Methods and Compositions Related to Anti-CD73 Antibodies and Variants

Abstract

Anti-CD73 antibodies, variants, and antigen-binding fragments thereof are provided that bind to human CD73 with high affinity, inhibit the enzymatic activity of CD73, and optionally induce internalization of CD73. Also provided are isolated nucleic acid molecules encoding the anti-CD73 antibodies, variants, and antigen-binding fragments thereof, as well as related expression vectors and host cells. Also provided are methods for making the anti-CD73 antibodies, variants, and antigen-binding fragments thereof. Also provided are related pharmaceutical compositions and methods for treating subjects using these pharmaceutical compositions.

Description

[Technical field]

[0001] The present invention relates generally to anti-CD73 antibodies, variants or mutants, or antigen-binding fragments thereof, and methods of using them to treat human cancers. [Background technology]

[0002] CD73 (called ecto-5'-nucleotidase (NT5E, EC 3.1.3.5)) is a glycosylphosphatidylinositol (GPI)-linked 70-kDa cell surface enzyme found in most tissues (Zhang B., Cancer Res., 70(16):6407-6411 (2010)). CD73, originally defined as a lymphocyte differentiation antigen, is thought to act as a co-signaling molecule for T lymphocytes and as an adhesion molecule required for lymphocyte binding to endothelium. Recent studies have shown that CD73 can regulate a variety of physiological responses, including epithelial cell ion and fluid transport, ischemic preconditioning, tissue injury, platelet function, hypoxia, and blood leakage (Zhang B., Cancer Res., 70(16):6407-6411 (2010)).

[0003] The immunosuppressive effect of extracellular adenosine has already been demonstrated (Stagg J. et al., Oncogene, 29(39):5346-5358(2010)). The interaction of adenosine with 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., 130(3):1350-1355(1983); Ohta A. et al., J.Immunol., 183(9):5487-5493(2009)). Blocking the transmission of adenosine signals can improve immunotherapy. CD73 is a key molecule involved in the transmission of adenosine signals. CD73 influences the tumor microenvironment by hydrolyzing adenosine monophosphate (AMP), a product derived from the pathway of adenosine triphosphate (ATP) hydrolysis by CD39, to adenosine (Allard B. et al., Immunol. Rev., 276(1):121-144 (2017); Resta R. et al., Immunol. Rev., 161:95-109 (1998)). The CD73-adenosine axis constitutes one of the most promising pathways in immuno-oncology.

[0004] Research reports have shown that CD73 is involved in cell-cell and cell-matrix interactions, and that CD73 is involved in drug resistance and promotion of carcinogenesis (Spychala J., Pharmacol. Ther. [Pharmacology and Therapeutics], 87(2-3):161-173(2000)). CD73 is overexpressed in several types of cancer cells (Gao ZW. et al., Biomed. Res. Int. [International Biomedical Research], 2014:460654 (2014)), and its expression has been shown to be associated with poor prognosis or patient survival in several cancers (Loi S. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences], 110(27):11091-11096 (2013); Turcotte M. et al., Cancer Res. [Cancer Research], 75(21):4494-4503 (2015); Xiong L. et al., Cell Tissue Res. [Cell Tissue Research], 355(2):365-374 (2014)). In some instances, treatment with anti-CD73 monoclonal antibodies (mAbs) can inhibit metastasis and tumor angiogenesis (Allard B. et al., Int. J. Cancer, 134(6):1466-1473 (2014); Terp MG. et al., J. Immunol., 191(8):4165-4173 (2013)). Researchers have also demonstrated the immunosuppressive effects of CD73-adenosine in cancer, and believe that targeting and blocking CD73 or adenosine receptors can effectively promote antitumor immunity and enhance the activity of first-generation immune checkpoint inhibitors (Allard D. et al., Immunotherapy, 8(2):145-163 (2016)).

[0005] Recently, targeting CD73 has achieved favorable antitumor effects in preclinical models, and combination therapy of CD73 blockade with the use of other immunomodulatory agents is a particularly attractive therapeutic option (Antonioli L. et al., Trends Cancer, 2(2):95-109 (2016)). Combination with anti-CD73 mAb can significantly enhance the antitumor 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., 19(20):5626-5635 (2013)).

[0006] However, despite advances in research into CD73 and the clinical benefits of utilizing the protein, there remains a need to develop effective, safe and potent antibody agents directed against CD73. The present invention meets these and other needs. Summary of the Invention

[0007] In accordance with one or more of the objects of the invention, as embodied and broadly described herein, the invention in one aspect relates to anti-CD73 antibodies, variants, mutants, and / or antigen-binding fragments thereof, methods for making them, pharmaceutical compositions comprising them, and methods for treating human subjects with them. For example, the disclosed anti-CD73 antibodies, and affinity variants and / or mutants, or antigen-binding fragments thereof, can be used alone or in combination with other agents in the treatment of diseases associated with CD73 expression, CD73 overexpression, and / or aberrant CD73 function (e.g., cancer or fibrosis). The antibodies provided herein are further used to detect CD73 protein in a patient or a patient sample by administering an anti-CD73 antibody, and / or affinity variant / mutant, or antigen-binding fragment thereof to a patient and detecting the anti-CD73 antibody, and / or variant / mutant, or antigen-binding fragment thereof bound to CD73 protein in a sample from the patient (e.g., in vivo or in vitro), or by contacting an anti-CD73 antibody, and / or variant / mutant, or antigen-binding fragment thereof with a sample from the patient and detecting, qualitatively or quantitatively, the anti-CD73 antibody, and / or affinity variant / mutant, or antigen-binding fragment thereof bound to CD73 protein.

[0008] The present invention provides anti-CD73 antibodies and variants, and / or antigen-binding fragments thereof. In some embodiments, the present invention provides at least 18 anti-CD73 antibodies, variants, and / or antigen-binding fragments thereof, namely, anti-CD73 antibodies and / or variants N1, variant N1 variants (N1#2, N1#2-P, N1#9, and N1#9-PH), variant N2, variant N4, variant 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 nucleic acid and / or nucleic acid-encoded amino acid sequences of the light and heavy chains of each of these (18) anti-CD73 antibodies and / or variants thereof are shown in the sequence listing below. Tables 1 and 2 below show the CDR sequences of each light chain (CDR-L1, CDR-L2 and CDR-L3) and each heavy chain (CDR-H1, CDR-H2 and CDR-H3) of each anti-CD73 antibody and / or variant thereof, respectively.

[0009] [Table 1]

[0010] [Table 2-1] [Table 2-2]

[0011] In some embodiments, according to (or as applied to) any of the above embodiments, an anti-CD73 antibody of the invention comprises one or more mutations in N-glycosylation sites of one or more CDRs of its variable domain.

[0012] The resulting deglycosylated antibody retains similar functionality as the parent non-deglycosylated antibody.

[0013] In some embodiments, according to (or as applied to) any of the above embodiments, the anti-CD73 antibody of the invention is a full-length IgG antibody, the light chain consisting of the light chain variable region described above and a human antibody light chain constant region, and the heavy chain consisting of the heavy chain variable region described above and a human antibody heavy chain constant region.

[0014] In some embodiments, according to (or as applied to) any of the above embodiments, the anti-CD73 antibody of the present invention is a full-length IgG antibody, the light chain consisting of the light chain variable region as described above and a human antibody light chain constant region as shown in SEQ ID NO.115, and the heavy chain consisting of the heavy chain variable region as described above and a human antibody heavy chain constant region as shown in SEQ ID NO.112, 113 or 114.

[0015] In some embodiments, according to (or as applied to) any of the above embodiments, the anti-CD73 antibody of the invention is a full-length IgG antibody, (Antibody variant N1#2-P) the light chain is as set forth in SEQ ID NO.93 and the heavy chain is as set forth in SEQ ID NO.116; or (Antibody variant N1#9-PH) the light chain is as shown in SEQ ID NO.94 and the heavy chain is as shown in SEQ ID NO.117; or ((Antibody variant N4#6-3-P) the light chain is as set forth in SEQ ID NO.95 and the heavy chain is as set forth in SEQ ID NO.118; or (Antibody variant N4#6-4-P) The light chain is as shown in SEQ ID NO.96 and the heavy chain is as shown in SEQ ID NO.119.

[0016] [Table 3-1] [Table 3-2] [Table 3-3]

[0017] In some embodiments, according to (or as applied to) any of the above embodiments, the anti-CD73 antibody comprises an Fc sequence of human IgG. In some embodiments, according to (or as applied to) any of the above embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single chain Fv (scFv), Fv fragment, diabody and linear antibody. In some embodiments, according to (or as applied to) any of the above embodiments, the antibody is a multispecific antibody.

[0018] 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 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 a radioisotope, a fluorescent dye, and an enzyme.

[0019] 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 embodiments. Further provided is an expression vector encoding a nucleic acid molecule according to (or as applied to) any of the above embodiments. Further provided is a cell comprising an expression vector according to (or as applied to) any of the above embodiments. The present invention provides a method for 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 embodiments and recovering the antibody or antigen-binding fragment thereof from the cell culture. In some embodiments according to (or as applied to) any of the above embodiments, the cell is a mammalian cell. In some embodiments according to (or as applied to) any of the above embodiments, the mammalian cell is a CHO cell. In some embodiments according to (or as applied to) any of the above embodiments, the cell is a stable mammalian cell line. In some embodiments according to (or as applied to) any of the above embodiments, the stable mammalian cell line is a CHO cell line.

[0020] In some embodiments, an isolated antibody is provided that specifically binds to human CD73 on the cell surface and is capable of neutralizing the ecto-5'-nucleotidase activity of soluble or membrane-bound human CD73 protein. The antibody is capable of inducing cellular internalization of CD73.

[0021] In some embodiments, antibodies are provided that are capable of binding to soluble or membrane-bound human CD73 protein and inhibiting its enzymatic activity, wherein the antibodies are not internalized into cells expressing CD73.

[0022] The 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 above embodiments, and a pharma- ceutically acceptable carrier.

[0023] The invention provides a method of detecting CD73 protein in a sample from a patient by contacting the sample with an anti-CD73 antibody, variant, mutant or antigen-binding fragment thereof according to (or as applied to) any of the above embodiments and detecting the anti-CD73 antibody bound to the CD73 protein. In some embodiments, the anti-CD73 antibody, variant or antigen-binding fragment thereof according to (or as applied to) any of the above embodiments is used in an immunohistochemical assay (IHC) or ELISA assay.

[0024] Further provided is 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 above embodiments. Further provided is a composition comprising an anti-CD73 antibody, variant, mutant or antigen-binding fragment thereof according to (or as applied to) any of the above embodiments for use in treating cancer. Provided is the use of an anti-CD73 antibody, variant, mutant or antigen-binding fragment thereof according to (or as applied to) any of the above embodiments in the manufacture of a medicament for treating cancer (e.g. a cancer associated with expression of CD73).

[0025] In some embodiments, according to (or as applied to) any of the above embodiments, the cancer is selected from melanoma, head and neck cancer, urothelial cancer, breast cancer (e.g., triple negative breast cancer, TNBC), gastric cancer, classical Hodgkin's lymphoma (cHL), non-Hodgkin's 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 carcinoma (NPC), biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, and salivary gland cancer. In some embodiments, according to (or as applied to) any of the above embodiments, the subject is further administered a therapeutic agent selected from the group consisting of antineoplastic agents, chemotherapeutic agents, growth inhibitory agents, and cytotoxic agents. In some embodiments, according to (or as applied to) any of the above embodiments, the subject is further administered radiation therapy and / or surgery.

[0026] Other systems, methods, features, and advantages of the present invention will be apparent or become apparent to one of ordinary skill in the art upon study of the following figures and detailed description. All such additional systems, methods, features, and advantages are intended to be included within this specification, be within the scope of the present invention, and be protected by the accompanying claims. Furthermore, all optional and preferred features and modifications of the described embodiments are applicable to all aspects of the present invention taught herein. Furthermore, the individual features of the appended claims, together with all optional and preferred features and modifications of the described embodiments, can be combined and substituted for each other. [Brief description of the drawings]

[0027] Many aspects of the invention can be better understood with reference to the following drawings. [Figure 1A] Amino acid sequence alignments of the light chain (FIG. 1A) (FIG. 1A shows SEQ ID NOs. 79-81, respectively, in order of appearance) and heavy chain variable regions (FIG. 1B) (FIG. 1B shows SEQ ID NOs. 97-99, respectively, in order of appearance) of anti-CD73 leads N1, N2 and N4 selected by naive phage panning are shown. Three selected antibody leads with CD73 enzyme binding and blocking activity were identified by screening a human Fab naive phage display library with human CD73-ECD / His. These selected variable sequences were then cloned into the L234F, L235E, P331S mutants of the main chain of human IgG1 Fc to produce full-length antibodies. The sequence alignments of the antibody leads are listed herein and the CDRs (complementarity determining regions) as defined by Kabat are underlined and labeled in bold. [Figure 1B]Amino acid sequence alignments of the light chain (FIG. 1A) (FIG. 1A shows SEQ ID NOs. 79-81, respectively, in order of appearance) and heavy chain variable regions (FIG. 1B) (FIG. 1B shows SEQ ID NOs. 97-99, respectively, in order of appearance) of anti-CD73 leads N1, N2 and N4 selected by naive phage panning are shown. Three selected antibody leads with CD73 enzyme binding and blocking activity were identified by screening a human Fab naive phage display library with human CD73-ECD / His. These selected variable sequences were then cloned into the L234F, L235E, P331S mutants of the main chain of human IgG1 Fc to produce full-length antibodies. The sequence alignments of the antibody leads are listed herein and the CDRs (complementarity determining regions) as defined by Kabat are underlined and labeled in bold. [Figure 2A] Binding of selected antibodies to CD73 is shown. Binding of selected antibodies to recombinant human CD73 protein (by ELISA (Figure 2A)), human tumor cells expressing CD73, MDA-MB-231 (human breast cancer) cells (Figure 2B), and NCI-H292 (human lung mucoepidermoid carcinoma) cells (Figure 2C) (by flow cytometry) was tested. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 2B] Binding of selected antibodies to CD73 is shown. Binding of selected antibodies to recombinant human CD73 protein (by ELISA (Figure 2A)), human tumor cells expressing CD73, MDA-MB-231 (human breast cancer) cells (Figure 2B), and NCI-H292 (human lung mucoepidermoid carcinoma) cells (Figure 2C) (by flow cytometry) was tested. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 2C]Binding of selected antibodies to CD73 is shown. Binding of selected antibodies to recombinant human CD73 protein (by ELISA (Figure 2A)), human tumor cells expressing CD73, MDA-MB-231 (human breast cancer) cells (Figure 2B), and NCI-H292 (human lung mucoepidermoid carcinoma) cells (Figure 2C) (by flow cytometry) was tested. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 3A] Figure 3 shows the effect of anti-CD73 leads on soluble and cellular CD73 enzymatic activity. The ability of anti-CD73 antibodies to inhibit the enzymatic activity of recombinant human CD73 protein and cell surface CD73 was tested. Recombinant CD73 protein (Figure 3A) and NCI-H292 cells (Figure 3B) were incubated with anti-CD73 antibodies, ATP and AMP. The AMP concentration in the samples was determined using the CellTiter-Glo assay. The luminescence readout indicates the CD73 enzymatic activity. Anti-CD73 ref-1 antibody and APCP were used as positive controls and HLX01 (anti-CD20) was used as a negative control. [Figure 3B] Figure 3 shows the effect of anti-CD73 leads on soluble and cellular CD73 enzymatic activity. The ability of anti-CD73 antibodies to inhibit the enzymatic activity of recombinant human CD73 protein and cell surface CD73 was tested. Recombinant CD73 protein (Figure 3A) and NCI-H292 cells (Figure 3B) were incubated with anti-CD73 antibodies, ATP and AMP. The AMP concentration in the samples was determined using the CellTiter-Glo assay. The luminescence readout indicates the CD73 enzymatic activity. Anti-CD73 ref-1 antibody and APCP were used as positive controls and HLX01 (anti-CD20) was used as a negative control. [Figure 4] Figure 1 shows CD73 internalization mediated by anti-CD73 lead. After anti-CD73 antibody treatment, cell surface expression of CD73 was measured by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 5A]Figure 5 shows the binding of selected antibodies with different IgG Fc regions to CD73. Furthermore, the variable sequences of N1, N2 and N4 were cloned into the backbone of human wild-type or C219S mutant IgG2 Fc. The binding of different IgG isotypes N1 (Figure 5A), N2 (Figure 5B) and N4 (Figure 5C) to human MDA-MB-231 cells expressing CD73 was tested by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 5B] Figure 5 shows the binding of selected antibodies with different IgG Fc regions to CD73. Furthermore, the variable sequences of N1, N2 and N4 were cloned into the backbone of human wild-type or C219S mutant IgG2 Fc. The binding of different IgG isotypes N1 (Figure 5A), N2 (Figure 5B) and N4 (Figure 5C) to human MDA-MB-231 cells expressing CD73 was tested by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 5C] Figure 5 shows the binding of selected antibodies with different IgG Fc regions to CD73. Furthermore, the variable sequences of N1, N2 and N4 were cloned into the backbone of human wild-type or C219S mutant IgG2 Fc. The binding of different IgG isotypes N1 (Figure 5A), N2 (Figure 5B) and N4 (Figure 5C) to human MDA-MB-231 cells expressing CD73 was tested by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 6A] Figure 6 shows the effect of anti-CD73 leads with different IgG Fc regions on cellular CD73 enzymatic activity. Anti-CD73 leads N1 (Figure 6A), N2 (Figure 6B), N4 (Figure 6C) with different IgG isotypes were tested for their ability to inhibit cellular CD73 enzymatic activity. MDA-MB-231 cells were incubated with anti-CD73 antibodies. ATP, AMP and CellTiter-Glo reagent were added and luminescence was recorded. APCP was used as a positive control in the enzymatic activity assay. [Figure 6B]Figure 6 shows the effect of anti-CD73 leads with different IgG Fc regions on cellular CD73 enzymatic activity. Anti-CD73 leads N1 (Figure 6A), N2 (Figure 6B), N4 (Figure 6C) with different IgG isotypes were tested for their ability to inhibit cellular CD73 enzymatic activity. MDA-MB-231 cells were incubated with anti-CD73 antibodies. ATP, AMP and CellTiter-Glo reagent were added and luminescence was recorded. APCP was used as a positive control in the enzymatic activity assay. [Figure 6C] Figure 6 shows the effect of anti-CD73 leads with different IgG Fc regions on cellular CD73 enzymatic activity. Anti-CD73 leads N1 (Figure 6A), N2 (Figure 6B), N4 (Figure 6C) with different IgG isotypes were tested for their ability to inhibit cellular CD73 enzymatic activity. MDA-MB-231 cells were incubated with anti-CD73 antibodies. ATP, AMP and CellTiter-Glo reagent were added and luminescence was recorded. APCP was used as a positive control in the enzymatic activity assay. [Figure 7A] Figure 7 shows CD73 internalization mediated by anti-CD73 antibodies with different IgG Fc regions. After incubating cells with cells of different IgG isotypes with N1 (Figure 7A), N2 (Figure 7B) and N4 (Figure 7C), cell surface expression of CD73 was measured by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 7B] Figure 7 shows CD73 internalization mediated by anti-CD73 antibodies with different IgG Fc regions. After incubating cells with cells of different IgG isotypes with N1 (Figure 7A), N2 (Figure 7B) and N4 (Figure 7C), cell surface expression of CD73 was measured by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 7C]Figure 7 shows CD73 internalization mediated by anti-CD73 antibodies with different IgG Fc regions. After incubating cells with cells of different IgG isotypes with N1 (Figure 7A), N2 (Figure 7B) and N4 (Figure 7C), cell surface expression of CD73 was measured by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. [Figure 8A] Tumor growth inhibitory activity of anti-CD73 leads in MDA-MB-231 (human triple-negative breast cancer) xenograft mouse model. Mice (n=5 mice / group) were implanted subcutaneously with MDA-MB-231 cells. 14 days after tumor inoculation, when the implanted tumors reached 150-300 mm3 in size, the first dose of test article was administered. Mice were treated intraperitoneally with 5 mg / kg antibody for the indicated times. Tumor growth curves are shown in Figure 8A. Individual tumor volumes at day 45 are shown in Figure 8B. All data points are mean ± SEM. [Figure 8B] Tumor growth inhibitory activity of anti-CD73 leads in MDA-MB-231 (human triple-negative breast cancer) xenograft mouse model. Mice (n=5 mice / group) were implanted subcutaneously with MDA-MB-231 cells. 14 days after tumor inoculation, when the implanted tumors reached 150-300 mm3 in size, the first dose of test article was administered. Mice were treated intraperitoneally with 5 mg / kg antibody for the indicated times. Tumor growth curves are shown in Figure 8A. Individual tumor volumes at day 45 are shown in Figure 8B. All data points are mean ± SEM. [Figure 9] Amino acid sequence alignment of light chain variable regions from affinity matured N1 and N4 variants. In vitro phage display based affinity maturation experiments identified N1 and N4 variants with superior CD73 binding affinity and soluble CD73 enzyme blocking activity. Kabat defined CDRs (complementarity determining regions) are underlined and labeled in bold. Figure 9 shows SEQ ID NOs. 82-85, 88, 86-87 and 89-92 in order of appearance, respectively. [Figure 10]Amino acid sequence alignment of heavy chain variable regions from affinity matured N1 and N4 mutants is shown. In vitro phage display based affinity maturation experiments identified N1 and N4 mutants with superior CD73 binding affinity and soluble CD73 enzyme blocking activity. Kabat defined CDRs (complementarity determining regions) are underlined and labeled in bold. These selected variable sequences were then cloned into the backbone of human IgG2 Fc to produce full length antibodies. FIG. 10 shows SEQ ID NOs. 100-103, 106, 104-105 and 107-110, respectively, in order of appearance. [Figure 11A] Binding of N1 and N4 mutants to CD73 is shown. Selected mutants were tested for binding to human MDA-MB-231 (FIG. 11A) and NCI-H292 cells (FIG. 11B) expressing CD73 by flow cytometry. HLX01 (anti-CD20) was used as a negative control. [Figure 11B] Binding of N1 and N4 mutants to CD73 is shown. Selected mutants were tested for binding to human MDA-MB-231 (FIG. 11A) and NCI-H292 cells (FIG. 11B) expressing CD73 by flow cytometry. HLX01 (anti-CD20) was used as a negative control. [Figure 12] Figure 1 shows the effect of N1 and N4 mutants on immobilized CD73 enzymatic activity. The ability of N1 and N4 mutants to inhibit immobilized CD73 enzymatic activity was tested. After incubation of anti-CD73 antibody and CD73 protein, ATP, AMP and CellTiter-Glo reagent were added and luminescence was recorded. Anti-CD73 ref-1 antibody and anti-CD73 ref-2 were used as positive controls and HLX01 (anti-CD20) was used as a negative control. [Figure 13] Figure 1 shows CD73 internalization mediated by N1 and N4 mutants. NCI-H292 cells were incubated with N1 and N4 mutants, and 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 a negative control. [Figure 14]Shown is an amino acid sequence alignment of the light chains of the N1 and N4 top variants. The CDRs (complementarity determining regions) as defined by Kabat are underlined and labeled in bold. Figure 14 shows SEQ ID NOs. 93-96 in order of appearance, respectively. [Figure 15] Amino acid sequence alignment of the heavy chain variable regions of the N1 and N4 top mutants is shown. The CDRs (complementarity determining regions) defined by Kabat are underlined and labeled in bold. These sequences were then cloned into the backbone of human IgG2 Fc to produce full-length antibodies. Figure 15 shows SEQ ID NOs. 111, 101, and 108-109, respectively, in order of appearance. [Figure 16A] The CD73 binding ability of N1 and N4 top mutants is shown. Anti-CD73 antibodies were tested for binding to recombinant human CD73 protein by ELISA (Figure 16A) and to CD73-expressing human tumor cells MDA-MB-231 (Figure 16B) and NCI-H292 (Figure 16C) 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 a negative control. [Figure 16B] The CD73 binding ability of N1 and N4 top mutants is shown. Anti-CD73 antibodies were tested for binding to recombinant human CD73 protein by ELISA (Figure 16A) and to CD73-expressing human tumor cells MDA-MB-231 (Figure 16B) and NCI-H292 (Figure 16C) 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 a negative control. [Figure 16C]The CD73 binding ability of N1 and N4 top mutants is shown. Anti-CD73 antibodies were tested for binding to recombinant human CD73 protein by ELISA (Figure 16A) and to CD73-expressing human tumor cells MDA-MB-231 (Figure 16B) and NCI-H292 (Figure 16C) 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 a negative control. [Figure 17A] The effect of N1 and N4 top mutants on soluble (Figure 17A) and cell surface (Figure 17B, Figure 17C) CD73 enzymatic activity is shown. The ability of anti-CD73 antibodies to inhibit recombinant human CD73 protein and cell surface CD73 enzymatic activity was tested. Recombinant CD73 protein (Figure 17A), MDA-MB-231 (Figure 17B), NCI-H292 (Figure 17C) cells were incubated with anti-CD73 antibodies. ATP, AMP and CellTiter-Glo were added and luminescence was recorded. 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. [Figure 17B] The effect of N1 and N4 top mutants on soluble (Figure 17A) and cell surface (Figure 17B, Figure 17C) CD73 enzymatic activity is shown. The ability of anti-CD73 antibodies to inhibit recombinant human CD73 protein and cell surface CD73 enzymatic activity was tested. Recombinant CD73 protein (Figure 17A), MDA-MB-231 (Figure 17B), NCI-H292 (Figure 17C) cells were incubated with anti-CD73 antibodies. ATP, AMP and CellTiter-Glo were added and luminescence was recorded. 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. [Figure 17C]The effect of N1 and N4 top mutants on soluble (Figure 17A) and cell surface (Figure 17B, Figure 17C) CD73 enzymatic activity is shown. The ability of anti-CD73 antibodies to inhibit recombinant human CD73 protein and cell surface CD73 enzymatic activity was tested. Recombinant CD73 protein (Figure 17A), MDA-MB-231 (Figure 17B), NCI-H292 (Figure 17C) cells were incubated with anti-CD73 antibodies. ATP, AMP and CellTiter-Glo were added and luminescence was recorded. 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. [Figure 18A] Figure 18 shows CD73 internalization mediated by N1 and N4 top mutants. MDA-MB-231 (Figure 18A) and NCI-H292 (Figure 18B) cells were incubated with anti-CD73 N1 and N4 mutants. After incubation, 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 a negative control. [Figure 18B] Figure 18 shows CD73 internalization mediated by N1 and N4 top mutants. MDA-MB-231 (Figure 18A) and NCI-H292 (Figure 18B) cells were incubated with anti-CD73 N1 and N4 mutants. After incubation, 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 a negative control. [Figure 19A]Figure 19 shows reversal of the inhibitory effect of AMP on T cell activity by N1 and N4 top mutants. Isolated human T cells were cultured with CD3 / CD28 beads, AMPs and serially diluted antibodies at 37°C for 4 days. CD3+ T cell expansion was followed by CellTiter-Glo assay (Figure 19A) and IFN-γ secretion was measured using human IFN-γ ELISA MAX® 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. [Figure 19B] Figure 19 shows reversal of the inhibitory effect of AMP on T cell activity by N1 and N4 top mutants. Isolated human T cells were cultured with CD3 / CD28 beads, AMPs and serially diluted antibodies at 37°C for 4 days. CD3+ T cell expansion was followed by CellTiter-Glo assay (Figure 19A) and IFN-γ secretion was measured using human IFN-γ ELISA MAX® 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. [Figure 20] Tumor growth inhibitory activity of N1 and N4 Top mutants in MDA-MB-231 (human triple-negative breast cancer) xenograft mouse model. Mice (n=5 mice / group) were implanted subcutaneously with MDA-MB-231 cells. Seven days after tumor inoculation, when the implanted tumors reached approximately 100 mm3 in size, the first dose of test article was administered. Mice were treated intraperitoneally with 10 mg / kg and 2 mg / kg of antibody twice weekly for 5-6 weeks. All data points are mean ± SEM. [Figure 21]Figure 1 shows the tumor growth inhibitory activity of N1 and N4 Top mutants in NCI-H292 (human lung mucoepidermoid carcinoma) xenograft mouse model. Mice (n=5 mice / group) were implanted subcutaneously with NCI-H292 cells. The first dose of test article was administered 3 days after tumor inoculation. Mice were treated intraperitoneally with 50, 10 and 2 mg / kg antibody twice a week for 3 weeks. All data points are mean ± SEM. [Figure 22] Figure 1 shows the effect of N1 and N4 top mutants on cellular CD73 enzyme activity in NCI-H292 xenograft tumors. The ability of N1 and N4 top mutants to inhibit CD73 enzyme activity in NCI-H292 xenograft model was tested. Mice (n=4 mice / group) were implanted with NCI-H292 cells subcutaneously 4 days before treatment with antibody. Antibody, APCP and placebo were injected intraperitoneally on day 0. Tumors were excised on days 1, 3 and 7 after antibody administration. CD73 enzyme activity in tumors was measured by CellTiter-Glo assay. [Figure 23A] Figure 23 shows the effect of N1 and N4 top mutants on CD73 expression and cellular CD73 enzymatic activity in MDA-MB-231 xenograft tumors. The ability of anti-CD73 antibodies to reduce surface CD73 expression and inhibit CD73 enzymatic activity in MDA-MB-231 xenograft models was tested. MDA-MB-231 cells were implanted subcutaneously in mice (n=4 mice / group) 7 days prior to treatment with antibodies. Antibodies and placebo were injected on day 0. Tumors were harvested on days 1, 3, and 7 after antibody administration. CD73 expression was measured by mean fluorescence intensity (MFI) of staining (Figure 23A) and CD73 enzymatic activity in tumors was measured by CellTiter-Glo assay (Figure 23B). [Figure 23B]Figure 23 shows the effect of N1 and N4 top mutants on CD73 expression and cellular CD73 enzymatic activity in MDA-MB-231 xenograft tumors. The ability of anti-CD73 antibodies to reduce surface CD73 expression and inhibit CD73 enzymatic activity in MDA-MB-231 xenograft models was tested. MDA-MB-231 cells were implanted subcutaneously in mice (n=4 mice / group) 7 days prior to treatment with antibodies. Antibodies and placebo were injected on day 0. Tumors were harvested on days 1, 3, and 7 after antibody administration. CD73 expression was measured by mean fluorescence intensity (MFI) of staining (Figure 23A) and CD73 enzymatic activity in tumors was measured by CellTiter-Glo assay (Figure 23B). [Figure 24A] Cross-binding of N1 and N4 top variants to mouse and monkey CD73 expressing cells is shown. Anti-CD73 antibodies were tested for binding to CD73 expressing mouse breast cancer 4T1 cells (FIG. 24A) and monkey kidney epithelial LLC-MK2 cells (FIG. 24B) by flow cytometry. HLX01 was used as a negative control. [Figure 24B] Cross-binding of N1 and N4 top variants to mouse and monkey CD73 expressing cells is shown. Anti-CD73 antibodies were tested for binding to CD73 expressing mouse breast cancer 4T1 cells (FIG. 24A) and monkey kidney epithelial LLC-MK2 cells (FIG. 24B) by flow cytometry. HLX01 was used as a negative control.

[0028] Other advantages of the invention will be set forth in part in the description which follows and in part will be obvious from the description or may be learned by the practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In one aspect, the present invention relates to anti-CD73 antibodies, variants, mutants, and / or antigen-binding fragments thereof, methods for making them, pharmaceutical compositions comprising them, and methods for treating human subjects with them. The present invention further provides anti-CD73 antibodies that specifically bind to CD73 and inhibit tumor growth by inducing macrophage-mediated phagocytosis. The disclosed antibodies exhibit superior efficacy and / or anti-tumor activity over common anti-CD73 monoclonal antibodies for treating clinical conditions (e.g., cancer or fibrosis). Further provided are immunoconjugates, nucleic acids encoding the novel anti-CD73 antibodies (described herein), their affinity variants, or antigen-binding fragments thereof, and compositions (e.g., pharmaceutical compositions).

[0030] In some embodiments, the present invention provides at least eighteen anti-CD73 antibodies, variants and / or antigen-binding fragments thereof, namely, the anti-CD73 antibodies, variants and / or mutants designated in Tables 1 and 2. The sequences disclosed below in the Sequence Listing section provide the nucleic acid and / or nucleic acid encoded amino acid sequences of the light and heavy chains of each of these anti-CD73 antibodies, variants and / or mutants. Tables 1 and 2 provide the CDR sequences of each light chain (CDR-L1, CDR-L2 and CDR-L3) and each heavy chain (CDR-H1, CDR-H2 and CDR-H3), respectively, of each anti-CD73 antibody, variant and / or mutant.

[0031] The invention further provides methods of using the novel anti-CD73 antibodies, affinity variants and / or mutants or antigen-binding fragments thereof to detect CD73 in a sample (e.g., an in vivo or in vitro sample) and / or compositions comprising such antibodies, variants and / or mutants or antigen-binding fragments thereof for use in treating a clinical condition or disease associated with CD73 expression, CD73 overexpression and / or aberrant CD73 function (e.g., cancer or fibrosis). In other embodiments, the disclosed anti-CD73 antibodies, affinity variants and / or mutants or antigen-binding fragments thereof can be used in combination with other agents in the treatment of a disease associated with CD73 expression, CD73 overexpression and / or aberrant CD73 function (e.g., cancer or fibrosis). The invention further provides the use of such antibodies, variants or antigen-binding fragments thereof in the manufacture of a medicament for use in the treatment of cancer.

[0032] The antibodies provided herein are further used to detect CD73 protein in a patient or a patient sample by administering an anti-CD73 antibody, and / or affinity variant / mutant thereof, or antigen-binding fragment thereof to a patient and detecting the anti-CD73 antibody, and / or variant / mutant, or antigen-binding fragment thereof bound to CD73 protein in a sample from the patient (e.g., in vivo or in vitro), or by contacting an anti-CD73 antibody, and / or variant / mutant, or antigen-binding fragment thereof with a sample from the patient and detecting, qualitatively or quantitatively, the anti-CD73 antibody, and / or affinity variant / mutant, or antigen-binding fragment thereof bound to CD73 protein.

[0033] Based on the teachings provided in the above description and the associated drawings, one skilled in the art to which the disclosed compositions and methods pertain will recognize numerous modifications and other embodiments of the present disclosure. Therefore, it is to be understood that the present invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments are within the scope of the appended claims. Those skilled in the art will recognize many variations and modifications of the embodiments described herein. These variations and modifications are intended to be encompassed by the teachings of the present invention and are within the scope of the present claims.

[0034] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0035] It will be apparent to one of skill in the art, upon reading the specification of the present invention, that the individual embodiments described and illustrated herein have discrete components and features which may be isolated or combined with the features of any of the other embodiments without departing from the scope or spirit of the present invention.

[0036] Any method described may be carried out in the order of events described, or in any other order that is logically feasible. That is, unless expressly stated otherwise, it is not intended that any method or aspect described herein be construed as performing its steps in a particular order. Thus, in a method claim, no aspect is intended to infer an order unless the claim or specification specifically recites those steps as being limited to a particular order. This applies to any possible non-expressive basis for interpretation, including any clear meaning derived from the logic, grammatical structure, or punctuation regarding the arrangement of steps or operational flow, or the number or type of aspects described in the specification.

[0037] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Only those publications discussed herein that are disclosed prior to the filing date of this application are provided. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of the prior invention. Additionally, publication dates provided herein may differ from the actual publication dates, which must be independently confirmed.

[0038] Although aspects of the invention may be claimed and described in specific statutory classifications (e.g., statutory types of systems), this is merely for convenience and one of ordinary skill in the art should understand that each aspect of the invention may be described and claimed in any statutory classification.

[0039] It should be further understood that the terms used herein are for the purpose of describing specific embodiments only and are not limiting.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosed compositions and methods belong.It should be further understood that terms (e.g., those terms defined in common dictionaries) should be interpreted as having the meaning consistent with the context of the specification and related art, and should not be interpreted as idealized or less formal unless expressly defined herein.

[0040] Prior to describing various aspects of the present invention, the following definitions are provided and should be used unless otherwise indicated. Other terms may be defined elsewhere in the present invention.

[0041] definition It should be understood that aspects and embodiments of the invention described herein include "including aspects and embodiments," "consisting of aspects and embodiments," and "consisting essentially of aspects and embodiments."

[0042] As used herein, "comprising" should be interpreted as specifying the presence of the stated features, integers, steps or components, but does not exclude the presence or addition of one or more features, integers, steps or components or groups thereof. Also, each of the terms "by," "comprising," "comprises," "comprised of," "including," "includes," "involving," "involves," and "for example" are used in their open, non-limiting sense and can be used interchangeably. Also, the term "comprising" is intended to include embodiments and aspects covered by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of" is intended to include embodiments covered by the term "consisting of."

[0043] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in the form of a range. It is further understood that the endpoints of each range are each significant relative to, and independent of, the other endpoint. It is further understood that a number of values ​​are disclosed herein, and each value is also disclosed herein as "about" that particular value, in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. As used herein, "about" can express a range from one particular value and / or to "about" another particular value. Similarly, when values ​​are expressed as approximations using the antecedent "about," it will be understood that the particular value forms another aspect. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0044] When a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. For example, Where a stated range includes one or two of the limits, ranges excluding one or two of those included limits are also included in the present invention, for example, the phrase "x to y" includes ranges from "x" to "y" and ranges greater than "x" and less than "y". The range may be expressed as an upper limit, for example, "less than about x, y, z", and should be interpreted to include the specific ranges of "about x", "about y", and "about z", the ranges "less than x", "less than y", and "less than z". Similarly, the phrase "more than about x, y, z" should be interpreted to include the specific ranges of "about x", "about y", and "about z", the ranges "greater than x", "greater than y", and "greater than z". Also, in the phrase "about "x" to "y", "x" and "y" are numerical values, including "about "x" to about "y".

[0045] It should be understood that this range format is used for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​explicitly stated as range limits, but also all individual numerical values ​​or subranges covered within the range as each numerical value and subrange is clearly described. For illustrative purposes, the numerical range "about 0.1% to 5%" should be interpreted not only to include the explicitly stated values ​​of about 0.1% to about 5%, but also to include individual values ​​(e.g., about 1%, about 2%, about 3% and about 4%) and subranges (e.g., about 0.5% to about 1.1%, about 0.5% to about 2.4%, about 0.5% to about 3.2% and about 0.5% to about 4.4%, as well as other possible subranges) within the specified range.

[0046] As used herein, the terms "about," "to the extent," "is or is about," and "basically" mean that the quantity or value discussed may be an exact value or a value that provides an equivalent result or effect as described in the claims or teachings. Reference herein to "about" a value or parameter means a typical error range of the corresponding value that is readily known to one of ordinary skill in the art. That is, it should be understood that amounts, sizes, formulation methods, parameters, and other numbers and properties are not and need not be exact, but are approximate (where appropriate) and / or may be greater or less than the value and may reflect tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those of ordinary skill in the art to achieve an equivalent result or effect. In some cases, a value that achieves an equivalent result or effect cannot be reasonably determined. In such instances, unless otherwise specified, "about" and "is or is about" as used herein mean a nominal value that may vary within ±10%, as is commonly understood. Generally, an amount, size, method of formulation, parameter, or other number or characteristic is "about," "in the order of," "is about," "is about," whether or not expressly described. It should be understood that when "about," "in the order of," "is about," "is about," "is" preceding a quantitative value, the parameter also includes the specific quantitative value itself, unless otherwise specified. That is, reference herein to "about" a value or parameter includes (and describes) aspects of the value or parameter itself. For example, a description of "about X" includes a description of "X."

[0047] As used herein, the terms "optional" or "optionally" mean that the event or circumstance described below may or may not occur, and the description includes embodiments in which the event or circumstance occurs and embodiments in which the event or circumstance does not occur.

[0048] As used herein, "CD73" and "CD-73" can be used interchangeably and refer to a cell surface enzyme encoded by a human gene containing eight exons, the cytogenetic location of which is 3q13.12 and the molecular base pair location is 85,449,584-85,495,791 on chromosome 6 (Homo sapiens Annotation Release 109, GRCh38.p12). The CD73 protein is a 5'-nucleotidase (Zn +2 CD73 is an enzyme (EC classification: 3.1.3.5) with a 5'-terminal cofactor (using 5'-nucleotidase as a cofactor). The protein is capable of hydrolyzing extracellular nucleotides to membrane-permeable nucleosides and is involved in the regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism. The protein is associated with its intracellular location on the plasma membrane, mainly via a GPI anchor. Human CD73 protein has two isotypes produced by alternative splicing. The typical isotype (UniProtKB identifier P21589) contains 574 amino acids and has a molecular weight of about 63 kDa. CD73 is also called ecto-5'-nucleotidase and 5'-nucleotidase. Mutations in the CD73 gene are associated with calcification of joints and arteries, i.e. hereditary arterial and joint multiple calcification syndrome.

[0049] As used herein, "treatment" or "treating" is a method for obtaining beneficial or desired results (including clinical results). For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following results: alleviation of one or more symptoms resulting from a disease, reduction in the extent of the disease, stabilization of the disease (e.g., prevention or delay of disease progression), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, relief of symptoms, remission of the disease (partial or total), reduction in the dose of one or more other drugs required to treat the disease, delay of disease progression, improvement or improvement in quality of life, weight gain, and / or prolonged survival. "Treatment" also encompasses reduction in pathological results of cancer (e.g., tumor volume, etc.). The methods provided herein contemplate any one or more of these therapeutic aspects.

[0050] The terms "recurrence," "relapse," or "flare-up" refer to the recurrence of cancer or disease after clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence may be considered a recurrence.

[0051] The terms "refractory" or "resistant" refer to a cancer or disease that does not respond to treatment.

[0052] The term "adjuvant therapy" refers to therapy given after the primary therapy, usually surgery. Adjuvant therapy for cancer or disease can include immunotherapy, chemotherapy, radiation therapy, or hormone therapy.

[0053] The term "maintenance therapy" refers to scheduled retreatment to help maintain the effects of a previous therapy. Typically, maintenance therapy is given to help keep cancer in remission or to prolong the response to a particular therapy despite disease progression.

[0054] The term "invasive cancer" refers to cancer that has spread beyond a tissue layer (started in the normal surrounding tissue). Invasive cancer may or may not be metastatic.

[0055] The term "non-invasive cancer" refers to a very early stage cancer or a cancer that has not spread beyond the tissue of origin.

[0056] The term "progression-free survival" in oncology refers to the length of time during and after treatment during which cancer does not grow. Progression-free survival includes the time during which a patient experiences a complete or partial response, as well as the time during which a patient experiences stable disease.

[0057] The term "progressive disease" in oncology can mean tumor growth of 20% or more since the start of treatment, due to either increased mass or spread in the tumor.

[0058] "Disorder" refers to any clinical condition associated with CD73 expression, CD73 overexpression and / or abnormal CD73 function in a subject (e.g., a mammal such as a human), which subject can benefit from treatment or prevention using the disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof, and / or pharmaceutical compositions provided herein. For example, a mammal suffers from or is in need of prevention of CD73 overexpression. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the discussed disorders. Non-limiting examples of disorders to be treated herein include the disclosed cancers (e.g., head and neck cancer, pharyngeal cancer, colorectal cancer, lung cancer, etc.) or fibrosis (including idiopathic pulmonary fibrosis).

[0059] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0060] The term "antibody" is used in the broadest sense and specifically covers, for example, single monoclonal antibodies (including agonists, antagonists and neutralizing antibodies), antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain antibodies and antibody fragments (see below), so long as they specifically bind to a native polypeptide and / or exhibit the biological or immunological activity of the invention. According to one embodiment, the antibody binds to an oligomeric form, such as a trimeric form, of a target protein. According to another embodiment, the antibody specifically binds to a protein and the monoclonal antibody of the invention (such as the depositing antibody of the invention) is capable of inhibiting said binding. A "functional fragment or analog" of the phrase antibody is a compound that has a qualitative biological activity similar to the referenced antibody. For example, a functional fragment or analog of an antibody of the invention may be an antibody capable of specifically binding to CD73. In one embodiment, the antibody is capable of inducing macrophage-mediated phagocytosis of cancer cells expressing CD73.

[0061] An "isolated antibody" is an antibody that has been identified, isolated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody, most preferably greater than 99% by weight, as determined, for example, by the Lowry method, (2) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (3) to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, however, an isolated antibody will be prepared by at least one purification step.

[0062] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains (IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, and therefore contain 10 antigen-binding sites, while secretory IgA antibodies can polymerize to form multivalent configurations containing 2-5 basic four-chain units and a J chain). For IgG, the four-chain unit is generally about 150,000 daltons. Each light (L) chain is linked to a heavy (H) chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds (depending on the H chain isotype). Each H and L chain further has regularly spaced interchain disulfide bridges. At the N-terminus, each H chain has a variable domain (VH), followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form an interface between the variable domains of the light and heavy chains. A VH and a VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Daniel P. Stites et al., Appleton & Lange, Norwalk, CT, 1994, p. 71 and chapter 6.

[0063] L chains from any vertebrate species can be divided into one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domain. The amino acid sequence of the constant domain of their heavy chains (CH) allows immunoglobulins to be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, γ, ε, and μ, respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes are further divided into subclasses; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0064] The term "variable" means that certain portions of the variable domains differ extensively in sequence between antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, variability is not uniformly distributed across the 110 amino acids of the variable domain. Instead, the V region consists of relatively invariant segments of 15-30 amino acids called framework regions (FRs), which are separated by short regions called "hypervariable regions" (each of which is 9-12 amino acids in length). Each variable domain of naturally occurring heavy and light chains mainly contains four FRs that use a β-sheet structure connected by three hypervariable regions, which together form loops that connect the β-sheet structure and in some cases form part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, when the hypervariable regions are from other chains, help form the antigen-binding site of the antibody (see Sequences of Proteins of Immunological Interest, 5th ed., Elvin A. Kabat et al., Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0065] The term "CDR" or "complementarity determining region" as used herein is intended to mean the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been previously described in Kabat E A. et al., J. Biol. Chem. 252(19):6609-6616 (1977), Sequences of Proteins of Immunological Interest, 5th ed., Elvin A. Kabat et al., Public Health Service, National Institutes of Health, Bethesda, MD, 1991, Chothia C. et al., J. Mol. Biol. 196(4):901-917 (1987), and MacCallum R M. et al., J. Mol. Biol. 262(5):732-745 (1996), and these definitions include overlapping or subgroups of amino acid residues when compared with each other. Nonetheless, application of either definition to refer to a CDR of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which comprise the CDRs as defined in each of the above references are set forth below in Table 4 for comparison.

[0066] [Table 4]

[0067] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody comprising the population is identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, in contrast to polyclonal antibody preparations (which contain different antibodies against different determinants (epitopes)), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention can be made by the hybridoma method first described in Kohler G. et al., Nature, 256(5517):495-497 (1975), or can be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson T. et al., Nature, 352(6336):624-628 (1991), Marks JD. et al., J. Mol. Biol., 222(3):581-597 (1991), and, for example, in the Examples below.

[0068] The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of this or these chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the biological activity of the invention (see U.S. Pat. No. 4,816,567, and Morrison S.L. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 81(21):6851-6855 (1984)). Chimeric antibodies for purposes herein include "primatized" antibodies which include variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.), and human constant region sequences.

[0069] An "intact" antibody is one which comprises an antigen binding site, a CL and at least heavy chain constant domains CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variants thereof. Preferably, an 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, with selectable heavy chain constant region specific sequences as shown in SEQ ID NO:112, 113 or 114, and with selectable light chain constant region specific sequences as shown in SEQ ID NO:115.

[0070] An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies (see example 2 of US Pat. No. 5,641,870, Zapata G. et al., Protein Eng. 8(10):1057-1062 (1995)), single-chain antibody molecules, multispecific antibodies formed from antibody fragments. The expression "linear antibody" generally refers to the antibodies described in Zapata G. et al., Protein Eng. 8(10):1057-1062 (1995). Briefly, 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.

[0071] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and the remaining "Fc" fragment (this name reflects their ability to crystallize easily). The Fab fragment consists of the entire L chain, the variable region domain (VH) of the H chain, and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of antibodies produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking to antigen. The Fab' fragment differs from the Fab fragment in that it has other residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear free thiol groups. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0072] The Fc fragment contains the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector functions of the antibody are determined by sequences in the Fc region, which is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0073] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least an "amino acid modification" as defined herein.

[0074] Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or the Fc region of a parent polypeptide, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide. In one embodiment, the variant Fc region herein has at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology, or at least about 99% homology to a native sequence Fc region. In another embodiment, the variant Fc region herein has at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology, or at least about 99% homology to the Fc region of a parent polypeptide.

[0075] The term "polypeptide comprising an Fc region" refers to a polypeptide, such as an antibody or immunoadhesin (see other definitions herein), that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may have been removed, for example, during purification of the polypeptide or by recombinantly engineering the nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide (including an antibody) having an Fc region of the present disclosure may include a polypeptide population having all K447 residues removed, a polypeptide population in which the K447 residue has not been removed, or a mixture of polypeptides with and without the K447 residue.

[0076] Antibody "effector function" refers to a biological activity attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and varies with antibody isotype. Examples of antibody effector functions include C1q binding, complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors, and B-cell activation. A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. Examples of Fc sequences are described, for example, but not limited to, in Sequences of Proteins of Immunological Interest, 5th ed., Elvin A. Kabat et al., Public Health Service, National Institutes of Health, Bethesda, MD., 1991.

[0077] "Fv" is the minimum antibody fragment that contains an intact antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. The folding of the two domains results in six hypervariable loops (three loops each from the H chain and L chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody.

[0078] However, even a single variable domain (or half of an Fv containing only three CDRs with specificity for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the intact binding site.

[0079] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of scFvs, see A. Pluckthun, "Antibodies from Escherichia coli" in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 1994, pp. 269-315, or Ahmad ZA. et al., Clin. Dev. Immunol., 2012:980250 (2012).

[0080] The term "diabody" refers to small antibody fragments made by constructing sFv fragments (see previous paragraph) with a short linker (about 5-10 residues) between the VH and VL domains, such that interchain, but not intrachain, pairing of the V domains is achieved, producing a bivalent fragment, i.e. 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 described in detail, for example, in EP 404097, WO 93 / 11161, and in Hollinger P. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90(14):6444-6448 (1993).

[0081] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Often, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from a recipient hypervariable region are replaced by residues from a hypervariable region (donor antibody) from a non-human species (e.g., mouse, rat, rabbit, or non-human primate) having the desired antigen specificity, affinity, and capacity. In some examples, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.

[0082] Humanized antibodies may also contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further enhance antibody performance. Generally, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody also optionally contains at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones PT. et al., Nature 321(6069):522-525 (1986); Riechmann L. et al., Nature 332(6162):323-329 (1988); and Presta LG, Curr. Opin. Biotechnol. 3(4):394-398 (1992).

[0083] "Percent amino acid sequence identity" or "homology" for the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared after aligning the sequences (taking into account any conservative substitutions as part of the sequence identity). For purposes of determining percent amino acid sequence identity, alignment can be achieved in a variety of ways in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of this specification, the sequence comparison computer program ALIGN-2 is used to generate percent 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 user documentation to the US Copyright Office, Washington DC, 20559 (registered under US Copyright Registration No. TXU510087). The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif. The ALIGN-2 program should be compiled for use on a UNIX operating system (preferably digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are not altered.

[0084] The term "Fc receptor" or "FcR" describes a receptor that binds to an antibody Fc region. In one embodiment, an FcR of the present invention is an FcR (gamma receptor) that binds an IgG antibody, and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see Daeron M, Annu. Rev. Immunol. [Annual Review of Immunology], 15:203-234 (1997)). The term includes allotypes, e.g., FcγRIIIA allotypes FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131 and / or FcγRIIA-H131. FcRs are reviewed in Ravetch JV. et al., Annu. Rev. Immunol., 9:457-492 (1991), Capel PJ. et al., Immunomethods, 4(1):25-34 (1994), and de Haas M. et al., J. Lab. Clin. Med., 126(4):330-341 (1995). As used herein, the term "FcR" covers other FcRs, including FcRs identified in the future. The term further includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (see Guyer RL. et al., J. Immunol., 117(2):587-593 (1976) and Kim JK. et al., Eur. J. Immunol., 24(10):2429-2434 (1994)).

[0085] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2-microglobulin. The various functions of the neonatal Fc receptor FcRn are reviewed in Ghetie V. et al., Annu. Rev. Immunol., 18:739-766 (2000). FcRn plays a role in the passive delivery of immunoglobulin IgG from the mother to the progeny and in regulating serum IgG levels. FcRn can act as a salvage receptor, binding and transporting pinocytic IgG in intact form within and across cells, rescuing them from the default degradative pathway.

[0086] The "CH1 domain" (also called "C1" for "H1" domain) of a human IgG Fc region generally extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0087] The "hinge region" is generally defined as the stretch from Glu216 to Pro230 of human IgG1 (Burton DR., Mol. Immunol. [Molecular Immunology], 22(3):161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S-S bonds in the same positions.

[0088] The "lower hinge region" of an Fc region is generally defined as the range of residues immediately C-terminal to the hinge region, i.e., residues 233 to 239 of the Fc region. In previous reports, FcR binding has generally been attributed to amino acid residues in the lower hinge region of IgG Fc regions.

[0089] The "CH2 domain" (also called "C2" for "H2" domain) of human IgG Fc region generally extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique because it does not pair closely with another domain. Rather, two N-linked branched hydrocarbon chains are inserted between the two CH2 domains in intact native IgG molecules. It has been predicted that carbohydrates can provide substitution for domain-domain pairing and aid in the stability of the CH2 domain (Burton DR., Mol. Immunol. [Molecular Immunology], 22(3):161-206 (1985)).

[0090] The "CH3 domain" (also called the "C2" or "H3" domain) comprises the range of residues in the Fc region from the C-terminus to the CH2 domain, i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence (typically amino acid residue 446 or 447 for IgG).

[0091] A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downstream regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions generally require combination of the Fc region with a binding domain (e.g., an antibody variable domain) and can be measured, for example, using the various assays disclosed herein.

[0092] "C1q" is a polypeptide that contains a binding site for the Fc region of immunoglobulins. C1q forms the complex C1, which is the first component of the complement-dependent cytotoxicity (CDC) pathway, together with two serine proteases C1r and C1s. Human C1q can be purchased commercially, for example, from Quidel, San Diego, CA.

[0093] The term "binding domain" refers to a region of a polypeptide that binds to another molecule. In the case of an FcR, the binding domain may include the part of its polypeptide chain (e.g., its alpha chain) that is responsible for binding the Fc region. One useful binding domain is the extracellular domain of the FcR alpha chain.

[0094] An antibody having a variant IgG Fc (with "altered" FcR binding affinity or ADCC activity) is one that has increased or decreased FcR binding activity (e.g., FcγR or FcRn) and / or ADCC activity compared to a parent polypeptide or a polypeptide comprising a native sequence Fc region. A variant Fc that "has increased binding" to an FcR binds to at least one FcR with a higher affinity (e.g., lower apparent Kd or IC50 value) than a parent polypeptide or a native sequence IgG Fc. In some embodiments, the improved 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, 500-fold, or about 25%-1000% improved binding. A polypeptide variant that "has reduced binding" to an FcR binds to at least one FcR with a lower affinity (e.g., higher apparent Kd or higher IC50 value) than a parent polypeptide. The reduction in binding compared to the parent polypeptide may be about 40% or more reduction in binding.

[0095] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch JV. et al., Annu. Rev. Immunol. [Annual Review of Immunology] 9:457-492 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay (e.g., as described in U.S. Pat. No. 5,500,362 or 5,821,337 or in the Examples below) may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo, e.g., in an animal model, such as that disclosed in Clynes R. et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 95(2):652-656 (1998).

[0096] A polypeptide comprising a variant Fc region (which "exhibits enhanced ADCC" or mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of human effector cells more effectively than a polypeptide having a wild-type IgG Fc or the parent polypeptide) is one that is substantially more effective at mediating ADCC in vitro or in vivo when the amounts of the polypeptide having the variant Fc region and the polypeptide having the wild-type Fc region (or the parent polypeptide) are substantially identical in the assay. Generally, such variants are identified using any in vitro ADCC assay known in the art, e.g., an assay or method for determining ADCC activity (such as in an animal model). In one example, a preferred variant is about 5 to about 100 times (e.g., about 25 to about 50 times) more effective at mediating ADCC than the wild-type Fc (or parent polypeptide).

[0097] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that is bound to its cognate antigen. To assess complement activation, a CDC assay (e.g., as described in Gazzano-Santoro H. et al., J. Immunol. Methods, 202(2):163-171 (1997)) may be performed. Polypeptide variants with altered Fc region amino acid sequences and enhanced or reduced C1q binding capacity are described in U.S. Pat. No. 6,194,551 B1 and WO 99 / 51642, the contents of which are expressly incorporated herein by reference (see further Idusogie EE. et al., J. Immunol., 164(8):4178-4184 (2000)).

[0098] An "effective amount" of an anti-CD73 antibody (or fragment thereof) or composition disclosed herein is an amount sufficient to achieve a specifically described purpose. An "effective amount" can be determined empirically for the described purpose by known methods. The term "therapeutically effective amount" refers to an amount of an anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition disclosed herein that is effective to "treat" a disease or disorder in a mammal (also referred to as a patient). In the case of cancer, a therapeutically effective amount of an anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition disclosed herein can reduce the number of cancer cells, reduce the size or weight of a tumor, inhibit (i.e., slow to a certain extent, and preferably stop) the invasion of cancer cells into peripheral organs, inhibit (i.e., slow to a certain extent, and preferably stop) the metastasis of a tumor, inhibit (i.e., slow to a certain extent, and preferably stop) the growth of a tumor, and / or alleviate to a certain extent one or more symptoms associated with the cancer. To some extent, the anti-CD73 antibodies (or variants or antigen-binding fragments thereof) or compositions disclosed herein can inhibit the growth of and / or kill existing cancer cells and can be cytoinhibitory and / or cytotoxic. In one embodiment, a therapeutically effective amount is a growth inhibitory amount. In another embodiment, a therapeutically effective amount is an amount that increases the survival rate of a patient. In another embodiment, a therapeutically effective amount is an amount that improves progression-free survival of a patient.

[0099] A "growth inhibitory amount" of an anti-CD73 antibody (or variant or antigen-binding fragment thereof) of the invention or composition disclosed herein is an amount capable of inhibiting cell (particularly tumor, e.g., cancer cell) proliferation in vitro or in vivo. A "growth inhibitory amount" of a polypeptide, antibody, antagonist or composition of the disclosure for inhibiting tumor cell proliferation can be determined empirically by known methods or by the examples provided herein.

[0100] A "cytotoxic amount" of an anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition of the invention is an amount capable of destroying a cell (particularly a tumor, e.g., a cancer cell) in vitro or in vivo. A "cytotoxic amount" of an anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition of the invention for inhibiting tumor cell growth can be determined empirically by known methods.

[0101] A "growth inhibitory amount" of an anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition of the invention is an amount capable of inhibiting cell (particularly tumor, e.g., cancer cell) proliferation in vitro or in vivo. A "growth inhibitory amount" of an anti-CD73 antibody (or variant or antigen-binding fragment thereof) or composition of the disclosure for inhibiting tumor cell proliferation can be determined empirically by known methods or by the examples provided herein.

[0102] As used herein, "pharmacologically acceptable" or "pharmacologically compatible" refers to a substance that is not biologically or undesirable, e.g., a substance that may be incorporated into a pharmaceutical composition administered to a patient without producing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition. Pharmaceutically acceptable carriers or excipients preferably meet standard standards of required toxicological and manufacturing testing and / or are included in the Inactive Ingredients Guide prepared by the US Food and Drug Administration.

[0103] The term "detection" is intended to include determining the presence or absence of a substance or quantifying the amount of a substance (e.g., CD73). The term thus refers to the use of the substances, compositions and methods of the invention for qualitative and quantitative determinations. In general, the particular technique used for detection is not critical to the practice of the invention.

[0104] For example, "detection" according to the present invention may include observing the presence or absence of a CD73 gene product, mRNA molecule, or CD73 polypeptide, changes in the level of CD73 polypeptide or the amount of binding to a target, and changes in the biological function / activity of a CD73 polypeptide. In some embodiments, "detection" may include detecting wild-type CD73 levels (e.g., mRNA or polypeptide levels). Detection may include quantifying a change (increase or decrease) of anywhere from 10% to 90%, or anywhere from 30% to 60%, or 100% or more compared to a control. Detection may include quantifying a change of anywhere from 2-fold to 10-fold (including the limit) or more (e.g., 100-fold).

[0105] The term "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0106] Anti-CD73 Antibodies and Affinity Variants / Mutants The present invention relates to antibodies that bind to the CD73 receptor 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. An anti-CD73 antibody is an antibody that has sufficient affinity and specificity to bind to CD73. In some embodiments, the disclosed anti-CD73 antibodies (or variants or mutants thereof, or antigen-binding fragments thereof) can be used as therapeutic agents in the process of targeting and interfering with diseases or conditions associated with CD73 expression. In various aspects, the disclosed anti-CD73 antibodies (or variants / mutants, or antigen-binding fragments thereof) exhibit minimal binding to other proteins. In some embodiments, preferably, the disclosed anti-CD73 antibodies (or variants / mutants, or antigen-binding fragments thereof) are human or recombinant humanized anti-CD73 monoclonal antibodies.

[0107] Without being bound to a particular theory, it is believed that the disclosed anti-CD73 antibodies, and affinity variants and / or mutants thereof, or antigen-binding fragments thereof, can increase internalization of CD73 in cells expressing CD73, thereby decreasing the amount of CD73 protein on the plasma membrane on cells that bind to the disclosed anti-CD73 antibodies, and affinity variants and / or mutants thereof, or antigen-binding fragments thereof. Thus, the decrease in the amount of CD73 protein on the plasma membrane is associated with a decrease in the activity of 5'-nucleotidase associated with CD73. In some embodiments, again without being bound to a particular theory, it is believed that the decrease in the activity of 5'-nucleotidase associated with CD73 (increased internalization) can decrease tumor cell proliferation due to enhanced immune cell function at the tumor site, since the activity of 5'-nucleotidase is decreased.

[0108] As disclosed herein, the disclosed anti-CD73 antibodies, and affinity variants and / or mutants thereof, or antigen-binding fragments thereof, have beneficial effects in other clinical conditions associated with CD73 expression (e.g., fibrosis, including idiopathic pulmonary fibrosis). Without being bound to a particular theory, it is believed that the disclosed anti-CD73 antibodies, and affinity variants and / or mutants thereof, or antigen-binding fragments thereof, have a positive clinical benefit to patients (e.g., fibrosis, including idiopathic pulmonary fibrosis) in other such clinical conditions due to a reduction in CD73 protein (and correspondingly reduced 5'-nucleotidase activity) in appropriate cells expressing CD73.

[0109] In some examples, the anti-CD73 antibody comprises the CDRs, variable heavy chain region and / or variable light chain region of any one of the antibodies disclosed herein.

[0110] The present invention provides anti-CD73 antibodies, their affinity variants / mutants, and / or antigen-binding fragments thereof. In some embodiments, a particular anti-CD73 antibody, its affinity variant(s) / mutant(s), and / or antigen-binding fragment(s) thereof of the invention comprises a light chain (LC) variable domain sequence (comprising the specific CDR-L1, CDR-L2 and CDR-L3 sequences listed in Table 1 above) and a heavy chain (HC) variable domain sequence (comprising the specific CDR-H1, CDR-H2 and CDR-H3 sequences listed in Table 2 above). The nucleic acid and amino acid sequences of each light and heavy chain of each particular anti-CD73 antibody, its affinity variant(s) / mutant(s), and / or antigen-binding fragment(s) thereof of the invention are provided in the sequence listing below. The present invention provides that the heavy and light chain variable domains and CDRs referred to herein are combined in all possible pairwise combinations to generate a large number of anti-CD73 antibodies, and one or more affinity variants / one or more mutants, and / or one or more antigen-binding fragments thereof.

[0111] In some examples, the one or more amino acid substitutions are one or more conservative amino acid substitutions. In some examples, the amino acid substitutions do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions provided herein) can be made that do not substantially reduce the binding affinity of the disclosed antibodies to the target CD73. The binding affinity of anti-CD73 antibody variants can be assessed using methods described in the Examples below.

[0112] Conservative substitutions are shown in Table 5 under the heading of "Conservative Substitutions." More substantial changes are provided in Table 4 under the heading of "Exemplary Substitutions," as further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity (e.g., retained / improved CD73 binding, reduced immunogenicity, or improved ADCC or CDC).

[0113] [Table 5]

[0114] Non-conservative substitutions require exchanging a member of one of these classes for another. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated using phage display-based affinity maturation techniques (such as the methods disclosed herein). Briefly, one or more CDR residues are mutated, and the mutant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity). Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such changes may be made in HVR "hot spots" (i.e., residues encoded by codons that are mutated frequently during the somatic maturation process (e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing secondary libraries and reselecting from the secondary libraries is described, for example, in Hoogenboom HR, Methods in Mol. Biol. 178:1-37 (2002), Antibody Phage Display (Philippa O'Brien, RA, Humana Press, Totowa, NJ, 2001).

[0115] In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then produced. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves the HVR-directed method, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified using, for example, alanine scanning mutagenesis or modeling.

[0116] In some examples, anti-CD73 antibodies, variants or mutants, or antigen-binding fragments thereof may lack N-glycosylation motifs in the heavy or light chain variable regions, which may cause differences in batches, resulting in changes in function, immunogenicity or stability. Methods for analyzing antibody glycosylation include, but are not limited to, chromatography (e.g., cation exchange chromatography (CEX) or liquid chromatography), mass spectrometry (e.g., electrospray ionization mass spectrometry), and capillary electrophoresis-sodium dodecyl sulfate.Such methods are described, for example, in Jung ST. et al., Curr. Opin. Biochnol., 22(6):858-67 (2011), Cummings RD, Etzler ME. Antibodies and Lectins in Glycan Analysis., in Essentials of Glycobiology, 2nd ed., Varki A, Cummings RD, Esko JD et al. eds., Cold Spring Harbor (New York): Cold Spring Harbor Laboratory Press, 2009, Chapter 45; Mulloy et al., Antibodies and Lectins in Glycan Analysis., in Essentials of Glycobiology, 2nd ed., Varki A, Cummings RD, Esko JD et al. eds., Cold Spring Harbor (New York): Cold Spring Harbor Laboratory Press, 2009, Chapter 47. B, Hart GW, Stanley P. Structural Analysis of Glycans., Leymarie N. et al., Anal. Chem., 84(7):3040-3048 (2012), Fernandez DD., European Biopharmaceutical Review, 106-110 (2005), and Raju TS, Methods Mol. Biol., 988:169-180 (2013).

[0117] In some examples, mutants of particular anti-CD73 antibodies contain one or more mutations at an N-glycosylation site in one or more CDR regions, e.g., the sequon NXS / T. Anti-CD73 antibody mutants with one or more mutations at an N-glycosylation site remove an N-glycosylation site but retain equivalent function.

[0118] In some embodiments, the binding affinity of an anti-CD73 antibody, variant and / or mutant, or antigen-binding fragment thereof, to CD73 is higher than the binding affinity to a CD73 homolog. Generally, an anti-CD73 antibody, variant and / or mutant, or antigen-binding fragment thereof will "specifically bind" to CD73 (i.e., bind to at least about 1×10 -7 M or less, preferably about 1×10 -8 Less than about 1×10 -9 M or less), but has a binding affinity to many non-CD73 antibodies that is at least about 50-fold, or at least about 500-fold, or at least about 1000-fold weaker than its binding affinity to CD73. Anti-CD73 antibodies that specifically bind to CD73 can be any of the various types of antibodies defined above, but are preferably humanized or human antibodies.

[0119] In some examples, the extent of binding of an anti-CD73 antibody to a non-target protein is less than about 10% of the extent of binding of the antibody to CD73, as determined by methods known in the art (e.g., ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA)). For example, specific binding can be measured by determining binding of a molecule relative to the binding of a control molecule (which is generally a molecule of similar structure that has no binding activity). For example, specific binding can be determined by competition with a control molecule that is similar to the target (e.g., an excess of unlabeled target). In this case, specific binding is indicated when binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the terms "specific binding" or "specifically binds to" or "is specific for" a particular polypeptide or epitope on a particular polypeptide target refers to, for example, binding of at least about 10% to the target. -4 M, alternatively at least about 10 -5 M, alternatively at least about 10 -6 M, alternatively at least about 10 -7 M, alternatively at least about 10 -8M, 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 The specific binding can be demonstrated by a molecule having a Kd of, M, or greater. In one embodiment, the term "specific binding" refers to binding of a molecule to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0120] 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) whose membrane surface antigens are bound by specific antibodies (such as, for example, the anti-CD73 antibodies and / or affinity variants described herein). In some examples, the anti-CD73 antibodies and / or affinity variants exhibit similar antibody-dependent cell-mediated cytotoxicity (ADCC) effector function as a control anti-CD73 monoclonal antibody, e.g., as shown by the assays described in the Examples.

[0121] For example, in some embodiments, the ADCC effector function activity of the anti-CD73 antibodies and / or affinity variants or mutants thereof described herein is at least about 80%, at least 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% of the ADCC effector function activity of a control anti-CD73 antibody. , at least about 98%, at least about 99%, at least about 100%, or greater 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 therebetween).

[0122] In some embodiments, the anti-CD73 antibodies, and / or affinity variants or mutants thereof, exhibit a binding affinity to CD73 similar to that of a control anti-CD73 antibody. In some embodiments, binding to CD73 is demonstrated by ELISA, as described in the Examples. For example, the binding affinity of the anti-CD73 antibodies and / or affinity variants or mutants thereof to CD73 is about 1%, about 5%, about 10%, 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% or less than the binding affinity of the control anti-CD73 antibody to CD73. , 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 more than about 125%) higher.

[0123] In some embodiments, anti-CD73 antibodies and / or affinity variants or mutants thereof bind to human CD73 with a Kd of 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 0.75 pM, about 1 pM, about 5 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, or greater than about 190 pM (including any range between these values)). In some examples, the binding affinity of an anti-CD73 antibody and / or affinity variant or mutant thereof to CD73 is about 1%, about 5%, about 10%, 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%) greater than the binding affinity of a control anti-CD73 antibody to CD73. In some examples, the binding affinity of an anti-CD73 antibody and / or its variant or mutant antibody to CD73 is about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2 fold, about 2.25 fold, about 2.5 fold, about 2.75 fold, about 3 fold, about 3.25 fold, about 3.5 fold, about 3.75 fold, about 4 fold, about 4.25 fold, about 4.5 fold, about 4.75 fold, or about 4.75 fold or more (including any range between these values) greater than the binding affinity of a control anti-CD73 antibody to CD73.

[0124] In some examples, the anti-CD73 antibodies provided herein and / or variants or mutants thereof have increased in vivo half-lives as compared to a control anti-CD73 antibody, hi some examples, the in vivo half-life of the anti-CD73 antibodies and / or variants or mutants thereof described herein is not shorter than the in vivo half-life of the control anti-CD73 antibody.

[0125] In some examples, the anti-CD73 antibodies and / or variants or mutants thereof provided herein exhibit pharmacokinetic properties similar to a control anti-CD73 antibody, hi some examples, the anti-CD73 antibodies and / or variants or mutants thereof provided herein exhibit an AUC (area under the curve) that is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than 95% (e.g., about 96%, about 97%, about 98%, about 99%, or more than 99%) of the serum concentration-time profile of the control anti-CD73 antibody (including any ranges between these values).

[0126] In some embodiments, the anti-CD73 antibodies and / or variants or mutants thereof of the present disclosure comprise an Fc sequence of human IgG (e.g., human IgG1 or human IgG4). In some embodiments, the Fc sequence is altered or otherwise modified to eliminate antibody-dependent cellular cytotoxicity (ADCC) effector functions, often associated with binding to their Fc receptors (FcRs). There are many examples of alterations or mutations to Fc sequences that can alter effector function. For example, WO00 / 42072 and Shields et al., J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with improved or reduced binding to FcRs. The contents of these publications are expressly incorporated herein by reference. The anti-CD73 antibodies and / or variants or mutants thereof of the present disclosure may be in the form of Fab, Fab', F(ab')2, single chain Fv (scFv), Fv fragments, diabodies and linear antibodies. The anti-CD73 antibodies and / or variants or mutants thereof of the present disclosure may also be multispecific antibodies and / or variants or mutants thereof that not only bind to CD73 but also bind to one or more other mutants and inhibit one or more functions thereof. The anti-CD73 antibodies and / or variants or mutants thereof of the present disclosure may be conjugated to therapeutic agents (e.g., cytotoxic drugs, radioisotopes, and chemotherapeutic agents) or labels (e.g., radioisotopes, fluorescent dyes, and enzymes) for detecting CD73 in patient samples or in vivo by imaging. Other modifications are also provided herein, including conjugation of toxins to the anti-CD73 antibodies and / or variants or mutants thereof.

[0127] Further contemplated herein are nucleic acid molecules encoding anti-CD73 antibodies and / or variants and / or mutants thereof, and expression vectors comprising nucleic acid molecules encoding the CDRs and / or heavy chain variable domains and / or light chain variable domains, as well as cells comprising these nucleic acid molecules. The anti-CD73 antibodies of the invention and / or variants or mutants thereof can be used in the therapeutic methods described herein to detect CD73 protein in patient samples or in patients (e.g., by FACS, immunohistochemistry (IHC), ELISA assays).

[0128] Monoclonal antibodies Monoclonal antibodies may be made, for example, using hybridoma methods (see, e.g., Kohler G. et al., Nature, 174(5):2453-2455 (2005)), or produced by recombinant DNA methods (U.S. Pat. No. 4,816,567), or produced by the methods described herein in the Examples below. In the hybridoma method, typically a hamster, mouse or other suitable host animal is immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0129] The immunizing agent typically comprises a polypeptide, a fusion protein of the protein of interest, or a composition comprising the protein. Generally, peripheral blood lymphocytes ("PBL") are used if human cells are desired, or spleen cells or lymph node cells are used if a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent (e.g., polyethylene glycol) to form a hybridoma cell (Monoclonal Antibodies: principles and practice, James W. Goding, New York: Academic Press, 1986, pp. 59-103). The immortalized cell line is generally a transformed mammalian cell, particularly a rodent, bovine, and human myeloma cell. Generally, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of the unfused immortalized cells. For example, if the parent cells are deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium typically contains hypoxanthine, aminopterin and thymidine ("HAT medium"), which substances inhibit the growth of HGPRT-deficient cells.

[0130] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to the medium (e.g., HAT medium). More preferred immortalized cell lines are mouse myeloma cell lines, such as those available from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor D. et al., J. Immunol., 133(6):3001-3005 (1984); Monoclonal Antibody Production Techniques and Applications, Brodeur et al., Marcel Dekker, Inc.: New York, 1987, pp. 51-63).

[0131] The medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the polypeptide. The binding specificity of the monoclonal antibodies produced by the 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 antibodies can be determined, for example, by the Scatchard analysis of Munson PJ. et al., Anal. Biochem., 107(1):220-239 (1980).

[0132] After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Monoclonal Antibodies: principles and practice, James W. Goding, New York: Academic Press, 1986, pp. 59-103). Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0133] The monoclonal antibodies secreted from the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification methods, such as protein A Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0134] Monoclonal antibodies can also be produced by recombinant DNA methods known in the art and / or later developed. DNA encoding the monoclonal antibodies provided herein can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells provided herein serve as a preferred source of such DNA. Once isolated, the DNA is incorporated into expression vectors, and these expression vectors are then transfected into host cells that do not specifically produce immunoglobulin proteins (e.g., monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to allow synthesis of monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by replacing the coding sequences for human heavy and light chain constant regions with the homologous mouse sequences (see Morrison et al., supra), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody provided herein or for the variable domains of one antigen-binding site of an antibody provided herein to produce a chimeric bivalent antibody.

[0135] In some embodiments, the anti-CD73 antibodies and / or variants or mutants thereof provided herein are expressed by a stable mammalian cell line. In some embodiments, the anti-CD73 antibodies and / or variants or mutants thereof provided herein are expressed by a stable mammalian cell line at a titer of about 2.0 grams / liter, about 2.5 grams / liter, about 3.0 grams / liter, about 3.5 grams / liter, about 4.0 grams / liter, about 4.5 grams / liter, about 5.0 grams / liter, about 5.5 grams / liter, about 6.0 grams / liter, about 6.5 grams / liter, about 7.0 grams / liter, or about 7.0 grams / liter or greater (including any ranges between these values). In some embodiments, the stable mammalian cell line expressing the anti-CD73 antibodies and / or variants or mutants thereof provided herein is a CHO cell line.

[0136] In some embodiments, the antibody is a monovalent antibody. Methods for producing monovalent antibodies are known in the art, for example, methods involving recombinant expression of immunoglobulin light chains and modified heavy chains. Typically, the heavy chain is truncated at some point in the Fc region to prevent cross-linking of the heavy chains. Alternatively, the relevant cysteine ​​residues are replaced with another amino acid residue or deleted to prevent cross-linking of the heavy chains.

[0137] In vitro methods are also suitable for producing monovalent antibodies. Digestion of antibodies can be carried out using techniques known in the art to produce fragments thereof, particularly, but not limited to, Fab fragments.

[0138] Human and humanized antibodies The anti-CD73 antibodies and / or variants or mutants thereof of the invention are human antibodies. They may be humanized antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that typically contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies comprise a human immunoglobulin (acceptor antibody) in which residues from the CDRs of the acceptor are replaced by residues from the CDRs of a non-human species (donor antibody) (e.g., mouse, rat or rabbit) having the desired specificity, affinity and capacity. In some examples, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues found neither in the acceptor antibody nor in the imported CDR or framework sequences. In general, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody also preferably comprises at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region (e.g., Jones PT. et al., Nature, 321(6069):522-525 (1986); Riechmann L. et al., Nature, 332(6162):323-327 (1988); Presta LG, Curr. Opin. Biochnol., 3(4):394-398 (1992)).

[0139] Typically, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are commonly referred to as "import" residues and are typically taken from an "import" variable domain. In one embodiment, humanization is performed according to the method of Winter and coworkers, essentially by substituting rodent CDR or CDR sequences for the corresponding sequences of a human antibody (Jones PT. et al., Nature, 321(6069):522-525 (1986); Riechmann L. et al., Nature, 332(6162):323-327 (1988); Verhoeyen M. et al., Science, 239(4847):1534-1536 (1988)). Thus, a "humanized" antibody as described herein is one in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0140] As an alternative to humanization, human antibodies can be produced. For example, it is now possible to generate transgenic animals (e.g., mice) that are capable, after immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in a complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene sequences into such germ-line mutant mice will result in the production of human antibodies following antigen challenge; see, e.g., Jakobovits A. et al., Proc. Natl. Acad. Sci. USA 90(6):2551-2555 (1993); Jakobovits A. et al., Nature 362(6417):255-258 (1993); Bruggemann M. et al., Year Immunol. 7:33-40 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO 97 / 17852.

[0141] Alternatively, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. The methods are known in the art, see, for example, Marks JD. et al., Biotechnology, 10(7):779-783 (1992); Lonberg N. et al., Nature, 368(6474):856-859 (1994); Morrison SL., Nature, 368(6474):812-813 (1994); Fishwild DM. et al., Nat. Biotechnol., 14(7):845-851 (1996); Neuberger M., Nat. Biotechnol., 14(7):826 (1996); Lonberg N. et al., Int. Rev. Immunol., 13(1):65-93 (1995).

[0142] Alternatively, phage display technology (McCafferty J. et al., Nature, 348(6301):552-554 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires derived from unimmunized donors. According to one embodiment of the technology, antibody V domain sequences are cloned in frame into a major or minor coat protein gene of a filamentous bacteriophage (e.g., M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Phage display can be performed in a variety of formats, e.g., as described in the Examples section below or reviewed in, e.g., Johnson KS. et al., Curr. Opin. Struct. Biol., 3(4):564-571 (1993). V gene fragments from several sources can be used for phage display. In Clackson T. et al., Nature 352(6336):624-628 (1991), a variety of anti-oxazolone antibodies are isolated from a small random combinatorial library of V genes derived from the spleens of immunized mice. Repertoires of V genes from unimmunized human donors can be constructed and antibodies against a variety of antigens (including self-antigens) can be isolated essentially following the techniques described in Marks JD. et al., J. Mol. Biol. 222(3):581-597 (1991) or Griffith AD. et al., EMBO J. 12(2):725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0143] As described above, human antibodies can also be generated by in vitro activated B cells by methods known in the art.

[0144] Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. See Hoogenboom HR. et al., J. Mol. Biol., 227(2):381-388 (1991); Marks JD. et al., J. Mol. Biol., 222(3):581-597 (1991). The techniques of Cole et al. and Boerner et al. can also be used to generate human monoclonal antibodies (Monoclonal Antibodies and Cancer Therapy, Cole et al., Alan Liss, Inc., 1985, p. 77; and Boerner P. et al., J. Immunol., 147(1):86-95 (1991)).

[0145] multispecific antibodies A multispecific antibody is a monoclonal antibody, preferably a human or humanized antibody, that has binding specificities for two or more different antigens (e.g. a bispecific antibody that has binding specificities for at least two antigens). For example, one of the binding specificities may be for the a5-1 protein and the other may be for any other antigen. According to one preferred embodiment, the other antigen is a cell surface protein or a receptor or receptor subunit. For example, the cell surface protein may be a natural killer (NK) cell receptor. Thus, according to one embodiment, a bispecific antibody of the invention is capable of binding to both CD73 and, for example, a second cell surface receptor.

[0146] Suitable methods for producing bispecific antibodies are known in the art. For example, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein C et al., Nature, 305(5934):537-540 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is generally performed by an affinity chromatography step. Similar methods are disclosed in the art, for example in WO93 / 08829 and Traunecker A. et al., EMBO J., 10(12):3655-3659 (1991).

[0147] Antibody variable domains with the desired binding specificities (antibody-antigen binding sites) can be fused to immunoglobulin constant domain sequences. Fusion to immunoglobulin heavy chain constant domains, including at least part of the hinge, CH2, and CH3 regions, is preferred. Preferably, in at least one of these fusions, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into a single expression vector and co-transfected into a suitable host organism. For further details on generating bispecific antibodies, see, e.g., Suresh MR. et al., Methods Enzymol. 121:210-228 (1986).

[0148] Various techniques have also been disclosed for producing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, leucine zippers are used to produce bispecific antibodies (Kostelny SA. et al., J. Immunol., 148(5):1547-1553 (1992)). Leucine zipper peptides derived from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by genetic fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "double antibody" technology described in Hollinger P. et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993) provides an alternative mechanism for generating bispecific antibody fragments. The fragments comprise a VH linked to a VL by a linker that is too short to allow pairing of the two domains in the same chain. Thus, the VH and VL domains of one fragment pair with the complementary VL and VH domains of another fragment to form two antigen-binding sites. Furthermore, another method for making bispecific antibody fragments using single-chain Fv (sFv) dimers has been reported. See Gruber M. et al., J. Immunol., 152(11):5368-5374 (1994).

[0149] Antibodies with more than one valency are contemplated, for example trispecific antibodies can be made (Tutt A. et al., J. Immunol., 147(1) 60-69 (1991)).

[0150] Heteroconjugate antibodies Heteroconjugate antibodies consist of two covalently linked antibodies. For example, such antibodies have been proposed to target immune system cells against unwanted cells and be used to treat HIV infection. It is contemplated that these antibodies can be produced in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using disulfide bond exchange reactions or by forming thioether bonds. Examples of suitable reagents for such purposes include iminothiolate, methyl-4-mercaptobutyrimidate, and others known in the art.

[0151] Effector function operation It may be desirable to modify the antibodies provided herein with respect to effector function, e.g., to enhance the efficacy of the antibody in treating cancer. For example, one or more cysteine ​​residues can be introduced into the Fc region to form interchain disulfide bonds in said region. Homodimeric antibodies thus generated can have improved internalization capability and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron PC. et al., J. Exp. Med., 176(4):1191-1195 (1992) and Shopes B., J. Immunol., 148(9):2918-2922 (1992). Additionally, heterobifunctional crosslinkers known in the art can be used to generate homodimeric antibodies with enhanced antitumor activity. Alternatively, antibodies can be engineered to have dual Fc regions to have enhanced complement lysis and ADCC capabilities. See, for example, Stevenson GT. et al., Anticancer Drug Des., 3(4):219-230 (1989).

[0152] Mutations or changes in the Fc region sequence may be made to improve FcR binding (e.g., FcγR, FcRn). According to one embodiment, the antibodies of the invention have at least one altered effector function selected from the group consisting of ADCC, CDC and improved FcRn binding compared to a native IgG or parent antibody. Examples of some useful specific mutations are described, for example, in Shields RL. et al., J. Biol. Chem., 276(6):6591-6604 (2001), Presta, LG. et al., Biochem. Soc. Trans., 30(4):487-490 (2002), and WO00 / 42072.

[0153] According to one embodiment, the mutations in the Fc receptor are 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, and at least one substitution at a position selected from the group consisting of 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, where the numbering of the residues in the Fc region is according to the EU numbering system. In some embodiments, the mutation in the Fc receptor is a D265A substitution. In some embodiments, the mutation in the Fc receptor is a N297 A substitution. Further suitable mutations are known in the art, for example, those shown in U.S. Pat. No. 7,332,581.

[0154] immunoconjugate The present invention also relates to immunoconjugates comprising the antibody or a variant or mutant thereof conjugated to a cytotoxic drug (e.g., a chemotherapeutic agent), a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin or a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[0155] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, aleurone proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the trichothecenes. A variety of radionuclides can be used for the production of radioconjugated antibodies. Examples are: 212 Bi, 131 I, 131 In, 90 Y, and 186 Exemplary chemotherapeutic agents useful in generating such immunoconjugates include those described elsewhere herein.

[0156] In some embodiments, the anti-CD73 antibodies of the invention and / or variants or mutants thereof are conjugated to a maytansine, a maytansinoid, or a calicheamicin, hi some embodiments, the anti-CD73 antibodies of the invention and / or variants or mutants thereof are conjugated to the maytansinoid DM1.

[0157] Conjugates of antibodies and cytotoxic drugs are made using a variety of bifunctional protein coupling agents (e.g., N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP)), iminothiolane (IT), imidoester bifunctional derivatives (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be made as described in Vitetta ES. et al., Science, 238(4830):1098-1104 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026.

[0158] In some examples, the anti-CD73 antibodies of the invention and / or variants or mutants thereof can be conjugated to a "receptor" (e.g., streptavidin) for pre-targeting of the tumor, and the antibody-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) that is conjugated to a cytotoxic drug (e.g., a radionucleotide).

[0159] covalent modification Covalent modifications of anti-CD73 antibodies, variants, mutants and fragments thereof are included within the scope of this disclosure. One type of covalent modification involves reacting targeted amino acid residues of a polypeptide with an organic derivatizing agent capable of reacting with selected side chains or N- or C-terminal residues of the polypeptide. Derivatization with bifunctional agents is useful, for example, in methods for crosslinking a polypeptide to a water-insoluble support matrix or surface to purify an antibody, and vice versa. Common crosslinkers include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid), homobifunctional imidoesters (including disuccinimidyl esters, e.g., 3,3'-dithiobis(succinimidyl-propionate)), bifunctional maleimides (e.g., bis-N-maleimido-1,8-octane), and agents such as methyl-3-[(p-azidophenyl)-dithio]propioimidate.

[0160] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (Proteins: Structure and Molecular Properties, Thomas E. Creighton, WH Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0161] Another type of covalent modification of a polypeptide involves linking the polypeptide to one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, by methods known in the art (e.g., methods described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337).

[0162] Chimeric molecules The anti-CD73 antibodies of the present invention and / or variants or mutants thereof may be modified where it is advantageous to form chimeric molecules comprising a polypeptide (e.g., an immunoadhesin or peptibody) fused to another, heterologous polypeptide or amino acid sequence.

[0163] In one embodiment, such a chimeric molecule comprises a fusion of a polypeptide with a protein transduction domain that provides targeted delivery of the polypeptide to various tissues, and more specifically across the blood-brain barrier, for example, the protein transduction domain of the human immunodeficiency virus TAT ​​protein (Schwarze SR. et al., Science 285(5433):1569-1572 (1999)).

[0164] In some examples, such a chimeric molecule comprises a fusion of the polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the polypeptide. The presence of such an epitope-tagged polypeptide can be detected using an antibody against the tag polypeptide. Also, by providing the epitope tag, the polypeptide can be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine (poly-His) or poly-histidine-glycine (poly-His-gly) tags, influenza HA tag polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol., 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., 5(12):3610-3616 (1985)), and herpes simplex virus glycoprotein D (gD) tag and its antibodies (Paborsky LR et al., Protein Eng., 3(6):547-553 (1990)). Other tag polypeptides include Flag-peptide (Hopp TP et al., Bio / Technology, 6:1204-1210 (1988)), KT3 epitope peptide (Martin GA., Science, 255(5041):192-194 (1992)), α-tubulin epitope peptide (Skinner MP. et al., J. Biol. Chem., 266(9):15163-15166 (1991)), and T7 gene 10 protein peptide tag (Lutz-Freyermuth C. et al., Proc. Natl. Acad. Sci. USA, 87(16):6393-6397 (1990)).

[0165] In alternative embodiments, the chimeric molecule may comprise a fusion of a polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (e.g., an "immunoadhesin"), such a fusion may be to the Fc region of an IgG molecule. The Ig fusions of the invention comprise a polypeptide comprising human residues 94-243, residues 33-53, or residues 33-52, around or only, in place of at least one variable region within an Ig molecule. In one particularly preferred embodiment, 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, US Pat. No. 5,428,130.

[0166] Immunoliposomes The anti-CD73 antibodies and / or variants or mutants thereof of the present invention can also be formulated as immunoliposomes. Liposomes containing the antibodies can be made by methods known in the art, such as those described in 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. U.S. Patent No. 5,013,556 discloses liposomes with extended blood circulation time.

[0167] Particularly useful liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to generate liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be conjugated to liposomes by disulfide bond exchange reaction as described in Martin FJ. et al., J. Biol. Chem., 257(1):286-288 (1982). Antitumor, growth inhibitory, or chemotherapeutic agents (e.g., doxorubicin) can also be optionally included in the liposomes. See Gabizon A. et al., J. Natl. Cancer Inst., 81(19):1484-1488 (1989).

[0168] Treatment with anti-CD73 antibodies, variants or mutants thereof The disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof, and / or pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal, such as a human) to treat a disease, disorder or clinical condition associated with CD73 expression, CD73 overexpression, or aberrant CD73 function. For example, a cancer associated with CD73 expression can be, but is not limited to, lymphoma, renal cancer, lung cancer, colon cancer, liver cancer, gastric cancer, primary effusion lymphoma, osteosarcoma, and non-Hodgkin's lymphoma. Another example of a disease, disorder, or clinical condition associated with CD73 expression can be fibrosis, including idiopathic pulmonary fibrosis. In some embodiments, the present invention provides the use of the anti-CD73 antibodies and / or variants / mutants (or fragments thereof) described herein in the manufacture of a medicament for treating cancer or fibrosis in a subject. In some embodiments, the subject to be treated is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a clinical patient, a clinical trial volunteer, a laboratory animal, etc. In some embodiments, the subject is suspected of or at risk for cancer or fibrosis.

[0169] In some examples, the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or 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 cancer associated with CD73 expression.

[0170] In some examples, the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal, such as a human) to treat a refractory or resistant cancer associated with CD73 expression.

[0171] In some examples, administration of the disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof, and / or pharmaceutical compositions provided herein to a subject (e.g., a mammal, such as a human) can provide adjuvant therapy for cancers associated with CD73 expression.

[0172] In some examples, administration of the disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof, and / or pharmaceutical compositions provided herein to a subject (e.g., a mammal such as a human) can provide a maintenance therapy for cancer associated with CD73 expression.

[0173] In some examples, invasive cancers associated with CD73 expression can be treated by administering the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions provided herein to a subject (e.g., a mammal, such as a human).

[0174] In some examples, the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions provided herein can be administered to a subject (e.g., a mammal, such as a human) to treat non-invasive cancers associated with CD73 expression.

[0175] In some examples, administration of the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions provided herein to a subject (e.g., a mammal, such as a human), can increase progression-free survival in a subject diagnosed with a cancer associated with CD73 expression.

[0176] In some embodiments, the cancers associated with CD73 expression treated with the disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof may be multi-drug resistant (MDR) or drug sensitive. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, hematological cancer and leukemia. In some embodiments, the cancers associated with CD73 expression 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, glioma, head and neck cancer, thyroid cancer, B-cell acute lymphocytic leukemia, medulloblastoma, atypical teratoid / rhabdoid tumor and oral squamous cell carcinoma. In another embodiment, cancers associated with CD73 expression that are treated with the disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof include, but are not limited to, blood (or hematopoietic tissue) cancer, bladder cancer, brain cancer, breast cancer (e.g., 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, genitourinary tract cancer, lymphoid hematopoietic tumors, myeloid lineage hematopoietic tumors, ovarian cancer, pancreatic cancer, rectal cancer, gastric cancer, prostate cancer, skin cancer (including squamous cell carcinoma), and thyroid cancer.

[0177] In some embodiments, the cancer associated with CD73 expression treated with the disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof may be a hematological cancer. In yet another aspect, the hematological cancer is selected from leukemia, myeloma, or lymphoma, including, but not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), Hodgkin's lymphoma, classical Hodgkin's lymphoma (cHL), non-Hodgkin's lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), non-Hodgkin's lymphoma, multiple myeloma, solitary myeloma, marginal myeloma, extramedullary myeloma, small lymphocytic lymphoma, B-cell non-Hodgkin's lymphoma, and large B-cell lymphoma. Exemplary hematopoietic tumors of the lymphatic system include, but are not limited to, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burkitt's lymphoma. Hematopoietic tumors of the myeloid lineage include, but are not limited to, acute and chronic myeloid leukemia, myelodysplastic syndromes and promyelocytic leukemia, tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma.

[0178] In other examples, the cancer associated with CD73 expression treated with the disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof may be a brain cancer or central nervous system cancer. Central and peripheral nervous system tumors include, but are not limited to, astrocytoma, neuroblastoma, glioma and schwannoma. Other 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, pineocytoma, pineoblastoma, atypical teratoid rhabdoid tumor, chondrosarcoma, chondroma, choroid plexus carcinoma, choroid plexus papilloma, craniopharyngioma, dysembryoplastic neuroepithelial tumor, gangliocytoma, germ cell tumor, hemangioblastoma, hemangiopericytoma, and metastatic brain tumors. In yet another aspect, the glioma is selected from ependymoma, astrocytoma, oligodendroglioma, and oligoastrocytoma. In yet another aspect, the glioma is selected from juvenile pilocytic astrocytoma, subependymal giant cell astrocytoma, ganglioglioma, subependymoma, pleomorphic xanthoastrocytoma, anaplastic astrocytic tumor, glioblastoma multiforme, brain stem glioma, oligodendroglioma, ependymoma, oligoastrocytoma, pilocytic astrocytoma, desmoplastic infantile astrocytoma, subependymal giant cell astrocytoma, diffuse astrocytoma, mixed glioma, optic nerve glioma, gliomatosis cerebri, multifocal gliomatous tumor, multicentric glioblastoma multiforme tumor, gangliocytoma, and ganglioglioma.

[0179] Other exemplary tumors associated with CD73 expression that have been 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 gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal tumor, nephroblastoma, cervical carcinoma, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, osteosarcoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, melanoma, teratocarcinoma, xenoderoma pigmentosum, keratoctanthoma, follicular thyroid carcinoma, and Kaposi's sarcoma.

[0180] Many diagnostic methods for cancer (e.g., melanoma, non-small cell lung cancer (NSCLC), head and neck cancer, urothelial cancer, breast cancer (e.g., triple-negative breast cancer TNBC), gastric cancer, classical 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 carcinoma (NPC), biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer) or other diseases exhibiting abnormal CD73 function and clinical criteria for these diseases are known in the art. Such methods include, but are not limited to, for example, immunohistochemistry, PCR, and fluorescent in situ hybridization (FISH). Further details regarding diagnostic methods for abnormal CD73 activity or expression are described in the art.

[0181] The disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered by any suitable route, including intravenously, intramuscularly, or subcutaneously. In some examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered by parenteral administration. By "parenteral" is meant intravenous, subcutaneous, and intramuscular administration.

[0182] In some examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with at least one agent (e.g., a second, third, or fourth agent), which may be any suitable agent known to treat cancer or fibrosis (including idiopathic pulmonary fibrosis).

[0183] In various embodiments, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with at least one agent used to treat cancer. In various embodiments, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with at least one agent used to treat cancer (e.g., oxaliplatin, leucovorin, fluorouracil (5-FU), gemcitabine, paclitaxel (protein-bound paclitaxel, such as, but not limited to, nanoparticle albumin-bound paclitaxel, sometimes referred to as "nab-paclitaxel"), or combinations thereof).

[0184] Other examples of the at least one drug used in cancer treatment include, but are not limited to, chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, antiangiogenic agents, cytokines, hormone therapy, gene therapy, biological therapy, and radiation therapy. For example, the at least one drug known for cancer treatment may be a known cell inhibitory or cytotoxic or anticancer agent (e.g., antitumor agent), growth inhibitor, cytotoxic drug, or chemotherapeutic agent (e.g., docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (anti-VEGF antibody), FOLFOX-4, infusion-administered fluorouracil, leucovorin, (leucovorin), as well as oxaliplatin, afatinib, gemcitabine, capecitabine, pemetrexed, tivantinib, everolimus, CpG-ODN, rapamycin, lenalidomide, vemurafenib, endostatin, lapatinib, PX-866, Imprime PGG, and irlotinibm.

[0185] In some embodiments, the anti-CD73 antibodies and / or variants / mutants (or fragments thereof) are conjugated to another agent. For example, the at least one agent may be selected from the group consisting of uracil mustard, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, temozolomide, thiotepa, altretamine, methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, and the like. phosphate), pentostatin, bortezomib, vinblastine, vincristine, vinorelbine, vindesine, bleomycin, actinomycin D, daunorubicin, doxorubicin, epirubicin, dexamethasone, clofarabine, cladribine, pemextresed, idarubicin, paclitaxel, docetaxel, ixabepilone , mithramycin, topotecan, irinotecan, pentostatin, mitomycin-C, L-asparaginase, interferon, etoposide, teniposide 17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionatepropionate, testolactone, megestrol acetate, tamoxifen, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, goserelin, cisplatin, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole vamisole), vinorelbine (navelbene), anastrozole, letrazole, capecitabine, reloxafine, droloxafine, hexamethylmelamine, oxaliplatin (Eloxatin®), Iressa (gefinitib, Zd1839), XELODA® (capecitabine), Tarceva® (erlotinib), azacitidine (5-azacitidine, 5-AzaC), temozolomide (Temodar®), gemcitabine (e.g., GEMZAR® (gemcitabine HCl)), and vasostatin.

[0186] In various embodiments, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with at least one agent, such as, for example, alkylating agents, such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramine ... ethylenimines and methylamelamines, including riethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogs HYCAMTIN®, CPT-Il (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopoletin, and 9-aminocamptothecin. ); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (specifically cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including synthetic analogs, KW-2189 and CBl-TML); eleutherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide amides, mechlorethamines such as uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma I1 and calicheamicin omega I1 (see, e.g., Nicolaou KC. et al., Angew Chem Int. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; esperamicin;Neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramicin, azaserine, bleomycins, cactinomycin, actinomycin C, carabicin, carminomycin, carzinophilin, chromomycin is), actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN(R), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL(R)) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin n), mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate;Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanol propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folate replenishers such as florinic acid; aceglatone; aldophosphamide glycosides glycoside);aminolevulinic acid;eniluracil;amsacrine;bestrabucil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;elfornithine;elliptinium acetate acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK(R) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid;Triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine (ELDISEME(R), FILDESIN(R)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipoproman; gacytosine; ara vinocid ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL(R)), albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE(TM)), and docetaxel (TAXOTERE(R)); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN(R)); platinum; etoposide (VP-16); ifosfamide (ifosf amide); mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunorubicin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylolnitine (DMFO); retinoids such as retinoic acid; pharmacologic acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above, such as CHOP (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone), and FOLFOX (abbreviation for treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin);

[0187] In various embodiments, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with at least one agent, which may be an angiogenesis inhibitor, such as angiostatin (plasminogen fragment), antiangiogenic antithrombin III, Angiozyme, ABT-627, Bay 12-9566, Benefin, bevazumab, BMS-275291, cartilage derived inhibitor (CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatin A-4, endostatin (collagen XVIII fragment), fibronectin fragment, gro-β, halofuginone, heparinases, heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin (hCG), IM-862, interferon α / β / γ, interferon-inducible protein (IP-10), interleukin-12, Kringle 5 (plasminogen fragment), marimastat, metalloproteinase inhibitor (TIMP), 2-methoxyestradiol, MMI 270 (CGS 27023A), MoAb IMC-1C11, Neovastat, NM-3, panzem, PI-88, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor 4 (PF4), prinomastat, prolactin 16 kD fragment, proliferin-related protein (PRP), PTK 787 / ZK 222594, retinoids, solimastat, squalamine, SS 3304, SU 5416, SU6668, SU11248, tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1 (TSP-1), TNP-470, transforming growth factor-β (TGF-b), vasculostatin, vasostatin (calreticulin fragment), ZD6126, ZD6474, farnesyltransferase inhibitors (FTIs), and bisphosphonates.

[0188] In various embodiments, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with at least one agent, which may be a DNA-interacting agent, such as cisplatin and doxorubicin; a topoisomerase II inhibitor, such as etoposide; a topoisomerase I inhibitor, such as CPT-11 or nogitecan; a tubulin-interacting agent, such as naturally occurring or synthetic paclitaxel, docetaxel, or an epothilone (e.g., ixabepilone); a hormonal agent, such as tamoxifen; a thymidilate synthase inhibitor, such as 5-fluorouracil; inhibitors); antimetabolites such as methotrexate, Iressa and other tyrosine kinase inhibitors such as OSI-774; angiogenesis inhibitors; BTK inhibitors, SYK inhibitors, ITK suppressors, 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 cetuximab (erbitux) (EGF) and herceptin (Her2) and other protein kinase modulators. These agents may be used in combination with differentiation agents such as ATRA, EZH2 inhibitors, DNMT inhibitors, corticosteroids, IDH1 inhibitors, IDH2 inhibitors and vitamin C.

[0189] In various examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with small molecules that enhance DNA killing effects in cancer cells, including PARP inhibitors, MDM2 inhibitors, NAMPT inhibitors, and HSP90 inhibitors.

[0190] In various embodiments, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with one or more immunotherapeutic agents (including antagonists, antibodies, and immunomodulators, including, but not limited to, HERCEPTINS, RITUXANS, OVAREX®, PANOREX@, BEC2, IMC-C225, VITAMIN, CAMPATH@I / H, Smart MI95, LYMPHOCIDE®, Smart I D10, and ONCOLYM®, rituximab, gemtuzumab, or trastuzumab). In other examples, the immunotherapeutic agent may be an antibody targeting 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. In some embodiments, the immunotherapeutic agent may be an antagonist targeting 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 thereof, an agonist targeting B7-1, B7-2, 4-1BBL, CD137, CD28, CD28H, GITR, GITRL, ICOS, ICOS-L, OX40, OX40L, CD70, CD27, CD40, DR3, or a combination thereof, or a combination of the foregoing antagonists and agonists.

[0191] In various examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with an antibody or combination of such antibodies that targets a cell surface molecule on an immune cell or cancer cell (including, but not limited to, EGFR, HER2, CD38, mesothelin, CD33, CD37, CD19, CD20, CD3, CD123, CD70, BAFFR, CD4, CD8, CD56, CD38). In some examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with an antibody or combination of such antibodies that targets a cell surface molecule on an immune cell or cancer cell (including, but not limited to, EGFR, HER2).

[0192] In various examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with at least one chemotherapeutic agent (e.g., alkylating agents, antimetabolites, platinum agents, paclitaxel, antihormones, topoisomerase inhibitors, tubulin agents, signal transduction inhibitors (e.g., kinase inhibitors), and other chemotherapeutic agents). A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including the synthetic analogs HYCAMTIN®, CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (specifically cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride mechlorethamines such as melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Nicolaou KC et al., Angew Chem Int. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramicin, azaserine, bleomycins, cactinomycin, actinomycin C, carabicin, carminomycin, carzinophilin lin), chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), doxorubicin liposome TLC D-99 (MYOCET®), pegylated doxorubicin liposomal (including CAELYX® and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, cin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU);folate analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folate replenishers such as floric acid; aceglatone; aldophosphamide glycosides glycoside);aminolevulinic acid;eniluracil;amsacrine;bestrabucil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;elfornithine;elliptinium acetate acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizophyllan; spirogermanium;tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan, dacarbazine; mannomustine; mitobronitol; mitolactol; pipoproman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), paclitaxel (ABRAXANE®) albumin-engineered nanoparticle formulations, and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin, and carboplatin; vincas that prevent the polymerization of tubulin to form microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunorubicin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylolnitine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®);bisphosphonates such as clodronic acid (e.g., BONEFOSO® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®) or risedronate (ACTONEL®); syn analogs; antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways associated with abnormal cell proliferation (e.g., PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R)); vaccines, such as THERATOOPE® vaccine and gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (So; Rafenib, Bayer; SU-11248 (Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; ipifarnib (R11577); sorafenib, ABT510; oblimersen sodium pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); pharmacologic acceptable salts, acids, or derivatives of the above; and combinations of two or more of the above, such as CHOP (abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone), and FOLFOX (abbreviation for the treatment regimen of oxaliplatin (ELOXATIN®) combined with 5-FU and leucovorin).

[0193] As used herein, chemotherapeutic agents include "anti-hormonal agents" or "endocrine therapeutic agents" that regulate, reduce, block or inhibit the action of hormones that promote the growth of cancer. They may themselves be hormones, and include antiestrogens with mixed agonist / antagonist profiles, including selective estrogen receptor modulators (SERMs) such as tamoxifen (NOLVADEX®), 4-hydroxytamoxifene, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxyphene, keoxifene, and SERM3; pure antiestrogens without agonist properties, such as fulvestrant (FASLODEX®) and EM800 (such agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and anastolol. Aromatase inhibitors, including nonsteroidal aromatase inhibitors such as rozole (ARIMIDEX®), letrozole (FEMARA®), and aminoglutethimide, and other aromatase inhibitors including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; leuprolide (LUPRON® and ELIGARD®), goserelide (ALPHA®), valproate ... luteinizing hormone releasing hormone agonists including luteinizing hormone, buserelin and tripterelin; progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and sex steroids including androgens / retinoids such as fluoxymesterone, all-trans retinoic acid and fenretinide; onapristone; antiprogesterones;Estrogen receptor downregulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide; pharma- ceutically acceptable salts, acids or derivatives of the foregoing; and combinations of two or more of the foregoing;

[0194] As used herein, a "taxoid" is a chemotherapeutic agent that inhibits microtubule depolymerization. Examples include paclitaxel (TAXOL®), paclitaxel (ABRAXANE®) albumin-engineered nanoparticle formulations, and docetaxel (TAXOTERE®). A preferred taxoid is paclitaxel.

[0195] The term "EGFR inhibitors" as used herein means compounds that bind to or otherwise directly interact with EGFR to prevent or reduce its signaling activity, and may be interchangeably referred to as "EGFR antagonists." Examples of such agents include EGFR-binding antibodies and small molecules. Examples of EGFR-binding antibodies include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimeric 225 (C225 or Cetuximab; ERBUTIX® and reshaped human 225 (H225) (WO 96 / 40210, Imclone Systems, Inc.). See, IMC-11F8, a fully human EGFR targeting antibody (Imclone), an antibody that binds to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR, as described in U.S. Pat. No. 5,891,996, and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see, WO 98 / 50433, (Abgenix / Amgen)); EMD 55900 (Stragliotto G. et al., Eur. J. Cancer 32A(4):636-640 (1996); EMD7200 (matuzumab), a humanized EGFR antibody against EGFR that competes with both EGF and TGF-α for EGFR binding (EMD / Merck); the human EGFR antibody, HuMax-EGFR (GenMab); the fully human antibodies designated E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns (TG. et al., J. Biol. Chem. 279(29):30375-30384 (2004)).Anti-EGFR antibodies can be conjugated to cytotoxic drugs to produce immunoconjugates (see, for example, EP659439A2 to Merck Patent GmbH). EGFR antagonists include small molecules and are described, for example, in U.S. Pat. Nos. 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, and 5,747,498, and PCT publication WO 2003 / 023636. These compounds are described in WO 98 / 14451, WO 98 / 50038, WO 99 / 09016 and WO 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholino)propoxyl]-6-quinazoline]-dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA J) 4-(3′-chloro-4′-fluoroaniline)-7-methoxy-6-(3-morpholinopropoxyl)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); Ingelheim; PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol; (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine; CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazoline]-2-butyramide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Sugen); AG1571 (SU 5271, Sugen); lapatinib (GW and dual EGFR / HER2 tyrosine kinase inhibitors such as 572016 or N-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-aminoquinazoline (Glaxo-SmithKline).

[0196] A "tyrosine kinase inhibitor" is a molecule that inhibits the tyrosine kinase activity of a tyrosine kinase, e.g., a HER receptor. Examples of such inhibitors include the EGFR-targeted drugs described in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, e.g., TAK165 (available from Takeda); CP-724, 714 (an oral selective inhibitor of ErbB2 receptor tyrosine kinase (available from Pfizer and OSI); dual HER inhibitors, e.g., EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits cells overexpressing both HER2 and EGFR; Lapatinib, a HER2 inhibitor, and a HER2 inhibitor. nib (GW572016, available from Glaxo-SmithKline, which is an oral HER2 and tyrosine kinase inhibitor); PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033, Pharmacia); Raf-1 inhibitors, such as the antisense reagent ISIS-5132 (ISIS Pharmaceuticals), which inhibits Raf-1 signaling. Pharmaceuticals); non-HER targeted TK inhibitors such as Imatinib mesylate (GLEEVAC J) (available from Glaxo); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines such as PD 153035, 4-(3-chloroaniline) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, such as 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroaniline)phthalimide); tyrphostines containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., antisense molecules bound to a HER-encoding nucleic acid);Quinoxalines (US Pat. No. 5,804,396); tryphostins (US Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors, e.g. CI-1033 (Pfizer); Affinitac (ISIS 3521, Isis / Lilly); imatinib mesylate (Gleevac, Novartis); PKI 166 (Novartis); GW2016 (GlaxoSmithKline SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), or other compounds disclosed in U.S. Pat. No. 5,804,396, International Patent Publication No. WO 99 / 09016 (American Cyanamid), WO 98 / 43960 (American Cyanamid), WO 97 / 38983 (Warner Lambert), WO 99 / 06378 (Warner Lambert), WO 99 / 06396 (Warner Lambert), WO 96 / 30347 (Pfizer Inc.), WO 96 / 33978 (Zeneca), WO 96 / 3397 (Zeneca), WO 96 / 33980 (Zeneca);

[0197] In various examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with actinomycin D, capecitabine, carboplatin, cisplatin, colchicine, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, melphalan, methotrexate, mitomycin C, mitoxantrone, paclitaxel, thalidomide, topotecan, vinblastine, and vincristine.

[0198] In various examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with one or more other therapies or clinical interventions (e.g., radiation therapy, surgery, chemotherapy, and / or targeted therapy). In some examples, the disclosed anti-CD73 antibodies and / or variants (or fragments thereof), and / or pharmaceutical compositions provided herein are administered in combination with radiation therapy for the disclosed cancers.

[0199] In some examples, the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be administered in combination with surgery (e.g., surgery may be performed prior to administration of the disclosed anti-CD73 antibodies, surgery may be performed after administration of the disclosed anti-CD73 antibodies, and / or surgery may be performed at or near the same time as administration of the disclosed anti-CD73 antibodies).

[0200] Depending on the indication being treated and factors related to administration well known to one of skill in the art, the anti-CD73 antibodies, variants, mutants, or fragments thereof provided herein are administered at an effective dose, e.g., a dose that effectively treats the indication while minimizing toxicity and side effects. In general, it is preferred to use the maximum dose, i.e., the highest safe dose based on reasonable medical judgment, of the pharmacological agents of the present disclosure (alone or in combination with other therapeutic agents). However, as will be appreciated by one of skill in the art, a patient may insist on a lower dose or a tolerable dose for medical, psychological, or virtually any other reason. In some embodiments, with an effective dose, the desired response consists of inhibiting the progression of the disease or condition, e.g., the progression of cancer. The desired response may only slow the progression of the disease temporarily, but more preferably, it is to halt the progression of the disease permanently. The desired response can be monitored by common diagnostic methods for any particular disease, known to one of skill in the art. The desired response to treatment of a disease or condition may be to delay or prevent the onset of the disease or condition.

[0201] The toxicity and therapeutic efficacy of the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein, e.g., LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of the patient population can be determined in cell cultures or experimental animals by standard pharmaceutical procedures. The ratio of toxic to therapeutic dose is the therapeutic index, and the ratio LD 50 / ED 50 The therapeutic index can be expressed as: A compound that exhibits a large therapeutic index may be desirable. It should be noted that although the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein that exhibit toxicity and side effects can be used, by designing a delivery system that targets such compounds to the site of affected tissue, thereby minimizing potential damage to uninfected cells and reducing side effects.

[0202] The data obtained from cell culture assays and animal studies can be used to determine a range of dosages for use in humans. The doses of the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein can be administered at ED 1 with little or no toxicity. 50 The dose will vary within that range depending on the dosage form employed and the route of administration used. For any of the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein, the therapeutically effective amount can be estimated initially from cell culture assays. Dosages can be determined in animal models to provide a therapeutically effective dose that is consistent with the IC50 determined in cell culture experiments. 50 The circulating plasma concentration range can be achieved that includes: 1) 0.5 to 1.0 mg / kg / day of the active ingredient; 2) 0.5 to 1.0 mg / kg / day of the active ingredient; 3) 0.5 to 1.0 mg / kg / day of the active ingredient; 4) 0.5 to 1.0 mg / kg / day of the active ingredient; 5) 0.5 to 1.0 mg / kg / day of the active ingredient; 6) 0.5 to 1.0 mg / kg / day of the active ingredient; 7) 0.5 to 1.0 mg / kg / day of the active ingredient; 8) 0.5 to 1.0 mg / kg / day of the active ingredient; 9) 0.5 to 1.0 mg / kg / day of the active ingredient; 10) 0.5 to 1.0 mg / kg / day of the active ingredient; 11) 0.5 to 1.0 mg / kg / day of the active

[0203] Various experimental animal models known in the art or described in Yamanaka K. et al., Microbiol. Immunol. 45(7):507-514 (2001) (e.g., SCID mouse models or nude mice bearing human tumor xenografts) can be used to determine the anti-cancer activity of the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein.

[0204] The disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein may be tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity prior to use in humans. For example, in vitro assays for determining whether administration of a particular therapeutic regimen is indicated include in vitro cell culture assays in which a patient tissue sample is grown in culture, exposed to or otherwise administered the therapeutic regimen, and the effect of such therapeutic regimen on the tissue sample is observed.

[0205] A low level of proliferation or survival of the contacted cells can indicate that the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein effectively treat the selected disorder in the subject. Alternatively, tumor or malignant cell lines (not cultured cells derived from the patient) can be used to screen treatment regimens. Such survival and / or proliferation can be assessed using a number of assays known in the art, for example, cell proliferation can be measured by measuring 3H-thymidine incorporation, by direct cell counting, or by detecting changes in transcriptional activity of known genes, such as proto-oncogenes or cell cycle markers, cell vitality can be assessed by trypan blue staining, and differentiation can be visually assessed based on changes in morphology, etc.

[0206] The disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof) for use in therapy, and / or pharmaceutical compositions provided herein may be tested in appropriate animal model systems (including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc.) prior to testing in humans. For example, the primary animal model of cancer known in the art and widely used involves mice, as described in Hann B. et al., Curr. Opin. Cell Biol. [New Perspectives in Cell Biology] 13(6):778-784 (2001), the entire contents of which are incorporated herein by reference.

[0207] Additionally, any assay known to one of skill in the art can be used to evaluate the prophylactic and / or therapeutic effectiveness of the disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof), and / or pharmaceutical compositions provided herein in treating, preventing, managing, or ameliorating symptoms associated with one or more of the above-mentioned diseases or disorders.

[0208] One of skill in the art can readily determine the appropriate daily dose for treating or preventing the disorders described herein. For the treatment of the disclosed cancers, typical doses can range, for example, from 0.001 μg to 1000 μg, although doses below or above this exemplary range are within the scope of the present disclosure. The daily dose is about 0.1 μg / kg to about 100 mg / kg of total body weight (e.g., about 5 μg / kg, about 10 μg / kg, about 100 μg / kg, about 500 μg / kg, about 1 mg / kg, about 50 mg / kg, or a range defined by any two of the aforementioned values), preferably about 0.3 μg / kg to about 10 mg / kg of total body weight (e.g., about 0.5 μg / kg, about 1 μg / kg, about 50 μg / kg, about 150 μg / kg, about 300 μg / kg, about 750 μg / kg, about 1.5 mg / kg, about 5 mg / kg, or a range defined by any two of the aforementioned values). The therapeutic or prophylactic efficacy may be monitored by periodic evaluation of the treated patient, as described above. For repeated administration over several days or more, depending on symptoms, treatment is repeated until the desired inhibition of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the present disclosure. The desired dose may be delivered by a single bolus administration of the composition, multiple boluses of the composition, or continuous infusion of the composition.

[0209] A pharmaceutical composition comprising an anti-CD73 antibody, variant, mutant, or fragment thereof may be administered once, twice, three or four times daily. The composition may also be administered less frequently than once daily, for example, six times per week, five times per week, four times per week, three times per week, twice per week, once per week, once per two weeks, once per three weeks, once per month, once per two months, once per three months or once per six months. The composition may also be administered in a sustained release formulation, for example, an implant that slowly releases the composition for use over a period of time, allowing for less frequent administration of the composition, for example, once per month, once every two to six months, once per year or once per month. A sustained release device (e.g., pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) may be administered by injection.

[0210] The antibody and / or variant or mutant (or fragment thereof) may be administered in a single daily dose, or the total daily dose may be administered in divided doses two, three or four times daily. The composition may also be administered less frequently than once a day, for example, six times a week, five times a week, four times a week, three times a week, two times 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 antibody (or fragment thereof) may also be administered in a sustained release formulation, for example, an implant that slowly releases the composition for use over a period of time, allowing for less frequent administration of the composition, for example, once a month, once every two to six months, once a year or once a day. Sustained release devices (eg, pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) may be administered by injection or surgically implanted at various locations.

[0211] Cancer treatment may be evaluated, for example, but not limited to, by tumor regression, reduction in tumor weight or size, time to progression, survival time, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Methods for determining efficacy of treatment can be used, including, for example, measuring response by radiological imaging.

[0212] In some embodiments, efficacy of treatment is measured as percent tumor growth inhibition (%TGI), calculated using the formula 100-(T / Cx100), where T is the mean relative tumor volume of treated tumors and C is the mean relative tumor volume of untreated tumors. In some embodiments, %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 greater. In some examples, the %TGI of an anti-CD73 antibody and / or variant or mutant thereof is the same as or greater than the %TGI of a control anti-CD73 antibody, e.g., 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 greater than or equal to about 2.7 times the %TGI of the control anti-CD73 antibody.

[0213] Pharmaceutical Compositions The disclosed anti-CD73 antibodies and / or variants or mutants (or fragments thereof) of the present invention can be made into pharmaceutical compositions containing suitable carriers or excipients for suitable administration. The antibody (or fragment thereof) having the desired purity is mixed with optional pharma- ceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. Ed. (1980)) to obtain a suitable pharmaceutical composition of the antibody in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosages and concentrations employed and include buffers (e.g., phosphates, citrates, and other organic acids); antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum. Proteins such as albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). Exemplary antibody formulations are described in WO 98 / 56418, which is incorporated herein by reference. Lyophilized pharmaceutical compositions suitable for subcutaneous administration are described in WO 97 / 04801. Such lyophilized pharmaceutical compositions may be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation may be administered subcutaneously to the mammal to be treated herein.

[0214] In some embodiments, the disclosed pharmaceutical compositions are suitable for parenteral or intravenous administration. In some embodiments, the disclosed pharmaceutical compositions suitable for parenteral administration may be aqueous and non-aqueous formulations that are isotonic with the blood of the desired subject, and aqueous and non-aqueous sterile suspensions may include suspension systems designed to target the compound to blood components of one or more organs. The formulations may be in unit-dose or multi-dose sealed containers (e.g., ampoules or vials). Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described. Parenteral and intravenous formulations may include minerals and other materials to be compatible with the type of injection or delivery system selected.

[0215] Common pharma- ceutically acceptable carriers for parenteral administration include water, suitable oil, saline, aqueous dextro-glucose (glucose) or related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizers and (if necessary) buffer substances, and antioxidants, either alone or in combination (e.g., sodium bisulfite, sodium sulfite or ascorbic acid), are suitable stabilizers. Citrate and sodium EDTA may be used as carriers. Parenteral solutions may also contain preservatives, such as benzalkonium chloride, methyl or propyl paraben, or chlorobutanol. Suitable pharmaceutical carriers are described in Remington, cited above.

[0216] Injectable preparations may be prepared in conventional forms, either as liquid solutions or suspensions, or as solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Preferably, sterile suspensions for injection are formulated using suitable pharma- ceutically acceptable carriers and optional components mentioned above, according to techniques known in the art.

[0217] Although parenteral administration can be accomplished in a variety of ways, parenteral administration of the formulations described herein is preferably accomplished using a syringe, catheter, or similar device. Systemic injection of the formulation results in delivery of the active agent throughout substantially the entire bloodstream.

[0218] It is also desirable to inject the formulation locally into a target location, for example, into a specific part of the body to treat cancer or fibrosis. The advantage of local administration by injection is that it limits or avoids systemic exposure to one or more active agents (e.g., inhibitors and / or therapeutic agents). It should be noted that in the context of the present invention, the term "local administration" includes regional administration, for example, administering the formulation to a part of the body by delivery to a blood vessel that serves that body region. Local delivery may be direct, for example, intratumoral delivery. Local delivery may be nearly direct, i.e., intralesional or intraperitoneal delivery, i.e., delivery to a region sufficiently close to the tumor or site of infection that the inhibitor exhibits the desired pharmacological activity. Thus, when local delivery is desired, it is preferable to deliver the drug formulation intralesional, intratumoral, or intraperitoneal.

[0219] In some embodiments, the pharmaceutical composition is in unit dosage form. In this form, the composition is divided into unit dosage forms containing appropriate amounts of the active ingredient. The unit dosage form may be a packaged preparation containing discrete quantities of the preparation, for example, a lyophilized powder in a vial or ampoule.

[0220] The pharmaceutical compositions herein may further comprise one or more active compounds necessary for the specific indication being treated, preferably active compounds with complement activity but which do not adversely affect each other. For example, it may be desirable to further provide an antitumor agent, a growth inhibitor, a cytotoxic drug or a chemotherapeutic agent. Such molecules are present in suitable combinations in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors as described above. These are generally used in the same doses and routes of administration as described herein, or about 1-99% of the doses conventionally used. The active ingredient may also be encapsulated in microcapsules, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively, produced, for example, by coacervation techniques or interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980).

[0221] Sustained release preparations may be manufactured. 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, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON). DEPOT® (injectable microspheres made of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxylbutyric acid. Polymers (e.g., ethylene-vinyl acetate and lactic acid-glycolic acid) can release molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time. Encapsulated antibodies may denature or aggregate as a result of exposure to moisture at 37°C if left in the body for an extended period of time, resulting in loss of biological activity and possible changes in immunogenicity. Rational methods can be devised for stability depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular SS bond formation via thiodisulfide conversion, stabilization can be achieved by modifying mercapto residues, lyophilizing from acidic solution, adjusting water content, using appropriate additives, and developing specific polymer matrix compositions.

[0222] Lipofectin or liposomes can be used to deliver the polypeptides and antibodies (or fragments thereof) or compositions of the present invention to cells. When antibody fragments are used, the smallest inhibitory fragments that specifically bind to the binding domain of the target protein are preferred. For example, based on the variable region sequences of the antibody, peptide molecules can be designed to retain the ability to bind to the target protein sequence. Such peptides are 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 of the United States of America] 90(16):7889-7893 (1993).

[0223] The active ingredient may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's PHARMACEUTICAL SCIENCES, supra.

[0224] Sustained release preparations may be manufactured. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody (or fragment thereof), which matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices are polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON DEPOT TM (injectable microspheres composed of poly(lactic-co-glycolic acid) and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers (e.g., ethylene-vinyl acetate and lactic-co-glycolic acid) can release molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time. Encapsulated antibodies may denature or aggregate as a result of exposure to moisture at 37°C if left in the body for an extended period of time, resulting in loss of biological activity and possible changes in immunogenicity. Rational methods can be devised for stability depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular SS bond formation via thiodisulfide conversion, stabilization can be achieved by modifying mercapto residues, lyophilizing from acidic solution, adjusting water content, using appropriate additives, and developing specific polymer matrix compositions.

[0225] In some examples, the formulation comprises an anti-CD73 antibody and / or variant or mutant thereof described herein at 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, greater than about 6 mg / mL, greater than about 7 mg / mL, greater than about 8 mg / mL, greater than about 9 mg / mL, greater than about 10 mg / mL, greater than about 11 mg / mL, greater than about 12 mg / mL, greater than about 13 mg / mL, greater than about 14 mg / mL, greater than about 15 mg / mL, greater than about 16 mg / mL, greater than about 17 mg / mL, greater than about 18 mg / mL, greater than about 19 mg / mL, greater than about 20 mg / mL, greater than about 21 mg / mL, greater than about 22 mg / mL, greater than about 23 mg / mL, greater than about 24 mg / mL, greater than about 25 mg / mL, greater than about 26 mg / mL, greater than about 27 mg / mL, greater than about 28 mg / mL, greater than about 29 mg / mL, greater than about 30 mg / mL, greater than about 31 mg / mL, greater than about 32 mg / mL, greater than about 33 mg / mL, greater than about 34 mg / mL, greater than about 35 mg / mL, greater than about 36 mg / mL, greater than about 37 mg / mL, greater than about 38 mg / mL, greater than about 39 mg / mL, greater than about 40 mg / mL, greater than about 41 mg / mL, greater than about 42 mg / mL, greater than about 43 mg / mL, greater than about 44 mg / mL, greater than about 45 mg / mL, greater than about 46 mg / mL, greater than about 47 mg / mL, greater than about 48 mg / mL, greater than about 4 and / or at a concentration of greater than about 15 mg / mL, greater than about 16 mg / mL, greater than about 17 mg / mL, greater than about 18 mg / mL, greater than about 19 mg / mL, greater than about 20 mg / mL, greater than about 21 mg / mL, greater than about 22 mg / mL, greater than about 23 mg / mL, greater than about 24 mg / mL, greater than about 25 mg / mL, greater than about 26 mg / mL, greater than about 27 mg / mL, greater than about 28 mg / mL, greater than about 29 mg / mL, or greater than about 30 mg / mL (including any range between these values).

[0226] Formulations containing the disclosed antibodies 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 formulation has a pH of 7, a pI of 8-9 or higher may be appropriate. Without being bound by theory, it is believed that a final formulation with improved stability can be provided, e.g., the antibody or fragment remains in solution. For example, in some embodiments, a disclosed drug formulation having a pH in the range of 4.0-7.0 may contain 1 mg / mL-200 mg / mL of the disclosed antibody and 1-100 mM buffer, and optionally one or more of (a) 0.001%-1% surfactant, (b) 10 mM-500 mM stabilizer, (c) 10 mM-500 mM stabilizer and 5 mM-500 mM tonicity agent, and / or (d) 5 mM-500 mM tonicity agent. Suitable buffers include, but are not limited to, citrate, glycine, phosphate, acetate, succinate, and bicarbonate. For example, a formulation having a pH of about 6 may contain 1-50 mg / mL antibody, 20 mM L-histidine HCl, 240 mM trehalose, and 0.02% polysorbate 20. Alternatively, a formulation having a pH of about 5.5 may contain 1-50 mg / mL antibody, 20 mM citrate buffer, 240 mM sucrose, 20 mM arginine, and 0.02% polysorbate 20.

[0227] In some examples, the anti-CD73 antibody and / or variant or mutant thereof is formulated in a buffer comprising citrate, NaCl, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination of the foregoing (e.g., at a concentration greater 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 15 mg / mL, greater than about 20 mg / mL, or greater than about 25 mg / mL (including any ranges between these values). In some examples, the anti-CD73 antibody and / or variant thereof is formulated in a buffer containing 100 mM to 150 mM glycine (e.g., at a concentration of greater 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 15 mg / mL, greater than about 20 mg / mL, or greater than about 25 mg / mL (including any range between these values)). In some examples, the anti-CD73 antibody and / or variant thereof is formulated in a buffer containing about 50 mM to about 100 mM NaCl. In some examples, the anti-CD73 antibody and / or variant or mutant thereof is formulated in a buffer containing about 10 mM to about 50 mM acetate (e.g., at a concentration greater 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 15 mg / mL, greater than about 20 mg / mL, or greater than about 25 mg / mL (including any range therebetween)). In some examples, the anti-CD73 antibody and / or variant thereof is formulated in a buffer containing about 10 mM to about 50 mM succinate. In some examples, the anti-CD73 antibody and / or variant or mutant thereof is formulated in a buffer containing about 0.005% to about 0.02% polysorbate 80 (e.g., at a concentration greater 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 15 mg / mL, greater than about 20 mg / mL, or greater than about 25 mg / mL (including any range between these values)).In some embodiments, the anti-CD73 antibody and / or variant or mutant thereof is formulated in a suitable buffer to provide a buffer pH of about 5.1 to 5.6. For example, the anti-CD73 antibody and / or variant or mutant thereof is formulated in a buffer containing 10 mM citric acid, 100 mM NaCl, 100 mM glycine, and 0.01% polysorbate 80, the formulation having a pH of 5.5.

[0228] In some examples, a formulation comprising an anti-CD73 antibody and / or variant or mutant thereof described herein (e.g., at a concentration of greater 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 15 mg / mL, greater than about 20 mg / mL, or greater than about 25 mg / mL (including any ranges between these values)) (e.g., a formulation comprising a buffer (the buffer comprising 10 mM citric acid, 100 mM NaCl, 100 mM glycine, and 0.01% polysorbate 80), wherein the formulation has a pH=5.5) is stable at room temperature (e.g., about 20-25° C.) for about 0.5 weeks, 1.0 week, 1.5 weeks, 2.0 weeks, 2.5 weeks, 3.5 weeks, 4.0 weeks, 4.5 weeks, or 5.0 weeks (including any ranges between these values). In some examples, a formulation comprising an anti-CD73 antibody and / or variant or mutant thereof described herein (e.g., at a concentration of greater 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 15 mg / mL, greater than about 20 mg / mL, or greater than about 25 mg / mL (including any ranges between these values)) (e.g., a formulation comprising a buffer (the buffer comprising 10 mM citric acid, 100 mM NaCl, 100 mM glycine, and 0.01% polysorbate 80), wherein the formulation has a pH=5.5) is stable under accelerated conditions (e.g., stored at about 37° C.) for about 0.5 weeks, 1.0 week, 1.5 weeks, 2.0 weeks, 2.5 weeks, 3.5 weeks, 4.0 weeks, 4.5 weeks, or 5.0 weeks (including any ranges between these values).

[0229] Size exclusion chromatography (SEC) is a well-known and widely used method in protein stabilization studies to detect potential fragmentation and aggregation corresponding to physicochemical instability. In some examples, formulations containing 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein exhibit an increase in high molecular weight species (HMWS) of 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) relative to the initial % HMWS after 1 week at 37°C, as measured using SEC. In some examples, formulations comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody described herein exhibit an increase in high molecular weight species of 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 any range between these values) relative to the initial % high molecular weight species as measured by SEC after 2 weeks at 37°C. In some examples, formulations comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein exhibit an increase in high molecular weight species of 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) relative to the initial % high molecular weight species as measured using SEC after 4 weeks at 37° C.

[0230] In some examples, formulations comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein exhibit an increase in low molecular weight species (LMWS) of 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) relative to the initial % low molecular weight species as measured using SEC after one week at 37°C. In some examples, formulations comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein exhibit an increase in low molecular weight species of 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 any range between these values) relative to the initial % low molecular weight species as measured using SEC after 2 weeks at 37°C. In some examples, formulations comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein exhibit an increase in low molecular weight species of less than about 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) relative to the initial % low molecular weight species as measured using SEC after 4 weeks at 37°C.

[0231] In some examples, formulations comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein exhibit an increase in monomer of 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) or less relative to the initial % monomer as measured using SEC after one week at 37° C. In some examples, formulations comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein exhibit an increase in monomer of 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) or less relative to the initial % monomer as measured using SEC after 2 weeks at 37° C. In some examples, formulations comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein exhibit an increase in monomer of 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) or less relative to the initial % monomer as measured using SEC after 2 weeks at 37° C.

[0232] Cation exchange chromatography (CEX) is a well-known and widely used method to detect proteolytic events such as deamidation or oxidation (Moorhouse KG. et al., J. Pharm. Biomed. Anal. 16(4):593-603 (1997)). The degradation products are typically referred to as acidic or basic substances. Acidic substances are variants that elute earlier than the main CEX peak, and basic substances are variants that elute later than the main CEX peak. In some examples, a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein has an acidic peak fraction of less than about 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% (including any range between these values) of total protein after 1 week at 37° C. as measured using CEX. In some examples, a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant thereof described herein has an acidic peak fraction of less than about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17% or 18% (including any range between these figures) of total protein after 2 weeks at 37° C. as measured using CEX. In some examples, a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant (or fragment) thereof described herein has an acidic peak fraction of less than about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26% or 27% (including any range between these figures) of total protein after 2 weeks at 37° C. as measured using CEX.

[0233] In some embodiments, the basic peak fraction of a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or 25 mg / mL of an anti-CD73 antibody described herein and / or a variant or mutant thereof (or fragment) is less than about 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46% (including any ranges between these values) of total protein after 1 week at 37° C., as measured using CEX. In some embodiments, the basic peak fraction of a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or 25 mg / mL of an anti-CD73 antibody described herein and / or a variant or mutant thereof (or fragment) is less than about 39%, 40%, 41%, 42%, 43%, 44%, 45%, or 46% (including any ranges between these values) of total protein after 2 weeks at 37° C., as measured using CEX. In some examples, a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant (or fragment) thereof described herein has a basic peak fraction of less than about 39%, 40%, 41%, 42%, 43%, 44%, 45% or 46% (including any range between these figures) of total protein after 4 weeks at 37° C. as measured using CEX.

[0234] In some examples, a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant (or fragment) thereof described herein has a major peak fraction of greater than or equal to about 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% or 46% (including any range between these figures) of total protein after 1 week at 37° C. as measured using CEX. In some examples, a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant (or fragment) thereof described herein has a basic peak fraction of greater than or equal to about 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% or 46% (including any range between these figures) of total protein after 2 weeks at 37° C. as measured using CEX. In some examples, a formulation comprising 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL or 25 mg / mL of an anti-CD73 antibody and / or variant or mutant (or fragment) thereof described herein has a basic peak fraction of greater than or equal to about 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% or 46% (including any range between these figures) of total protein after 4 weeks at 37° C. as measured using CEX.

[0235] Formulations to be used for in vivo administration must be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0236] Diagnostic and Imaging Methods Using Anti-CD73 Antibodies and Variants / Mutants Thereof 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 diseases and / or disorders associated with CD73 expression, aberrant expression, and / or activity. For example, the anti-CD73 antibodies and / or variants or mutants thereof (or fragments thereof) disclosed herein can be used in in situ, in vivo, ex vivo, and in vitro diagnostic or imaging assays. Methods for detecting CD73 expression include (a) measuring expression of a polypeptide in cells (e.g., tissues) or body fluids of an individual using one or more antibodies of the present disclosure, and (b) comparing the gene expression level to a standard gene expression level, where an increase or decrease in the measured gene expression level compared to the standard expression level indicates aberrant expression.

[0237] Further examples provided herein include a method for diagnosing a disease or disorder associated with expression or aberrant expression of CD73 in an animal (e.g., a mammal, such as a human). The method includes detecting a CD73 molecule in the mammal. In some examples, the diagnosis includes (a) administering an effective amount of a labeled anti-CD73 antibody and / or variant or mutant (or fragment) thereof to the mammal, (b) waiting a period of time after administration to allow the labeled anti-CD73 antibody and / or variant or mutant (or fragment) thereof to preferentially concentrate at sites in the subject where CD73 molecules are expressed (and to remove unbound labeled molecule to background levels), (c) determining a background level, and (d) detecting the labeled molecule in the subject, where detection of the labeled molecule above the background level indicates that the subject suffers from a particular disease or disorder associated with expression or aberrant expression of CD73. The background level can be determined by a variety of methods, including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

[0238] The anti-CD73 antibodies and / or variants or mutants (or fragments) thereof provided herein can be used to measure protein levels in biological samples using conventional immunohistochemistry known to those of skill in the art (see, e.g., Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and 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 ( 186 Re, 188 Re), praseodymium (142 Pr), Rhodium ( 105 Rh), ruthenium ( 97 Ru); luminol; and fluorescent labels (eg, fluorescein, rhodamine and biotin).

[0239] Techniques known in the art may be applied to the labeled antibodies (or fragments thereof) provided herein, including, but not limited to, the use of bifunctional conjugation agents (see, e.g., U.S. Patent Nos. 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, and 5,808,003).

[0240] Alternatively or additionally, the level of nucleic acid encoding CD73 or mRNA in the cells can be measured, for example by fluorescent in situ hybridization using a probe based on a nucleic acid corresponding to a nucleic acid encoding CD73 or a complementary sequence thereof (FISH, see WO 98 / 45479 published October 1998), Southern blotting, Northern blotting or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). Also, overexpression of CD73 can be studied by measuring shed antigens in biological fluids (e.g., serum) using, for example, antibody-based assays (see, e.g., U.S. Pat. No. 4,933,294, issued Jun. 12, 1990; WO 91 / 05264, issued Apr. 18, 1991; U.S. Pat. No. 5,401,638, issued Mar. 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the above assays, a variety of in vivo and ex vivo assays are available to those skilled in the art. For example, cells within a mammal can be exposed to an antibody that is optionally labeled with a detectable label (e.g., a radioisotope), and binding of the antibody to the cells can be assessed, for example, by external scanning for radioactivity or by analyzing a sample (e.g., a biopsy or other biological sample) obtained from a mammal previously exposed to the antibody.

[0241] Products and Kits In various embodiments, the invention relates to kits comprising the disclosed anti-CD73 antibodies, variants, mutants, or fragments thereof, and / or pharmaceutical compositions provided herein, and (a) at least one known agent for treating cancer, or (b) instructions for treating cancer.

[0242] In another embodiment, the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions provided herein are formulated with at least one agent known to treat cancer.

[0243] In yet another embodiment, the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions provided herein are packaged with at least one agent known to treat cancer.

[0244] In another embodiment, the known drug for treating cancer is a hormonal therapy agent. In yet another embodiment, the hormonal therapy agent is one or more selected from the group consisting of leuprolide, tamoxifen, raloxifene, megestrol, fulvestrant, triptorelin, medroxyprogesterone, letrozole, anastrozole, exemestane, bicalutamide, goserelin, histrelin, fluoxymesterone, estramustine, flutamide, toremifene, degarelix, nilutamide, abarelix, and testolactone, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

[0245] In another embodiment, the at least one known agent for treating cancer is a chemotherapeutic agent, hi yet another embodiment, the chemotherapeutic agent is one or more selected from the group consisting of an alkylating agent, an antimetabolite agent, an antitumor antibiotic agent, a mitotic inhibitor, an mTOR inhibitor, or other chemotherapeutic agent.

[0246] In another embodiment, the antitumor antibiotic agent is one or more selected from the group consisting of doxorubicin, mitoxantrone, bleomycin, daunorubicin, actinomycin D, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

[0247] In another embodiment, the antimetabolite agent is one or more selected from the group consisting of gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

[0248] In another embodiment, the alkylating agent is one or more selected from the group consisting of carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or a pharma- ceutically acceptable salt, hydrate, solvate, or polymorph thereof.

[0249] In another embodiment, the mitotic inhibitor is one or more selected from the group consisting of irinotecan, nogitecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etoposide, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

[0250] In another embodiment, the mTOR inhibitor is one or more selected from the group consisting of everolimus, siroliumus, and temsirolimus, or a pharma- ceutically acceptable salt, hydrate, solvate, or polymorph thereof.

[0251] In another embodiment, the kit further comprises instructions for combining a disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or a pharmaceutical composition provided herein with surgery. In yet another embodiment, the kit further comprises instructions for combining a disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or a pharmaceutical composition provided herein with surgery, the instructions indicating performing the surgery prior to administering the at least one compound. In yet another embodiment, the kit further comprises instructions for combining a disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or a pharmaceutical composition provided herein with surgery, the instructions indicating performing the surgery after administering the disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or a pharmaceutical composition provided herein. In yet another embodiment, the kit further comprises instructions for combining a disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or a pharmaceutical composition provided herein with surgery, the instructions indicating administering the disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or pharmaceutical composition followed by surgery, the instructions indicating administering the disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or pharmaceutical composition provided herein to achieve pre-operative tumor shrinkage. In yet another embodiment, the kit further comprises instructions for combining a disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or pharmaceutical composition provided herein with surgery, the instructions indicating administering the disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or pharmaceutical composition provided herein followed by surgery, the instructions indicating administering the disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or pharmaceutical composition provided herein at about the same time as administering the disclosed anti-CD73 antibody, variant, mutant, and / or fragment thereof, and / or pharmaceutical composition provided herein.

[0252] In another embodiment, the kit further comprises instructions for combining a disclosed anti-CD73 antibody, variant, mutant and / or fragment thereof, and / or pharmaceutical composition provided herein with radiation therapy. In yet another embodiment, the kit further comprises instructions for combining a disclosed anti-CD73 antibody, variant, mutant and / or fragment thereof, and / or pharmaceutical composition with radiation therapy, the instructions indicating administering radiation therapy prior to administering the disclosed anti-CD73 antibody, variant, mutant and / or fragment thereof, and / or pharmaceutical composition. In yet another embodiment, the kit further comprises instructions for combining a disclosed anti-CD73 antibody, variant, mutant and / or fragment thereof, and / or pharmaceutical composition with radiation therapy, the instructions indicating administering radiation therapy following the step of administering the disclosed anti-CD73 antibody, variant, mutant and / or fragment thereof, and / or pharmaceutical composition. In yet another embodiment, the kit further comprises instructions for combining the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions with radiation therapy, the instructions indicating administering the radiation therapy substantially simultaneously with administering the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions.

[0253] In another embodiment, the kit further comprises one or more dosage forms, each of which 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 agent. In yet another embodiment, the kit further comprises one or more dosage forms, each of which 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 agent, and each dose of the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions is formulated with at least one agent known to treat cancer. In yet another embodiment, the kit further comprises one or more dosage forms, each dosage containing a therapeutically effective amount of the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions, and at least one agent, and each dosage of the disclosed anti-CD73 antibodies, variants, mutants, and / or fragments thereof, and / or pharmaceutical compositions is packaged with at least one agent known to treat cancer.

[0254] In another example, the kit further comprises one or more dosage forms, each of which 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 agent (formulated for oral and / or intravenous administration). In yet another example, the kit further comprises one or more dosage forms, each of which 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 agent (formulated for oral administration). In yet another example, the kit further comprises one or more dosage forms, each of which 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 agent (formulated for intravenous administration).

[0255] The disclosed anti-CD73 antibodies, variants, mutants and / or fragments thereof, and / or pharmaceutical compositions can be readily provided as a kit showing two or more components (which may be active or inactive ingredients, carriers, diluents, etc.) and instructions for use by the patient or the person administering the drug to the patient to prepare the actual dosage form. Such a kit may have all necessary materials and components included therein, or the kit may include instructions for use to use or prepare materials or components that must be independently obtained by the patient or the person administering the drug to the patient. In another example, the kit may include optional components that aid in administration of a unit dose to the patient, such as vials for reconstituting a powder form, syringes for injection, customized IV delivery systems, inhalers, etc. The kit may also include instructions for preparing and administering the composition. The kit may be manufactured as a single-use unit dose for use in one subject, as a multi-use unit dose for use in a particular subject (wherein the potency of a single compound may vary at a fixed dose or as therapy progresses), or the kit may contain multi-dose doses suitable for administration to multiple patients ("bulk packaging"). The components of the kit may be contained in a cardboard box, blister pack, bottle, tube, or the like.

[0256] In another embodiment, the disclosed kits may be packaged according to a daily dosing regimen (e.g., packaged on cards, packaged with dosing cards, blister or blow molded plastic packaging, etc.). Such packaging can facilitate product sales and improve patient compliance with the drug regimen. Such packaging can reduce patient confusion. The disclosure features kits that further include instructions for use.

[0257] In another embodiment, the present invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the present invention. Associated with such containers may be a label in a form prescribed by a governmental agency regulating the manufacture, use, or sale of a drug or biological product, the label reflecting the authorization of the manufacturer, use, or sale agency for administration to humans.

[0258] In various embodiments, the disclosed kits may further include compounds and / or products that are packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, drug reseller, physician, compounding shop, or pharmacist can provide a kit that includes the disclosed compounds and / or products and another component to be delivered to a patient.

[0259] It is contemplated that the disclosed kits can be used in conjunction with the disclosed methods of manufacture, the disclosed uses or methods of treatment, and / or the disclosed compositions.

[0260] Kits for various purposes are further provided, for example, kits for isolating or detecting CD73 in a patient, optionally in combination with an article of manufacture. For isolating and purifying CD73, the kit may include an anti-CD73 antibody provided herein and / or a variant or mutant (or fragment) thereof coupled to beads (e.g., SEPHAROSE® beads). For example, a kit can be provided that includes an antibody (or fragment) for detecting and quantifying CD73 in vitro in ELISA or Western blotting. As with an article of manufacture, the kit can include a container and a label or package insert on or associated with the container. For example, the container holds a composition that includes at least one anti-CD73 antibody provided herein and / or a variant or mutant (or fragment) thereof. Other containers that can be included hold, for example, diluents and buffers, control antibodies. The label and package insert can provide a description of the composition and instructions for the intended in vitro or diagnostic use.

[0261] Another embodiment provided herein is an article of manufacture containing the disclosed materials for treating cancer. The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of a variety of materials, such as glass and plastic. Typically, the container holds a composition that effectively treats a condition and may have a sterile access port (e.g., 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 of the composition is an anti-CD73 antibody and / or variant or mutant (or fragment) thereof provided herein. The label or package insert indicates that the composition is used to treat a particular condition. The label or package insert further includes instructions for administering the antibody composition to a patient. Articles of manufacture and kits of combination therapies described herein are also contemplated.

[0262] Package insert refers to instructions typically included in the sales packaging of a therapeutic product that contain information about the indications, usage, dosage, administration, contraindications and / or warnings associated with the use of such therapeutic product. In one embodiment, the package insert indicates that the composition is used to treat cancer (e.g., head and neck cancer, lung cancer, or colorectal cancer).

[0263] Additionally, the article of manufacture may further include a second container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0264] Having now generally described aspects of the present invention, the following examples illustrate some other aspects of the present invention. Although aspects of the present invention have been described with reference to the following examples and corresponding text and drawings, it is not intended to limit the aspects of the present invention to this description. On the contrary, it is intended to cover all alternatives, modifications, and equivalents that are included within the spirit and scope of the present invention.

[0265] Working Example The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention, but provide those of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the compounds, compositions, products, devices and / or methods claimed herein. Every effort has been made to ensure accuracy of numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Also, unless otherwise indicated, parts are parts by weight, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric pressure. [Table 6] EXAMPLES

[0266] Generation of anti-CD73 antibodies and mutants A human Fab naive phage display library was panned with human CD73-ECD / His. After four rounds of panning with biotinylated huCD73-ECD / His coupled to streptavidin-coated magnetic Dynabeads® M-280 (Thermo Fisher Scientific #11205D), 52 possible clones with CD73 binding activity and immobilized CD73 enzyme blocking activity were selected and four sequences (N1-N4) were determined. The selected variable sequences were then cloned into the L234F, L235E, P331S mutants of the human IgG1 Fc backbone to form full-length antibodies used for in vitro functional assays (CD73 binding, CD73 enzyme blocking activity, antibody-mediated CD73 internalization). The N1, N2 and N4 leads were further cloned into human wild-type or C219S mutant IgG2 Fc backbone. Based on in vitro and in vivo efficacy studies (MDA-MB-231 xenograft model), N1 and N4 were selected for following affinity maturation and the wtIgG2 backbone was decided upon.

[0267] Antibody lead N1 and lead N4 were used in in vitro phage display-based affinity maturation experiments to generate additional clones with improved CD73 binding activity performance and CD73 enzyme blocking activity performance. A CDR-L1 / CDR-L3 / CDR-H3 (focused on three CDRs) nucleic acid library of lead N1 was generated via PCR, cloned into a phage display vector, and transformed into E. coli TG1 cells to generate a phage library. After three rounds of panning with biotinylated huCD73-ECD / His, 15 Fab clones with different sequences were screened via binding assay and enzyme blocking assay, and two Fabs (N1#2 and N1#9) were found to have higher efficacy. For affinity maturation of lead N4, a CDR-L3 / CDR-H1 / CDR-H3 nucleic acid library was first generated to generate a phage library. After three rounds of panning, six Fab clones (N4#1-N4#6) with different sequences were screened via binding assay and enzyme blocking assay. Then, the CDR-L1 / CDR-L2 / CDR-H2 nucleic acid library of clones N4#1, N4#4, N4#5 and N4#6 was prepared to prepare a phage library. After three rounds of panning, clones N4#4-3, N4#6-2, N4#6-3, N4#6-4 and N4#6-5 with better binding activity and enzyme blocking activity were selected.

[0268] After affinity maturation, N1#2, N1#9, N4#5, N4#6, N4#4-3, N4#6-2, N4#6-3, N4#6-4, and N4#6-5 were cloned into IgG2 Fc backbone to produce full-length antibodies, and in vitro efficacy tests (CD73 protein binding ELISA, CD73-expressing tumor cell binding, immobilization and cellular CD73 enzyme blockade, antibody-mediated CD73 internalization) and in vivo efficacy tests (NCI-H292 xenograft model) were performed to select clones N1#2, N1#9, N4#6-3, and N4#6-4. Based on sequence alignment and CMC stability, these four clones were modified to generate N1#2-P, N1#9-PH, N4#6-3-P, and N4#6-4-P. N1#9-PH has higher soluble CD73 enzyme blocking efficacy, 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 inhibition and have antitumor activity against MDA-MB-231 xenografts in vivo. N4#6-4-P can also induce CD73 downregulation and block CD73 enzyme activity in xenograft tumors, and has higher antitumor activity than other anti-CD73 antibodies in NCI-H292 xenograft model.

[0269] The amino acid sequence alignment of the light and heavy chain variable regions of anti-CD73 leads N1, N2 and N4 selected by panning of naive phage is shown in Figure 1A and Figure 1B, respectively. The amino acid sequence alignment of the light chain variable regions from affinity matured N1 and N4 mutants is shown in Figure 9, where the CDRs (complementarity determining regions) defined in Kabat are underlined and labeled in bold, and the amino acid sequence alignment of the heavy chain variable regions from affinity matured N1 and N4 mutants is shown in Figure 10, where the CDRs (complementarity determining regions) defined in Kabat are underlined and labeled in bold. These selected variable sequences were then cloned into the backbone of human IgG2 Fc to produce full-length antibodies. The amino acids of the light and heavy chain constant regions of the following human antibodies can be selected (not limiting the amino acid sequences of the light and heavy chain variable regions 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) Light chain constant region (SEQ ID NO:115) N1#2-P heavy chain (SEQ ID NO:116) N1#9-PH heavy chain (SEQ ID NO:117) N4#6-3-P_heavy chain (SEQ ID NO:118) N4#6-4-P heavy chain (SEQ ID NO:119).

[0270] Figure 14 shows an amino acid sequence alignment of the light chains of the top variants N1 and N4, where the CDRs (complementarity determining regions) as defined in Kabat are underlined and labeled in bold, and Figure 15 shows an amino acid sequence alignment of the heavy chain variable regions of the top variants N1 and N4, where the CDRs (complementarity determining regions) as defined in Kabat are underlined and labeled in bold. These sequences were then cloned into the backbone of human IgG2 Fc to produce full length antibodies. EXAMPLES

[0271] CD73 binding ability of selected antibodies An ELISA assay was performed to evaluate the binding of selected antibodies to recombinant human CD73-ECD / His protein. Goat anti-human IgG Fd (or Fc) antibodies were coated overnight at 4°C on 96-well EIA microplates at 30 ng per well. After blocking with 5% skim milk (in PBS), serially diluted antibodies were added and incubated at room temperature for 1 h. Unbound antibodies were removed and wells were washed 3 times with 1x PBST containing 0.05% Tween 20. Biotin-labeled CD73-ECD / His protein was added to the wells at 30 ng per well and incubated for an additional hour at room temperature. Unbound biotin-labeled CD73-ECD / His was washed 3 times with 1x PBST containing 0.05% Tween 20. Avidin-HRP was then added to the wells and incubated for 30 min at room temperature. Excess second antibody was removed by washing 3 times with 1x PBST containing 0.05% Tween 20. After washing, the plate was developed with TMB and the absorbance was read at 450 nm wavelength in a microplate reader.

[0272] The anti-CD73 antibodies selected by ELISA assay bound to recombinant human CD73 protein, and the binding data are shown in FIG. 2A.

[0273] 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 with 2% FBS) for 30 min at 4°C. Cells were washed with FACS buffer and binding was detected with FITC-labeled goat anti-human IgG (H+L) antibody (or PE-labeled goat anti-human IgG Fcγ) for another 30 min at 4°C. The binding of antibodies to the cell surface was measured by the mean fluorescence intensity (MFI) of the staining. Flow cytometry analysis was performed using a Cytomics FC500 or CytoFLEX flow cytometer (Beckman Coulter Inc.). Figure 2B and Figure 2C show that the selected anti-CD73 antibodies bound to human tumor cells MDA-MB-231 (human breast cancer) cells (Figure 2B) and NCI-H292 (human lung mucoepidermoid carcinoma) cells (Figure 2C) expressing CD73.

[0274] In these studies, anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively. These data demonstrate that all selected anti-CD73 antibodies are capable of binding to human CD73 recombinant protein and human tumor cells expressing CD73.

[0275] Figures 11A-11B further show the binding of N1 and N4 mutants to CD73. The binding of selected mutants to CD73-expressing MDA-MB-231 (Figure 11A) and NCI-H292 (Figure 11B) cells was tested by flow cytometry. HLX01 (anti-CD20) was used as a negative control. These data demonstrate that after affinity maturation, all N1 and N4 mutants can bind to human tumor cells expressing CD73. In these two cell lines, N4 mutants have better CD73 binding ability than N1 mutants. The ranking of total cell binding ability is N4#5, N4#6-4, N4#6-5>N4#6-2>N4#4-3, N4#6, N4#6-3, N1#9>N1#2.

[0276] Figures 16A-16C show the CD73 binding ability of N1 and N4 top mutants. Anti-CD73 antibodies were tested for binding to recombinant human CD73 protein by ELISA (Figure 16A), and to CD73-expressing human tumor cells MDA-MB-231 (Figure 16B) and NCI-H292 (Figure 16C) 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 a negative control. These data demonstrate that N4#6-3-P, N4#6-4-P, and N1#9-PH bind to recombinant CD73 and surface CD73 on MDA-MB-231 cells with comparable affinity. The binding of N1#9-PH to NCI-H292 cells expressing CD73 is slightly better than that of N4#6-3-P and N4#6-4-P to the same cells. EXAMPLES

[0277] Effect of anti-CD73 leads on soluble and cellular CD73 enzymatic activity The ability of anti-CD73 antibodies to inhibit the enzymatic activity of human recombinant CD73 protein and cell surface CD73 was tested. CD73-catalyzed hydrolysis of AMP to adenosine and inorganic phosphate was analyzed by measuring the ATP-dependent oxidation of fluorescein inhibited by AMP (CellTiter-Glo Assay, Promega). 15 ng of recombinant human CD73-ECD / His protein was incubated with serially diluted anti-CD73 antibodies, ATP (final concentration 100 μM) and AMP (final concentration 300 μM) in assay buffer (25 mM Tris, 5 mM MgCl2, pH 7.46) at 37°C. The reaction was mixed for 2 min with the same volume of CellTiter-Glo reagent in a white 96-well microplate. After 10 min of incubation, luminescence was measured.

[0278] Cells expressing CD73 (5E3-1E4 cells / well) were resuspended in medium and placed in 96-well microplates (or pre-seeded for 24 h). 3-fold (or 5-fold) serially diluted antibodies were added to each culture. After 15 min incubation at 37°C, AMP (final concentration 225 μM) was added and incubated for 4-20 h. After centrifugation, culture supernatants were collected and placed in 96-well microplates, ATP was added to a final concentration of 100 μM, and incubated for 2 min. The same volume of CellTiter-Glo reagent was added to each well and mixed for 2 min. Luminescence was measured after 10 min incubation.

[0279] Data showing the effect of anti-CD73 leads on soluble and cellular CD73 enzymatic activity are shown in Figures 3A and 3B, respectively. These data demonstrate that all selected antibodies are able to inhibit the enzymatic activity of both human soluble and surface CD73. In the soluble CD73 enzymatic activity assay, loss of inhibition was observed when IgG was in stoichiometric excess over sCD73. Such a so-called "hook effect" may result from monovalent antibody binding driven by Fab arms on the same IgG molecule competing for limited binding sites on the target antigen.

[0280] The ability of anti-CD73 leads N1, N2, N4 with different IgG isotypes to inhibit the enzymatic activity of cellular CD73 was tested. MDA-MB-231 cells were incubated with anti-CD73 antibodies. ATP, AMP and CellTiter-Glo reagent were added and luminescence was recorded. APCP was used as a positive control in the enzymatic activity assay. The effect of these anti-CD73 leads with different IgG Fc regions on cellular CD73 enzymatic activity is shown in Figure 6A (Lead N1), 6B (Lead N2) and 6C (Lead N4).

[0281] These data show that: Clone N1, wtIgG2 isotype, has better blocking activity than tmtIgG1 and mtIgG2 isotypes; Clone N2, tmtIgG1 isotype, has better blocking activity than wtIgG2 and mtIgG2 isotypes; N4, with wtIgG2 isotype, shows optimal blocking activity than tmtIgG1 and mtIgG2 isotypes; tmtIgG1 isotype has Fc site specific changes of L234F, L235E and P331S.

[0282] Figures 17A-17C further show the effect of N1 and N4 top mutants on soluble (Figure 17A) and cell surface (Figure 17B and Figure 17C) CD73 enzymatic activity. The ability of anti-CD73 antibodies to inhibit human recombinant CD73 protein and cell surface CD73 enzymatic activity was tested. Recombinant CD73 protein (Figure 17A), MDA-MB-231 (Figure 17B), NCI-H292 (Figure 17C) cells were incubated with anti-CD73 antibodies. ATP, AMP and CellTiter-Glo were added and luminescence was recorded. 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.

[0283] The data shown in Figures 17A to 17C demonstrate that the ability of N1#9-PH to inhibit soluble CD73 enzyme activity is superior to that of N4#6-3-P and N4#6-4P, but that N1#9-PH and N4#6-4-P exhibit equivalent blocking activity against cell surface CD73 enzyme activity. EXAMPLES

[0284] CD73 internalization mediated by anti-CD73 lead Tumor cells (5E4–1E5 cells / well) were suspended in culture medium and incubated with different concentrations of anti-CD73 antibody for 4 h. After removing the culture medium, the cells were suspended in FACS buffer (PBS containing 2% FBS) and incubated with mouse anti-human CD73 antibody (4G4) for 30 min at 4°C. 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 Fcγ antibody. Flow cytometry analysis was performed using a Cytomics FC500 or CytoFLEX flow cytometer (Beckman Coulter Inc.). Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively.

[0285] The data presented in FIG. 4 show that only N4-tmtIgG1, but not N1-tmtIgG1 and N2-tmtIgG1, is able to induce CD73 internalization.

[0286] Figure 7 further shows CD73 internalization mediated by anti-CD73 antibodies with different IgG Fc regions. After incubating cells with different IgG isotypes, 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 and negative controls, respectively. The data show that N4 (but not N1 and N2) can induce CD73 internalization, and there is no obvious difference between the different IgG isotypes.

[0287] Figure 13 further shows CD73 internalization mediated by N1 and N4 mutants. NCI-H292 cells were incubated with N1 and N4 mutants and 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 a negative control. The data show that after affinity maturation, N4 mutant can induce CD73 internalization and N1 mutant still cannot induce CD73 internalization.

[0288] Figures 18A and 18B further show CD73 internalization mediated by N1 and N4 top mutants. MDA-MB-231 (Figure 18A), NCI-H292 (Figure 18B) cells were incubated with anti-CD73 N1 and N4 mutants. After incubation, 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 a negative control. These data indicate that N4#6-3-P and N4#6-4-P can induce CD73 internalization in two types of cancer cells expressing CD73. EXAMPLES

[0289] Binding of selected antibodies with different IgG Fc regions to CD73 Binding of anti-CD73 antibodies to cell surface CD73 was tested by incubating MDA-MB-231 cells with serially diluted biotin-labeled anti-CD73 antibodies in FACS buffer (PBS with 2% FBS) for 30 min at 4 °C. Cells were washed with FACS buffer and binding was detected with streptavidin-FITC for 30 min at 4 °C. Flow cytometry analysis was performed using a Cytomics FC500 (Beckman Coulter Inc.).

[0290] The variable sequences of N1, N2 and N4 were cloned into the backbone of human wild-type or C219S mutant IgG2 Fc. The binding of different IgG isotypes, N1 (Figure 5A), N2 (Figure 5B) and N4 (Figure 5C), to human MDA-MB-231 cells expressing CD73 was tested by flow cytometry. Anti-CD73 ref-1 antibody and HLX01 (anti-CD20) were used as positive and negative controls, respectively.

[0291] 5A-5C show that there is no difference between the different IgG isotypes of N1 in CD73 binding ability. The data further show that clone N2 (tmtIgG1 isotype) has superior CD73 binding activity to the wtIgG2 and mtIgG2 isotypes, and clone N4 (tmtIgG1 isotype) has superior CD73 binding activity to the wtIgG2 and mtIgG2 isotypes. EXAMPLES

[0292] Tumor growth inhibitory activity of anti-CD73 leads in MDA-MB-231 (human triple-negative breast cancer) xenograft mouse models We used immune-compromised NOD / SCID (non-obese diabetic / severe combined immunodeficiency) mice to study the in vivo activity of anti-human CD73 antibodies in a xenograft mouse model. A total of 1E7 human triple-negative breast cancer MDA-MB-231 cells expressing CD73 in 100 μL PBS were mixed (1:1 ratio) with 100 μL Matrigel (Corning, CA, USA) and implanted subcutaneously into both flanks of female NOD / SCID mice (Biolasco, Taipei, Taiwan). Tumors with sizes between 150 and 300 mm were cultured in a xenograft mouse model. 3 When the tumor size reached 14 days after tumor inoculation, 5 mg / kg of anti-CD73 antibody or 10 mL / kg of placebo was administered intraperitoneally twice a week or at the specified time. The tumors were observed and measured until the 45th day. The tumor volume was calculated as TV (tumor volume) = (length × width). 2 ) / 2.

[0293] Tumor growth curves are shown in Figure 8A. Individual tumor volumes at day 45 are shown in Figure 8B. All data points are mean ± SEM. The data demonstrate that, except for the tmtIgG1 isotype of clones N1, N2 and N4, all anti-CD73 antibodies have anti-tumor activity (TGI > 50%) in the MDA-MB-231 xenograft model, with N1-wtIgG2, N1-mtIgG2, N2-wtIgG2, N2-mtIgG2 almost completely inhibiting tumor growth.

[0294] Figure 20 further shows the tumor growth inhibitory activity of N1 and N4 Top mutants in MDA-MB-231 (human triple-negative breast cancer) xenograft mouse model. Mice (n=5 mice / group) were subcutaneously implanted with MDA-MB-231 cells. Seven days after tumor inoculation, the size of the implanted tumors was approximately 100 mm 3 Upon reaching CD73, the first dose of test article was administered. Mice were treated intraperitoneally with 10 mg / kg and 2 mg / kg antibody twice weekly for 5-6 weeks. All data points are mean ± SEM. These data demonstrate that all N1 and N4 top variants have antitumor activity against MDA-MB-231 xenografts in vivo, with N4#6-3-P, N4#6-4-P and N1#9-PH having superior antitumor activity to the anti-CD73 control antibody. EXAMPLES

[0295] Effect of N1 and N4 mutants on immobilized CD73 enzyme activity The ability of N1 and N4 mutants to inhibit immobilized CD73 enzymatic activity was tested. Goat anti-human IgG Fd antibody was coated overnight at 4°C with high affinity at 300ng per well onto white 96-well microplates. 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 wells were washed 3 times with 1x PBST containing 0.05% Tween 20. 25μL of 100ng / mL CD73-ECD / His protein in assay buffer (25mM Tris, 5mM MgCl2, pH 7.46) was added to the wells and incubated for an additional hour at room temperature. 25μL of a mixture of 66μM ATP and 200μM AMP was added and incubated for 9 minutes at 37°C. The same volume of CellTiter-Glo reagent was added to the reaction mixture (to a white 96-well microplate) and the contents were mixed for 2min. Luminescence was measured after 10 min incubation. Anti-CD73 ref-1 and anti-CD73 ref-2 antibodies were used as positive controls, and HLX01 (anti-CD20) was used as a negative control.

[0296] The effect of N1 and N4 mutants on immobilized CD73 enzyme activity is shown in FIG. 12. These data show that after affinity maturation, all N1 and N4 mutants can inhibit CD73 enzyme activity. The CD73 enzyme blocking activity of N1 mutants is superior to N4 mutants and anti-CD73 ref-2 antibody, but is comparable to anti-CD73 ref-1 antibody. For the activity of blocking immobilized CD73 enzyme, N1#2, N1#9>N4#6-4, N4#6-5>N4#4-3, N4#6-2>N4#6-3, N4#5>N4#6. EXAMPLES

[0297] Reversal of the inhibitory effect of AMP on T cell activity by N1 and N4 top mutants CD73 catalyzes adenosine monophosphate (AMP) to generate adenosine extracellularly. Adenosine inhibits immune responses (including immune responses of T cells, NK cells, and dendritic cells) by activating A2aR and A2Q receptors. In addition to CD73 enzyme inhibition and internalization, we further detected the functional effect of CD73 inhibition by anti-CD73 antibody on T cell proliferation.

[0298] Human T cells were isolated from peripheral blood mononuclear cells (PBMCs) using the MagniSort Human T Cell Enrichment Kit (eBioscience, Inc.) and suspended in complete medium (RPMI-1640 with 10% FBS) containing 50 IU / mL recombinant human IL-2 (eBioscience, Inc.). A volume of 100 μL of T cells (5E4 cells / reaction) was incubated with Dynabeads® human T-activating factor CD3 / CD28 (Thermo Fisher Scientific, Inc.) for 1 h. The CD3 / CD28 beads (Agilent Scientific) were mixed with AMP (1 mM) at a ratio of 1:0.5 to cell beads. AMP (1 mM) and serially diluted antibodies were added to each reaction in a volume of 100 μL. Reactions without activation beads (unstimulated group), with only CD3 / CD28 beads (stimulated group) or with only 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. Cells were cultured at 37°C for 4 days. On the fourth day, cells were harvested, washed and resuspended in fresh RPMI-1640 medium. T cell proliferation was analyzed by CellTiter-Glo assay. Culture supernatants were collected and used for cytokine measurements. Human IFN-γ ELISA MAX® IFN-γ levels were measured using a Deluxe kit (BioLegend, Inc.).

[0299] The reversal of the inhibitory effect of AMP on T cell activity by the N1 and N4 top mutants is shown in Figures 19A and 19B. +After T cell proliferation, CellTiter-Glo assay was performed (Figure 19A) and IFN-γ secretion was measured using human IFN-γ ELISA MAX® 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 negative control. The data shown in Figures 19A and 19B demonstrate that N1 and N4 top mutants can enhance T cell proliferation and IFN-γ secretion in a dose-dependent manner. The data further show that N4#6-3-P, N4#6-4-P and N1#9-PH can alleviate AMP-mediated T cell inhibition. EXAMPLES

[0300] Tumor growth inhibitory activity of N1 and N4 top mutants in NCI-H292 (human pulmonary mucoepidermoid carcinoma) xenograft mouse model The in vivo activity of anti-human CD73 antibodies was studied in a xenograft mouse model using immune-compromised BALB / c nude mice. A total of 5E6 NCI-H292 cells in 100 μL PBS were mixed (1:1 ratio) with 100 μL Matrigel (Corning, CA, USA) and implanted subcutaneously into both flanks of male BALB / c nude mice (Biolasco, Taipei, Taiwan). Tumors with sizes ranging from 150 to 300 mm were cultured in a 1:1 ratio. 3 When the tumor volume reached 100 mm Hg, anti-CD73 antibody or placebo was administered intraperitoneally twice a week for 3 weeks. The tumors were observed and measured twice a week. The tumor volume was calculated as TV (tumor volume) = (length × width). 2 ) / 2.

[0301] Figure 21 shows the tumor growth inhibitory activity of N1 and N4 top mutants in NCI-H292 (human lung mucoepidermoid carcinoma) xenograft mouse model. Mice (n=5 mice / group) were subcutaneously implanted with NCI-H292 cells. The first dose of test article was administered 3 days after tumor inoculation. Mice were treated intraperitoneally with 50, 10 and 2 mg / kg antibody twice a week for 3 weeks. All data points are mean ± SEM. The data demonstrate that N4#6-4-P has higher antitumor activity than other anti-CD73 antibodies in NCI-H292 xenograft model. EXAMPLES

[0302] Effects of N1 and N4 top mutants on cellular CD73 enzyme activity in NCI-H292 xenograft tumors A total of 5E6 NCI-H292 cells in 100 μL PBS were mixed (1:1 ratio) with 100 μL Matrigel (Corning, CA, USA) and implanted subcutaneously into both flanks of male BALB / c nude mice (Biolasco, Taipei, Taiwan) 4 days before antibody administration. Mice were treated intraperitoneally with 30 mg / kg antibody, APCP and placebo on day 0, and tumors were harvested on days 1, 3 and 7. Tumor cells (2E4) were resuspended in assay buffer (25 mM Tris, 5 mM MgCl2, pH 7.46) and treated with ATP (final concentration 100 μM) and AMP (final concentration 300 μM) for 45 min at 37°C. An equal volume of CellTiter-Glo reagent was added to the reaction mixture (in a white 96-well microplate) and the contents were mixed for 2 min. Luminescence was measured after a 10 min incubation.

[0303] Figure 22 shows the effect of N1 and N4 top mutants on cellular CD73 enzyme activity in NCI-H292 xenograft tumors. The ability of N1 and N4 top mutants to inhibit CD73 enzyme activity in NCI-H292 xenograft model was tested. Mice (n=4 mice / group) were implanted with NCI-H292 cells subcutaneously 4 days before treatment with antibody. Antibody, APCP and placebo were injected intraperitoneally on day 0. Tumors were excised on days 1, 3 and 7 after antibody administration. CD73 enzyme activity in tumors was measured by CellTiter-Glo assay.

[0304] The data demonstrate that N1 and N4 top mutants can inhibit CD73 enzyme activity in the NCI-H292 xenograft model, and that N4#6-3-P and N4#6-4-P are superior to N1#9-PH and an anti-CD73 control antibody in blocking CD73 enzyme in tumors. EXAMPLES

[0305] Effects of N1 and N4 top mutants on CD73 expression and cellular CD73 enzyme activity in MDA-MB-231 xenograft tumors A total of 1E7 MDA-MB-231 cells in 100 μL PBS were mixed (1:1 ratio) with 100 μL Matrigel (Corning, CA, USA) and implanted subcutaneously into both flanks of female NOD / SCID mice (Biolasco, Taipei, Taiwan) 7 days before antibody administration. Mice were treated intraperitoneally with 2 mg / kg antibody and 10 mL / kg placebo on day 0. Tumors were harvested on days 1, 3, and 7. To measure surface CD73 expression levels, 5E4 tumor cells were resuspended in FACS buffer (PBS with 2% FBS) and treated with anti-human and anti-mouse FcR blocking reagents for 30 min at 4°C. After centrifugation, cells were incubated with mouse anti-human CD73 antibody (4G4) for 30 min at 4°C. Cells were washed with FACS buffer and incubated with Alexa 488-labeled goat anti-mouse IgG Fcγ antibody for an additional 30 min at 4°C. Flow cytometric analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter Inc.). A total of 2E4 tumor cells were resuspended in assay buffer (25 mM Tris, 5 mM MgCl2, pH 7.46) for measuring CD73 enzyme activity. The cell suspension was treated with ATP (final concentration 100 μM) and AMP (final concentration 300 μM) for 45 min at 37°C. An equal volume of CellTiter-Glo reagent was added to the reaction mixture (in a white 96-well microplate) and mixed for 2 min. Luminescence was measured after 10 min incubation.

[0306] Figures 23A and 23B show the effect of N1 and N4 top mutants on CD73 expression and cellular CD73 enzyme activity in MDA-MB-231 xenograft tumors. CD73 expression was measured by mean fluorescence intensity (MFI) of staining (Figure 23A), and CD73 enzyme activity in tumors was measured by CellTiter-Glo assay (Figure 23B). The data demonstrate that N4#6-4-P can downregulate surface CD73 expression and inhibit CD73 enzyme activity in MDA-MB-231 xenograft mouse model compared to placebo group. EXAMPLES

[0307] Cross-binding of N1 and N4 top variants to mouse and monkey CD73-expressing cells Anti-CD73 antibody binding was assessed by incubating 4T1 or LLC-MK2 cells (1E5 cells / test) with serially diluted antibodies in FACS buffer (PBS with 2% FBS) for 30 min at 4°C. Cells were washed with FACS buffer and binding was detected with FITC-labeled goat anti-human IgG (H+L) antibody for an additional 30 min at 4°C. Antibody binding to the cell surface was measured by the mean fluorescence intensity (MFI) of the staining. Flow cytometric analysis was performed using a Cytomics FC500 or CytoFLEX flow cytometer (Beckman Coulter Inc.).

[0308] Figures 24A and 24B show cross-binding of N1 and N4 top variants to cells expressing mouse and monkey CD73. Anti-CD73 antibodies were tested for binding to mouse breast cancer 4T1 cells (Figure 24A) and monkey kidney epithelial LLC-MK2 cells (Figure 24B) expressing CD73 by flow cytometry. HLX01 was used as a negative control. These data show that N1 and N4 top variants have cross-reactivity with cells expressing monkey CD73, and N1 and N4 top variants cannot cross-bind to mouse CD73.

[0309] The foregoing examples are merely possible examples of embodiments used for illustrative purposes and provided to clearly understand the principles of the present invention, and are not intended to limit the scope of the present invention in any manner. Many changes and modifications can be made to one or more of the above examples without fundamentally departing from the spirit and principles of the present invention. Such modifications and variations are within the scope of the present invention and are protected by the appended claims.

Claims

1. Anti-CD73 antibodies, including: (i) a light chain (LC) variable domain sequence comprising a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:3, b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:13, and c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:19; and a heavy chain variable domain (VH) sequence comprising a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

41. (ii) a light chain variable domain (VL) sequence comprising a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 3, b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, and a heavy chain variable domain (VH) sequence comprising a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41; (iii) a light chain variable domain (VL) sequence comprising a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 3, b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain variable domain (VH) sequence comprising a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41; (iv) a light chain variable domain (VL) sequence comprising a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 3, b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23, and a heavy chain variable domain (VH) sequence comprising a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 29, b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 44; (v) a light chain variable domain (VL) sequence comprising a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 3, b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain variable domain (VH) sequence comprising a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; (vi) a light chain variable domain (VL) sequence comprising a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6, b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain variable domain (VH) sequence comprising a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 41; (vii) a light chain variable domain (VL) sequence comprising a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, and c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain variable domain (VH) sequence comprising a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; (viii) a light chain variable domain (VL) sequence comprising a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain variable domain (VH) sequence comprising a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45; (ix) a light chain (LC) variable domain sequence comprising a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:15, and c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:24, and a heavy chain variable domain (VH) sequence comprising a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:45; or (x) a light chain (LC) variable domain sequence comprising: a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:9; b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:16; and c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:24; and a heavy chain variable domain (VH) sequence comprising: a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:27; b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:37; and c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

45.

2. (i) the LC of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 81, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 99; (ii) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 84, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 102; (iii) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 85, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 103; (iv) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 86, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 104; (v) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 87, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 105; (vi) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 88, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 106; (vii) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 89, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 107; (viii) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 90, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 108; (ix) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 91, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 109; (x) the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 92, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 110; (xi) the LC of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 95, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 108; or (xii) the LC of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 96, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 109; 2. The anti-CD73 antibody of claim 1.

3. 2. The anti-CD73 antibody of claim 1, comprising a light chain variable domain (VL) sequence comprising: a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6, b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain variable domain (VH) sequence comprising: a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:

41.

4. The anti-CD73 antibody of claim 1 or 2, wherein the VL of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 88, and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO:

106.

5. 2. The anti-CD73 antibody of claim 1, comprising a light chain (LC) variable domain sequence comprising: a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 9, b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain variable domain (VH) sequence comprising: a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:

45.

6. The anti-CD73 antibody of claim 1 or 2, wherein the LC of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO: 96 and the VH of the anti-CD73 antibody comprises the amino acid sequence of SEQ ID NO:

109.

7. 3. The anti-CD73 antibody of claim 1 or 2, comprising an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab')2, single chain Fv (scFv), Fv fragment, bibody and linear antibody.

8. The anti-CD73 antibody of claim 1 or 2, wherein the anti-CD73 antibody is a multispecific antibody.

9. An anti-CD73 antibody as described in claim 1 or 2, conjugated to a therapeutic agent or label.

10. An anti-CD73 antibody described in claim 1 or 2, conjugated to a radioisotope, a fluorescent dye, or an enzyme.

11. An isolated nucleic acid molecule encoding an anti-CD73 antibody according to any one of claims 1 to 10.

12. An expression vector comprising the nucleic acid molecule of claim 11.

13. A cell comprising the expression vector of claim 12.

14. 14. A method for producing an anti-CD73 antibody according to any one of claims 1 to 8, comprising culturing a cell according to claim 13 and recovering the anti-CD73 antibody from the cell culture.

15. A composition comprising the anti-CD73 antibody of any one of claims 1 to 8 and a pharma- ceutically acceptable carrier.

16. An agent comprising an anti-CD73 antibody described in any one of claims 1 to 8 for use in a method for detecting CD73 protein in a patient-derived sample by detecting anti-CD73 antibody bound to CD37 protein.

17. The method of claim 16, wherein the anti-CD73 antibody is used in an immunohistochemical assay (IHC) or an ELISA assay.

18. 16. The composition of claim 15 for use in treating cancer in a subject.

19. The composition for use of claim 18, wherein the cancer is selected from the group consisting of melanoma, NSCLC, head and neck cancer, urothelial carcinoma, triple negative breast cancer (TNBC), gastric cancer, classical Hodgkin lymphoma (cHL), non-Hodgkin lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer, esophageal cancer, nasopharyngeal carcinoma (NPC), biliary tract cancer, colorectal cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, bladder cancer, pancreatic cancer, salivary gland cancer and CD73-expressing malignant tumors.

20. 16. The composition of claim 15 for use in treating fibrosis in a subject.

21. 21. The composition for use of claim 18, 19 or 20, wherein the subject is further administered a therapeutic agent selected from the group consisting of anti-tumor agents, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents and immunotherapeutic agents.

22. The composition for use according to claim 18, 19 or 20, wherein the subject is further administered radiation therapy and / or surgery.

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