体的重组产生是基于两个免疫球蛋白重链/轻链对的共表达，其中这两条重链具有不同的特异性(Milstein C等人,Nature[自然],305(5934):537-540(1983))。由于免疫球蛋白重链和轻链的随机分类，这些杂交瘤(quadromas)产生十种不同抗体分子的潜在混合物，其中只有一种具有正确的双特异性结构。通常通过亲和色谱法步骤完成正确分子的纯化。本领域披露了类似的方法，例如在WO93/08829中和Traunecker A.等人,EMBO J.[欧洲分子生物学学会杂志],10(12):3655-3659(1991)中。
具有所希望的结合特异性的抗体可变结构域(抗体-抗原结合位点)可以与免疫球蛋白恒定结构域序列融合。融合优选是与包含至少部分铰链、CH2和CH3区的免疫球蛋白重链恒定结构域一起。优选是在这些融合体的至少一种中存在含有轻链结合所需位点的第一重链恒定区(CH1)。将编码免疫球蛋白重链融合体和(如果需要)免疫球蛋白轻链的DNA插入单独的表达载体中，并共转染到合适的宿主生物体中。关于生成双特异性抗体的进一步细节，参见例如Suresh MR.等人,Methods Enzymol.[酶学方法],121:210-228(1986)。
还描述了直接从重组细胞培养物制备和分离双特异性抗体片段的各种技术。例如，使用亮氨酸拉链产生双特异性抗体(Kostelny SA.等人,J.Immunol.[免疫学杂志],148(5):1547-1553(1992))。将来自Fos和Jun蛋白的亮氨酸拉链肽通过基因融合与两种不同抗体的Fab'部分连接。将抗体同源二聚体在铰链区还原以形成单体，然后再氧化以形成抗体异源二聚体。该方法也可用于产生抗体同源二聚体。描述于Hollinger P.等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],90(14):6444-6448(1993)的“双抗体”技术提供了制备双特异性抗体片段的替代机制。片段包含通过接头与VL连接的VH，该接头太短而不允许同一链上的两个结构域之间配对。因此，迫使一个片段的VH和VL结构域与另一个片段的互补VL和VH结构域配对，从而形成两个抗原结合位点。还报道了通过使用单链Fv(sFv)二聚体制备双特异性抗体片段的另一种策略。参见Gruber M.等人,J.Immunol.[免疫学杂志],152(11):5368-5374(1994)。
考虑了具有多于两个化合价的抗体。例如，可以制备三特异性抗体(Tutt A.等人,J.Immunol.[免疫学杂志],147(1)60-69(1991))。
异源缀合抗体
异源缀合抗体由两个共价连接的抗体组成。例如，已经提出此类抗体将免疫系统细胞靶向不需要的细胞，并用于治疗HIV感染。考虑了可以使用合成蛋白化学中的已知方法(包括涉及交联剂的那些方法)在体外制备这些抗体。例如，可以使用二硫键交换反应或通过形成硫醚键来构建免疫毒素。用于该目的的合 适的试剂的实施例包括亚氨基硫醇酯(iminothiolate)和4-巯基丁亚氨酸甲酯(methyl-4-mercaptobutyrimidate)和本领域已知的那些。
效应子功能工程化
可以希望的是在效应子功能方面修饰本文提供的抗体，以增强例如抗体在治疗癌症中的有效性。例如，可以将一个或多个半胱氨酸残基引入Fc区，从而允许在该区域中形成链间二硫键。由此生成的同二聚体抗体可以具有改善的内化能力和/或增加的补体介导的细胞杀伤和抗体依赖的细胞毒性(ADCC)。参见Caron PC.等人,J.Exp.Med.[实验医学杂志],176(4):1191-1195(1992)和Shopes B.,J.Immunol.[免疫学杂志],148(9):2918-2922(1992)。还可以使用本领域已知的异源双功能交联剂制备具有增强的抗肿瘤活性的同源二聚体抗体。可替代地，可以工程化抗体使得具有双Fc区，从而可以具有增强的补体裂解和ADCC能力。参见，例如Stevenson GT.等人,Anticancer Drug Des.[抗癌药物设计],3(4):219-230(1989)。
可以进行Fc区序列中的突变或改变以改善FcR结合(例如，FcγR，FcRn)。根据一个实施例，与天然IgG或亲本抗体相比，本发明的抗体具有选自下组的至少一个改变的效应子功能，该组由以下组成：ADCC、CDC、和改善的FcRn结合。若干个有用的特异性突变的实施例描述于，例如，Shields RL.等人,J.Biol.Chem.[生物化学杂志],276(6):6591-6604(2001)；Presta,LG.等人,Biochem.Soc.Trans.[生物化学学会汇报]30(4):487-490(2002)；以及WO00/42072。
根据一个实施例，Fc受体突变是在选自下组的至少一个位置处的取代，该组由以下组成：Fc区的238、239、246、248、249、252、254、255、256、258、265、267、268、269、270、272、276、278、280、283、285、286、289、290、292、293、294、295、296、298、301、303、305、307、309、312、315、320、322、324、326、327、329、330、331、332、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、434、435、437、438或439，其中Fc区中残基的编号是根据EU编号系统进行的。在一些实施例中，Fc受体突变是D265A取代。在一些实施例中，Fc受体突变是N297A取代。另外的合适的突变是本领域已知的，例如，如美国专利号7,332,581中列出的。
免疫缀合物
本发明还涉及包含与细胞毒性剂(如化学治疗剂)、毒素(例如，细菌、真菌、植物或动物来源的酶活性毒素或其片段)、或放射性同位素(即放射缀合物)缀合的抗体、或其变体或突变体的免疫缀合物。
可以使用的酶活性毒素及其片段包括白喉A链、白喉毒素的非结合活性片段、外毒素A链(来自铜绿假单胞菌(Pseudomonas aeruginosa)、蓖麻毒蛋白A链、相思豆毒蛋白A链、蒴莲根毒蛋白A链、α-帚曲毒蛋白、油桐蛋白、香石竹毒蛋白、美洲商陆(Phytolacaamericana)蛋白(PAPI、PAPII和PAP-S)、苦瓜(momordicacharantia)抑制剂、麻疯树毒蛋白、巴豆毒蛋白、肥阜草(sapaonaria officinalis)抑制剂、多花白树毒蛋白、丝林霉素(mitogellin)、局限曲菌素(restrictocin)、酿霉素(phenomycin)、依诺霉素(enomycin)和单端抱菌素(tricothecenes)。多种放射性核素可用于产生放射缀合的抗体。实施例包括 212Bi、 131I、 131In、 90Y和 186Re。有用于生成此类免疫缀合物的示例性化学治疗剂包括本文其他地方描述的那些。
在某些实施例中，本发明的抗CD73抗体、和/或其变体或其突变体与美登素(maytansine)、美登木素生物碱(maytansinoid)或卡里奇霉素(calicheamicin)缀合。在某些实施例中，本发明的抗CD73抗体、和/或其变体或其突变体与美登木素生物碱DM1缀合。
使用多种双功能蛋白偶联剂(例如，N-琥珀酰亚胺基-3-(2-吡啶二巯基)丙酸酯(SPDP)、亚氨基硫烷(IT)、亚氨酸酯的双功能衍生物(如二甲基己二亚氨酸酯HCl)、活性酯(如辛二酸二琥珀酰亚胺酯)、醛类(如戊二醛)、双叠氮化合物(如双(对叠氮苯甲酰基)己二胺)、双重氮衍生物(如双-(对重氮苯甲酰基)-乙二胺)、二异氰酸酯(如甲代亚苯基2,6-二异氰酸酯)、和双活性氟化合物(如1,5-二氟-2,4-二硝基苯))制备抗体和细胞毒性剂的缀合物。例如，可以如Vitetta ES.等人,Science[科学],238(4830):1098-1104(1987)描述的制备蓖麻毒蛋白免疫毒素。碳-14-标记的1-异硫氰酸基苄基-3-甲基二亚乙基三胺-五乙酸(MX-DTPA)是用于将放射性核苷酸与抗体缀合的示例性螯合剂。参见，WO 94/11026。
在某些实施例中，本发明的抗CD73抗体、和/或其变体或其突变体可以与“受体”(如链霉抗生物素蛋白)缀合，用于肿瘤预靶向，其中向患者给予抗体-受体缀合物，随后使用清除剂从循环中除去未结合的缀合物，然后给予与细胞毒性剂(例如，放射性核苷酸)缀合的“配体”(例如，抗生物素蛋白)。
共价修饰
抗CD73抗体、其变体、突变体、和片段的共价修饰包括在本披露的范围内。一种类型的共价修饰包括使多肽的靶氨基酸残基与能够和多肽的选择的侧链或N末端或C末端残基反应的有机衍生剂反应。用双功能剂进行衍生化是有用的，例如，用于将多肽与水不溶性支持基质或表面交联以用于纯化抗体的方法，反之亦然。常用的交联剂包括，例如，1,1-双(重氮乙酰基)-2-苯基乙烷、戊二醛、 N-羟基琥珀酰亚胺酯(例如，与4-叠氮水杨酸的酯)、同双功能亚氨酸酯(包括二琥珀酰亚胺酯如3,3'-二硫代双(琥珀酰亚胺基-丙酸酯))、双功能马来酰亚胺(如双-N-马来酰亚胺基-1,8-辛烷)和药剂(如甲基-3-[(对叠氮苯基)-二硫代]丙酰亚氨酸酯)。
其他修饰包括谷氨酰胺酰和天冬酰胺酰残基分别脱酰胺为相应的谷氨酰和天冬氨酰残基，脯氨酸和赖氨酸的羟基化、丝氨酰或苏氨酰残基的羟基磷酸化，赖氨酸、精氨酸和组氨酸侧链的α-氨基基团的甲基化(Proteins:Structure and Molecular Properties[蛋白质：结构和分子特性],Thomas E.Creighton,W.H.Freeman&Co.[W.H.弗里曼公司],San Francisco[旧金山],1983,第79-86页)，N末端胺的乙酰化和任何C末端羧基的酰胺化。
对多肽的另一种类型的共价修饰的包括以本领域已知的方式(例如，在美国专利号4,640,835；4,496,689；4,301,144；4,670,417；4,791,192或4,179,337中列出的)将多肽连接到多种非蛋白质聚合物中的一种，例如聚乙二醇(PEG)、聚丙二醇或聚氧化烯。
嵌合分子
如果有利于形成包含与另一种异源多肽或氨基酸序列融合的多肽(例如，免疫黏附蛋白或肽体)的嵌合分子，本发明的抗CD73抗体、和/或其变体或其突变体也可以被修饰。
在一个实施例中，这种嵌合分子包含多肽与蛋白质转导结构域的融合体，该蛋白质转导结构域将多肽靶向递送至各种组织，并且更具体为穿过脑血屏障，其中使用例如人免疫缺陷病毒TAT蛋白的蛋白质转导结构域(Schwarze SR.等人,Science[科学]285(5433):1569-1572(1999))。
在某些实施例中，此类嵌合分子包含带有标签多肽的多肽的融合体，该标签多肽提供了抗标签抗体可选择性结合的表位。表位标签通常位于多肽的氨基或羧基末端。此类带表位标签形式的多肽的存在可使用针对该标签多肽的抗体来检测。此外，表位标签的提供使多肽易于使用抗标签抗体或另一类型结合该表位标签的亲和基质通过亲和纯化来纯化。多种标签多肽及其各自抗体是本领域已知的。实施例包括多组氨酸(poly-His)或多组氨酸-甘氨酸(poly-His-gly)标签；流感HA标签多肽及其抗体12CA5(Field J.等人,Mol.Cell.Biol.[分子细胞生物学杂志],8(5):2159-2165(1988))；c-myc标签及其8F9、3C7、6E10、G4、B7和9E10抗体(Evan GI.等人,Mol.Cell.Biol.[分子细胞生物学杂志],5(12):3610-3616(1985))；及单纯疱疹病毒糖蛋白D(gD)标签及其抗体(Paborsky LR.等人,Protein Eng.[蛋白质工程],3(6):547-553(1990))。其他标签多肽包括Flag肽(Hopp TP.等人,Bio/Technology[生物 技术],6:1204-1210(1988))；KT3表位肽(Martin GA.等人,Science[科学],255(5041):192-194(1992))；α-微管蛋白表位肽(Skinner MP.等人,J.Biol.Chem.[生物化学杂志],266(9):15163-15166(1991))；和T7基因10蛋白肽标签(Lutz-Freyermuth C.等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],87(16):6393-6397(1990))。
在替代性实施例中，嵌合分子可以包含多肽与免疫球蛋白或免疫球蛋白特定区域的融合体。对于二价形式的嵌合分子(例如“免疫黏附蛋白”)，此类融合体可以是与IgG分子Fc区的融合。本发明的Ig融合体包括大约包含或只包含人的残基94-243、残基33-53或残基33-52以取代Ig分子内的至少一个可变区的多肽。在一个特别优选的实施例中，免疫球蛋白融合体包含IgG1分子的铰链、CH2和CH3，或者铰链、CH1、CH2和CH3区。免疫球蛋白融合体的产生是本领域已知的，参见例如美国专利号5,428,130。
免疫脂质体
还可以将本发明的抗CD73抗体、和/或其变体或其突变体配制为免疫脂质体。通过本领域已知的方法制备含有抗体的脂质体，如以下描述的：Epstein DA.等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],82(11):3688-3692(1985)；和Hwang KJ.等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊],77(7):4030-4034(1980)；和美国专利号4,485,045和4,544,545。美国专利号5,013,556披露了具有增强的循环时间的脂质体。
特别有用的脂质体可以通过反相蒸发法用包含磷脂酰胆碱、胆固醇和PEG衍生的磷脂酰乙醇胺(PEG-PE)的脂质组合物生成。将脂质体挤出通过具有限定孔径的过滤器以产生具有所希望的直径的脂质体。本披露的抗体的Fab’片段可以经由二硫键交换反应与脂质体缀合，如Martin FJ.等人,J.Biol.Chem.[生物化学杂志],257(1):286-288(1982)中描述的。脂质体内还任选地含有抗肿瘤剂、生长抑制剂或化学治疗剂(如多柔比星(doxorubicin))。参见，Gabizon A.等人,J.Natl.Cancer Inst.[国家癌症研究所杂志],81(19):1484-1488(1989)。
使用抗CD73抗体、其变体或突变体进行治疗
可以向受试者(例如，哺乳动物如人)给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以治疗与CD73表达、CD73过表达或异常CD73功能相关的疾病、障碍、或临床病症。例如，与CD73的表达相关的癌症可以是但不限于：如淋巴瘤、肾癌、肺癌、结肠癌、肝癌、胃癌、原发性渗出性淋巴瘤、骨肉瘤和非霍奇金淋巴瘤。与CD73表达相关的疾病、障 碍或临床病症的另外的实施例可以是纤维化，包括特发性肺纤维化。在某些实施例中，本发明提供了本文描述的抗CD73抗体和/或变体/突变体(或其片段)，用于制造用于在受试者中治疗癌症或纤维化的药物。在某些实施例中，待治疗的受试者是哺乳动物(例如，人，非人灵长类、大鼠、小鼠、牛、马、猪、绵羊、山羊、狗、猫等)。在某些实施例中，受试者是人。在某些实施例中，受试者是临床患者、临床试验志愿者、实验动物等。在某些实施例中，怀疑受试者患有癌症或纤维化或有此风险。
在一些实施例中，可以向受试者(例如，哺乳动物如人)给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以治疗与CD73表达相关的癌症的癌症重现或复发。
在一些实施例中，可以向受试者(例如，哺乳动物如人)给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以治疗与CD73表达相关的难治性或抗性癌症。
在一些实施例中，可以向受试者(例如，哺乳动物如人)给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以提供用于与CD73表达相关的癌症的辅助疗法。
在一些实施例中，可以向受试者(例如，哺乳动物如人)给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以提供用于与CD73表达相关的癌症的维持疗法。
在一些实施例中，可以向受试者(例如，哺乳动物如人)给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以治疗与CD73表达相关的侵袭性癌症。
在一些实施例中，可以向受试者(例如，哺乳动物如人)给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以治疗与CD73表达相关的非侵袭性癌症。
在一些实施例中，可以向受试者(例如，哺乳动物如人)给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以增加诊断为与CD73表达相关的癌症的受试者的无进展存活。
在一些实施例中，可以使用所披露的抗CD73抗体、变体、突变体和/或其片段治疗的与CD73表达相关的癌症可以是多药抗性(MDR)或药物敏感的。癌症的实施例包括但不限于癌、淋巴瘤、母细胞瘤、肉瘤、血液学癌症和白血病。在一些实施例中，可以使用所披露的抗CD73抗体、变体、突变体和/或其片段治疗的与CD73表达相关的癌症包括但不限于：结肠直肠癌、卵巢癌、胃癌、胆囊癌、白血病、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、前列腺癌、黑色素瘤、乳腺癌、三阴性乳腺癌(TNBC)、膀胱癌、非小细胞肺癌(NSCLC)、 胰腺癌、胶质瘤、头颈癌、甲状腺癌、B-细胞急性淋巴细胞白血病、髓母细胞瘤、非典型畸胎样/横纹肌样瘤和口腔鳞状细胞癌。在另外的实施例中，可以使用所披露的抗CD73抗体、变体、突变体和/或其片段治疗的与CD73表达相关的癌症包括但不限于：血液(或造血组织)癌、膀胱癌、脑癌、乳腺癌(例如，三阴性乳腺癌，TNBC)、中枢和周围神经系统癌、宫颈癌、结肠癌、头颈癌、肾癌、肝癌、肺癌(包括小细胞肺癌)、食管癌、胆囊癌、胃肠道癌、泌尿生殖道的癌症、淋巴谱系淋巴系统造血肿瘤、骨髓谱系造血肿瘤、卵巢癌、胰腺癌、直肠癌、胃癌、前列腺癌、皮肤癌(包括鳞状细胞癌)和甲状腺癌。
在一些实施例中，可以使用所披露的抗CD73抗体、变体、突变体和/或其片段治疗的与CD73表达相关的癌症可以是血液学癌症。在仍另外的方面，血液学癌症选自白血病、骨髓瘤、或淋巴瘤，包括但不限于：急性髓细胞性白血病(AML)、急性淋巴母细胞白血病(ALL)、慢性髓细胞性白血病(CML)、慢性淋巴细胞白血病(CLL)、毛细胞白血病、慢性粒-单核细胞白血病(CMML)、幼年型粒-单核细胞白血病(JMML)、霍奇金淋巴瘤、经典型霍奇金淋巴瘤(cHL)、非霍奇金淋巴瘤原发性纵隔B细胞淋巴瘤(NHL PMBCL)、非霍奇金淋巴瘤、多发性骨髓瘤、单发性骨髓瘤、局限性骨髓瘤、髓外骨髓瘤、小淋巴细胞淋巴瘤、B细胞非霍奇金淋巴瘤、和大B细胞淋巴瘤。淋巴谱系的示例性造血肿瘤包括但不限于：白血病、急性淋巴细胞白血病、急性淋巴母细胞白血病、B细胞淋巴瘤、T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、毛细胞淋巴瘤和伯基特淋巴瘤。髓细胞谱系的造血肿瘤包括但不限于：急性和慢性髓细胞性白血病，骨髓增生异常综合征和早幼粒细胞白血病；间充质来源的肿瘤，包括纤维肉瘤和横纹肌肉瘤。
在其他实施例中，可以使用所披露的抗CD73抗体、变体、突变体和/或其片段治疗的与CD73表达相关的癌症可以是脑或中枢神经系统的癌症。中枢和周围神经系统肿瘤包括但不限于：星形细胞瘤、成神经细胞瘤、胶质瘤和神经鞘瘤。另外的示例性脑癌或中枢神经系统癌包括：胶质瘤、髓母细胞瘤、原始神经外胚瘤(PNET)、听神经瘤、胶质瘤、脑膜瘤、垂体腺瘤、神经鞘瘤、CNS淋巴瘤、原始神经外胚瘤、颅咽管瘤、脊索瘤、髓母细胞瘤、中枢成神经细胞瘤、中枢神经细胞瘤、成松果体细胞瘤、松果体母细胞瘤(pineoblastoma)、非典型畸胎样横纹肌样瘤、软骨肉瘤、软骨瘤、脉络丛癌(choroid plexus carcinoma)、脉络丛乳头状瘤、颅咽管瘤、胚胎发育不良性神经上皮瘤、神经节细胞瘤、生殖细胞瘤、血管母细胞瘤、血管周细胞瘤和转移性脑肿瘤。在又另外的方面，胶质瘤选自室管膜细胞瘤、星形细胞瘤、少突神经胶质瘤、和少突星形细胞瘤。在甚至另外的方面，胶质瘤选自幼年型毛细胞型星形细胞瘤、室管膜下巨细胞星形细胞瘤、神经节神经胶质瘤、室管膜下瘤、多形性黄色瘤形星形细胞瘤(pleomorphic xanthoastrocytoma)、间变性星形细胞瘤、多形性胶质母细胞 瘤、脑干胶质瘤、少突神经胶质瘤、室管膜细胞瘤、少突星形细胞瘤、小脑星形细胞瘤、结缔组织增生性小儿星形细胞瘤、室管膜下巨细胞星形细胞瘤、弥散型星形细胞瘤、混合型胶质瘤、视神经胶质瘤、大脑胶质瘤病(gliomatosis cerebri)、多灶性神经胶质瘤(multifocal gliomatous tumor)、多中心多形性胶质母细胞瘤(multicentric glioblastoma multiforme tumor)、神经节细胞瘤和神经节神经胶质瘤。
可以使用所披露的抗CD73抗体、变体、突变体和/或其片段治疗的与CD73表达相关的其他示例性肿瘤包括但不限于：鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、囊腺癌、髓样癌、支气管肺癌、肾细胞癌、肝癌、胆管癌、绒毛膜癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、睾丸肿瘤、肺癌、小细胞肺癌、膀胱癌、上皮癌、骨肉瘤、星形细胞瘤、髓母细胞瘤、颅咽管瘤、室管膜细胞瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突神经胶质瘤、脑膜瘤、黑色素瘤、成神经细胞瘤、视网膜母细胞瘤、黑色素瘤、畸胎癌、异皮着色性干皮病(xenoderoma pigmentosum)、角质棘皮瘤(keratoctanthoma)、滤泡性甲状腺癌和卡波西肉瘤。
针对癌症(如黑色素瘤、非小细胞肺癌(NSCLC)、头颈癌、尿路上皮癌、乳腺癌(例如，三阴性乳腺癌，TNBC)、胃癌、经典型霍奇金淋巴瘤(cHL)、非霍奇金淋巴瘤原发性纵隔B细胞淋巴瘤(NHL PMBCL)、间皮瘤、卵巢癌、肺癌(例如，小-细胞肺癌)、食管癌、鼻咽癌(NPC)、胆道癌、结肠直肠癌、宫颈癌、甲状腺癌)或任何其他表现出异常CD73功能和这些疾病的临床描述的疾病的许多诊断方法是本领域已知的。此类方法包括但不限于例如免疫组织化学、PCR、荧光原位杂交(FISH)。关于异常CD活性或表达的诊断方法的其他细节在本领域中描述。
所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以通过任何合适的途径给予，包括，例如静脉内、肌肉内、或皮下。在一些实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以通过肠胃外给予来进行给予。“肠胃外”是指静脉内、皮下和肌肉内给予。
在一些实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与至少一种药剂(如第二、第三或第四药剂)组合给予。该至少一种药剂可以是已知治疗癌症或纤维化(包括特发性肺纤维化)的任何合适的药剂。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与用于癌症治疗的至少一种药剂组合给予。在具体的实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本 文提供的药物组合物可以与用于癌症治疗的至少一种药剂(如奥沙利铂、甲酰四氢叶酸、氟尿嘧啶(5-FU)、吉西他滨、紫杉醇(包括蛋白质结合型紫杉醇(例如但不限于纳米颗粒白蛋白结合型紫杉醇，其有时被称为“白蛋白结合型紫杉醇(nab-paclitaxel)”)、或其组合)组合给予。
用于癌症治疗的至少一种药剂的另外的实施例包括但不限于：化学治疗剂、免疫治疗剂、癌症疫苗、抗血管生成剂、细胞因子、激素疗法、基因疗法、生物疗法和放射疗法。例如，已知治疗癌症的至少一种药剂可以是已知的细胞抑制性或细胞毒性或抗癌剂(如抗肿瘤剂)、生长抑制剂、细胞毒性剂或化学治疗剂(如多西他赛(docetaxel)、吉非替尼(gefitinib)、FOLFIRI(伊立替康(irinotecan)、5-氟脲嘧啶、和亚叶酸(leucovorin))、伊立替康、顺铂、卡铂、紫杉醇、贝伐单抗(bevacizumab)(抗VEGF抗体)、FOLFOX-4、输注的氟脲嘧啶、甲酰四氢叶酸(leucovorin)、和奥沙利铂(oxaliplatin)、阿法替尼(afatinib)、吉西他滨(gemcitabine)、卡培他滨(capecitabine)、培美曲塞(pemetrexed)、替万替尼(tivantinib)、依维莫司(everolimus)、CpG-ODN、雷帕霉素(rapamycin)、来那度胺(lenalidomide)、维莫非尼(vemurafenib)、内皮抑制蛋白、拉帕替尼(lapatinib)、PX-866、Imprime PGG和irlotinibm。
在一些实施例中，抗CD73抗体和/或变体/突变体(或其片段)与另外的药剂缀合。例如，该至少一种药剂包括乌拉莫司汀(uracil mustard)、氮芥、环磷酰胺、异环磷酰胺、美法仑(melphalan)、苯丁酸氮芥(chlorambucil)、哌泊溴烷(pipobroman)、曲他胺(triethylenemelamine)、三亚乙基硫代磷酰胺(triethylenethiophosphoramine)、白消安、卡莫司汀、环己亚硝脲(lomustine)、链脲菌素(链脲菌素)、达卡巴嗪(dacarbazine)、替莫唑胺(temozolomide)、噻替派(thiotepa)、六甲蜜胺(altretamine)、甲氨蝶呤(methotrexate)、5-氟脲嘧啶、氟尿苷、阿糖孢苷、6-巯嘌呤、6-硫鸟嘌呤、磷酸氟达拉滨(fludarabine phosphate)、喷司他丁(pentostatin)、硼替佐米(bortezomib)、长春花碱、长春新碱、长春瑞滨、长春地辛、博莱霉素(bleomycin)、放线菌素D、道诺霉素(daunorubicin)、多柔比星、表柔比星(epirubicin)、地塞米松、氯法拉滨、克拉屈滨、pemextresed、伊达比星(idarubicin)、紫杉醇、多西他赛、伊沙匹隆(ixabepilone)、光神霉素(mithramycin)、拓扑替康(topotecan)、伊立替康、脱氧助间型霉素(deoxycoformycin)、丝裂霉素-C、L-天冬酰胺酶、干扰素、依托泊苷(etoposide)、替尼泊苷17α-炔雌醇(ethinylestradiol)、己烯雌酚(diethylstilbestrol)、睾酮、强的松、氟甲睾酮、屈他雄酮丙酸酯(dromostanolone propionate)、睾内酯、醋酸甲地孕酮、三苯氧胺(tamoxifen)、甲基强的松龙、甲基睾酮、泼尼松龙、曲安奈德(triamcinolone)、氯烯雌醚 (chlorotrianisene)、羟孕酮、氨鲁米特、雌莫司汀(estramustine)、醋酸甲羟孕酮(medroxyprogesteroneacetate)、亮丙瑞林、氟他胺(flutamide)、托瑞米芬、戈舍瑞林、顺铂、卡铂、羟基脲(hydroxyurea)、安吖啶、丙卡巴肼(procarbazine)、米托坦(mitotane)、米托蒽醌(mitoxantrone)、左旋咪唑(levamisole)、诺维本(navelbene)、阿那曲唑(anastrazole)、来曲唑(letrazole)、卡培他滨、雷罗色分(reloxafine)、卓罗沙吩(droloxafine)、六甲嘧胺(hexamethylmelamine)、奥沙利铂
Figure PCTCN2020102678-appb-000006
易瑞沙(iressa)(吉非替尼(gefinitib)、Zd1839)、
Figure PCTCN2020102678-appb-000007
(卡培他滨)、
Figure PCTCN2020102678-appb-000008
(埃罗替尼(erlotinib)、阿扎胞苷(5-氮胞啶；5-AzaC)、替莫唑胺
Figure PCTCN2020102678-appb-000009
吉西他滨(例如、
Figure PCTCN2020102678-appb-000010
(吉西他滨HCl))和血管抑制因子(vasostatin)。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与至少一种药剂组合给予，该至少一种药剂为例如烷化剂，如噻替派和CYTOXAN(R)环磷酰胺；烷基磺酸盐，如白消安、英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶类(aziridines)，如苯并多巴(benzodopa)、卡巴醌(carboquone)、美妥替哌(meturedopa)和脲多巴(uredopa)；乙烯亚胺和甲基蜜胺类(methylamelamines)，包括六甲蜜胺、曲他胺、三亚乙基磷酰胺(trietylenephosphoramide)、三亚乙基硫代磷酰胺(triethiylenethiophosphoramide)和三羟甲蜜胺(trimethylolomelamine)；多聚乙酰类(acetogenins)(尤其是布拉它辛(bullatacin)和布拉他辛酮(bullatacinone)；δ-9-四氢大麻酚(屈大麻酚，MARINOL(R))；β-拉帕醌；拉帕醇；秋水仙碱；桦木酸；喜树碱(包括合成类似物拓扑替康(HYCAMTIN(R))、CPT-Il(伊立替康，CAMPTOSAR(R))、乙酰基喜树碱(acetylcamptothecin)、莨菪亭(scopolectin)和9-氨基喜树碱(aminocamptothecin))；苔藓虫素；callystatin；CC-1065(包括其阿多来新(adozelesin)、卡折来新(carzelesin)和比折来新(bizelesin)合成类似物)；鬼臼毒素(podophyllotoxin)；鬼臼酸(podophyllinic acid)；替尼泊苷；念珠藻素类(cryptophycins)(具体是念珠藻素1和念珠藻素8)；尾海兔素(dolastatin)；倍癌霉素(duocarmycin)(包括合成类似物，KW-2189和CBl-TML)；五加素(eleutherobin)；水鬼蕉碱(pancratistatin)；匍枝珊瑚醇(sarcodictyin)；海绵抑制素(spongistatin)；氮芥类，如苯丁酸氮芥、萘氮芥、胆怜酸胺(cholophosphamide)、雌莫司汀、异环磷酰胺、氮芥、盐酸氧氮芥(mechlorethamine oxide hydrochloride)、美法仑、新恩比兴(novembichin)、苯芥胆甾醇(phenesterine)、泼尼氮芥(prednimustine)、曲磷胺(trofosf amide)、乌拉莫司汀；亚硝基脲类(nitrosureas)，如卡莫司汀、氯脲霉素(chlorozotocin)、福莫司汀(fotemustine)、环己亚硝脲、尼莫司汀(nimustine)、和雷莫司汀 (ranimnustine)；抗生素类，如烯二炔抗生素(例如，卡里奇霉素，尤其是卡里奇霉素γll和卡里奇霉素ωll(参见，例如，Nicolaou KC.等人,Angew.Chem.Int.Ed.Engl.[应用化学英文国际版],33:183-186(1994))；dynemicin，包括dynemicin A；埃斯佩拉霉素(esperamicin)；以及新制癌菌素发色团(neocarzinostatin chromophore)和相关的色蛋白烯二炔抗生素发色团)、阿克拉霉素类(aclacinomysins)、放线菌素、氨茴霉素(authramycin)、重氮丝氨酸(azaserine)、博莱霉素、放线菌素C、卡柔比星(carabicin)、洋红霉素、嗜癌霉素(carzinophilin)、色霉素(chromomycinis)、放线菌素D、道诺霉素、地托比星(detorubicin)、6-重氮-5-氧代-L-正亮氨酸、多柔比星(包括ADRIAMYCIN(R)、吗啉代-多柔比星、氰基吗啉代-多柔比星、2-吡咯啉代-多柔比星、多柔比星HCl脂质体注射(DOXIL(R))和去氧多柔比星)、表柔比星、依索比星(esorubicin)、伊达比星、麻西罗霉素(marcellomycin)、丝裂霉素如丝裂霉素C、霉酚酸、诺加霉素、橄榄霉素类(olivomycins)、培洛霉素(peplomycin)、泊非霉素(potfiromycin)、嘌呤霉素(puromycin)、三铁阿霉素(quelamycin)、罗多比星(rodorubicin)、链黑菌素(streptonigrin)、链脲菌素(streptozocin)、杀结核菌素(tubercidin)、乌苯美司(ubenimex)、新制癌菌素(zinostatin)、佐柔比星(zorubicin)；抗代谢物，如甲氨蝶呤、吉西他滨(GEMZAR(R))、喃氟啶(tegafur)(UFTORAL(R))、卡培他滨(XELODA(R))、埃博霉素和5-氟脲嘧啶(5-FU)；叶酸类似物，如二甲叶酸、甲氨蝶呤、蝶罗呤(pteropterin)、三甲曲沙(trimetrexate)；嘌呤类似物，如氟达拉滨，6-巯嘌呤、硫咪嘌呤(thiamiprine)、硫鸟嘌呤；嘧啶类似物，如环胞苷(ancitabine)、阿扎胞苷、6-氮尿苷(azauridine)、卡莫氟(carmofur)、阿糖孢苷、二脱氧尿苷(dideoxyuridine)、去氧氟尿苷(doxifluridine)、依诺他滨、氟尿苷；雄激素，如卡普睾酮(calusterone)、屈他雄酮丙酸酯、环硫雄醇、美雄烷、睾内酯；抗肾上腺类，如氨鲁米特、米托坦、曲洛司坦；叶酸补充剂，如亚叶酸；醋葡醛内酯(aceglatone)；醒磷酰胺糖苷(aldophosphamide glycoside)；氨基乙酰丙酸；恩尿嘧啶(eniluracil)；安吖啶；bestrabucil；比生群(bisantrene)；依达曲沙(edatraxate)；地佛法明(defofamine)；地美可辛(demecolcine)；地吖醌(diaziquone)；依洛尼塞(elfornithine)；依利醋铵(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖；洛尼代宁(lonidainine)；美登木素生物碱，如美登素和安丝菌素(ansamitocins)；米托胍腙(mitoguazone)；米托蒽醌；莫匹丹莫(mopidanmol)；二胺硝吖啶(nitraerine)；喷司他丁；苯来美特(phenamet)；吡柔比星(pirarubicin)；洛索蒽醌(losoxantrone)；2-乙基酰肼(ethylhydrazide)；丙卡巴肼；PSK(R)多糖复合物(JHS自然产品公司(JHS Natural Products)， 尤金市(Eugene)，俄勒冈州(OR))；丙亚胺(razoxane)；根霉素；西佐喃；锗螺胺(spirogermanium)；细交链孢菌酮酸(tenuazonic acid)；三亚胺醌；2,2',2"-三氯三乙胺；单端孢霉烯(trichothecenes)(尤其是T-2毒素、维拉库林(verracurin)A、杆孢菌素(roridin)A和蛇形菌素(anguidine)；乌拉坦(urethane)；长春地辛(ELDISEME(R)、FILDESIN(R))；达卡巴嗪；甘露醇氮芥(mannomustine)；二溴甘露醇；二溴卫矛醇；哌泊溴烷；加西托新(gacytosine)；阿拉伯糖苷("Ara-C")；噻替派；紫杉烷类，例如紫杉醇(TAXOL(R))、紫杉醇(ABRAXANE(TM))的白蛋白工程化的纳米颗粒配制品、和多西他赛(doxetaxel)(TAXOTERE(R))；苯丁酸氮芥(chloranbucil)；6-硫鸟嘌呤；巯嘌呤；甲氨蝶呤；铂类似物，如顺铂和卡铂；长春花碱(VELBAN(R))；铂；依托泊苷(VP-16)；异环磷酰胺(ifosf amide)；米托蒽醌；长春新碱(ONCOVIN(R))；奥沙利铂；甲酰四氢叶酸；长春瑞滨(NAVELBINE(R))；能灭瘤(novantrone)；依达曲沙(edatrexate)；道诺霉素(daunomycin)；氨基蝶呤；伊班膦酸钠(ibandronate)；拓扑异构酶抑制剂RFS 2000；二氟甲基鸟胺酸(DMFO)；类视黄醇，如视黄酸；任何上述的药学上可接受的盐、酸或衍生物；以及上述两种或多种的组合，如CHOP(环磷酰胺、多柔比星、长春新碱和泼尼松龙组合疗法的缩写)，以及FOLFOX(奥沙利铂(ELOXATIN(TM))组合5-FU和甲酰四氢叶酸的治疗方案的缩写)。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与至少一种药剂组合给予，该至少一种药剂为血管发生抑制剂，例如但不限于：血管抑素(纤溶酶原片段)；抗血管生成抗凝血酶III；Angiozyme；ABT-627；Bay 12-9566；氟草胺(Benefin)；贝伐单抗；BMS-275291；软骨衍生的抑制剂(CDI)；CAI；CD59补体片段；CEP-7055；Col 3；康普瑞汀(combretastatin)A-4；内皮抑制蛋白(胶原蛋白XVIII片段)；纤连蛋白片段；gro-β；常山酮(halofuginone)；肝素酶(heparinases)；肝素多聚己糖片段；HMV833；人绒毛膜促性腺素(hCG)；IM-862；干扰素α/β/γ；干扰素诱导蛋白(IP-10)；白介素-12；Kringle 5(纤溶酶原片段)；马立马司他(Marimastat)；金属蛋白酶抑制剂(TIMP)；2-甲氧雌二醇(2-methoxyestradiol)；MMI 270(CGS 27023A)；MoAb IMC-1C11；癌立消(Neovastat)；NM-3；panzem；PI-88；胎盘核糖核酸酶抑制剂；纤溶酶原激活物抑制剂；血小板因子-4(PF4)；普马司他(prinomastat)；催乳素16kD片段；增殖蛋白相关的蛋白(PRP)；PTK 787/ZK 222594；类视黄醇；索利司他(solimastat)；角鲨胺；SS 3304；SU 5416；SU6668；SU11248；四氢可的松-S；四硫钼酸盐；沙利度胺；血小板应答蛋白-1(TSP-1)；TNP-470；转化生长因子-β(TGF-b)；血管抑制素(vasculostatin)；血管抑制因子(钙网蛋白片 段)；ZD6126；ZD 6474；法尼基转移酶抑制剂(FTI)；和双膦酸盐。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与至少一种药剂组合给予，该至少一种药剂选自DNA相互作用剂，如顺铂或多柔比星；拓扑异构酶II抑制剂，如依托泊苷；拓扑异构酶I抑制剂，如CPT-11或拓扑替康；微管蛋白相互作用剂，如天然存在的或合成的紫杉醇、多西他赛或埃博霉素(例如伊沙匹隆)；激素剂，如三苯氧胺；胸苷酸合酶抑制剂(thymidilate synthase inhibitor)，如5-氟脲嘧啶；和抗代谢物，如甲氨蝶呤，其他酪氨酸激酶抑制剂，如易瑞沙和OSI-774；血管发生抑制剂；BTK抑制剂、SYK抑制剂、ITK抑制剂、PI3-激酶抑制剂、FLT3抑制剂、EGF抑制剂；PAK抑制剂、VEGF抑制剂；CDK抑制剂；SRC抑制剂；c-Kit抑制剂；Her1/2抑制剂和针对生长因子受体的单克隆抗体，如爱必妥(erbitux)(EGF)和赫塞汀(herceptin)(Her2)以及其他蛋白激酶调节剂。这些药剂可以与分化剂如ATRA、EZH2抑制剂、DNMT抑制剂、皮质类固醇、IDH1抑制剂、IDH2抑制剂和维生素C组合使用。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与在癌细胞中增强DNA损伤杀伤的小分子(包括PARP抑制剂、MDM2抑制剂、NAMPT抑制剂和HSP90抑制剂)组合给予。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与一种或多种免疫治疗剂(包括拮抗剂、抗体和免疫调节剂，包括但不限于：HERCEPTINS、RITUXANS、OVAREX TM、PANOREX@、BEC2、IMC-C225、VITAMIN、CAMPATH@I/H、Smart MI95、LYMPHOCIDE TM、Smart I D10和ONCOLYM TM、利妥昔单抗、吉妥单抗(gemtuzumab)或曲妥单抗(trastuzumab))组合给予。在其他实施例中，免疫治疗剂可以是靶向PD-1、PD-L1、PD-L2、CTLA4、LAG3、TIGIT、TIM3、CEACAM-1、半乳凝素-1、半乳凝素-9、BLTA、CD69、CD113、GPR56、2B4、CD48、PD1H、LAIR1、TIM-1、TIM-4、VISTA、GARP、CD73、CD39、A2AR、B7-1、B7-2、4-1BBL、CD137、CD28、CD28H、GITR、GITRL、ICOS、ICOS-L、OX40、OX40L、CD70、CD27、CD40、DR3的抗体，或针对此类靶标的抗体的组合。在一些实施例中，免疫治疗剂可以是靶向PD-1、PD-L1、PD-L2、CTLA4、LAG3、TIGIT、TIM3、CEACAM-1、半乳凝素-1、半乳凝素-9、BLTA、CD69、CD113、GPR56、2B4、CD48、PD1H、LAIR1、TIM-1、TIM-4、VISTA、GARP、CD73、CD39、A2AR、或其组合的拮抗剂；靶向B7-1、B7-2、4-1BBL、CD137、CD28、CD28H、GITR、GITRL、ICOS、ICOS-L、OX40、OX40L、CD70、CD27、CD40、DR3、或其组合的激动剂；或前述拮抗剂和激动剂的组合。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与靶向免疫细胞或癌细胞上的细胞表面分子(包 括但不限于：EGFR、HER2、CD38、间皮素、CD33、CD37、CD19、CD20、CD3、CD123、CD70、BAFFR、CD4、CD8、CD56、CD38)的抗体或此类抗体的组合进行组合给予。在一些实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与靶向免疫细胞或癌细胞上的细胞表面分子(包括但不限于：EGFR、HER2)的抗体或此类抗体的组合进行组合给予。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与至少一种化学治疗剂(如烷化剂、抗代谢物、铂酸盐剂、紫杉醇、抗激素剂、拓扑异构酶抑制剂、微管蛋白药剂、信号传导抑制剂(例如，激酶抑制剂)和其他化学治疗剂)组合给予。“化学治疗剂”是在癌症治疗中有用的化学化合物。化学治疗剂的实施例包括：烷化剂，如噻替派和环磷酰胺
Figure PCTCN2020102678-appb-000011
烷基磺酸盐，如白消安、英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)；氮丙啶类(aziridines)，如苯并多巴(benzodopa)、卡巴醌(carboquone)、美妥替哌(meturedopa)和脲多巴(uredopa)；乙烯亚胺和甲基蜜胺类(methylamelamines)，包括六甲蜜胺、曲他胺、三亚乙基磷酰胺(trietylenephosphoramide)、三亚乙基硫代磷酰胺(triethiylenethiophosphoramide)和三羟甲蜜胺(trimethylolomelamine)；多聚乙酰类(acetogenins)(尤其是布拉它辛(bullatacin)和布拉他辛酮(bullatacinone)；δ-9-四氢大麻酚(屈大麻酚，
Figure PCTCN2020102678-appb-000012
)；β-拉帕醌；拉帕醇；秋水仙碱；桦木酸；喜树碱(包括合成类似物拓扑替康
Figure PCTCN2020102678-appb-000013
CPT-11(伊立替康、
Figure PCTCN2020102678-appb-000014
)、乙酰基喜树碱(acetylcamptothecin)、莨菪亭(scopolectin)和9-氨基喜树碱(aminocamptothecin))；苔藓虫素；callystatin；CC-1065(包括其阿多来新(adozelesin)、卡折来新(carzelesin)和比折来新(bizelesin)合成类似物)；鬼臼毒素(podophyllotoxin)；鬼臼酸(podophyllinic acid)；替尼泊苷；念珠藻素类(cryptophycins)(具体是念珠藻素1和念珠藻素8)；尾海兔素(dolastatin)；倍癌霉素(duocarmycin)(包括合成类似物，KW-2189和CB1-TM1)；五加素(eleutherobin)；水鬼蕉碱(pancratistatin)；匍枝珊瑚醇(sarcodictyin)；海绵抑制素(spongistatin)；氮芥类，如苯丁酸氮芥、萘氮芥、胆怜酸胺(cholophosphamide)、雌莫司汀、异环磷酰胺、氮芥、盐酸氧氮芥(mechlorethamine oxide hydrochloride)、美法仑、新恩比兴(novembichin)、苯芥胆甾醇(phenesterine)、泼尼氮芥(prednimustine)、曲磷胺(trofosf amide)、乌拉莫司汀；亚硝基脲类(nitrosureas)，如卡莫司汀、氯脲霉素(chlorozotocin)、福莫司汀(fotemustine)、环己亚硝脲、尼莫司汀(nimustine)、和雷莫司汀(ranimnustine)；抗生素类，如烯二炔抗生素(例如，卡里奇霉素，尤其是卡里奇霉素γ1I和卡里奇霉素ωI1(参见，例如，Nicolaou KC.等人,Angew.Chem. Int.Ed.Engl.[应用化学英文国际版],33:183-186(1994))；dynemicin，包括dynemicin A；埃斯佩拉霉素(esperamicin)；以及新制癌菌素发色团(neocarzinostatin chromophore)和相关的色蛋白烯二炔抗生素发色团)、阿克拉霉素类(aclacinomysins)、放线菌素、氨茴霉素(authramycin)、重氮丝氨酸(azaserine)、博莱霉素、放线菌素C、卡柔比星(carabicin)、洋红霉素、嗜癌霉素(carzinophilin)、色霉素(chromomycinis)、放线菌素D、道诺霉素、地托比星(detorubicin)、6-重氮-5-氧代-L-正亮氨酸、多柔比星(包括
Figure PCTCN2020102678-appb-000015
吗啉代-多柔比星、氰基吗啉代-多柔比星、2-吡咯啉代-多柔比星、多柔比星HCl脂质体注射
Figure PCTCN2020102678-appb-000016
多柔比星脂质体TLC D-99
Figure PCTCN2020102678-appb-000017
聚乙二醇化脂质体多柔比星
Figure PCTCN2020102678-appb-000018
和去氧多柔比星)、表柔比星、依索比星(esorubicin)、伊达比星、麻西罗霉素(marcellomycin)、丝裂霉素如丝裂霉素C、霉酚酸、诺加霉素、橄榄霉素类(olivomycins)、培洛霉素(peplomycin)、泊非霉素(potfiromycin)、嘌呤霉素(puromycin)、三铁阿霉素(quelamycin)、罗多比星(rodorubicin)、链黑菌素(streptonigrin)、链脲菌素(streptozocin)、杀结核菌素(tubercidin)、乌苯美司(ubenimex)、新制癌菌素(zinostatin)、佐柔比星(zorubicin)；抗代谢物，如甲氨蝶呤、吉西他滨
Figure PCTCN2020102678-appb-000019
喃氟啶(tegafur)
Figure PCTCN2020102678-appb-000020
卡培他滨
Figure PCTCN2020102678-appb-000021
埃博霉素和5-氟脲嘧啶(5-FU)；叶酸类似物，如二甲叶酸、甲氨蝶呤、蝶罗呤(pteropterin)、三甲曲沙(trimetrexate)；嘌呤类似物，如氟达拉滨，6-巯嘌呤、硫咪嘌呤(thiamiprine)、硫鸟嘌呤；嘧啶类似物，如环胞苷(ancitabine)、阿扎胞苷、6-氮尿苷(azauridine)、卡莫氟(carmofur)、阿糖孢苷、二脱氧尿苷(dideoxyuridine)、去氧氟尿苷(doxifluridine)、依诺他滨、氟尿苷；抗肾上腺类，如氨鲁米特、米托坦、曲洛司坦；叶酸补充剂，如亚叶酸；醋葡醛内酯(aceglatone)；醒磷酰胺糖苷(aldophosphamide glycoside)；氨基乙酰丙酸；恩尿嘧啶(eniluracil)；安吖啶；bestrabucil；比生群(bisantrene)；依达曲沙(edatraxate)；地佛法明(defofamine)；地美可辛(demecolcine)；地吖醌(diaziquone)；依洛尼塞(elfornithine)；依利醋铵(elliptinium acetate)；依托格鲁(etoglucid)；硝酸镓；羟基脲；香菇多糖；洛尼代宁(lonidainine)；美登木素生物碱，如美登素和安丝菌素(ansamitocins)；米托胍腙(mitoguazone)；米托蒽醌；莫匹丹莫(mopidanmol)；二胺硝吖啶(nitraerine)；喷司他丁；苯来美特(phenamet)；吡柔比星(pirarubicin)；洛索蒽醌(losoxantrone)；2-乙基酰肼(ethylhydrazide)；丙卡巴肼；
Figure PCTCN2020102678-appb-000022
多糖复合物(JHS自然产品公司(JHS Natural Products)，尤金市(Eugene)，俄勒冈州(Oreg))；丙亚胺(razoxane)；根霉素；西佐喃；锗螺胺(spirogermanium)；细交链孢菌酮酸(tenuazonic acid)；三亚胺醌；2,2′,2″-三氯三乙胺；单端孢霉烯(trichothecenes)(尤其是T-2 毒素、维拉库林(verracurin)A、杆孢菌素(roridin)A和蛇形菌素(anguidine)；乌拉坦(urethan)；达卡巴嗪；甘露醇氮芥(mannomustine)；二溴甘露醇；二溴卫矛醇；哌泊溴烷；加西托新(gacytosine)；阿拉伯糖苷(“Ara-C”)；噻替派；紫杉烷类，例如紫杉醇
Figure PCTCN2020102678-appb-000023
紫杉醇(ABRAXANE TM)的白蛋白工程化的纳米颗粒配制品、和多西他赛
Figure PCTCN2020102678-appb-000024
苯丁酸氮芥(chloranbucil)；6-硫鸟嘌呤；巯嘌呤；甲氨蝶呤；铂剂，如顺铂、奥沙利铂和卡铂；预防微管蛋白聚合形成微管的长春花类(vincas)，包括长春花碱
Figure PCTCN2020102678-appb-000025
长春新碱
Figure PCTCN2020102678-appb-000026
长春地辛
Figure PCTCN2020102678-appb-000027
和长春瑞滨
Figure PCTCN2020102678-appb-000028
依托泊苷(VP-16)；异环磷酰胺；米托蒽醌；甲酰四氢叶酸；能灭瘤(novantrone)；依达曲沙(edatrexate)；道诺霉素(daunomycin)；氨基蝶呤；伊班膦酸钠(ibandronate)；拓扑异构酶抑制剂RFS 2000；二氟甲基鸟胺酸(DMFO)；类视黄醇，如视黄酸，包括蓓萨罗丁(bexarotene)
Figure PCTCN2020102678-appb-000029
双膦酸盐，如氯膦酸盐(例如，
Figure PCTCN2020102678-appb-000030
或
Figure PCTCN2020102678-appb-000031
)、依替膦酸钠(etidronate)
Figure PCTCN2020102678-appb-000032
NE-58095、唑来膦酸/唑来膦酸盐
Figure PCTCN2020102678-appb-000033
阿仑膦酸盐
Figure PCTCN2020102678-appb-000034
帕米膦酸二钠(pamidronate)
Figure PCTCN2020102678-appb-000035
替鲁膦酸盐(tiludronate)
Figure PCTCN2020102678-appb-000036
或利塞膦酸盐
Figure PCTCN2020102678-appb-000037
曲沙他滨(troxacitabine)(1,3-二氧杂环戊烷核苷胞嘧啶类似物)；反义寡核苷酸，特别是那些抑制与异常细胞增殖有关的信号传导途径中基因(例如PKC-α、Raf、H-Ras和表皮生长因子受体(EGF-R))表达的反义寡核苷酸；疫苗，如
Figure PCTCN2020102678-appb-000038
疫苗和基因疗法疫苗，例如
Figure PCTCN2020102678-appb-000039
疫苗、
Figure PCTCN2020102678-appb-000040
疫苗和
Figure PCTCN2020102678-appb-000041
疫苗；拓扑异构酶1抑制剂(例如，
Figure PCTCN2020102678-appb-000042
)；rmRH(例如，
Figure PCTCN2020102678-appb-000043
)；BAY439006(索拉非尼；拜耳公司(Bayer))；SU-11248(辉瑞公司(Pfizer))；哌立福辛(perifosine)、COX-2抑制剂(例如，塞来昔布(celecoxib)或依托考昔(etoricoxib))、蛋白酶体抑制剂(例如，PS341)；硼替佐米
Figure PCTCN2020102678-appb-000044
CCI-779；替吡法尼(ipifarnib)(R11577)；orafenib、ABT510；Bcl-2抑制剂，如奥利默森钠(blimersen sodium)
Figure PCTCN2020102678-appb-000045
匹克生琼(pixantrone)；EGFR抑制剂(参见下文定义)；酪氨酸激酶抑制剂(参见下文定义)；以及任何上述的药学上可接受的盐、酸或衍生物；以及上述两种或多种的组合，如CHOP(环磷酰胺、多柔比星、长春新碱和泼尼松龙组合疗法的缩写)，以及FOLFOX(奥沙利铂(ELOXATIN TM)组合5-FU和甲酰四氢叶酸的治疗方案的缩写)。
本文中，化学治疗剂包括用于调节、减少、阻断或抑制可促进癌症生长的激素的作用的“抗激素剂”或“内分泌治疗剂”。它们本身可能是激素，包括但不限于：具有混合激动剂/拮抗剂谱的抗雌激素，包括三苯氧胺
Figure PCTCN2020102678-appb-000046
4-羟基三苯氧胺，托瑞米芬
Figure PCTCN2020102678-appb-000047
艾多昔芬(idoxifene)、屈洛昔芬(droloxifene)、雷洛昔芬
Figure PCTCN2020102678-appb-000048
曲沃昔芬、雷洛西芬(keoxifene)和 选择性雌激素受体调节剂(SERM)如SERM3；不具有激动剂特性的纯抗雌激素，如氟维司群(fulvestrant)
Figure PCTCN2020102678-appb-000049
和EM800(此类药剂可以封闭雌激素受体(ER)二聚化、抑制DNA结合、增加ER周转和/或抑制ER水平)；芳香酶抑制剂，包括甾体芳香酶抑制剂如福美斯坦(formestane)和依西美坦
Figure PCTCN2020102678-appb-000050
和非甾体芳香酶抑制剂如阿那曲唑
Figure PCTCN2020102678-appb-000051
来曲唑
Figure PCTCN2020102678-appb-000052
和氨鲁米特，以及其他芳香酶抑制剂包括伏氯唑(vorozole)
Figure PCTCN2020102678-appb-000053
醋酸甲地孕酮
Figure PCTCN2020102678-appb-000054
法倔唑(fadrozole)和4(5)-咪唑；促黄体激素释放激素激动剂，包括亮丙瑞林(
Figure PCTCN2020102678-appb-000055
和
Figure PCTCN2020102678-appb-000056
)、戈舍瑞林、布舍瑞林(buserelin)和曲普瑞林(tripterelin)；性类固醇，包括孕激素如醋酸甲地孕酮和醋酸甲羟孕酮，雌激素如己烯雌酚和结合雌激素(premarin)，以及雄激素/类视黄醇如氟甲睾酮，全反视黄酸(和芬维A胺；奥那司酮(onapristone)；抗孕酮；雌激素受体下调剂(ERD)；抗雄激素，如氟他胺、尼鲁米特(nilutamide)和比卡鲁胺(bicalutamide)；以及任何上述的药学上可接受的盐、酸或衍生物；以及上述两种或更多种的组合。
本文中，“紫杉烷”是具有抑制微管解聚功能的化学治疗剂。实施例包括紫杉醇
Figure PCTCN2020102678-appb-000057
紫杉醇(ABRAXANE TM)的白蛋白工程化的纳米颗粒配制品、和多西他赛
Figure PCTCN2020102678-appb-000058
优选的紫杉烷是紫杉醇。
如本文使用的，术语“EGFR抑制剂”是指与EGFR结合或以其他方式直接与EGFR相互作用并防止或降低其信号传导活性的化合物，并且可替代地称为“EGFR拮抗剂”。此类药剂的实施例包括与EGFR结合的抗体和小分子。与EGFR结合的抗体的实施例包括MAb 579(ATCC CRL HB 8506)、MAb 455(ATCC CRL HB8507)、MAb 225(ATCC CRL 8508)、MAb 528(ATCC CRL 8509)(参见，美国专利号4,943,533，Mendelsohn等人)及其变体，如嵌合的225(C225或西妥昔单抗(Cetuximab)；
Figure PCTCN2020102678-appb-000059
)和重新成形的人225(H225)(参见，WO96/40210，埃克隆系统公司(Imclone Systems Inc.))；IMC-11F8(完全人EGFR靶向抗体(埃克隆公司(Imclone)))结合II型突变体EGFR的抗体(美国专利号5,212,290)；如美国专利号5,891,996中描述的，结合EGFR的人源化和嵌合抗体；以及结合EGFR的人抗体，如ABX-EGF或帕尼单抗(参见WO 98/50433，(安根尼克斯公司(Abgenix)/安进公司(Amgen)))；EMD 55900(Stragliotto G.等人,Eur.J.Cancer[欧洲癌症杂志]32A(4):636-640(1996))；针对EGFR且与EGF和TGF-α两者竞争EGFR结合的EMD7200(马妥珠单抗(matuzumab))人源化EGFR抗体(EMD公司/默克公司(Merck))；人EGFR抗体，HuMax-EGFR(GenMab公司)；称作E1.1、E2.4、E2.5、E6.2、E6.4、E2.11、E6.3和E7.6.3并且在美国专利号6,235,883中描述的完全人抗体；MDX-447(梅达雷克斯公司(Medarex Inc))；和mAb 806或人源化mAb 806(Johns TG.等人,J.Biol.Chem.[生物化学杂志] 279(29):30375-30384(2004))。抗EGFR抗体可以与细胞毒性剂缀合，由此生成免疫缀合物(参见，例如，EP659,439A2，默克专利有限公司(Merck Patent GmbH))。EGFR拮抗剂包括小分子，例如在以下中描述的化合物：美国专利号：5,616,582、5,457,105、5,475,001、5,654,307、5,679,683、6,084,095、6,265,410、6,455,534、6,521,620、6,596,726、6,713,484、5,770,599、6,140,332、5,866,572、6,399,602、6,344,459、6,602,863、6,391,874、6,344,455、5,760,041、6,002,008和5,747,498，以及以下PCT出版物：WO98/14451、WO 98/50038、WO 99/09016和WO 99/24037。特定小分子EGFR拮抗剂包括OSI-774(CP-358774、埃罗替尼、
Figure PCTCN2020102678-appb-000060
基因泰克公司(Genentech)/OSI制药公司(OSI Pharmaceuticals))；PD 183805(CI 1033、2-丙烯酰胺，N-[4-[(3-氯-4--氟苯基)氨基]-7-[3-(4-吗啉基)丙氧基]-6-喹唑啉基]-，二盐酸盐，辉瑞有限公司(Pfizer Inc.))；ZD1839，吉非替尼(易瑞沙(IRESSA)J)4-(3′-氯-4′-氟苯胺基)-7-甲氧基-6-(3-吗啉代丙氧基)喹唑啉，阿斯利康公司(AstraZeneca))；ZM 105180((6-氨基-4-(3-甲基苯基-氨基)-喹唑啉，捷利康公司(Zeneca))；BIBX-1382(N8-(3-氯-4-氟-苯基)-N2-(1-甲基-哌啶-4-基)-嘧啶并[5,4-d]嘧啶-2,8-二胺，勃林格殷格翰公司(Boehringer Ingelheim))；PKI-166((R)-4-[4-[(1-苯乙基)氨基]-1H-吡咯并[2,3-d]嘧啶-6-基]-苯酚)；(R)-6-(4-羟基苯基)-4-[(1-苯乙基)氨基]-7H-吡咯并[2,3-d]嘧啶)；CL-387785(N-[4-[(3-溴苯基)氨基]-6-喹唑啉基]-2-丁炔酰胺)；EKB-569(N-[4-[(3-氯-4-氟苯基)氨基]-3-氰基-7-乙氧基-6-喹啉基]-4-(二甲基氨基)-2-丁酰胺)(惠氏公司(Wyeth))；AG1478(苏根公司(Sugen))；AG1571(SU 5271；苏根公司(Sugen))；双重EGFR/HER2酪氨酸激酶抑制剂，如拉帕替尼(GW 572016或N-[3-氯-4-[(3氟苯基)甲氧基]苯基]6[5[[[2甲磺酰基)乙基]氨基]甲基]-2-呋喃基]-4-氨基喹唑啉；葛兰素史克公司(Glaxo-SmithKline))。
“酪氨酸激酶抑制剂”是抑制酪氨酸激酶如HER受体的酪氨酸激酶活性的分子。此类抑制剂的实施例包括前述段落中提到的EGFR-靶向药物；小分子HER2酪氨酸激酶抑制剂，例如TAK165(可获得自武田药品公司(Takeda))；CP-724,714(ErbB2受体酪氨酸激酶的口服选择性抑制剂(辉瑞公司(Pfizer)和OSI公司)；双重HER抑制剂，如优先结合EGFR但抑制过表达HER2和EGFR两者的细胞的EKB-569(可获得自惠氏公司(Wyeth))；拉帕替尼(GW572016；可获得自葛兰素史克公司(Glaxo-SmithKline))，一种口服HER2和EGFR酪氨酸激酶抑制剂；PKI-166(可获得自诺华公司(Novartis))；泛HER抑制剂，如卡奈替尼(canertinib)(CI-1033；法玛西亚公司(Pharmacia))；Raf-1抑制剂，如抑制Raf-1信号传导的反义试剂ISIS-5132(可获得自ISIS制药公司(ISIS  Pharmaceuticals))；非HER靶向TK抑制剂，如甲磺酸伊马替尼(Imatinib mesylate)(格列卫(GLEEVAC)J)，可获得自葛兰素公司(Glaxo)；MAPK细胞外调节激酶I抑制剂CI-1040(可获得自法玛西亚公司(Pharmacia))；喹唑啉类，如PD 153035、4-(3-氯苯胺基)喹唑啉；吡啶并嘧啶类；嘧啶并嘧啶类；吡咯并嘧啶类，如CGP 59326、CGP 60261和CGP 62706；吡唑并嘧啶类，4-(苯基氨基)-7H-吡咯并[2,3-d]嘧啶；姜黄素(二阿魏酰甲烷，4,5-双(4-氟苯胺基)邻苯二甲酰亚胺)；含有硝基噻吩部分的酪氨酸磷酸化抑制剂(tyrphostines)；PD-0183805(华纳兰勃特公司(Warner-Lamber))；反义分子(例如，与HER-编码核酸结合的那些反义分子)；喹喔啉类(美国专利号5,804,396)；酪氨酸磷酸化抑制剂(tryphostins)(美国专利号5,804,396)；ZD6474(阿斯利康公司(Astra Zeneca))；PTK-787(诺华公司(Novartis)/先灵公司(Schering AG))；泛HER抑制剂，如CI-1033(辉瑞公司(Pfizer))；Affinitac(ISIS 3521；Isis公司/礼来公司(Lilly))；甲磺酸伊马替尼(格列卫(Gleevac)；诺华公司(Novartis))；PKI 166(诺华公司(Novartis))；GW2016(葛兰素史克公司(Glaxo SmithKline))；CI-1033(辉瑞公司(Pfizer))；EKB-569(惠氏公司(Wyeth))；Semaxinib(苏根公司(Sugen))；ZD6474(阿斯利康公司(AstraZeneca))；PTK-787(诺华公司(Novartis)/先灵公司(Schering AG))；INC-1C11(埃克隆公司(Imclone))；或如以下专利出版物中任一个所述：美国专利号5,804,396；WO99/09016(美国氰胺公司(American Cyanamid))；WO 98/43960(美国氰胺公司(American Cyanamid))；WO 97/38983(华纳兰勃特公司(Warner Lambert))；WO 99/06378(华纳兰勃特公司(Warner Lambert))；WO 99/06396(华纳兰勃特公司(Warner Lambert))；WO 96/30347(辉瑞有限公司(Pfizer Inc.))；WO 96/33978(捷利康公司(Zeneca))；WO 96/3397(捷利康公司(Zeneca))；和WO 96/33980(捷利康公司(Zeneca))。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与放线菌素D、卡培他滨、卡铂、顺铂、秋水仙碱、道诺霉素、多西他赛、多柔比星、表柔比星、依托泊苷、5-氟脲嘧啶、吉西他滨、美法仑、甲氨蝶呤、丝裂霉素C、米托蒽醌、紫杉醇、萨力多胺、拓扑替康、长春花碱、和长春新碱组合给予。
在各种实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可以与一种或多种另外的疗法或临床干预(如放射疗法、手术、化学疗法和/或靶向疗法)组合给予。在某些实施例中，所披露的抗CD73抗体和/或变体(或其片段)和/或本文提供的药物组合物与针对所披露的癌症的放射疗法组合给予。
在一些实施例中，所披露的抗CD73抗体和/或变体或突变体(或其片段)和 /或本文提供的药物组合物可以与手术(例如，可以在给予所披露的抗CD73抗体之前进行手术、可以在给予所披露的抗CD73抗体之后进行手术、和/或可以与给予所披露的抗CD73抗体同时进行或几乎同时进行手术)联合给予。
取决于待治疗的适应症和本领域技术人员熟悉的与给药相关的因素，本文提供的抗CD73抗体、变体、突变体或其片段将以有效剂量给予，例如在最小化毒性和副作用的同时有效治疗该适应症的剂量。通常优选使用最大剂量的本披露的药理学药剂(单独或与其他治疗剂组合)，即根据合理的医学判断的最高安全剂量。然而，本领域的普通技术人员将理解，出于医学原因、心理原因或几乎任何其他原因，患者可坚持较低剂量或可耐受剂量。在一些实施例中，有效剂量可以使得所希望的应答是抑制疾病或病症的进展，例如癌症的进展。这可能仅涉及暂时减缓疾病的进展，但更优选地，这涉及永久地停止疾病的进展。这可以通过本领域的普通技术人员已知的针对任何特定疾病的常规诊断方法来监测。对疾病或病症的治疗的所希望的应答也可以是延迟发作或甚至预防疾病或病症的发作。
可以通过细胞培养物或实验动物中的标准药物程序确定所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物的毒性和治疗功效，例如确定LD 50(使50％的群体致死的剂量)和ED 50(在50％的患者群体中治疗有效的剂量)。在毒性和治疗效果之间的剂量比率为治疗指数，并且它可以表示为比率LD 50/ED 50。可以希望表现出大治疗指数的化合物。尽管可以使用表现出毒副作用的所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物，但是应该小心设计将此类化合物靶向至受影响的组织的部位的递送系统，从而将对未感染的细胞的潜在损伤最小化，并且由此减少副作用。
从细胞培养测定和动物研究获得的数据可以用于配制一系列的用于在人中使用的剂量。所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物的剂量优选地在包括ED 50且具有很小毒性或无毒性的循环浓度范围内。剂量可根据所采用的剂型和所使用的给予途径在该范围内变化。对于任何所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物，最初可以从细胞培养测定来评估治疗有效量。可以在动物模型中配制剂量以达到包括如细胞培养实验中确定的IC 50的循环血浆浓度范围。该信息可用于更精确地确定在人中有用的剂量。可以例如通过高效液相色谱法来测量血浆中的水平。
可以通过使用本领域已知的或在Yamanaka K.等人,Microbiol.Immunol.[微生物免疫]45(7):507-514(2001)中描述的各种实验动物模型(例如SCID小鼠模型或具有人肿瘤移植物的裸鼠)确定所披露的抗CD73抗体和/或 变体或突变体(或其片段)和/或本文提供的药物组合物的抗癌活性。
在用于人之前，可以在体外测试、然后在体内测试所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物的所希望的治疗或预防活性。例如，可以用于确定是否指示给予特定治疗方案的体外测定包括体外细胞培养测定，其中患者组织样品在培养中生长、并暴露于方案或以其他方式给予方案，且观察这种方案对该组织样品的作用。
接触的细胞的较低水平的增殖或存活可以指示所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物可在受试者中有效治疗选择的障碍。可替代地，可以使用肿瘤细胞系或恶性细胞系(而不是从患者培养细胞)来筛选方案。可以使用本领域已知的许多测定来评估这种存活和/或生长；例如，可以通过测量3H-胸苷掺入、通过直接细胞计数、通过检测已知基因如原癌基因或细胞周期标志物的转录活性的变化来测定细胞增殖；可以通过台盼蓝染色来评估细胞活力，同时可以基于形态学等的变化来目测评估分化。
在人中测试之前，可以在合适的动物模型系统(括但不限于大鼠、小鼠、鸡、牛、猴、兔等)中测试用于疗法的所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物。本领域已知并广泛使用的癌症的主要动物模型包括小鼠，如Hann B.等人,Curr.Opin.Cell Biol.[细胞生物学新观点]13(6):778-784(2001)中描述的，其全部内容通过引用并入本文。
此外，本领域技术人员已知的任何测定可用于评估所披露的抗CD73抗体和/或变体或突变体(或其片段)和/或本文提供的药物组合物用于治疗、预防、管理或改善一种或多种与如上所述的疾病或障碍相关的症状的预防和/或治疗效用。
本领域技术人员可以容易地确定用于治疗或预防本文描述的障碍的合适的日剂量。对于所披露的癌症的治疗，典型的剂量可以是例如在0.001μg至1000μg的范围内；然而，低于或高于该示例性范围的剂量在本披露的范围内。日剂量可以是约0.1μg/kg总体重至约100mg/kg总体重(例如，约5μg/kg、约10μg/kg、约100μg/kg、约500μg/kg、约1mg/kg、约50mg/kg，或由前述值中的任两个所定义的范围)，优选地从约0.3μg/kg总体重至约10mg/kg总体重(例如，约0.5μg/kg、约1μg/kg、约50μg/kg、约150μg/kg、约300μg/kg、约750μg/kg、约1.5mg/kg、约5mg/kg，或由前述值中的任两个所定义的范围)，更优选地从约1μg/kg总体重至1mg/kg总体重(例如，约3μg/kg、约15μg/kg、约75μg/kg、约300μg/kg、约900μg/kg，或由前述值中的任两个所定义的范围)，并且甚至更优选地从约0.5mg/kg总体重/天至10mg/kg总体重/天(例如，约2mg/kg、约4mg/kg、约7mg/kg、约9mg/kg，或由前述值中的任两个所定义的范围，包括前述值之间的任何范围)。如上提到的，可以通 过定期评估治疗的患者来监测治疗或预防功效。对于经若干天或更长时间的重复给予，取决于病症，重复进行治疗直至发生所希望的疾病症状抑制。然而，其他剂量方案可能是有用的并且在本披露的范围内。所希望的剂量可通过单次推注给予组合物、通过多次推注给予组合物、或通过连续输注给予组合物来递送。
包含抗CD73抗体、变体、突变体或其片段的药物组合物可以每天给予一次、两次、三次或四次。组合物也能以低于每日给予的频率进行给予，例如，每周六次、每周五次、每周四次、每周三次、每周两次、每周一次、每两周一次、每三周一次、每月一次、每两个月一次、每三个月一次或每六个月一次。组合物也可以例如在植入物中以缓释配制品给予，该植入物逐渐释放该组合物以在一段时间内使用，并且允许该组合物以较低频率给予，例如每月一次、每2-6个月一次、每年一次、或甚至单次给予。缓释装置(例如圆粒剂型(pellets)、纳米颗粒、微粒、纳米球、微球等)可以通过注射进行给予。
抗体和/或变体或突变体(或其片段)能以单次日剂量给予，或者总日剂量能以每日两次、三次或四次的分剂量给予。组合物也能以低于每日给予的频率进行给予，例如，每周六次、每周五次、每周四次、每周三次、每周两次、每周一次、每两周一次、每三周一次、每月一次、每两个月一次、每三个月一次或每六个月一次。抗体(或其片段)也可以例如在植入物中以缓释配制品给予，该植入物逐渐释放该组合物以在一段时间内使用，并且允许该组合物以较低频率给予，例如每月一次、每2-6个月一次、每年一次、或甚至单次给予。缓释装置(例如圆粒剂型(pellets)、纳米颗粒、微粒、纳米球、微球等)可以通过注射或手术植入各种位置进行给予。
可以通过例如但不限于肿瘤消退、肿瘤重量或尺寸缩小、进展时间、存活持续时间、无进展存活、总体反应率、应答持续时间、生活质量、蛋白质表达和/或活性来评估癌症治疗。可以采用确定疗法功效的方法，包括例如通过放射成像测量反应。
在一些实施例中，将治疗功效测量为肿瘤生长抑制百分比(％TGI)，使用等式100-(T/C x 100)进行计算，其中T是治疗的肿瘤的平均相对肿瘤体积，并且C是未治疗的肿瘤的平均相对肿瘤体积。在某些实施例中，％TGI是约10％、约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、或多于95％。在某些实施例中，抗CD73和/或其变体或突变体的％TGI与参照抗CD73抗体的％TGI相同或比其高例如约1.1倍、约1.2倍、约1.3倍、约1.4倍、约1.5倍、约1.6倍、约1.7倍、约1.8倍、约1.9倍、约2倍、约2.1倍、约2.2倍、约2.3倍、约2.4倍、约2.5倍、约2.6倍、约2.7倍(包括这些值之间的任何范围)，或比参照抗CD73抗体的％TGI 高约2.7倍。
药物组合物
可以将本发明的所披露的抗CD73抗体和/或变体或突变体(或其片段)制备成包含合适的载体或赋形剂的药物组合物，使得它们适于给予。通过将具有所希望的纯度的抗体(或其片段)与任选的药学上可接受的载体、赋形剂或稳定剂(Remington's Pharmaceutical Sciences[雷明顿药物科学]第16版,Osol,A.编辑(1980))混合，以冻干的配制品或水溶液的形式获得抗体的合适的药物组合物。可接受的载体、赋形剂或稳定剂在所采用的剂量和浓度下对受体无毒，并且包括缓冲液(如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和蛋氨酸；防腐剂(如十八烷基二甲基苄基氯化铵；六甲基氯化铵；苯扎氯铵、苄索氯铵；苯酚、丁基或苄醇；烷基对羟基苯甲酸酯，如甲基对羟基苯甲酸酯或丙基对羟基苯甲酸酯；儿茶酚；间苯二酚；环己醇；3-戊醇；和间甲酚)；低分子量(少于约10个残基的)多肽；蛋白质，如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，如聚乙烯吡咯烷酮；氨基酸，如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸、或赖氨酸；单糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖、或糊精；螯合剂，如EDTA；糖类，如蔗糖、甘露醇、海藻糖或山梨糖醇；成盐反离子，如钠；金属络合物(例如锌-蛋白质络合物)；和/或非离子表面活性剂，如TWEEN TM、PLURONICS TM或聚乙二醇(PEG)。示例性抗体配制品描述于WO 98/56418中，其通过引用明确地并入本文。适于皮下给予的冻干的药物组合物描述于WO 97/04801中。此类冻干的药物组合物可以用合适的稀释剂重构成高蛋白质浓度，并且重构的配制品可以皮下给予至本文中待治疗的哺乳动物。
在一些实施例中，所披露的药物组合物是适于肠胃外或静脉内给予。在一些实施例中，所披露的适于肠胃外给予的药物组合物可以是与预期受试者的血液等渗的水性和非水性配制品；水性和非水性无菌悬浮液可包括悬浮系统，其设计用于将化合物靶向一个或多个器官的血液组分。配制品可以存在于单位剂量或多剂量密封容器(例如安瓿或小瓶)中。临时注射溶液和悬浮液可以由先前描述类型的无菌粉末、颗粒和片剂制备。肠胃外和静脉内配制品可以包括矿物质和其他材料，以使它们与选择的注射或递送系统类型相容。
用于肠胃外给予的常用的药学上可接受的载体包括水、合适的油、盐水、右旋糖(葡萄糖)水溶液或相关的糖溶液和二醇如丙二醇或聚乙二醇。用于肠胃外给予的溶液优选地含有活性成分的水溶性盐、合适的稳定剂和(如果必要)缓冲物质，单独或组合的抗氧化剂(如亚硫酸氢钠、亚硫酸钠或抗坏血酸)是合适的稳定剂。柠檬酸盐和EDTA钠也可用作载体。此外，肠胃外溶液可以含有 防腐剂，例如苯扎氯铵、甲基对羟基苯甲酸酯或丙基对羟基苯甲酸酯、或氯丁醇。合适的药物载体描述于上文引用的Remington[雷明顿]中。
可注射配制品能以常规形式来制备，或者作为液体溶液或悬浮液；作为适于在注射前溶解或悬浮在液体中的固体形式；或作为乳液。优选地，根据本领域已知的技术，使用合适的药学上可接受的载体和如上所述的其他任选的组分配制无菌可注射悬浮液。
肠胃外给予能以许多方式进行，但优选使用注射器、导管或类似装置来实现本文描述的配制品的肠胃外给予。可以全身注射配制品，使得活性剂传播基本上遍布整个血流。
此外，还可以将配制品局部注射到靶位点，例如，注射到身体的特定部分以治疗癌症或纤维化是希望的。经由注射的局部给予的优点是其限制或避免整个身体暴露于一种或多种活性剂(例如，抑制剂和/或其他治疗剂)。必须注意的是，在本发明的上下文中，术语局部给予包括区域给予，例如，通过递送至服务于该身体区域的血管来给予针对身体的一部分的配制品。局部递送可以是直接的，例如，瘤内递送。局部递送也可以是几乎直接的，即病灶内或腹膜内，也就是说，递送至足够接近肿瘤或感染部位的区域，使得抑制剂表现出所希望的药理学活性。因此，当希望局部递送时，药物配制品优选地在病灶内、瘤内或腹膜内递送。
在某些实施例中，药物组合物处于单位剂型。在这种形式中，将组合物分成含有适量活性组分的单位剂量。单位剂型可以是包装的制剂，该包装含有离散量的制剂，例如小瓶或安瓿中的冻干的粉末。
本文的药物组合物还可含有所治疗的具体适应症所必需的一种以上的活性化合物，优选那些具有互补活性而不会相互产生不利影响的活性化合物。例如，可以希望的是进一步提供抗肿瘤剂、生长抑制剂、细胞毒性剂或化学治疗剂。此类分子合适地以对预期目的有效的量组合存在。此类其他药剂的有效量取决于配制品中存在的抗体的量、疾病或障碍或治疗的类型、以及上文讨论的其他因素。这些通常以与本文描述相同的剂量和给予途径使用，或者以迄今采用的剂量的从约1％至99％使用。活性成分也可以包埋在例如通过凝聚技术或通过界面聚合制备的微胶囊中，例如分别在胶体药物递送系统中(例如，脂质体、白蛋白微球、微乳液、纳米颗粒和纳米胶囊)或在粗乳液中的羟甲基纤维素或明胶-微胶囊和聚(甲基丙烯酸甲酯)微胶囊。此类技术披露于Remington's Pharmaceutical Sciences[雷明顿药物科学],第16版,Osol,A.编辑(1980)中。
可以制备缓释制剂。缓释制剂的合适的实施例包括含有拮抗剂的固体疏水聚合物的半透性基质，这些基质处于成型制品的形式，例如薄膜或微胶囊。缓释 基质的实施例包括聚酯，水凝胶(例如，聚(2-羟乙基-甲基丙烯酸酯)、或聚(乙烯醇))，聚丙交酯(美国专利号3,773,919)，L-谷氨酸和乙基-L-谷氨酸的共聚物，不可降解的乙烯-乙烯基，可降解的乳酸-乙醇酸共聚物(如LUPRON DEPOT TM(由乳酸-乙醇酸共聚物和醋酸亮丙瑞林组成的可注射微球))以及聚-D-(-)-3-羟基丁酸。虽然聚合物(如乙烯-乙酸乙烯酯和乳酸-乙醇酸)的能够释放分子超过100天，但某些水凝胶在更短的时间段内释放蛋白质。当包封的抗体长时间保留在体内时，它们会因暴露于37℃的湿气而变性或聚集，导致生物活性的丧失和免疫原性的可能变化。可以根据所涉及的机制设计合理的策略以实现稳定。例如，如果发现聚集机理是通过硫代-二硫键交换形成分子间S-S键，则可以通过修饰巯基残基、从酸性溶液中冻干、控制水分含量、使用适当的添加剂并且开发特定的聚合物基质组合物来实现稳定化。
脂质转染体(lipofectin)或脂质体可用于将本发明的多肽和抗体(或其片段)或组合物递送到细胞中。在使用抗体片段的情况下，优选特异性结合靶蛋白结合域的最小抑制片段。例如，基于抗体的可变区序列，可以设计肽分子使其保留结合靶蛋白序列的能力。此类肽可以是化学合成地和/或通过重组DNA技术产生。参见，例如，Marasco WA.等人,Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]90(16):7889-7893(1993)。
活性成分也可以包埋在例如通过凝聚技术或通过界面聚合制备的微胶囊中，例如分别在胶体药物递送系统中(例如，脂质体、白蛋白微球、微乳液、纳米颗粒和纳米胶囊)或在粗乳液中的羟甲基纤维素或明胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊。此类技术披露于Remington's PHARMACEUTICAL SCIENCES[雷明顿药物科学]，同上。
可以制备缓释制剂。缓释制剂的合适的实施例包括含有抗体(或其片段)的固体疏水聚合物的半透性基质，这些基质处于成型制品的形式，例如薄膜或微胶囊。缓释基质的实施例包括聚酯，水凝胶(例如，聚(2-羟乙基-甲基丙烯酸酯)、或聚(乙烯醇))，聚丙交酯(美国专利号3,773,919)，L-谷氨酸和乙基-L-谷氨酸的共聚物，不可降解的乙烯-乙酸乙烯酯，可降解的乳酸-乙醇酸共聚物(如LUPRON DEPOT TM(由乳酸-乙醇酸共聚物和醋酸亮丙瑞林组成的可注射微球))以及聚-D-(-)-3-羟基丁酸。虽然聚合物(如乙烯-乙酸乙烯酯和乳酸-乙醇酸)的能够释放分子超过100天，但某些水凝胶在更短的时间段内释放蛋白质。当包封的抗体长时间保留在体内时，它们会因暴露于37℃的湿气而变性或聚集，导致生物活性的丧失和免疫原性的可能变化。可以根据所涉及的机制设计合理的策略以实现稳定。例如，如果发现聚集机理是通过硫代-二硫键交换形成分子间S-S键，则可以通过修饰巯基残基、从酸性溶液中冻干、控制水分含量、使用适当的添加剂并且开发特定的聚合物基质组合物来实现稳定化。
在某些实施例中，配制品包含高于约0.5mg/mL、高于约1mg/mL、高于约2mg/mL、高于约3mg/mL、高于约4mg/mL、高于约5mg/mL、高于约6mg/mL、高于约7mg/mL、高于约8mg/mL、高于约9mg/mL、高于约10mg/mL、高于约11mg/mL、高于约12mg/mL、高于约13mg/mL、高于约14mg/mL、高于约15mg/mL、高于约16mg/mL、高于约17mg/mL、高于约18mg/mL、高于约19mg/mL、高于约20mg/mL、高于约21mg/mL、高于约22mg/mL、高于约23mg/mL、高于约24mg/mL、高于约25mg/mL、高于约26mg/mL、高于约27mg/mL、高于约28mg/mL、高于约29mg/mL、或高于约30mg/mL(包括这些值之间的任何范围)浓度的本文描述的抗CD73抗体和/或其变体或突变体。
包含所披露的抗体的配制品应该具有与该抗体或片段的等电点值不相似的pH(在最终配制品中)，例如，如果配制品的pH为7，则pI为从8至9或更高可能是适当的。尽管不希望受理论的束缚，但据认为这可能最终提供具有改善的稳定性的最终配制品，例如抗体或片段保持在溶液中。例如，在某些实施例中，pH在4.0至7.0的范围内的所披露的药物配制品可以包含：1mg/mL至200mg/mL的根据本披露的抗体和1至100mM的缓冲液，以及任选地以下中的一种或多种：(a)0.001％至1％的表面活性剂，(b)10mM至500mM的稳定剂，(c)10mM至500mM的稳定剂和5mM至500mM的张度剂，和/或(d)5mM至500mM的张度剂。合适的缓冲液包括但不限于柠檬酸盐、甘氨酸、磷酸盐、乙酸盐、琥珀酸盐和碳酸氢盐。例如，大约pH 6的配制品可以包含1至50mg/mL的抗体、20mM的L-组氨酸HCl、240mM的海藻糖和0.02％的聚山梨醇酯20。可替代地，大约pH 5.5的配制品可以包含1至50mg/mL的抗体、20mM柠檬酸盐缓冲液、240mM蔗糖、20mM精氨酸和0.02％聚山梨醇酯20。
在某些实施例中，抗CD73抗体和/或其变体或突变体(例如，以高于约0.5mg/mL、高于约1mg/mL、高于约5mg/mL、高于约10mg/mL、高于约15mg/mL、高于约20mg/mL、或高于约25mg/mL(包括这些值之间的任何范围)的浓度)配制于包含柠檬酸盐、NaCl、乙酸盐、琥珀酸盐、甘氨酸、聚山梨醇酯80(Tween80)或前述的任何组合的缓冲液中。在某些实施例中，抗CD73抗体和/或其变体(例如，以高于约0.5mg/mL、高于约1mg/mL、高于约5mg/mL、高于约10mg/mL、高于约15mg/mL、高于约20mg/mL、或高于约25mg/mL(包括这些值之间的任何范围)的浓度)配制于包含约100mM至约150mM甘氨酸的缓冲液中。在某些实施例中，将抗CD73抗体和/或其变体配制于包含约50mM至约100mM NaCl的缓冲液中。在某些实施例中，将抗CD73抗体和/或其变体或突变体(例如，以高于约0.5mg/mL、高于约1mg/mL、高于约5mg/mL、高于约10mg/mL、高于约15mg/mL、高于约20mg/mL、或高于约25mg/mL(包括这些值之间的任何范围)的浓度)配制于包含约10mM至约50mM乙酸盐的缓冲液中。在某些 实施例中，将抗CD73抗体和/或其变体配制于包含约10mM至约50mM琥珀酸盐的缓冲液中。在某些实施例中，将抗CD73抗体和/或其变体或突变体(例如，以高于约0.5mg/mL、高于约1mg/mL、高于约5mg/mL、高于约10mg/mL、高于约15mg/mL、高于约20mg/mL、或高于约25mg/mL(包括这些值之间的任何范围)的浓度)配制于包含约0.005％至约0.02％聚山梨醇酯80的缓冲液中。在某些实施例中，将抗CD73抗体和/或其变体或突变体配制于合适的缓冲液中，以提供在约5.1至5.6之间的缓冲pH。例如，可以将抗CD73抗体和/或其变体或突变体配制于包含10mM柠檬酸盐、100mM NaCl、100mM甘氨酸和0.01％聚山梨醇酯80的缓冲液中，其中该配制品处于pH＝5.5。
在某些实施例中，包含本文描述的抗CD73抗体和/或其变体或突变体(例如，以高于约0.5mg/mL、高于约1mg/mL、高于约5mg/mL、高于约10mg/mL、高于约15mg/mL、高于约20mg/mL、或高于约25mg/mL(包括这些值之间的任何范围)的浓度)的配制品(如包含缓冲液(该缓冲液包含10mM柠檬酸盐、100mM NaCl、100mM甘氨酸和0.01％聚山梨醇酯80)的配制品，其中该配制品处于pH＝5.5)在室温(如在约20℃-25℃)稳定持续约0.5周、1.0周、1.5周、2.0周、2.5周、3.5周、4.0周、4.5周、或5.0周(包括这些值之间的任何范围)。在某些实施例中，包含本文描述的抗CD73抗体和/或其变体或突变体(例如，以高于约0.5mg/mL、高于约1mg/mL、高于约5mg/mL、高于约10mg/mL、高于约15mg/mL、高于约20mg/mL、或高于约25mg/mL(包括这些值之间的任何范围)的浓度)的配制品(如包含缓冲液(该缓冲液包含10mM柠檬酸盐、100mM NaCl、100mM甘氨酸和0.01％聚山梨醇酯80)的配制品，其中该配制品处于pH＝5.5)在加速条件下(如在约37℃储存)稳定持续约0.5周、1.0周、1.5周、2.0周、2.5周、3.5周、4.0周、4.5周、或5.0周(包括这些值之间的任何范围)。
尺寸排阻色谱法(SEC)是用于蛋白质稳定性研究的熟知且广泛使用的方法，以检测对应于物理和化学不稳定性的潜在片段化和聚集。在某些实施例中，如使用SEC测量的，在37℃下1周后，相对于初始高分子量种类的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体的配制品显示小于约1.6％、1.4％、1.2％、1.0％、0.8％、0.6％、0.4％、0.2％、或0.1％(包括这些值之间的任何范围)的高分子量种类(HMWS)的增加。在某些实施例中，如使用SEC测量的，在37℃下2周后，相对于初始高分子量种类的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体的配制品显示小于约2.0％、1.8％1.6％、1.4％、1.2％、1.0％、0.8％、0.6％、0.4％、0.2％、或0.1％(包括这些值之间的任何范围)的高分子量种类的增加。在某些实施例中，如使用SEC测量的，在37℃下4周后， 相对于初始高分子量种类的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体的配制品显示小于约3.3％、3.2％、3.1％、3.0％、2.9％、2.8％、2.7％、2.6％、2.5％、2.4％、2.2％、2.0％、1.8％、1.6％、1.4％、1.2％、1.0％、0.8％、0.6％、0.4％、0.2％、或0.1％(包括这些值之间的任何范围)的高分子量种类的增加。
在某些实施例中，如使用SEC测量的，在37℃下1周后，相对于初始低分子量种类的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体的配制品显示小于约1.6％、1.4％、1.2％、1.0％、0.8％、0.6％、0.4％、0.2％、或0.1％(包括这些值之间的任何范围)的低分子量种类(LMWS)的增加。在某些实施例中，如使用SEC测量的，在37℃下2周后，相对于初始低分子量种类的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体的配制品显示小于约2.0％、1.8％、1.6％、1.4％、1.2％、1.0％、0.8％、0.6％、0.4％、0.2％、或0.1％(包括这些值之间的任何范围)的低分子量种类的增加。在某些实施例中，如使用SEC测量的，在37℃下4周后，相对于初始低分子量种类的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体的配制品显示小于约2.4％、2.2％、2.0％、1.8％、1.6％、1.4％、1.2％、1.0％、0.8％、0.6％、0.4％、0.2％、或0.1％(包括这些值之间的任何范围)的低分子量种类的增加。
在某些实施例中，如使用SEC测量的，在37℃下1周后，相对于初始单体的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或其突变体的配制品显示不超过约0.2％、0.4％、0.6％、0.8％、0.9％、1.0％、1.1％、1.2％、1.3％、1.4％、1.6％、1.7％、1.8％、1.9％、2.0％、2.1％、2.2％、2.3％、2.4％、2.5％、2.6％、2.7％、2.8％、2.9％、3.0％、3.1％、3.2％、3.3％、3.4％、或3.5％(包括这些值之间的任何范围)的单体的减少。在某些实施例中，如使用SEC测量的，在37℃下2周后，相对于初始单体的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或其突变体的配制品显示不超过约0.2％、0.4％、0.6％、0.8％、0.9％、1.0％、1.1％、1.2％、1.3％、1.4％、1.6％、1.7％、1.8％、1.9％、2.0％、2.1％、2.2％、2.3％、2.4％、2.5％、2.6％、2.7％、2.8％、2.9％、3.0％、3.1％、3.2％、3.3％、3.4％、或3.5％(包括这些值之间的任何范围)的单体的减少。在某些实施例中，如使用SEC测量的，在37℃下2周后，相对于初始单体的％，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或其突变体的配制品显示不超过约0.2％、0.4％、0.6％、0.8％、0.9％、1.0％、1.1％、1.2％、1.3％、1.4％、1.6％、1.7％、1.8％、1.9％、2.0％、2.1％、2.2％、2.3％、2.4％、 2.5％、2.6％、2.7％、2.8％、2.9％、3.0％、3.1％、3.2％、3.3％、3.4％、或3.5％(包括这些值之间的任何范围)的单体的减少。
阳离子交换色谱法(CEX)是熟知且广泛使用的工具，以检测蛋白质降解事件，例如脱酰胺或氧化(Moorhouse KG.等人,J.Pharm.Biomed.Anal.[药学和生物医学分析杂志]16(4):593-603(1997))。降解产物典型地称为酸性或碱性物质。酸性物质是早于CEX的主峰洗脱出来的变体，而碱性种类是晚于CEX的主峰洗脱出来的变体。在某些实施例中，如使用CEX测量的，在37℃下1周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或其突变体的配制品的酸性峰部分不超过总蛋白的约7％、8％、9％、10％、11％、12％、13％、14％、或15％(包括这些值之间的任何范围)。在某些实施例中，如使用CEX测量的，在37℃下2周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或其突变体的配制品的酸性峰部分不超过总蛋白的约8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、或18％(包括这些值之间的任何范围)。在某些实施例中，如使用CEX测量的，在37℃下2周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体(或片段)的配制品的酸性峰部分不超过总蛋白的约8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、或27％(包括这些值之间的任何范围)。
在某些实施例中，如使用CEX测量的，在37℃下1周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体(或片段)的配制品的碱性峰部分不超过总蛋白的约39％、40％、41％、42％、43％、44％、45％、或46％(包括这些值之间的任何范围)。在某些实施例中，如使用CEX测量的，在37℃下2周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体(或片段)的配制品的碱性峰部分不超过总蛋白的约39％、40％、41％、42％、43％、44％、45％、或46％(包括这些值之间的任何范围)。在某些实施例中，如使用CEX测量的，在37℃下4周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体(或片段)的配制品的碱性峰部分不超过总蛋白的约39％、40％、41％、42％、43％、44％、45％、或46％(包括这些值之间的任何范围)。
在某些实施例中，如使用CEX测量的，在37℃下1周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体(或片段)的配制品的主峰部分不小于总蛋白的约32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、或46％(包括这些值之 间的任何范围)。在某些实施例中，如使用CEX测量的，在37℃下2周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体(或片段)的配制品的碱性峰部分不小于总蛋白的约32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、或46％(包括这些值之间的任何范围)。在某些实施例中，如使用CEX测量的，在37℃下4周后，包含5mg/mL、10mg/mL、15mg/mL、20mg/mL、或25mg/mL的本文描述的抗CD73抗体和/或其变体或突变体(或片段)的配制品的碱性峰部分不小于总蛋白的约32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、或46％(包括这些值之间的任何范围)。
用于体内给予的配制品必须是无菌的。这可以通过例如穿过无菌过滤膜过滤而容易地实现。
使用抗CD73抗体及其变体/突变体的诊断和成像方法
与CD73特异性结合的经标记的抗CD73抗体、变体、突变体、其片段，以及其衍生物和类似物可用于诊断目的，以检测、诊断或监测与CD73的表达、异常表达和/或活性相关的疾病和/或障碍。例如，本文提供的所披露的抗CD73抗体和/或变体或突变体(或其片段)可以用于原位、体内、离体和体外诊断测定或成像测定。用于检测CD73表达的方法，包括(a)使用本披露的一种或多种抗体测定多肽在个体的细胞(例如组织)或体液中的表达，和(b)比较该基因表达水平和标准基因表达水平，其中与标准表达水平相比，测定的基因表达水平的增加或减少指示异常表达。
本文提供的另外的实施例包括在动物(例如哺乳动物，如人)中诊断与CD73的表达或异常表达相关的疾病或障碍的方法。这些方法包含在哺乳动物中检测CD73分子。在某些实施例中，诊断包括：(a)向哺乳动物给予有效量的经标记的抗CD73抗体和/或其变体或突变体(或片段)；(b)在给予后等待一段时间间隔，以允许经标记的抗CD73抗体和/或其变体或突变体(或片段)优先浓缩在受试者的表达CD73的部位(并且将未结合的经标记的分子清除至背景水平)；(c)确定背景水平；和(d)检测受试者中经标记的分子，使得检测到高于背景水平的经标记的分子指示受试者患有与CD73表达或异常表达相关的特定疾病或障碍。背景水平可以通过不同方法确定，这些方法包括将所检测到的标记的分子的量与先前针对一个具体系统确定的标准值进行对比。
本文提供的抗CD73抗体和/或其变体或突变体(或片段)可以用于使用本领域技术人员已知的经典免疫组织学方法来测定生物样品中蛋白质水平(例如，参见Jalkanen等人,J.Cell.Biol.[细胞生物学杂志]101:976-985(1985)；Jalkanen等人,J.Cell.Biol.[细胞生物学杂志]105:3087-3096(1987))。 有用于检测蛋白质基因表达的其他基于抗体的方法包括免疫测定，例如酶联免疫吸附测定(ELISA)和放射免疫测定(RIA)。合适的抗体测定标记是本领域中已知的并且包括酶标记(如，葡萄糖氧化酶)；放射性同位素，如碘( 131I、 125I、 123I、 121I)、碳( 14C)、硫( 35S)、氚( 3H)、铟( 115mIn、 113mIn、 112In、 111In)、锝( 99Tc、 99mTc)、铊( 201Ti)、镓( 68Ga、 67Ga)、钯( 103Pd)、钼( 99Mo)、氙( 133Xe)、氟( 18F)、钐( 153Sm)、镥( 177Lu)、钆( 159Gd)、钷( 149Pm)、镧( 140La)、镱( 175Yb)、钬( 166Ho)、钇( 90Y)、钪( 47Sc)、铼( 186Re、 188Re)、镨( 142Pr)、铑( 105Rh)、钌( 97Ru)；鲁米诺；以及荧光标记(如荧光素及罗丹明及生物素)。
本领域已知的技术可以应用于本文提供的经标记的抗体(或其片段)。此类技术包括但不限于使用双功能缀合剂(参见例如，美国专利号5,756,065；5,714,631；5,696,239；5,652,361；5,505,931；5,489,425；5,435,990；5,428,139；5,342,604；5,274,119；4,994,560；和5,808,003)。
可替代地，或另外地，可以测量细胞中编码CD73的核酸或mRNA的水平，例如经由使用对应于编码CD73的核酸或其互补序列的基于核酸的探针的荧光原位杂交；(FISH；参见1998年10月公布的WO 98/45479)，DNA印迹法、RNA印迹法或聚合酶链反应(PCR)技术，例如实时定量PCR(RT-PCR)。还可以通过测量生物流体(如血清)中的脱落抗原来研究CD73过表达，例如使用基于抗体的测定(还参见，例如，1990年6月12日发布的美国专利号4,933,294；1991年4月18日发布的WO 91/05264；1995年3月28日发布的美国专利号5,401,638；以及Sias等人,J.Immunol.Methods[免疫方法杂志]132:73-80(1990))。除了上述测定之外，本领域技术人员可获得各种体内和离体测定。例如，可以将哺乳动物体内的细胞暴露于抗体，该抗体任选地用可检测标记(例如放射性同位素)进行标记，并且可以例如通过外部扫描放射性或者通过分析取自先前暴露于该抗体的哺乳动物的样品(例如，活体标本检查或其他生物样品)来评估抗体与细胞的结合。
制品和试剂盒
在各种实施例中，本发明涉及如下试剂盒，该试剂盒包含所披露的抗CD73抗体、变体、突变体和/或其片段，和/或本文提供的药物组合物，以及以下中的一个或多个：(a)已知治疗癌症的至少一种药剂；或(b)治疗癌症的说明书。
在另外的实施例中，将所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物，以及已知治疗癌症的至少一种药剂进行共同配制。
在仍另外的实施例中，将所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物，以及已知治疗癌症的至少一种药剂进行共同包装。
在另外的实施例中，已知治疗癌症的一种药剂是激素治疗剂。在仍另外的实 施例中，激素治疗剂选自下组中的一个或多个，该组由以下组成：亮丙瑞林、三苯氧胺、雷洛昔芬、甲地孕酮、氟维司群、曲普瑞林(triptorelin)、甲羟孕酮、来曲唑、阿那曲唑(anastrozole)、依西美坦、比卡鲁胺、戈舍瑞林、组氨瑞林(histrelin)、氟甲睾酮、雌莫司汀、氟他胺、托瑞米芬、地加瑞克(degarelix)、尼鲁米特、阿巴瑞克(abarelix)和睾内酯，或其药学上可接受的盐、水合物、溶剂合物或多晶型物。
在另外的实施例中，已知治疗癌症的至少一种药剂是化学治疗剂。在仍另外的实施例中，该化学治疗剂选自下组中的一个或多个，该组由以下组成：烷化剂、抗代谢物剂、抗肿瘤抗生素剂、有丝分裂抑制剂、mTOR抑制剂或其他化学治疗剂。
在另外的实施例中，该抗肿瘤抗生素剂选自下组中的一个或多个，该组由以下组成：多柔比星、米托蒽醌、博莱霉素、道诺霉素、放线菌素D、表柔比星、伊达比星、普卡霉素、丝裂霉素、喷司他丁和戊柔比星(valrubicin)，或其药学上可接受的盐、水合物、溶剂合物或多晶型物。
在另外的实施例中，该抗代谢物剂选自下组中的一个或多个，该组由以下组成：吉西他滨、5-氟脲嘧啶、卡培他滨、羟基脲、巯嘌呤、培美曲塞、氟达拉滨、奈拉滨(nelarabine)、克拉屈滨、氯法拉滨、阿糖孢苷、地西他滨(decitabine)、普拉曲沙(pralatrexate)、氟尿苷、甲氨蝶呤和硫鸟嘌呤，或其药学上可接受的盐、水合物、溶剂合物或多晶型物。
在另外的实施例中，该烷化剂选自下组中的一个或多个，该组由以下组成：卡铂、顺铂、环磷酰胺、苯丁酸氮芥、美法仑、卡莫司汀、白消安、环己亚硝脲、达卡巴嗪、奥沙利铂、异环磷酰胺、氮芥、替莫唑胺、噻替派、苯达莫司汀(bendamustine)和链脲菌素，或其药学上可接受的盐、水合物、溶剂合物或多晶型物。
在另外的实施例中，该有丝分裂抑制剂选自下组中的一个或多个，该组由以下组成：伊立替康、拓扑替康、鲁比替康(rubitecan)、卡巴他赛(cabazitaxel)、多西他赛、紫杉醇、依托泊甙(etopside)、长春新碱、伊沙匹隆、长春瑞滨、长春花碱和替尼泊苷，或其药学上可接受的盐、水合物、溶剂合物或多晶型物。
在另外的实施例中，该mTOR抑制剂选自下组中的一个或多个，该组由以下组成：依维莫司、西罗莫司(siroliumus)和替西罗莫司(temsirolimus)，或其药学上可接受的盐、水合物、溶剂合物或多晶型物。
在另外的实施例中，试剂盒进一步包含提供所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物与手术联合的说明书。在仍另外的实施例中，试剂盒进一步包含提供所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物与手术联合的说明书，其中这些说明 书提供了在给予至少一种化合物之前进行手术。在又另外的实施例中，试剂盒进一步包含提供所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物与手术联合的说明书，其中这些说明书提供了在给予所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物之后进行手术。在甚至另外的实施例中，试剂盒进一步包含提供所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物与手术联合的说明书，其中这些说明书提供了在给予所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物之后进行手术，并且其中这些说明书提供了给予所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物是用于实现肿瘤的术前减积。在仍另外的实施例中，试剂盒进一步包含提供化合物与手术联合的说明书，其中这些说明书提供了在给予所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物之后进行手术，并且其中这些说明书提供了与给予所披露的抗CD73抗体、其变体、突变体和/或片段，和/或本文提供的药物组合物几乎同时进行手术。
在另外的实施例中，试剂盒进一步包含提供所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物与放射治疗联合的说明书。在仍另外的实施例中，试剂盒进一步包含提供所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物与放射治疗联合的说明书，其中这些说明书提供了在给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物之前进行放射治疗。在又另外的实施例中，试剂盒进一步包含提供所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物与放射治疗联合的说明书，其中这些说明书提供了在给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物的步骤之后进行放射治疗。在甚至另外的实施例中，试剂盒进一步包含提供所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物与放射治疗联合的说明书，其中这些说明书提供了与给予所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物的步骤几乎同时进行放射治疗。
在另外的实施例中，试剂盒进一步包含多个剂型，该多个包含一个或多个剂量；其中每个剂量包含治疗有效量的所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物和至少一种药剂。在仍另外的实施例中，试剂盒进一步包含多个剂型，该多个包含一个或多个剂量；其中每个剂量包含治疗有效量的所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物和至少一种药剂，并且其中将所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物的每个剂量和已知治疗癌症的至少一种药剂共同配制。在又另外的实施例中，试剂盒进一步包含多个剂型，该多个包含一个或多个剂量；其 中每个剂量包含治疗有效量的所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物和至少一种药剂，并且其中将所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物的每个剂量和已知治疗癌症的至少一种药剂共同包装。
在另外的实施例中，试剂盒进一步包含多个剂型，该多个包含一个或多个剂量；其中每个剂量包含治疗有效量的所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物(配制用于静脉内给予)，和至少一种药剂(配制用于口服给予和/或静脉内给予)。在仍另外的实施例中，试剂盒进一步包含多个剂型，该多个包含一个或多个剂量；其中每个剂量包含治疗有效量的所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物(配制用于静脉内给予)，和至少一种药剂(配制用于口服给予)。在又另外的实施例中，试剂盒进一步包含多个剂型，该多个包含一个或多个剂量；其中每个剂量包含治疗有效量的所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物(配制用于静脉内给予)，和至少一种药剂(配制用于静脉内给予)。
所披露的抗CD73抗体、变体、突变体和/或其片段，和/或药物组合物可以方便地作为试剂盒提供，其中提供了两种或更多种组分(可以是活性或非活性成分、载体、稀释剂等)与用于使患者或将药物给予至患者的人制备实际剂型的说明书。此类试剂盒可以具有包含在其中的所有必需的材料和成分，或者这些试剂盒可以含有用于使用或制造必须由患者或将药物给予至患者的人独立获得的材料或组分的说明书。在另外的实施例中，试剂盒可以包括有助于向患者给予单位剂量的任选的组分，如用于重构粉末形式的小瓶、用于注射的注射器、定制的IV递送系统、吸入器等。另外地，试剂盒可以包含用于制备和给予组合物的说明书。该试剂盒可以作为用于一个受试者的单一使用单位剂量，用于特定受试者的多次使用(以恒定剂量或者其中随疗法进展单一化合物的效力可以变化)而制造；或者该试剂盒可以含有适于给予至多个患者的多个剂量(“散装包装”)。试剂盒组分可以装在纸箱、泡罩包装、瓶、管等中。
在另外的实施例中，所披露的试剂盒能以每日给药方案包装(例如，卡片包装(packaged on cards)、给药卡片包装(packaged with dosing cards)、泡罩包装或吹塑塑料包装等)。此类包装促销产品并增加患者对药物方案的依从性。此类包装还可以减少患者的困惑。本披露的特征在于进一步包含使用说明书的试剂盒。
在另外的实施例中，本发明还提供了药物包装或试剂盒，该药物包装或试剂盒包含填充有本发明的药物组合物的一种或多种成分的一个或多个容器。与这种容器或此类容器相关的可以是处于管理药物或生物制品制造、使用或销售的政府机构所规足的形式的标识，该标识反映制造、使用或销售机构批准用于人 给予。
在各种实施例中，所披露的试剂盒还可以包含与其他组分共同包装、共同配制、和/或共同递送的化合物和/或产品。例如，药物制造商、药物经销商、医师、配药店(compounding shop)或药剂师可以提供如下试剂盒，该试剂盒包含所披露的化合物和/或产品、以及另一种组分用于递送至患者。
考虑了所披露的试剂盒可以与所披露的制备方法、所披露的使用或治疗方法和/或所披露的组合物一起使用。
还提供了可用于各种目的的试剂盒，例如任选地与制品组合的用于在患者中分离或检测CD73的试剂盒。为了分离和纯化CD73，试剂盒可以含有与珠(例如，SEPHAROSE TM珠)偶联的本文提供的抗CD73抗体和/或其变体或突变体(或片段)。可以提供含有抗体(或其片段)的试剂盒，用于例如在ELISA或蛋白质印迹法中体外检测和定量CD73。与制品一样，试剂盒包含容器和在容器上或与容器相关的标记或包装插入物。例如，容器容纳包含至少一种本文提供的抗CD73抗体和/或其变体或突变体(或片段)的组合物。可以包括的另外的容器含有，例如，稀释剂和缓冲液、对照抗体。标记或包装插入物可以提供对组合物的描述以及用于预期的体外或诊断用途的说明书。
本文提供的另一个实施例是含有用于治疗所披露的癌症的材料的制品。制品可以包含容器和该容器上或与该容器相关的标记或包装插入物。适合的容器包括例如，瓶子、小瓶、注射器等。容器可以由多种材料制成，例如玻璃或塑料。通常，容器容纳有效治疗病症的组合物，并且可具有无菌进入口(例如，容器可以是静脉内溶液袋或具有可由皮下注射针刺穿的塞子的小瓶)。组合物中的至少一种活性剂是本文提供的抗CD73抗体和/或其变体或突变体(或片段)。标记或包装插入物指示组合物用于治疗特定病症。标记或包装插入物将进一步包含向患者给予抗体组合物的说明书。还考虑了包含本文描述的组合疗法的制品和试剂盒。
包装插入物是指通常包括在治疗产品的商业包装中的说明书，这些说明书含有关于使用此类治疗产品的适应症、用法、剂量、给予、禁忌症和/或警告的信息。在一个实施例中，包装插入物指示组合物用于治疗癌症(如头颈癌、肺癌或结肠直肠癌)。
另外地，制品可以进一步包含第二容器，该容器包含药学上可接受的缓冲液，如抑菌性注射用水(BWFI)、磷酸盐缓冲盐水、林格氏溶液和右旋糖溶液。其可以进一步包括从商业和用户角度所希望的其他材料，包括其他缓冲液、稀释剂、过滤器、针头和注射器。
现在已经总体上描述了本发明的方面，以下实施例描述了本发明的一些另外的方面。虽然结合以下实施例和相应的文本和附图描述了本发明的方面，但是 并不旨在将本发明的方面限制于此描述。相反，旨在涵盖包括于本发明的精神和范围内的所有替代物、修改和等效物。
实施例
提出以下实施例以向本领域的普通技术人员提供如何制备和评估本文要求保护的化合物、组合物、制品、装置和/或方法的完整披露和描述，并且旨在纯粹是本发明的示例，而并非旨在限制诸位发明人认为的本发明的范围。虽然已尽力确保数字(例如量、温度等)的准确性，但仍应考虑一些误差和偏差。除非另外指示，否则份数是重量份数，温度是℃或环境温度，并且压力是大气压或接近大气压。
Figure PCTCN2020102678-appb-000061
实施例1.抗CD73抗体和变体的制备。
用人CD73-ECD/His淘选人Fab初始噬菌体展示文库。用与链霉抗生物素蛋白包衣的磁性
Figure PCTCN2020102678-appb-000062
M-280(赛默飞世尔科技公司(Thermo Fisher Scientific)#11205D)偶联的生物素化的huCD73-ECD/His进行四轮淘选后，选择了具有CD73结合活性和固定化的CD73酶阻断活性的五十二个可能克隆并确定四种序列(N1至N4)。然后将选择的可变序列克隆到人IgG1 Fc主链的L234F、L235E、P331S突变体中，以成为用于体外功能分析(CD73结合、CD73酶活性阻断、抗体介导的CD73内化)的全长抗体。还将N1、N2和N4先导克隆到人野生型或C219S突变体IgG2 Fc主链中。根据体外功效和体内功效测试(MDA-MB-231异种移植模型)，选择N1和N4用于以下亲和力成熟并确定wtIgG2主链。
将抗体先导克隆N1和先导克隆N4用于基于体外噬菌体展示的亲和力成熟实验，以生成具有改善的CD73结合活性性能和CD73酶阻断活性性能的另外的克隆。经由PCR生成N1先导克隆的CDR-L1/CDR-L3/CDR-H3(集中于3个CDR)核 酸文库，将其克隆到噬菌体展示载体中并转化到大肠杆菌TG1细胞中，以产生噬菌体文库。用生物素化的huCD73-ECD/His进行三轮淘选后，经由结合测定和酶阻断测定筛选出具有不同序列的十五个Fab克隆，并且发现两个Fab(N1#2和N1#9)具有更好的功效。关于N4先导克隆的亲和力成熟，首先生成CDR-L3/CDR-H1/CDR-H3核酸文库以产生噬菌体文库。经过三轮淘选后，经由结合测定和酶阻断测定筛选出具有不同序列的六个Fab克隆(N4#1至N4#6)。然后，生成N4#1、N4#4、N4#5和N4#6克隆的CDR-L1/CDR-L2/CDR-H2核酸文库以产生噬菌体文库。经过三轮淘选后，选择具有更好的结合活性和酶阻断活性的N4#4-3、N4#6-2、N4#6-3、N4#6-4、N4#6-5克隆。
亲和力成熟后，将N1#2、N1#9、N4#5、N4#6、N4#4-3、N4#6-2、N4#6-3、N4#6-4、N4#6-5克隆到IgG2 Fc主链中，以成为全长抗体并进行体外功效测试(包括CD73蛋白结合ELISA、表达CD73的肿瘤细胞结合、固定化和细胞CD73酶阻断、抗体介导的CD73内化)和体内功效测试(NCI-H292异种移植模型)，并选择N1#2、N1#9、N4#6-3、N4#6-4克隆。根据序列比对和CMC稳定性，修饰这四个克隆以产生N1#2-P、N1#9-PH、N4#6-3-P、N4#6-4-P。N1#9-PH具有更好的可溶性CD73酶阻断功效，并且N4#6-3-P、N4#6-4-P可以诱导表面CD73内化。N1#9-PH、N4#6-3-P、N4#6-4-P可以减轻AMP介导的T细胞抑制并且在体内具有针对MDA-MB-231异种移植的抗肿瘤活性。此外，N4#6-4-P可在异种移植肿瘤中诱导CD73下调并阻断CD73酶活性，并且在NCI-H292异种移植模型中具有比其他抗CD73抗体更好的抗肿瘤活性。
从初始噬菌体淘选中选择的抗CD73先导克隆N1、N2和N4的轻链和重链可变区的氨基酸序列比对分别示于图1A和图1B中。衍生自亲和力成熟的N1和N4变体的轻链可变区的氨基酸序列比对示于图9中，其中将Kabat定义的CDR(互补决定区)加下划线并用粗体标记，并且将衍生自亲和力成熟的N1和N4变体的重链可变区的氨基酸序列比对示于图10中，其中将Kabat定义的CDR(互补决定区)加下划线并用粗体标记。然后将这些选择的可变序列克隆到人IgG2 Fc主链中，以变成全长抗体。可选择人抗体轻链恒定区和重链恒定区氨基酸如下(并非对本文轻重链可变区氨基酸序列的限制)：
wtIgG2同种型重链恒定区(SEQ ID NO：112)
mtIgG2同种型重链恒定区(SEQ ID NO：113)
tmtIgG1同种型重链恒定区(SEQ ID NO：114)
轻链恒定区(SEQ ID NO：115)
N1#2-P重链(SEQ ID NO：116)
N1#9-PH重链(SEQ ID NO：117)
N4#6-3-P_重链(SEQ ID NO：118)
N4#6-4-P重链(SEQ ID NO：119)
图14显示N1和N4靠前变体的轻链的氨基酸序列比对，其中将Kabat定义的CDR(互补决定区)加下划线并用粗体标记，并且图15显示了N1和N4靠前变体的重链可变区的氨基酸序列比对，其中将Kabat定义的CDR(互补决定区)加下划线并用粗体标记。然后将这些序列克隆到人IgG2 Fc主链中，以变成全长抗体。
实施例2.选择的抗体的CD73结合能力。
进行ELISA测定以评估选择的抗体与重组人CD73-ECD/His蛋白的结合。将山羊抗人IgG Fd(或Fc)抗体以每孔三十纳克在4℃在96孔EIA微孔板上涂覆过夜。用(在PBS中的)5％脱脂乳阻断后，添加连续稀释的抗体并将其在室温孵育1小时。除去未结合的抗体，将孔用含有0.05％Tween20的1×PBST洗涤三次。将生物素标记的CD73-ECD/His蛋白以每孔三十纳克添加到孔中，并在室温再孵育1小时。将未结合的生物素标记的CD73-ECD/His用含有0.05％Tween20的1×PBST洗涤三次。然后将抗生物素蛋白-HRP添加到孔中并在室温孵育30分钟。通过用含有0.05％Tween20的1×PBST洗涤三次而来除去过量的第二抗体。洗涤后，将板使用TMB进行显色。通过酶标仪读取450nm的波长处的吸光度。
通过ELISA测试选择的抗CD73抗体与重组人CD73蛋白的结合，并且结合数据示于图2A中。
通过将MDA-MB-231或NCI-H292细胞与连续稀释的抗体在FACS缓冲液(含有2％FBS的PBS)中在4℃一起孵育30分钟来测试抗CD73抗体的全细胞结合能力。将细胞用FACS缓冲液洗涤，并在4℃用FITC标记的山羊抗人IgG(H+L)抗体(或PE标记的山羊抗人IgG Fcγ)对结合再进行30分钟检测。通过染色的平均荧光强度(MFI)测量抗体与细胞表面的结合。使用Cytomics FC500或CytoFLEX流式细胞仪(贝克曼库尔特公司(Beckman Coulter Inc.))进行流式细胞术分析。图2B和2C显示选择的抗CD73抗体结合表达CD73的人肿瘤细胞MDA-MB-231(人乳腺癌)细胞(图2B)和NCI-H292(人黏液表皮样肺癌)细胞(图2C)。
在这些研究中，将抗CD73 ref-1抗体和HLX01(抗CD20)分别用作阳性对照和阴性对照。这些数据表明，所有选择的抗CD73抗体都可以与人CD73重组蛋白和表达CD73的人肿瘤细胞结合。
图11A-11B还显示了N1和N4变体的CD73结合。通过流式细胞术来测试选择的变体与表达CD73的MDA-MB-231(图11A)和NCI-H292(图11B)细胞的结合。将HLX01(抗CD20)用作阴性对照。这些数据指示，在亲和力成熟后，所有N1和N4变体都可以与表达CD73的人肿瘤细胞结合。在这两种细胞系中，N4变体具有比N1变体更好的CD73结合能力。全细胞结合能力的排序是：N4#5、 N4#6-4、N4#6-5>N4#6-2>N4#4-3、N4#6、N4#6-3、N1#9>N1#2。
图16A-16C显示N1和N4靠前变体的CD73结合能力。通过ELISA测试抗CD73抗体与重组人CD73蛋白的结合(图16A)、通过流式细胞术测试抗CD73抗体与表达CD73的人肿瘤细胞MDA-MB-231(图16B)和NCI-H292(图16C)的结合。将抗CD73 ref-1抗体和抗CD73 ref-2抗体用作阳性对照，而将HLX01(抗CD20)用作阴性对照。这些数据指示，N4#6-3-P、N4#6-4-P和N1#9-PH以可比的亲和力与重组CD73和MDA-MB-231细胞上的表面CD73结合。N1#9-PH与表达CD73的NCI-H292细胞的结合略好于N4#6-3-P和N4#6-4-P与该细胞的结合。
实施例3.抗CD73先导克隆对可溶性CD73酶活性和细胞CD73酶活性的作用。
测试抗CD73抗体抑制人重组CD73蛋白和细胞表面CD73的酶活性的能力。通过测量AMP抑制的荧光素的ATP依赖性氧化(CellTiter-Glo测定；普洛麦格公司(Promega))，分析CD73催化AMP水解成腺苷和无机磷酸盐。将十五纳克的重组人CD73-ECD/His蛋白与连续稀释的抗CD73抗体、ATP(终浓度100μM)和AMP(终浓度300μM)在测定缓冲液(25mM Tris，5mM MgCl 2，pH 7.46)中在37℃孵育。将相同体积的CellTiter-Glo试剂添加到反应中并在白色96孔微孔板中混合2min。孵育10min后，测量冷光。
将表达CD73的细胞(5E3-1E4细胞/孔)重悬于培养基中，并置于96孔微孔板中(或预接种24小时)。向每种培养物中添加三倍(或五倍)连续稀释的抗体。在37℃孵育15分钟后，添加AMP(终浓度225μM)并孵育4-20小时。离心后，收集培养上清液，置于96孔微孔板中，并添加ATP至终浓度为100μM，用于孵育2min。将相同体积的CellTiter-Glo试剂添加到每个孔中并混合2min。孵育10min后，测量冷光。
显示抗CD73先导克隆对可溶性CD73酶活性和细胞CD73酶活性的作用的数据分别示于图3A和3B中。这些数据表明，所有选择的抗体都可以抑制人可溶性CD73和表面CD73两者的酶活性。在可溶性CD73酶活性测定中，当IgG相对于sCD73处于化学计量过量时，观察到抑制丧失。这种所谓的“钩状效应(hook effect)”可以产生自相同IgG分子上的Fab臂为竞争靶抗原上有限的结合位点而驱动的单价抗体结合。
测试具有不同IgG同种型的抗CD73先导克隆N1、N2、N4抑制细胞CD73酶活性的能力。将MDA-MB-231细胞与抗CD73抗体一起孵育。添加ATP、AMP和CellTiter-Glo试剂并记录冷光。在酶活性测定中将APCP用作阳性对照。具有不同IgG Fc区的这些抗CD73先导克隆对细胞CD73酶活性的作用显示于图6A(先导克隆N1)、6B(先导克隆N2)和6C(先导克隆N4)中。
这些数据指示，N1克隆的wtIgG2同种型具有比tmtIgG1和mtIgG2同种型更好的阻断活性。N2克隆的tmtIgG1同种型具有比wtIgG2和mtIgG2同种型更 好的阻断活性。具有wtIgG2同种型的N4显示出比tmtIgG1和mtIgG2同种型最佳的阻断活性。tmtIgG1同种型具有以下Fc位点特异性变化：L234F、L235E和P331S。
图17A-17C还显示N1和N4靠前变体对可溶性CD73酶活性(图17A)和细胞表面CD73酶活性(图17B和图17C)的作用。测试抗CD73抗体抑制人重组CD73蛋白和细胞表面CD73酶活性的能力。将重组CD73蛋白(图17A)、MDA-MB-231(图17B)和NCI-H292(图17C)细胞与抗CD73抗体一起孵育。添加ATP、AMP和CellTiter-Glo并记录冷光。将抗CD73 ref-1抗体、抗CD73 ref-2抗体和APCP用作阳性对照。将HLX01(抗CD20)用作阴性对照。
图17A-17C中展示的数据显示N1#9-PH抑制可溶性CD73酶活性的能力优于N4#6-3-P和N4#6-4P。然而，N1#9-PH和N4#6-4-P对细胞表面CD73酶活性显示出可比的阻断活性。
实施例4.抗CD73先导克隆的抗体介导的CD73内化。
将肿瘤细胞(5E4至1E5细胞/孔)悬浮于培养基中，并与不同浓度的抗CD73抗体一起孵育4小时。除去培养基后，将细胞悬浮于FACS缓冲液(含有2％FBS的PBS)中，并与小鼠抗人CD73抗体(4G4)在4℃一起孵育30分钟。将细胞用FACS缓冲液洗涤，并与FITC标记的山羊抗小鼠IgG(H+L)抗体或Alexa488标记的山羊抗小鼠IgG Fcγ抗体一起孵育。使用Cytomics FC500或CytoFLEX流式细胞仪(贝克曼库尔特公司(Beckman Coulter Inc.))进行流式细胞术分析。将抗CD73 ref-1抗体和HLX01(抗CD20)分别用作阳性对照和阴性对照。
图4中展示的数据指示仅N4-tmtIgG1可以诱导CD73内化。N1-tmtIgG1和N2-tmtIgG1不能诱导CD73内化。
图7还显示具有不同IgG Fc区的抗CD73抗体的抗体介导的CD73内化。在将细胞与N1(图7A)、N2(图7B)和N4(图7C)的不同IgG同种型孵育后，通过流式细胞术测量CD73的细胞表面表达。将抗CD73 ref-1抗体和HLX01(抗CD20)分别用作阳性对照和阴性对照。数据显示N4(而不是N1和N2)可以诱导CD73内化。不同IgG同种型之间没有明显差异。
图13还显示N1和N4变体的抗体介导的CD73内化。将NCI-H292细胞与N1和N4变体一起孵育，并通过流式细胞术测量CD73的细胞表面表达。将抗CD73 ref-1抗体和抗CD73 ref-2抗体用作阳性对照，而将HLX01(抗CD20)用作阴性对照。数据指示，在亲和力成熟后，N4变体可以诱导CD73内化，并且N1变体仍然不能诱导CD73内化。
图18A和18B还显示N1和N4靠前变体的抗体介导的CD73内化。将MDA-MB-231(图18A)和NCI-H292(图18B)细胞与抗CD73 N1和N4变体一起孵育。孵育后，通过流式细胞术测量CD73的细胞表面表达。将抗CD73 ref-1 和抗CD73 ref-2抗体用作阳性对照，而将HLX01(抗CD20)用作阴性对照。这些数据指示，N4#6-3-P和N4#6-4-P可在两种表达CD73的癌细胞中诱导CD73内化。
实施例5.具有不同IgG Fc区的选择的抗体的CD73结合。
通过将MDA-MB-231细胞与连续稀释的生物素标记的抗CD73抗体在FACS缓冲液(含2％FBS的PBS)中在4℃一起孵育30分钟来测试抗CD73抗体与细胞表面CD73的结合。将细胞用FACS缓冲液洗涤，并用链霉抗生物素蛋白-FITC在4℃对结合进行30分钟检测。使用Cytomics FC500(贝克曼库尔特公司(Beckman Coulter Inc.))进行流式细胞术分析。
将N1、N2和N4的可变序列克隆到人野生型或C219S突变体IgG2 Fc主链中。通过流式细胞术测试N1(图5A)、N2(图5B)、N4(图5C)的不同IgG同种型与表达CD73的人MDA-MB-231细胞的结合。将抗CD73 ref-1抗体和HLX01(抗CD20)分别用作阳性对照和阴性对照。
示于图5A-5C中的数据指示在CD73结合能力方面N1的不同IgG同种型之间不存在差异。数据还显示N2克隆(tmtIgG1同种型)具有比wtIgG2和mtIgG2同种型更好的CD73结合活性，并且N4克隆(tmtIgG1同种型)具有比wtIgG2和mtIgG2同种型更好的CD73结合活性。
实施例6.MDA-MB-231(人三阴性乳腺癌)异种移植小鼠模型中抗CD73先导 克隆的肿瘤生长抑制活性。
使用免疫受损的NOD/SCID(非肥胖糖尿病/严重联合免疫缺陷)小鼠，在异种移植小鼠模型中研究抗人CD73抗体的体内活性。将100μL的PBS中总共1E7个表达CD73的人三阴性乳腺癌MDA-MB-231细胞与100μL的人工基膜(Matrigel)(科宁公司(Corning)，加利福尼亚州(CA)，美国)(以1:1比率)混合，并皮下植入雌性NOD/SCID小鼠(乐斯科公司(Biolasco)，台北，台湾)的两侧胁腹。当肿瘤大小达到150-300mm 3(肿瘤接种后第14天)时，每周两次或在指定时间腹膜内给予5mg/kg抗CD73抗体或10mL/kg安慰剂。观察肿瘤并进行测量，直至第45天。将肿瘤体积定义为TV(肿瘤体积)＝(长度x宽度 2)/2。
肿瘤生长曲线示于图8A中。第45天的个体肿瘤体积示于图8B中。所有数据点均为平均值±SEM。数据证明，除了N1、N2和N4克隆的tmtIgG1同种型外，所有抗CD73抗体在MDA-MB-231异种移植模型中具有抗肿瘤活性(TGI≥50％)。N1-wtIgG2、N1-mtIgG2、N2-wtIgG2和N2-mtIgG2几乎完全抑制肿瘤生长。
图20还显示MDA-MB-231(人三阴性乳腺癌)异种移植小鼠模型中N1和N4靠前变体的肿瘤生长抑制活性。将小鼠(n＝5只小鼠/组)皮下移植MDA-MB-231细胞。在肿瘤接种后7天，当移植的肿瘤大小达到约100mm 3时，给予测试制品 的第一剂量。每周两次用10mg/kg和2mg/kg的抗体腹膜内处理小鼠，持续5-6周。所有数据点均为平均值±SEM。这些数据证明，所有N1和N4靠前变体针对体内MDA-MB-231异种移植具有抗肿瘤活性。N4#6-3-P、N4#6-4-P和N1#9-PH具有比抗CD73参考抗体更好的抗肿瘤活性。
实施例7.N1和N4变体对固定化CD73酶活性的作用。
测试N1和N4变体抑制固定化CD73酶活性的能力。将山羊抗人IgG Fd抗体以每孔三百纳克在4℃在白色高亲和力96孔微孔板上涂覆过夜。用(在PBS中的)5％脱脂乳阻断后，添加连续稀释的抗体并将其在室温孵育1小时。除去未结合的抗体，将孔用含有0.05％Tween 20的1×PBST洗涤三次。将在测定缓冲液(25mM Tris，5mM MgCl 2，pH 7.46)中的二十五微升的100ng/mL CD73-ECD/His蛋白添加到孔中，并在室温再孵育1小时。添加二十五微升的66μM ATP和200μM AMP混合物，并在37℃孵育9分钟。将相同体积的CellTiter-Glo试剂添加到反应混合物中并(在白色96孔微孔板中)混合内容物2min。孵育10min后，测量冷光。将抗CD73 ref-1抗体、抗CD73 ref-2抗体用作阳性对照，而将HLX01(抗CD20)用作阴性对照。
N1和N4变体对固定化CD73酶活性的作用示于图12中。这些数据指示，在亲和力成熟后，所有N1和N4变体都可以抑制CD73酶活性。N1变体的CD73酶阻断活性优于N4变体和抗CD73 ref-2抗体，但与抗CD73 ref-1抗体是可比的。阻断固定化CD73酶活性：N1#2、N1#9>N4#6-4、N4#6-5>N4#4-3、N4#6-2>N4#6-3、N4#5>N4#6。
实施例8.通过N1和N4靠前变体逆转AMP对T细胞活性的抑制作用。
CD73催化腺苷一磷酸(AMP)胞外产生腺苷。腺苷通过激活A2aR和A2bR受体抑制免疫应答(包括T细胞、NK细胞和树突细胞的免疫应答)。除了CD73酶抑制和内化之外，还检测了抗CD73抗体的CD73抑制对T细胞增殖的功能性作用。
将人T细胞使用MagniSort人T细胞富集试剂盒(eBioscience公司(eBioscience,Inc.))从外周血单核细胞(PBMC)分离，并悬浮于含有50IU/mL的重组人IL-2(eBioscience公司(eBioscience,Inc.))的完全培养基(含有10％FBS的RPMI-1640)中。将体积为100μL的T细胞(5E4细胞/反应)与Dynabeads TM人T-活化因子CD3/CD28(赛默飞世尔科技公司(Thermo Fisher Scientific))以1:0.5的细胞比珠的比率进行混合。将AMP(1mM)和连续稀释的抗体以100μL的体积添加到每个反应中。将不含活化珠(未刺激组)、仅含有CD3/CD28珠(刺激组)、或含有CD3/CD28珠和AMP(AMP介导的抑制组)的反应用作测定对照。将抗CD73 ref-1抗体、抗CD73 ref-2抗体和APCP用作阳性对照。将HLX04(抗VEGF)用作阴性对照。将细胞在37℃培养4天。在第 4天，收集细胞、洗涤并重悬于新鲜的RPMI-1640培养基中。通过CellTiter-Glo测定来分析T细胞增殖。收集培养物上清液用于细胞因子测量。使用人IFN-γELISA MAX TM Deluxe试剂盒(Biolegend有限公司(BioLegend,Inc.))测量IFN-γ的水平。
通过N1和N4靠前变体逆转AMP对T细胞活性的抑制作用显示于图19A和19B中。CD3 +T细胞的增殖之后进行CellTiter-Glo测定(图19A)，并使用人IFN-γELISA MAX TM Deluxe试剂盒(图19B)测量IFN-γ分泌。将抗CD73 ref-1抗体、抗CD73 ref-2抗体和APCP用作阳性对照。将HLX04(抗VEGF)用作阴性对照。图19A和19B中显示的数据表明N1和N4靠前变体能够以剂量依赖性方式增强T细胞增殖和IFN-γ分泌。数据进一步指示N4#6-3-P、N4#6-4-P和N1#9-PH可以减轻AMP介导的T细胞抑制。
实施例9.NCI-H292(人黏液表皮样肺癌)异种移植小鼠模型中N1和N4靠 前变体的肿瘤生长抑制活性。
使用免疫受损的BALB/c裸鼠，在异种移植小鼠模型中研究抗人CD73抗体的体内活性。将100μL的PBS中的总共5E6NCI-H292细胞与100μL的人工基膜(Matrigel)(科宁公司(Corning)，加利福尼亚州(CA)，美国)(以1:1比率)混合，并皮下植入雄性BALB/c裸鼠(乐斯科公司(Biolasco)，台北，台湾)的两侧胁腹。当肿瘤大小达到150-300mm 3，每周两次腹膜内给抗予CD73抗体或安慰剂，持续3周。观察肿瘤并每周测量两次。将肿瘤体积定义为TV(肿瘤体积)＝(长度x宽度 2)/2。
图21显示NCI-H292(人黏液表皮样肺癌)异种移植小鼠模型中N1和N4靠前变体的肿瘤生长抑制活性。将小鼠(n＝5只小鼠/组)皮下移植NCI-H292细胞。肿瘤接种后3天给予测试制品的第一剂量。每周两次用50、10和2mg/kg的抗体腹膜内处理小鼠，持续3周。所有数据点均为平均值±SEM。数据证明，N4#6-4-P在NCI-H292异种移植模型中具有比其他抗CD73抗体更好的抗肿瘤活性。
实施例10.NCI-H292异种移植肿瘤中N1和N4靠前变体对细胞CD73酶活性 的作用。
将100μL的PBS中的总共5E6NCI-H292细胞与100μL的人工基膜(Matrigel)(科宁公司(Corning)，加利福尼亚州(CA)，美国)(以1:1比率)混合，并在给予抗体前4天，皮下植入雄性BALB/c裸鼠(乐斯科公司(Biolasco)，台北，台湾)的两侧胁腹。在第0天用30mg/kg抗体、APCP和安慰剂腹膜内处理小鼠，并在第1、3和7天收集肿瘤。将肿瘤细胞(2E4)重悬于测定缓冲液(25mM Tris，5mM MgCl 2，pH 7.46)中，并用ATP(终浓度100μM)和AMP(终浓度300μM)在37℃处理45分钟。将相同体积的CellTiter-Glo试剂添 加到反应混合物中并(在白色96孔微孔板中)混合内容物2min。孵育10min后，测量冷光。
图22显示NCI-H292异种移植肿瘤中N1和N4靠前变体对细胞CD73酶活性的作用。测试N1和N4靠前变体在NCI-H292异种移植模型中抑制CD73酶活性的能力。在抗体处理前4天，将小鼠(n＝4只小鼠/组)皮下移植NCI-H292细胞。在第0天腹膜内注射抗体、APCP和安慰剂。在抗体给予后第1、3和7天切除肿瘤。通过CellTiter-Glo测定来测量肿瘤中的CD73酶活性。
数据证明，N1和N4靠前变体可以抑制NCI-H292异种移植模型中的CD73酶活性。N4#6-3-P和N4#6-4-P在肿瘤中阻断CD73酶活性优于N1#9-PH和抗CD73参照抗体。
实施例11.MDA-MB-231异种移植肿瘤中N1和N4靠前变体对CD73表达和细 胞CD73酶活性的作用。
将100μL的PBS中的总共1E7MDA-MB-231细胞与100μL的人工基膜(Matrigel)(科宁公司(Corning)，加利福尼亚州(CA)，美国)(以1:1比率)混合，并在给予抗体前7天，皮下植入雌性NOD/SCID小鼠(乐斯科公司(Biolasco)，台北，台湾)的两侧胁腹。在第0天用2mg/kg抗体和10mL/kg安慰剂腹膜内处理小鼠。在第1、3和7天收集肿瘤。为了测量表面CD73表达水平，将5E4肿瘤细胞重悬于FACS缓冲液(含2％FBS的PBS)中，并用抗人和抗小鼠FcR阻断试剂在4℃处理30分钟。离心后，然后将细胞与小鼠抗人CD73抗体(4G4)在4℃孵育30分钟。将细胞用FACS缓冲液洗涤，并与Alexa 488标记的山羊抗小鼠IgG Fcγ抗体一起在4℃再孵育30分钟。使用CytoFLEX流式细胞仪(贝克曼库尔特公司(Beckman Coulter Inc.))进行流式细胞术分析。将总共2E4肿瘤细胞重悬于测定缓冲液(25mM Tris，5mM MgCl 2，pH 7.46)中用于CD73酶活性测量。将细胞悬浮液用ATP(终浓度100μM)和AMP(终浓度300μM)在37℃处理45分钟。将相同体积的CellTiter-Glo试剂添加到反应混合物中并混合2min(在白色96孔微孔板中)。孵育10min后，测量冷光。
图23A和23B显示MDA-MB-231异种移植肿瘤中N1和N4靠前变体对CD73表达和细胞CD73酶活性的作用。通过染色的平均荧光强度(MFI)来测量CD73表达(图23A)，并通过CellTiter-Glo测定来测量肿瘤中的CD73酶活性(图23B)。数据证明，与安慰剂组相比，N4#6-4-P可以下调MDA-MB-231异种移植小鼠模型中表面CD73表达并抑制CD73酶活性。
实施例12.N1和N4靠前变体与小鼠和猴的表达CD73的细胞的交叉结合。
通过将4T1或LLC-MK2细胞(1E5细胞/测试)与连续稀释的抗体在FACS缓冲液(含有2％FBS的PBS)中在4℃孵育30分钟来评估抗CD73抗体的结合。将细胞用FACS缓冲液洗涤，并在4℃用FITC标记的山羊抗人IgG(H+L)抗体对 结合再进行30分钟检测。通过染色的平均荧光强度(MFI)测量抗体与细胞表面的结合。使用Cytomics FC500或CytoFLEX流式细胞仪(贝克曼库尔特公司(Beckman Coulter Inc.))进行流式细胞术分析。
图24A和24B显示N1和N4靠前变体与小鼠和猴的表达CD73的细胞的交叉结合。通过流式细胞术测试抗CD73抗体与表达CD73的小鼠乳腺癌4T1细胞(图24A)和猴肾上皮LLC-MK2细胞(图24B)的结合。将HLX01用作阴性对照。这些数据指示，N1和N4靠前变体与猴的表达CD73的细胞具有交叉反应性。N1和N4靠前变体不能与小鼠的CD73交叉结合。
提供前述实施例仅用于说明目的，并且仅是为了清楚理解本发明的原理而提出的实施方式的可能实施例，并且不旨在以任何方式限制本发明的范围。在基本上不背离本发明的精神和原理的情况下，可以对上述的一个或多个实施例进行许多变化和修改。所有此类修改和变化旨在包括在本发明的范围内并且由所附权利要求保护。

Claims (54)

  1. 一种抗CD73抗体(N1)，该抗体包含基本上由以下组成的轻链(LC)可变结构域序列：a)包含SEQ ID NO:1的氨基酸序列的CDR-L1，b)包含SEQ ID NO:11的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:17的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:25的氨基酸序列的CDR-H1，b)包含SEQ ID NO:30的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:39的氨基酸序列的CDR-H3。
  2. 如权利要求1所述的抗CD73抗体，其中该抗CD73抗体N1的该LC包含SEQ ID NO:46的核酸序列编码的SEQ ID NO:79的氨基酸序列，并且其中该抗CD73抗体N1的该VH包含SEQ ID NO:64的核酸序列编码的SEQ ID NO:97的氨基酸序列。
  3. 一种抗CD73抗体(N2)，该抗体包含基本上由以下组成的轻链(LC)可变结构域序列：a)包含SEQ ID NO:2的氨基酸序列的CDR-L1，b)包含SEQ ID NO:12的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:18的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:26的氨基酸序列的CDR-H1，b)包含SEQ ID NO:31的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:40的氨基酸序列的CDR-H3。
  4. 如权利要求3所述的抗CD73抗体，其中该抗CD73抗体N2的该LC包含SEQ ID NO:47的核酸序列编码的SEQ ID NO:80的氨基酸序列，并且其中该抗CD73抗体N2的该VH包含SEQ ID NO:65的核酸序列编码的SEQ ID NO:98的氨基酸序列。
  5. 一种抗CD73抗体(N4)，该抗体包含基本上由以下组成的轻链(LC)可变结构域序列：a)包含SEQ ID NO:3的氨基酸序列的CDR-L1，b)包含SEQ ID NO:13的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:19的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:32的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:41的氨基酸序列的CDR-H3。
  6. 如权利要求5所述的抗CD73抗体，其中该抗CD73抗体N4的该LC包含SEQ ID NO:48的核酸序列编码的SEQ ID NO:81的氨基酸序列，并且其中该抗CD73抗体N4的该VH包含SEQ ID NO:66的核酸序列编码的SEQ ID NO:99的氨基酸序列。
  7. 一种抗CD73抗体变体(N1#2)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:4的氨基酸序列的CDR-L1，b)包含SEQ ID NO:11的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:17的氨基 酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:25的氨基酸序列的CDR-H1，b)包含SEQ ID NO:33的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:42的氨基酸序列的CDR-H3。
  8. 如权利要求7所述的抗CD73抗体变体，其中该抗CD73抗体变体N1#2的该VL包含SEQ ID NO:49的核酸序列编码的SEQ ID NO:82的氨基酸序列，并且其中该抗CD73抗体变体N1#2的该VH包含SEQ ID NO:67的核酸序列编码的SEQ ID NO:100的氨基酸序列。
  9. 一种抗CD73抗体变体(N1#9)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:5的氨基酸序列的CDR-L1，b)包含SEQ ID NO:11的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:20的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:25的氨基酸序列的CDR-H1，b)包含SEQ ID NO:30的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:43的氨基酸序列的CDR-H3。
  10. 如权利要求9所述的抗CD73抗体变体，其中该抗CD73抗体变体N1#9的该VL包含SEQ ID NO:50的核酸序列编码的SEQ ID NO:83的氨基酸序列，并且其中该抗CD73抗体变体N1#9的该VH包含SEQ ID NO:68的核酸序列编码的SEQ ID NO:101的氨基酸序列。
  11. 一种抗CD73抗体变体(N4#1)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:3的氨基酸序列的CDR-L1，b)包含SEQ ID NO:13的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:21的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:28的氨基酸序列的CDR-H1，b)包含SEQ ID NO:32的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:41的氨基酸序列的CDR-H3。
  12. 如权利要求11所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#1的该VL包含SEQ ID NO:51的核酸序列编码的SEQ ID NO:84的氨基酸序列，并且其中该抗CD73抗体变体N4#1的该VH包含SEQ ID NO:69的核酸序列编码的SEQ ID NO:102的氨基酸序列。
  13. 一种抗CD73抗体变体(N4#4)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:3的氨基酸序列的CDR-L1，b)包含SEQ ID NO:13的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:22的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:34的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:41的氨基酸序列的CDR-H3。
  14. 如权利要求13所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#4的该VL包含SEQ ID NO:52的核酸序列编码的SEQ ID NO:85的氨基酸序列，
    并且其中该抗CD73抗体变体N4#4的该VH包含SEQ ID NO:70的核酸序列编码的SEQ ID NO:103的氨基酸序列。
  15. 一种抗CD73抗体变体(N4#5)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:3的氨基酸序列的CDR-L1，b)包含SEQ ID NO:13的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:23的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:29的氨基酸序列的CDR-H1，b)包含SEQ ID NO:35的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:44的氨基酸序列的CDR-H3。
  16. 如权利要求15所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#5的该VL包含SEQ ID NO:53的核酸序列编码的SEQ ID NO:86的氨基酸序列，并且其中该抗CD73抗体变体N4#5的该VH包含SEQ ID NO:71的核酸序列编码的SEQ ID NO:104的氨基酸序列。
  17. 一种抗CD73抗体变体(N4#6)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:3的氨基酸序列的CDR-L1，b)包含SEQ ID NO:13的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:24的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:34的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:45的氨基酸序列的CDR-H3。
  18. 如权利要求17所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#6的该VL包含SEQ ID NO:54的核酸序列编码的SEQ ID NO:87的氨基酸序列，并且其中该抗CD73抗体变体N4#6的该VH包含SEQ ID NO:72的核酸序列编码的SEQ ID NO:105的氨基酸序列。
  19. 一种抗CD73抗体变体(N4#4-3)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:6的氨基酸序列的CDR-L1，b)包含SEQ ID NO:13的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:22的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:35的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:41的氨基酸序列的CDR-H3。
  20. 如权利要求19所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#4-3的该VL包含SEQ ID NO:55的核酸编码的SEQ ID NO:88的氨基酸序列，并且其中该抗CD73抗体变体N4#4-3的该VH包含SEQ ID NO:73的核酸编码的SEQ ID NO:106的氨基酸序列。
  21. 一种抗CD73抗体变体(N4#6-2)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:7的氨基酸序列的CDR-L1，b)包含SEQ ID NO:14的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:24的 氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:36的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:45的氨基酸序列的CDR-H3。
  22. 如权利要求21所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#6-2的该VL包含SEQ ID NO:56的核酸序列编码的SEQ ID NO:89的氨基酸序列，并且其中该抗CD73抗体变体N4#6-2的该VH包含SEQ ID NO:74的核酸序列编码的SEQ ID NO:107的氨基酸序列。
  23. 一种抗CD73抗体变体(N4#6-3)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:8的氨基酸序列的CDR-L1，b)包含SEQ ID NO:15的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:24的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:32的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:45的氨基酸序列的CDR-H3。
  24. 如权利要求23所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#6-3的该VL包含SEQ ID NO:57的核酸序列编码的SEQ ID NO:90的氨基酸序列，并且其中该抗CD73抗体变体N4#6-3的该VH包含SEQ ID NO:75的核酸序列编码的SEQ ID NO:108的氨基酸序列。
  25. 一种抗CD73抗体变体(N4#6-4)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:9的氨基酸序列的CDR-L1，b)包含SEQ ID NO:16的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:24的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:37的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:45的氨基酸序列的CDR-H3。
  26. 如权利要求25所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#6-4的该VL包含SEQ ID NO:58的核酸序列编码的SEQ ID NO:91的氨基酸序列，并且其中该抗CD73抗体变体N4#6-4的该VH包含SEQ ID NO:76的核酸序列编码的SEQ ID NO:109的氨基酸序列。
  27. 一种抗CD73抗体变体(N4#6-5)，该抗体变体包含基本上由以下组成的轻链可变结构域(VL)序列：a)包含SEQ ID NO:10的氨基酸序列的CDR-L1，b)包含SEQ ID NO:13的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:24的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:36的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:45的氨基酸序列的CDR-H3。
  28. 如权利要求27所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#6-5的该VL包含SEQ ID NO:59的核酸序列编码的92的氨基酸序列，并且其中该 抗CD73抗体变体N4#6-5的该VH包含SEQ ID NO:77的核酸序列编码的SEQ ID NO:110的氨基酸序列。
  29. 一种抗CD73抗体变体(N1#2-P)，该抗体变体包含基本上由以下组成的轻链(LC)可变结构域序列：a)包含SEQ ID NO:4的氨基酸序列的CDR-L1，b)包含SEQ ID NO:11的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:17的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:25的氨基酸序列的CDR-H1，b)包含SEQ ID NO:38的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:42的氨基酸序列的CDR-H3。
  30. 如权利要求29所述的抗CD73抗体变体，其中该抗CD73抗体变体N1#2-P的该LC包含SEQ ID NO:60的核酸序列编码的SEQ ID NO:93的氨基酸序列，并且其中该抗CD73抗体变体N1#2-P的该VH包含SEQ ID NO:78的核酸序列编码的SEQ ID NO:111的氨基酸序列。
  31. 一种抗CD73抗体变体(N1#9-PH)，该抗体变体包含基本上由以下组成的轻链(LC)可变结构域序列：a)包含SEQ ID NO:5的氨基酸序列的CDR-L1，b)包含SEQ ID NO:11的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:20的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:25的氨基酸序列的CDR-H1，b)包含SEQ ID NO:30的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:43的氨基酸序列的CDR-H3。
  32. 如权利要求31所述的抗CD73抗体变体，其中该抗CD73抗体变体N1#9-PH的该LC包含SEQ ID NO:61的核酸序列编码的SEQ ID NO:94的氨基酸序列，并且其中该抗CD73抗体变体N1#9-PH的该VH包含SEQ ID NO:68的核酸序列编码的SEQ ID NO:101的氨基酸序列。
  33. 一种抗CD73抗体变体(N4#6-3-P)，该抗体变体包含基本上由以下组成的轻链(LC)可变结构域序列：a)包含SEQ ID NO:8的氨基酸序列的CDR-L1，b)包含SEQ ID NO:15的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:24的氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:32的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:45的氨基酸序列的CDR-H3。
  34. 如权利要求33所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#6-3-P的该LC包含SEQ ID NO:62的核酸序列编码的SEQ ID NO:95的氨基酸序列，并且其中该抗CD73抗体变体N4#6-3-P的该VH包含SEQ ID NO:75的核酸序列编码的SEQ ID NO:108的氨基酸序列。
  35. 一种抗CD73抗体变体(N4#6-4-P)，该抗体变体包含基本上由以下组成的轻链(LC)可变结构域序列：a)包含SEQ ID NO:9的氨基酸序列的CDR-L1，b)包含SEQ ID NO:16的氨基酸序列的CDR-L2，和c)包含SEQ ID NO:24的 氨基酸序列的CDR-L3，和基本上由以下组成的重链可变结构域(VH)序列：a)包含SEQ ID NO:27的氨基酸序列的CDR-H1，b)包含SEQ ID NO:37的氨基酸序列的CDR-H2，和c)包含SEQ ID NO:45的氨基酸序列的CDR-H3。
  36. 如权利要求35所述的抗CD73抗体变体，其中该抗CD73抗体变体N4#6-4-P的该LC包含SEQ ID NO:63的核酸序列编码的SEQ ID NO:96的氨基酸序列，并且其中该抗CD73抗体变体N4#6-4-P的该VH包含SEQ ID NO:76的核酸序列编码的SEQ ID NO:109的氨基酸序列。
  37. 根据权利要求1-36中任一项所述的抗CD73抗体或变体，其中所述抗CD73抗体或变体在N-糖基化位点处包含一个或多个突变。
  38. 根据权利要求1-37中任一项所述的抗CD73抗体或变体的抗原结合片段，其中该抗原结合片段选自下组，该组由以下组成：Fab、Fab’、F(ab')2、单链Fv(scFv)、Fv片段、双抗体、和线性抗体。
  39. 根据权利要求1-38中任一项所述的抗CD73抗体、变体或其抗原结合片段，其中该抗CD73抗体、变体、或抗原结合片段是多特异性抗体、变体、或抗原结合片段。
  40. 根据权利要求1-39中任一项所述的抗CD73抗体、变体、或其抗原结合片段，该抗CD73抗体、变体、或其抗原结合片段与治疗剂缀合。
  41. 根据权利要求1-39中任一项所述的抗CD73抗体、变体、或其抗原结合片段，该抗CD73抗体、变体、或其抗原结合片段与标记缀合。
  42. 根据权利要求41所述的抗CD73抗体、变体、或其抗原结合片段，其中该标记选自下组，该组由以下组成：放射性同位素、荧光染料和酶。
  43. 一种分离的核酸分子，该分离的核酸分子编码根据权利要求1-38中任一项所述的抗CD73抗体、变体、或其抗原结合片段。
  44. 一种表达载体，该表达载体包含根据权利要求43所述的核酸分子。
  45. 一种细胞，该细胞包含根据权利要求44所述的表达载体。
  46. 一种产生根据权利要求1-38中任一项所述的抗CD73抗体、变体、或其抗原结合片段的方法，该方法包括培养根据权利要求45所述的细胞和从该细胞培养物回收该抗CD73抗体、变体、或其抗原结合片段。
  47. 一种组合物，该组合物包含根据权利要求1-38中任一项所述的抗CD73抗体、变体、或其抗原结合片段和药学上可接受的载体。
  48. 根据权利要求1-38中任一项所述的抗CD73抗体、变体、或其抗原结合片段在制备如下药物中的用途，该药物用于通过检测与CD73蛋白结合的抗CD73抗体、变体、或其抗原结合片段而在来自患者的样品中检测CD73蛋白。
  49. 根据权利要求48所述的用途，其中该抗CD73抗体、变体、或其抗原结合片段在免疫组织化学测定(IHC)或在ELISA测定中使用。
  50. 根据权利要求1-38中任一项所述的抗CD73抗体、变体、或其抗原结合片段或根据权利要求47所述的组合物在制造用于在受试者中治疗癌症的药物中的用途。
  51. 根据权利要求50所述的用途，其中该癌症选自下组，该组由以下组成：黑色素瘤、非小细胞肺癌(NSCLC)、头颈癌、尿路上皮癌、三阴性乳腺癌(TNBC)、胃癌、经典型霍奇金淋巴瘤(cHL)、非霍奇金淋巴瘤原发性纵隔B-细胞淋巴瘤(NHL PMBCL)、间皮瘤、卵巢癌、肺癌、食管癌、鼻咽癌(NPC)、胆道癌、结肠直肠癌、乳腺癌、宫颈癌、甲状腺癌、前列腺癌、膀胱癌、胰腺癌、唾液腺癌和表达CD73的恶性肿瘤。
  52. 根据权利要求1-38中任一项所述的抗CD73抗体、变体、或其抗原结合片段或根据权利要求47所述的组合物在制造用于在受试者中治疗纤维化的药物中的用途。
  53. 根据权利要求50-52中任一项所述的用途，其中向该受试者进一步给予选自下组的治疗剂，该组由以下组成：抗肿瘤剂、化学治疗剂、生长抑制剂、细胞毒性剂、和免疫治疗剂。
  54. 根据权利要求50-52中任一项所述的用途，其中向该受试者进一步给予放射疗法或外科手术。
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WO2022214677A1 (en) * 2021-04-09 2022-10-13 Cancer Research Technology Limited Anti-cd73 antibodies
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

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