Anti-CD73 Antibody

Abstract

The present invention relates to anti-CD73 antibodies and methods of using them in the treatment of diseases and conditions associated with CD73 activity, such as cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 896,908, filed September 6, 2019, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The electronic copy of the sequence listing, created on September 1, 2020, is named 022675_WO055_SL.txt and is 30355 bytes in size. [Background technology]

[0003] Tumor cells are surrounded by a complex microenvironment (tumor microenvironment or TME) that influences each stage of tumorigenesis. In the TME, increased concentrations of immune modulators such as adenosine help tumor cells overcome the host anti-tumor immune response.

[0004] Adenosine is a CD4 + and CD8 + Adenosine binds to four different receptors (ARs) expressed on various immune cells, such as T cells and natural killer (NK) cells. Each of these purinergic G protein-coupled receptors, A1R, A2AR, A2BR, and A3R, exhibits different properties and cellular and tissue distribution. Adenosine binding to ARs suppresses the antitumor response of T cells and NK cells, and also promotes the development and activation of immune suppressive cells, such as regulatory T cells (Tregs) and myeloid-derived immune suppressor cells (MDSCs), thus promoting cancer progression. In addition to the immunomodulatory effects of adenosine receptors, their signaling can also directly affect the survival and proliferation of cancer cells.

[0005] The accumulation of adenosine in the TME is mediated by the cell surface enzymes CD73 and CD39, which act in a pathway that converts extracellular ATP to adenosine. CD73, also known as 5'-NT, is an extracellular enzyme consisting of two 65 kD subunits linked into a homodimer by an α-helical linker. When overexpressed by many cancer cells, CD73 mediates the hydrolysis of 5'-AMP to adenosine, especially under tumor hypoxic conditions. Studies have shown that high expression of CD73 leads to poor prognosis in various cancers, such as triple-negative breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, and melanoma. Inhibition of CD73 may be a powerful approach to cancer therapy. Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure relates to novel recombinant antibodies targeting CD73, as well as pharmaceutical compositions comprising one or more of these antibodies, and uses of the antibodies and pharmaceutical compositions to treat cancer. It is contemplated that, compared to currently available treatments for such cancers, including antibody treatments, the antibodies and compositions described herein may provide superior clinical responses, either alone or in combination with another cancer therapeutic. [Means for solving the problem]

[0007] In some aspects, the present disclosure provides a method for producing a method for treating a cancer cell comprising: a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 9 and 41 and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 13 and 42; b) a HC comprising the amino acid sequences of SEQ ID NOs: 10 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 14 and 42; c) a HC comprising the amino acid sequences of SEQ ID NOs: 11 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 15 and 42; or d) HC comprising the amino acid sequences of SEQ ID NOs: 12 and 41 and LC comprising the amino acid sequences of SEQ ID NOs: 16 and 42 The present invention provides an anti-CD73 antibody, or an antigen-binding portion thereof, that binds to the same epitope of human CD73 as an antibody comprising the compound comprising:

[0008] In a particular embodiment, the heavy chain of the antibody comprises: i) heavy chain complementarity determining regions (H-CDRs) 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 17 to 19; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:9; iii) a VH comprising the amino acid sequence of SEQ ID NO: 9; or iv) Heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 9 and 41 Includes; the light chain of the antibody i) light chain complementarity determining regions (L-CDRs) 1 to 3 each containing the amino acid sequences of SEQ ID NOs: 20 to 22; ii) a light chain variable domain (VL) comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13; iii) a VL comprising the amino acid sequence of SEQ ID NO: 13; or iv) Light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 13 and 42 Includes.

[0009] In a particular embodiment, the heavy chain of the antibody comprises: i) heavy chain complementarity determining regions (H-CDRs) 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 23 to 25; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 10; iii) a VH comprising the amino acid sequence of SEQ ID NO: 10; or iv) Heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 10 and 41 Includes; the light chain of the antibody i) light chain complementarity determining regions (L-CDRs) 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 26 to 28; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 14; iii) a VL comprising the amino acid sequence of SEQ ID NO: 14; or iv) Light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 14 and 42 Includes.

[0010] In a particular embodiment, the heavy chain of the antibody comprises: i) heavy chain complementarity determining regions (H-CDRs) 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 29 to 31; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:11; iii) a VH comprising the amino acid sequence of SEQ ID NO: 11; or iv) Heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 11 and 41 Includes; the light chain of the antibody i) light chain complementarity determining regions (L-CDRs) 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 32 to 34; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 15; iii) a VL comprising the amino acid sequence of SEQ ID NO: 15; or iv) Light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 15 and 42 Includes.

[0011] In a particular embodiment, the heavy chain of the antibody comprises: i) heavy chain complementarity determining regions (H-CDRs) 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 35 to 37; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; iii) a VH comprising the amino acid sequence of SEQ ID NO: 12; or iv) Heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 12 and 41 Includes; the light chain of the antibody i) light chain complementarity determining regions (L-CDRs) 1 to 3 each comprising the amino acid sequences of SEQ ID NOs: 38 to 40; ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16; iii) a VL comprising the amino acid sequence of SEQ ID NO: 16; or iv) Light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 16 and 42 Includes.

[0012] In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) SEQ ID NOs: 17 to 22, respectively; b) SEQ ID NOs: 23 to 28, respectively; c) SEQ ID NOs: 29 to 34, respectively; or d) SEQ ID NOs: 35 to 40, respectively The present invention provides an anti-CD73 antibody or an antigen-binding portion thereof, which comprises the amino acid sequences of H-CDR1 to 3 and L-CDR1 to 3 of the above.

[0013] In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) SEQ ID NOs: 9 and 13, respectively; b) SEQ ID NOs: 10 and 14, respectively; c) SEQ ID NOs: 11 and 15, respectively; or d) SEQ ID NOs: 12 and 16, respectively The present invention provides an anti-CD73 antibody or antigen-binding portion thereof, comprising a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence that are at least 90% identical to the amino acid sequence of

[0014] In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) SEQ ID NOs: 9 and 13, respectively; b) SEQ ID NOs: 10 and 14, respectively; c) SEQ ID NOs: 11 and 15, respectively; or d) SEQ ID NOs: 12 and 16, respectively and a light chain variable domain comprising the amino acid sequence of:

[0015] In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 9 and 41 and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 13 and 42; b) a HC comprising the amino acid sequences of SEQ ID NOs: 10 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 14 and 42; c) a HC comprising the amino acid sequences of SEQ ID NOs: 11 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 15 and 42; or d) HC comprising the amino acid sequences of SEQ ID NOs: 12 and 41 and LC comprising the amino acid sequences of SEQ ID NOs: 16 and 42 The present invention provides an anti-CD73 antibody comprising:

[0016] In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) amino acid residues R73, R109, and D168 of SEQ ID NO:43; b) amino acid residue R109 of SEQ ID NO: 43; or c) amino acid residues I301, S302, and H304 of SEQ ID NO: 43 The present invention provides an anti-CD73 antibody, or an antigen-binding portion thereof, that binds to an epitope on human CD73, comprising: In some embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) amino acid residues 27-31, 61-75, and 161-170 of SEQ ID NO:43; b) amino acid residues 61 to 70 and 161 to 170 of SEQ ID NO: 43; or c) amino acid residues 27 to 31, 266 to 270, and 291 to 305 of SEQ ID NO: 43 The present invention provides an anti-CD73 antibody, or an antigen-binding portion thereof, that binds to an epitope on human CD73, comprising:

[0017] In certain embodiments, the anti-CD73 antibodies or antigen-binding portions described herein are a) inhibits the activity of soluble CD73 in vitro; b) inhibiting the activity of CD73 on Calu-6 cells in vitro; c) inhibiting the activity of CD73 on H292 cells in vitro; d) specifically binds to human and cynomolgus CD73 expressed on CHO-S cells; e) K of 1 nM or less as measured by SPR D binds to the ECD of human CD73; f) K of 0.7 nM or less as measured by SPR D binds to the ECD of cynomolgus monkey CD73; g) does not bind to the same epitope on CD73 as oleclumab, CPX006, and / or 11E1; h) binds to an epitope on the CD73 homodimer in a manner that yields a 1:1 complex; i) inhibits soluble CD73 activity more effectively than oleculab in vitro; j) inhibits CD73 activity on Calu-6, H292, and Cynom-K1 cells in vitro; k) inhibits CD73 activity on Calu-6, NCI-H1775, KYSE-30, and Capan-2 cells in vitro; l) inhibiting the proliferation of MDA-MB-231 and MDA-MB-468 cells in vitro; m) In vitro primary CD4 + and CD8 + T cells and CD19 + Inhibits CD73 activity on B cells; n) In vitro CD4 + restores T-cell proliferation; o) In vitro CD4 + and CD8 + Activating T cells; p) in combination with anti-PD-1 antibodies, restores T-cell proliferation in a one-way mixed lymphocyte reaction (MLR) in the presence of AMPs; q) in combination with anti-PD-1 antibodies in a unidirectional MLR to enhance T cell activation in the presence of AMPs; r) does not stimulate B cell activation in vitro; s) does not reduce the levels of CD73 in H292 cells in vitro by more than 25%; t) inhibit CD73 activity in tumors harvested from PBMC-humanized mice implanted with A375 cells; u) inhibiting in vivo tumor growth in NOD-scid mice implanted with MDA-MB-231 cells; v) inhibiting in vivo tumor growth in PBMC-humanized mice engrafted with Calu-6 cells; and w) Inhibits in vivo tumor growth in PBMC-humanized mice implanted with A375 cells The present invention has at least one characteristic selected from the following: In specific embodiments, the antibody or antigen-binding portion has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all 23 of the above properties.

[0018] In certain embodiments, the anti-CD73 antibodies or antigen-binding portions described herein are IgG, such as IgG1. C For example, the antibody may be an IgG1 and may include a mutation in one or more of the heavy chain amino acid positions 234 and 235, numbered according to the IMGT® numbering scheme. In a specific embodiment, one or both of the amino acid residues at positions 234 and 235 are mutated from Leu to Ala.

[0019] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-CD73 antibody or antigen-binding portion thereof described herein and a pharma- ceutical acceptable excipient. In certain embodiments, the pharmaceutical composition may further comprise one or more of an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, and a CD73 pathway inhibitor.

[0020] In some embodiments, the disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof, or a nucleotide sequence encoding the light chain or antigen-binding portion thereof, of an anti-CD73 antibody or antigen-binding portion described herein, or both. In certain embodiments, the nucleic acid molecule may comprise the nucleotide sequence of any one of SEQ ID NOs: 1-8.

[0021] In some embodiments, the disclosure provides a vector comprising an isolated nucleic acid molecule described herein, the vector further comprising an expression control sequence.

[0022] In some embodiments, the disclosure provides a host cell comprising a nucleotide sequence encoding the heavy chain, or antigen-binding portion thereof, and a nucleotide sequence encoding the light chain, or antigen-binding portion thereof, of an anti-CD73 antibody or antigen-binding portion described herein.

[0023] In some embodiments, the disclosure provides methods for making an anti-CD73 antibody, or antigen-binding portion thereof, comprising providing a host cell as described herein, culturing the host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion.

[0024] In some embodiments, the disclosure provides bispecific binding molecules comprising antigen-binding portions of one or two distinct anti-CD73 antibodies described herein.

[0025] In some embodiments, the disclosure provides a method for reducing CD73 activity in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-CD73 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule described herein.

[0026] In some embodiments, the present disclosure provides a method for detecting CD4 + Methods for increasing T cell proliferation are provided, comprising administering to a patient a therapeutically effective amount of an anti-CD73 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule described herein.

[0027] In some embodiments, the disclosure provides a method for stimulating the immune system of a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-CD73 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule described herein.

[0028] In some embodiments, the disclosure provides a method for treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of an anti-CD73 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule described herein. In certain embodiments, the cancer originates in a tissue selected from the group consisting of skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas. In certain embodiments, the cancer is melanoma, head and neck cancer, breast cancer, bladder cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, colorectal cancer, cholangiocarcinoma, thyroid cancer, or testicular cancer.

[0029] In certain embodiments, the treatments described herein further comprise administering to the patient an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, a CD73 pathway inhibitor, or radiation therapy.

[0030] In some aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) reducing CD73 activity in patients; b) The patient's CD4 + Increase T-cell proliferation; c) stimulating the patient's immune system; or d) Treating cancer in patients The present invention provides the use of an anti-CD73 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule described herein for the manufacture of a medicament for treating a patient with atopic dermatitis, comprising:

[0031] In some aspects, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) reducing CD73 activity in patients; b) The patient's CD4 + Increase T-cell proliferation; c) stimulating the patient's immune system; or d) Treating cancer in patients The present invention provides an anti-CD73 antibody or antigen-binding portion, pharmaceutical composition, or bispecific binding molecule described herein for use in treating a patient with a mycobacterial inflammatory disease.

[0032] Other features, objects, and advantages of the present invention will become apparent in the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0033] [Figure 1]

[0023] Figure 1 is a graph showing the activity of soluble CD73 following treatment with the indicated anti-CD73 antibodies. Data were normalized to untreated controls and are presented as the mean ± SEM. [Diagram 2] FIG. 1 is a pair of graphs showing activity of CD73 expressed on Calu-6 cells (top) or H292 cells (bottom) after treatment with the indicated anti-CD73 antibodies. Data were normalized to untreated controls and are presented as mean±SEM. [Diagram 3] 1 is a set of graphs showing binding of the indicated anti-CD73 antibodies and a reference antibody (oleculab) analog to human and cynomolgus CD73 expressed on CHO-S cells. Mock-transfected CHO-S cells were used as a negative control. "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Data are presented as mean ± SEM. [Figure 4] FIG. 1 is a schematic diagram showing the competition patterns and epitope bins identified for the indicated anti-CD73 antibodies. Connecting black lines indicate cross-blocking activity. Connecting dotted lines indicate antibodies that block in only one direction. Antibodies are grouped according to their competition patterns with other anti-CD73 antibodies. [Diagram 5]FIG. 1 shows the binding epitopes of antibodies 21127 (panel A), 21163 (panel B), 21046 (panel C), 11E1 analog (panel D), oleculab analog (panel E), and CPX006 analog (panel F) mapped onto the crystal structure of the open CD73 dimer (4H2G). The structures are shown as surface representations with the N-terminal domains in light grey and the C-terminal domains in grey. Adenosines are shown as white sticks (indicated by arrows). Linear epitopes are shown in dark grey with contact residues in black. [Figure 6A] Figure 1 shows a series of SEC-MALS profiles of antibodies 21127, 21163, 21046, and 11E1, oleculab, and CPX006 analog mixed with CD73 in the ratio mAb:CD73 1:1 (black line), 0.5:1 (dark grey line), 0.1:1 (light grey line), 0:1 (dashed grey line) and 1:0 (dashed black line). The calculated sizes of the various peaks are shown in Table 10. [Figure 6B] Figure 1 shows a series of SEC-MALS profiles of antibodies 21127, 21163, 21046, and 11E1, oleculab, and CPX006 analog mixed with CD73 in the ratio mAb:CD73 1:1 (black line), 0.5:1 (dark grey line), 0.1:1 (light grey line), 0:1 (dashed grey line) and 1:0 (dashed black line). The calculated sizes of the various peaks are shown in Table 10. [Figure 7] Figure 1 is a graph showing the activity of soluble CD73 after treatment with the indicated anti-CD73 antibodies and reference antibody (oleclumab) analogs. "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Data are normalized to untreated controls and presented as mean ± SEM. Vertical dotted lines indicate antibody concentrations equimolar to those of soluble recombinant CD73. [Figure 8]

[0023] Figure 1 is a set of graphs showing activity of CD73 expressed on Calu-6 cells (top), H292 cells (middle), and Cynom-K1 cells (bottom) after treatment with the indicated anti-CD73 antibodies and reference antibody (oleclumab) analogs. "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Data are normalized to untreated controls and presented as mean ± SEM. [Figure 9] 1 is a set of graphs showing activity of CD73 expressed on H292 cells after treatment with the indicated anti-CD73 antibodies and a reference antibody analog (oleclumab) for 3 hours (top), 6 hours (middle), or 24 hours (bottom). Data are normalized to untreated controls and are presented as the mean ± SEM. [Figure 10] Graph showing the activity of CD73 expressed by 20 different cancer cell lines in the presence of the indicated antibodies as measured by CellTiter-Glo assay. "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Each data point represents the average of three technical replicates. [Figure 11] This is a pair of graphs showing the effect of blocking CD73 activity on the viability of two triple-negative breast cancer cell lines, MBA-MB-231 (top panel) and MBA-MB-468 (bottom panel), grown in the presence of 300 μM AMP. "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. All counts were normalized to untreated cells and data are presented as the mean ± SEM of three technical replicates. [Figure 12] 1 is a set of graphs showing the activity of CD73 expressed by primary CD4+ and CD8+ T cells and CD19+ B cells from healthy human donors in the presence of the indicated antibodies as measured by CellTiter-Glo assay. Activity is normalized to untreated cells (100%). "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Data are presented as the mean ± SEM of three technical replicates. [Figure 13]Graph showing proliferation of CD4+ T cells treated with anti-CD3 / CD28 beads, AMPs, and the indicated anti-CD73 and reference antibody (oleculab) analogs. Data are normalized to non-AMP treated controls and presented as mean±SEM. [Figure 14] 1 is a pair of graphs showing activation of CD4+ (upper panel) and CD8+ (lower panel) T cells treated with anti-CD3 / CD28 beads, AMP, and 21127 or reference antibody (oleclumab) analogs. "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Data are presented as mean ± SEM. [Figure 15] Graph showing T cell proliferation in a one-way mixed lymphocyte reaction (MLR) upon incubation with anti-PD-1 antibody (12819), AMP, and the indicated anti-CD73 antibody or reference antibody (oleclumab) analogs. "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Data are normalized to non-AMP treated controls and presented as mean ± SEM. [Figure 16] FIG. 1 is a pair of graphs showing T cell proliferation in a one-way MLR upon incubation with anti-PD-1 and / or anti-CD73 antibodies at the indicated concentrations, with (lower panel) or without (upper panel) AMP. The 12819+21127 combination is a 1:1 mixture of the two antibodies, and the concentrations shown indicate the total concentration of the mixture. "Control Ab" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Data are normalized to non-AMP treated controls and are presented as mean±SEM. [Figure 17A] Figure 17 shows B cell activation in PBMCs from healthy donors stimulated overnight with the indicated antibodies (10 μg / mL) and CD40 ligand (0.5 μg / mL). Figure 17A is a series of plots showing the levels of CD69 on B cells (CD20+). [Figure 17B]Figure 17B is a series of graphs showing mean fluorescence intensity (MFI) for antibody staining of B cell activation markers CD25, CD69, and CD83. "IgG1-LALA control" is a non-CD73 specific FcγR deficient IgG1-LALA isotype antibody. Data are replicates from one donor and are presented as mean ± SEM. [Figure 18] Graph showing levels of CD73 expressed in H292 cells after 24 hours of treatment with the indicated anti-CD73 antibodies or reference antibody (oleclumab) analogs. Data were normalized to untreated controls and are presented as mean±SEM. [Figure 19]

[0023] Figure 1 is a pair of graphs showing activity of CD73 in the human xenograft melanoma model A375 after treatment with the indicated antibodies. Top panel: PBMC-humanized mice implanted with A375 were dosed with various doses of 21127 three times per week for two weeks. Tumors were harvested one day after the last dose and analyzed for CD73 activity. Bottom panel: NOD-scid mice implanted subcutaneously with A375 were treated with the indicated antibodies or combinations three times per week for one week. Tumors were harvested 3, 7, 10, 17, and 29 days after the last treatment and analyzed for CD73 activity. Data are normalized to untreated controls and presented as mean ± SEM. [Figure 20] 1 is a graph showing tumor growth in NOD-scid mice subcutaneously implanted with the human triple-negative breast cancer cell line MDA-MB-231. Mice were treated twice weekly with 10 mg / kg of antibody 21127 or a reference antibody (oleculab) analog. The grey area indicates the treatment period. Data are presented as mean±SEM. [Figure 21] FIG. 1 is a pair of graphs showing the effect of treatment with anti-CD73 antibody 21127 or vehicle on tumor growth in PBMC-humanized mice implanted with Calu-6 or A375 tumor cells. The grey area indicates the treatment period. Data are presented as mean±SEM. *P<0.05 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present disclosure provides novel anti-human CD73 antibodies that can be used to suppress CD73 activity in patients, such as cancer patients. Unless otherwise specified, as used herein, "CD73" refers to human CD73. The human CD73 polypeptide sequence is available at UniProt accession number P21589 (5NTD_HUMAN) (SEQ ID NO: 43), as shown below. 10 20 30 40 50 MCPRAARAPA TLLLALGAVL WPAAGAWELT ILHTNDVHSR LEQTSEDSSK 60 70 80 90 100 CVNASRCMGG VARLFTKVQQ IRRAEPNVLL LDAGDQYQGT IWFTVYKGAE 110 120 130 140 150 VAHFMNALRY DAMALGNHEF DNGVEGLIEP LLKEAKFPIL SANIKAKGPL 160 170 180 190 200 ASQISGLYLP YKVLPVGDEV VGIVGYTSKE TPFLSNPGTN LVFEDEITAL 210 220 230 240 250 QPEVDKLKTL NVNKIIALGH SGFEMDKLIA QKVRGVDVVV GGHSNTFLYT 260 270 280 290 300 GNPPSKEVPA GKYPFIVTSD DGRKVPVVQA YAFGKYLGYL KIEFDERGNV 310 320 330 340 350 ISSHGNPILL NSSIPEDPSI KADINKWRIK LDNYSTQELG KTIVYLDGSS 360 370 380 390 400 QSCRFRECNM GNLICDAMIN NNLRHTDEMF WNHVSMCILN GGGIRSPIDE 410 420 430 440 450 RNNGTITWEN LAAVLPFGGT FDLVQLKGST LKKAFEHSVH RYGQSTGEFL 460 470 480 490 500 QVGGIHVVYD LSRKPGDRVV KLDVLCTKCR VPSYDPLKMD EVYKVILPNF 510 520 530 540 550 LANGGDGFQM IKDELLRHDS GDQDINVVST YISKMKVIYP AVEGRIKFST 560 570 GSHCHGSFSL IFLSLWAVIF VLYQ

[0035] The term "antibody" (Ab) or "immunoglobulin" (Ig) as used herein refers to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be further subdivided into hypervariable regions called "complementarity determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDRs (H-CDRs herein refer to CDRs derived from the heavy chain; L-CDRs herein refer to CDRs derived from the light chain) and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid numbering in the heavy or light chain, and the assignment of FR and CDR regions may follow the IMGT® definition (EU numbering; Lefranc et al., Dev Comp Immunol 27(1):55-77(2003)); or the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917(1987); Chothia et al., Nature 342:878-883(1989); MacCallum et al., J. Mol. Biol. 262:732-745(1996); or Honegger and Pluckthun, J. Mol. Biol. 309(3):657-70(2001).

[0036] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line that contains a nucleotide sequence encoding the antibody, where said nucleotide sequence is not naturally associated with the cell.

[0037] The term "isolated protein," "isolated polypeptide," or "isolated antibody" refers to a protein, polypeptide, or antibody that, by virtue of its origin or source of derivation, (1) is not associated with naturally associated components with which it is associated in its natural state, (2) is free of other proteins from the same species, (3) is expressed by cells from a different species, and / or (4) is not naturally occurring. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the cell in which it is naturally derived is "isolated" from its naturally associated components. A protein can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

[0038] The term "affinity" refers to a measure of the attraction between an antigen and an antibody. The intrinsic attractiveness of an antibody to an antigen is typically measured by the binding affinity equilibrium constant (K D The antibody is expressed as K D When the K is less than 1 mM, preferably less than 100 nM, it is said to specifically bind to an antigen. D Binding affinity constants can be measured, for example, by surface plasmon resonance (BIAcore™) or biolayer interferometry, using, for example, an IBIS MX96 SPR system from IBIS Technologies or an Octet™ system from ForteBio.

[0039] The term "epitope" as used herein refers to a portion (determinant) of an antigen that specifically binds to an associated molecule, such as an antibody or bispecific binding molecule. Epitope determinants generally consist of chemically active surface groups of molecules, such as amino acids or carbohydrate or sugar side chains, and generally have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes can be "linear" or "conformational". In a linear epitope, all of the interaction points between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction points occur across amino acid residues on the protein that are separated from each other in the primary amino acid sequence. Once a desired epitope on an antigen is determined, it is possible to generate antibodies against that epitope using techniques well known in the art. For example, antibodies against a linear epitope can be generated, for example, by immunizing an animal with a peptide having amino acid residues of the linear epitope. Antibodies to conformational epitopes can be generated, for example, by immunizing animals with a minidomain containing the relevant amino acid residues of the conformational epitope. Antibodies to a particular epitope can also be generated, for example, by immunizing an animal with a target molecule of interest (e.g., CD73) or a relevant portion thereof, and then screening for binding to the epitope.

[0040] Methods known in the art, including but not limited to competitive assays, epitope binning, and alanine scanning, can be used to determine whether an antibody binds to the same epitope as an anti-CD73 antibody of the present disclosure, or competes for binding with it. In some embodiments, an anti-CD73 antibody of the present disclosure is bound to CD73 under saturating conditions, and then the ability of the test antibody to bind to CD73 is measured. If the test antibody can bind to CD73 simultaneously with the reference anti-CD73 antibody, the test antibody binds to a different epitope than the reference anti-CD73 antibody. However, if the test antibody cannot bind to CD73 simultaneously, the test antibody binds to the same epitope as, overlapping with, or adjacent to the epitope bound by the anti-CD73 antibody of the present disclosure. This experiment can be performed, for example, using ELISA, RIA, BIACORE™, SPR, biolayer interferometry, or flow cytometry. To test whether an anti-CD73 antibody cross-competes with another anti-CD73 antibody, the above competition method can be used in two ways, i.e., to determine whether the known antibody blocks the test antibody, and vice versa. Such cross-competition experiments can be performed, for example, using an IBIS MX96 SPR instrument or an Octet™ system.

[0041] The term "human antibody" refers to an antibody in which the variable domain and constant region sequences are derived from human sequences. The term encompasses antibodies that are derived from human genes but have sequences that have been modified to, for example, reduce immunogenicity, increase affinity, and / or increase stability. In addition, the term encompasses antibodies recombinantly produced in non-human cells that may confer glycosylation that is not typical of human cells. The term also encompasses antibodies produced in transgenic non-human organisms (e.g., OmniRat® rats) that have human antibody genes.

[0042] The term "antigen-binding portion" of an antibody (or simply "antibody portion") as used herein refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human CD73, or a portion thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" include: (i) Fab fragment: a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragment consisting of the VH domain; and (vi) isolated complementarity determining regions (CDRs) capable of specifically binding to an antigen. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that allows the VL and VH domains to be produced as a single protein chain that pairs to form a monovalent molecule (known as single-chain Fv (scFv)). Antigen-binding molecules comprising VH and / or VL are also within the scope of the present disclosure. In the case of VH, the molecule may also include one or more of the CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but are too short to allow pairing between the two domains on the same chain, and thus use a linker that allows the domains to pair with complementary domains on another chain, creating two antigen-binding sites.

[0043] Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, for example, as described herein.

[0044] The class (isotype) and subclass of an anti-CD73 antibody may be determined by any method known in the art. Generally, the class and subclass of an antibody may be determined using antibodies specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass may be determined by ELISA or Western blot and other techniques. Alternatively, the class and subclass may be determined by sequencing all or part of the constant regions of the heavy and / or light chains of the antibody and comparing those amino acid sequences to known amino acid sequences of various classes and subclasses of immunoglobulins to determine the class and subclass of the antibody.

[0045] Unless otherwise indicated, all antibody amino acid residue numbers referred to in this disclosure are based on the IMGT® numbering scheme (EU numbering).

[0046] Anti-CD73 antibody The present disclosure provides antibodies against CD73, and antigen-binding portions thereof. In a specific embodiment, the antibodies disclosed herein are human antibodies generated from transgenic animals (e.g., rats) capable of producing antibodies encoded by rearranged human antibody genes. In certain embodiments, the human antibodies may contain specific mutations, for example, to change primer-derived mutations back to germline sequences (see, e.g., "Symplex correction" mutants in Table 1).

[0047] In some embodiments, the anti-CD73 antibodies of the present disclosure have "LALA" mutations (L234A / L235A) in the Fc region. These mutations weaken the binding of the antibody to human FcγR (Fc gamma receptor). Such antibodies are advantageous because they have low levels of secondary effector function and do not deplete effector T cells or target other non-malignant cells.

[0048] In some embodiments, the anti-CD73 antibody or antigen-binding portion comprises: a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 9 and 41 and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 13 and 42; b) a HC comprising the amino acid sequences of SEQ ID NOs: 10 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 14 and 42; c) a HC comprising the amino acid sequences of SEQ ID NOs: 11 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 15 and 42; or d) HC comprising the amino acid sequences of SEQ ID NOs: 12 and 41 and LC comprising the amino acid sequences of SEQ ID NOs: 16 and 42 competes or cross-competes for binding to human CD73 with an antibody comprising the same, or binds to the same epitope on human CD73 as an antibody comprising the same.

[0049] In some embodiments, the anti-CD73 antibody or antigen-binding portion has the heavy chain CDR3 (H-CDR3) amino acid sequence of SEQ ID NO: 19, 25, 31, or 37.

[0050] In some embodiments, an anti-CD73 antibody or antigen-binding portion has heavy chain CDR1-3 (H-CDR1-3) comprising the amino acid sequences of SEQ ID NOs: 17-19, 23-25, 29-31, or 35-37, respectively.

[0051] In some embodiments, the anti-CD73 antibody or antigen-binding portion has a heavy chain variable domain (VH) amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs:9-12.

[0052] In some embodiments, the anti-CD73 antibody or antigen-binding portion has a VH comprising the amino acid sequence of any one of SEQ ID NOs:9-12.

[0053] In some embodiments, the anti-CD73 antibody has a VH amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs:9-12; and a heavy chain constant region amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:41.

[0054] In some embodiments, the anti-CD73 antibody comprises a VH amino acid sequence of any one of SEQ ID NOs:9-12 and a heavy chain constant region amino acid sequence of SEQ ID NO:41.

[0055] In some embodiments, the anti-CD73 antibody or antigen-binding portion has the light chain CDR3 (L-CDR3) amino acid sequence of SEQ ID NO: 22, 28, 34, or 40.

[0056] In some embodiments, an anti-CD73 antibody or antigen-binding portion has light chain CDR1-3 (L-CDR1-3) comprising the amino acid sequences of SEQ ID NOs: 20-22, 26-28, 32-34, or 38-40, respectively.

[0057] In some embodiments, the anti-CD73 antibody or antigen-binding portion has a light chain variable domain (VL) amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 13-16.

[0058] In some embodiments, the anti-CD73 antibody or antigen-binding portion has a VL comprising the amino acid sequence of any one of SEQ ID NOs:13-16.

[0059] In some embodiments, the anti-CD73 antibody has a VL amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 13-16; and a light chain constant region amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42.

[0060] In some embodiments, the anti-CD73 antibody comprises the VL amino acid sequence of any one of SEQ ID NOs:13-16 and the light chain constant region amino acid sequence of SEQ ID NO:42.

[0061] In certain embodiments, the anti-CD73 antibody comprises any one of the heavy chains described above and any one of the light chains described above.

[0062] In some embodiments, the anti-CD73 antibody or antigen-binding portion of the disclosure comprises: a) SEQ ID NOs: 17 to 22, respectively; b) SEQ ID NOs: 23 to 28, respectively; c) SEQ ID NOs: 29 to 34, respectively; or d) SEQ ID NOs: 35 to 40, respectively It contains the amino acid sequences of H-CDR1 to 3 and L-CDR1 to 3.

[0063] In some embodiments, the anti-CD73 antibody or antigen-binding portion of the disclosure comprises: a) SEQ ID NOs: 9 and 13, respectively; b) SEQ ID NOs: 10 and 14, respectively; c) SEQ ID NOs: 11 and 15, respectively; or d) SEQ ID NOs: 12 and 16, respectively The VH and VL are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of

[0064] In some embodiments, the anti-CD73 antibody or antigen-binding portion of the disclosure comprises: a) SEQ ID NOs: 9 and 13, respectively; b) SEQ ID NOs: 10 and 14, respectively; c) SEQ ID NOs: 11 and 15, respectively; or d) SEQ ID NOs: 12 and 16, respectively The VH and VL comprise the amino acid sequence of

[0065] In some embodiments, the anti-CD73 antibodies of the disclosure are a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 9 and 41 and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 13 and 42; b) a HC comprising the amino acid sequences of SEQ ID NOs: 10 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 14 and 42; c) a HC comprising the amino acid sequences of SEQ ID NOs: 11 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 15 and 42; or d) HC comprising the amino acid sequences of SEQ ID NOs: 12 and 41 and LC comprising the amino acid sequences of SEQ ID NOs: 16 and 42 Includes.

[0066] The disclosure also provides anti-CD73 antibodies, or antigen-binding portions thereof, that compete or cross-compete for binding with, or bind to the same epitope as, antibody 21028, 21046, 21127, or 21163.

[0067] In some embodiments, an anti-CD73 antibody or antigen-binding portion of the disclosure comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 21028, 21046, 21127, or 21163.

[0068] In some embodiments, an anti-CD73 antibody or antigen-binding portion of the disclosure comprises a VH and VL that are at least 90% identical in amino acid sequence to the VH and VL of antibody 21028, 21046, 21127, or 21163, respectively.

[0069] In some embodiments, an anti-CD73 antibody or antigen-binding portion of the disclosure comprises a VH and VL that are the VH and VL, respectively, of antibody 21028, 21046, 21127, or 21163.

[0070] In some embodiments, the anti-CD73 antibody of the disclosure is antibody 21028, 21046, 21127, or 21163, or an antibody that comprises the same amino acid sequence as said antibodies.

[0071] The class of the anti-CD73 antibody obtained by the methods described herein can be changed or switched to another class or subclass. In some embodiments of the present disclosure, the nucleic acid molecule encoding the VL or VH is isolated using methods well known in the art so that it does not contain a nucleic acid sequence encoding a CL or CH, respectively. The nucleic acid molecule encoding the VL or VH is then operably linked to a nucleic acid sequence encoding a CL or CH, respectively, from a different class of immunoglobulin molecule. This can be achieved using a vector or nucleic acid molecule containing a CL or CH sequence, as described above. For example, an anti-CD73 antibody that was originally IgM can be class switched to an IgG. Furthermore, class switching can be used to convert one IgG subclass to another, for example from IgG1 to IgG2. The kappa light chain constant region can be changed to, for example, a lambda light chain constant region, or vice versa. An exemplary method for producing an antibody of the disclosure having a desired Ig isotype includes isolating a nucleic acid molecule encoding a heavy chain of an anti-CD73 antibody and a nucleic acid molecule encoding a light chain of an anti-CD73 antibody, obtaining the variable domain of the heavy chain, linking the coding sequence for the variable domain of the heavy chain to a coding sequence for a constant region of a heavy chain of the desired isotype, expressing the light and heavy chains encoded by the linked sequences in a cell, and recovering the anti-CD73 antibody having the desired isotype.

[0072] The anti-CD73 antibodies of the disclosure may be IgG, IgM, IgE, IgA, or IgD molecules, but are typically of the IgG isotype, e.g., IgG subclasses IgG1, IgG2a, or IgG2b, IgG3, or IgG4. In some embodiments, the antibodies are of isotype subclass IgG1. In some embodiments, the antibodies are of isotype subclass IgG2.

[0073] In some embodiments, the anti-CD73 antibody may comprise at least one mutation in the Fc region. Several different Fc mutations are known, which alter the effector function of the antibody. For example, in many cases, such as when ligand / receptor interactions are undesirable, or in the case of antibody-drug conjugates, it may be desirable to reduce or eliminate effector function.

[0074] In some embodiments, the anti-CD73 antibody comprises at least one mutation in the Fc region that reduces effector function, e.g., one or more mutations at positions 228, 233, 234, and 235, where amino acid positions are numbered according to the IMGT® numbering scheme.

[0075] In some embodiments, for example, when the antibody is of the IgG1 subclass, one or both of the amino acid residues at positions 234 and 235 may be mutated, for example, from Leu to Ala (L234A / L235A). These mutations reduce the effector function of the Fc region of an IgG1 antibody. The amino acid positions are numbered according to the IMGT® numbering scheme.

[0076] In some embodiments, for example, where the antibody is of the IgG4 subclass, it may contain the mutation S228P, where the amino acid positions are numbered according to the IMGT® numbering scheme, which is known to reduce undesired Fab arm exchange.

[0077] In some embodiments, an anti-CD73 antibody, or antigen-binding portion thereof, of the invention binds to an epitope of CD73 that includes at least one (e.g., at least one, at least two, at least three, at least four, or at least five) of the following residues of SEQ ID NO: 43: R73, R109, D168, I301, S302, and H304. In certain embodiments, the antibody or antigen-binding portion binds to an epitope of CD73 that includes or consists of residues R73, R109, and D168 (such as antibody 21127). In certain embodiments, the antibody or antigen-binding portion binds to an epitope of CD73 that includes or consists of residues R109 (such as antibody 21163). In certain embodiments, the antibody or antigen-binding portion binds to an epitope of CD73 that includes or consists of residues I301, S302, and H304 (such as antibody 21046).

[0078] In some embodiments, an anti-CD73 antibody of the invention, or antigen-binding portion thereof, binds to an epitope of CD73 that includes residues 27-31, 61-70, 61-75, 161-170, 266-270, and / or 291-305 of SEQ ID NO:43. In certain embodiments, the antibody or antigen-binding portion binds to an epitope of CD73 that includes, or consists of, residues 27-31, 61-75, and 161-170 (such as antibody 21127). In certain embodiments, the antibody or antigen-binding portion binds to an epitope of CD73 that includes, or consists of, residues 61-70 and 161-170 (such as antibody 21163). In certain embodiments, the antibody or antigen-binding portion binds to an epitope of CD73 that includes, or consists of, residues 27-31, 266-270, and 291-305 (such as antibody 21046).

[0079] In some embodiments, the antibody or moiety binds to an epitope that includes residues 27-31 of SEQ ID NO:43 (or a fragment thereof, such as a 1, 2, 3, or 4 residue fragment) (such as antibodies 21127 and 21046). In some embodiments, the antibody or moiety binds to an epitope that includes residues 61-75 of SEQ ID NO:43 (or a fragment thereof, such as a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residue fragment, such as residues 61-70) (such as antibodies 21127 and 21163). In some embodiments, the antibody or moiety binds to an epitope that includes residues 161-170 of SEQ ID NO:43 (or a fragment thereof, such as a 1, 2, 3, 4, 5, 6, 7, 8, or 9 residue fragment) (such as antibodies 21127 and 21163). In some embodiments, the antibody or portion binds to an epitope that includes residues 266-270 of SEQ ID NO: 43 (or a fragment thereof, such as a 1, 2, 3, or 4 residue fragment) (such as antibody 21046). In some embodiments, the antibody or portion binds to an epitope that includes residues 291-305 of SEQ ID NO: 43 (or a fragment thereof, such as a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residue fragment) (such as antibody 21046).

[0080] Epitopes comprising any combination of the above residues or those shown in Table 9 are also contemplated.

[0081] In some embodiments, an amino acid sequence comprising a CD73 epitope described herein can be used as an immunogen (e.g., as an antigen for administration to an animal or for screening an antibody library) to generate or identify anti-CD73 antibodies, or antigen-binding portions thereof, that bind to the epitope.

[0082] In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibodies or antigen-binding portions described herein inhibit the activity of soluble CD73 by at least 40%, 50%, 60%, 70%, 80%, 90%, or 100% (e.g., at least 40%). In some embodiments, at a concentration of 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 50, or 100 μg / mL, the anti-CD73 antibodies or antigen-binding portions described herein inhibit the activity of soluble CD73 by at least 90%.

[0083] In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibodies or antigen-binding portions described herein inhibit the activity of CD73 on Calu-6 cells by at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 75%).

[0084] In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibodies or antigen-binding portions described herein inhibit the activity of CD73 on H292 cells by at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 80%).

[0085] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein specifically bind to human CD73, cynomolgus monkey CD73, or both, expressed on CHO-S cells.

[0086] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein have a K of 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM or less (e.g., 1 nM or less) as measured by surface plasmon resonance (SPR). D It binds to the extracellular domain (ECD) of human CD73.

[0087] In some embodiments, the anti-CD73 antibodies or antigen binding portions described herein have a K of 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM or less (e.g., 0.7 nM or less) as measured by surface plasmon resonance (SPR). D It binds to the extracellular domain (ECD) of cynomolgus monkey CD73.

[0088] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein do not bind to the same epitope of CD73 as oleclumab, CPX006, 11E1, or any combination thereof.

[0089] In some embodiments, an anti-CD73 antibody or antigen-binding portion described herein binds to an epitope on a CD73 homodimer in a manner that results in a 1:1 complex. In certain embodiments, antibody / CD73 binding results in only a 1:1 complex. In certain embodiments, antibody / CD73 binding results in primarily a 1:1 complex. In specific embodiments, antibody or antigen-binding portion binding to CD73 results in a 1:1 complex independent of CD73 concentration.

[0090] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein inhibit soluble CD73 activity, e.g., at concentrations of 1, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL or less (e.g., at concentrations of 10 μg / mL or less). In certain embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibodies or antigen-binding portions inhibit the CD73 activity more effectively than oleclumab. In specific embodiments (e.g., using an antibody or antigen-binding portion at one of the above concentrations), CD73 activity is inhibited by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%. For example, the antibodies or antigen-binding portions may inhibit 100% of CD73 activity at a concentration of 3 μg / mL.

[0091] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein inhibit CD73 activity on Calu-6, H292, Cynom-K1 cells, or any combination thereof, at concentrations of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, or 100 μg / mL or less (such as at concentrations of 3 μg / mL or less). In certain embodiments, the anti-CD73 antibodies or antigen-binding portions inhibit the CD73 activity more effectively than oleclumab. In certain embodiments, the anti-CD73 antibodies or antigen-binding portions remain more effective than oleclumab after 3, 6, or 24 hours of additional incubation. In specific embodiments (e.g., using one of the above concentrations of the antibody or antigen-binding portion), CD73 activity is inhibited by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0092] In some embodiments (e.g., at a concentration of 25 μg / mL), the anti-CD73 antibodies or antigen binding portions described herein inhibit CD73 activity on any cell line or combination of cell lines shown in Table 11. In certain embodiments, the anti-CD73 antibodies or antigen binding portions inhibit CD73 activity on Calu-6, NCI-H1775 cells, KYSE-30 cells, Capan-2 cells, or any combination thereof. In certain embodiments, the anti-CD73 antibodies or antigen binding portions inhibit said CD73 activity more effectively than oleclumab.

[0093] In some embodiments, for example, at concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, or 25 μg / mL or less (such as at concentrations of 0.01 μg / mL or less), an anti-CD73 antibody or antigen-binding portion described herein inhibits survival and / or proliferation of MDA-MB-231 cells, MDA-MB-468 cells, or both in vitro. In certain embodiments (e.g., at concentrations of 0.5 μg / mL or more), an anti-CD73 antibody or antigen-binding portion inhibits said survival / proliferation more effectively than oleclumab. In specific embodiments (e.g., using an antibody or antigen-binding portion at one of the above concentrations), the number of viable cells is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0094] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein inhibit primary CD4 + cells, CD8 + T cells, CD19 + In a specific embodiment (e.g., using one of the antibodies or antigen-binding portions at a concentration as described above), CD73 activity is inhibited by, for example, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0095] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein inhibit CD4 + In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein, e.g., at a concentration of 0.1, 0.5, 1, 10, 15, 20, 25, 30, 40, 50, or 100 μg / mL or less (e.g., at a concentration of 25 μg / mL or less), restore proliferation of CD4 T cells. + In some embodiments (e.g., at a concentration of 0.1 μg / mL or greater), the anti-CD73 antibody or antigen-binding portion thereof inhibits CD4+ restores T cell proliferation more effectively than oleclumab. In certain embodiments (e.g., using one of the antibodies or antigen-binding portions at the above concentrations), T cell proliferation is restored by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0096] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein inhibit CD4 + and CD8 + In some embodiments (e.g., at a concentration of 0.1 μg / mL or greater), the anti-CD73 antibody or antigen-binding portion activates CD4 + and CD8 + It activates T cells more effectively than oleculab.

[0097] In some embodiments, for example at concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μg / mL or less (such as at concentrations of 0.01 μg / mL or less), an anti-CD73 antibody or antigen binding portion described herein, in combination with an anti-PD-1 antibody, restores T cell proliferation in the presence of AMP in a one-way mixed lymphocyte reaction (MLR). In certain embodiments (e.g., at concentrations of 1 μg / mL or less), an anti-CD73 antibody or antigen binding portion, in combination with an anti-PD-1 antibody, fully restores T cell proliferation. In certain embodiments (e.g., at concentrations of 0.1 μg / mL or more), an anti-CD73 antibody or antigen binding portion, in combination with an anti-PD-1 antibody, restores T cell proliferation more effectively than oleclumab. In specific embodiments (e.g., using one of the above concentrations of an antibody or antigen-binding portion in combination with an anti-PD-1 antibody), T cell proliferation is restored to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0098] In some embodiments, the anti-CD73 antibodies or antigen binding portions described herein enhance T cell activation in the presence of AMPs in a unidirectional MLR, e.g., at concentrations of 0.5, 1, 5, 10, 15, or 25 μg / mL or less (e.g., at concentrations of 1 μg / mL or less). In some embodiments, the anti-CD73 antibodies or antigen binding portions, in combination with an anti-PD-1 antibody (e.g., 12819), enhance T cell activation in the presence of AMPs in a unidirectional MLR.

[0099] In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibodies or antigen-binding portions described herein do not stimulate B cell activation in vitro. In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibodies or antigen-binding portions described herein stimulate B cell activation in vitro. B cell activation can be determined, for example, by looking at markers such as CD25, CD69, and / or CD83.

[0100] In some embodiments (e.g., at a concentration of 25 μg / mL), the anti-CD73 antibodies or antigen-binding portions described herein do not reduce the level of CD73 in H292 cells in vitro. In some embodiments (e.g., at a concentration of 25 μg / mL), the anti-CD73 antibodies or antigen-binding portions described herein reduce the level of CD73 in H292 cells in vitro by, for example, 40%, 50%, 60%, 70%, 80%, or 90% or less compared to an untreated control. In some embodiments (e.g., at a concentration of 25 μg / mL), the anti-CD73 antibodies or antigen-binding portions described herein only slightly reduce the level of CD73 in H292 cells in vitro. For example, in certain embodiments, the level of CD73 is reduced by, for example, 5%, 10%, 15%, 20%, 25%, 30%, or 35% (e.g., 25%) or less.

[0101] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein inhibit CD73 activity in tumors harvested from PBMC-humanized mice engrafted with A375 cells (e.g., when mice are administered 5 mg / kg, 20 mg / kg, or 50 mg / kg of the antibody or antigen-binding portion three times per week for one or two weeks). In certain embodiments, inhibition of CD73 activity is maintained for at least 20, 24, 28, 32, 36, 40, 44, 50, 75, 100, 125, 150, 175, or 200 days (e.g., at least 28 days) after treatment has ended.

[0102] In some embodiments, an anti-CD73 antibody or antigen-binding portion described herein inhibits tumor growth in NOD-scid mice implanted with MDA-MB-231 cells (e.g., when the mice are administered 10 mg / kg of the antibody or antigen-binding portion twice a week for a total of 16 treatments). In certain embodiments, tumor growth inhibition is maintained with limited increase in tumor volume (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, or 200 days (e.g., at least 60 days) after treatment has ended. 3 less than).

[0103] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein inhibit tumor growth in PBMC-humanized mice engrafted with Calu-6 or A375 cells (e.g., when mice are dosed with 10 mg / kg of the antibody or antigen-binding portion three times per week for a total of six treatments).

[0104] The present disclosure also contemplates anti-CD73 antibodies or antigen-binding portions having any combination of the above properties.

[0105] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein have the following characteristics: a) inhibits the activity of soluble CD73 in vitro; b) inhibiting the activity of CD73 on Calu-6 cells in vitro; c) inhibiting the activity of CD73 on H292 cells in vitro; d) specifically binds to human and cynomolgus CD73 expressed on CHO-S cells; e) K of 1 nM or less as measured by SPR D binds to the ECD of human CD73; f) K of 0.7 nM or less as measured by SPR D binds to the ECD of cynomolgus monkey CD73; g) does not bind to the same epitope on CD73 as oleclumab, CPX006, and / or 11E1; h) binds to an epitope on the CD73 homodimer in a manner that yields a 1:1 complex; i) inhibits soluble CD73 activity more effectively than oleculab in vitro; j) inhibits CD73 activity on Calu-6, H292, and Cynom-K1 cells in vitro; k) inhibits CD73 activity on Calu-6, NCI-H1775, KYSE-30, and Capan-2 cells in vitro; l) inhibiting the proliferation of MDA-MB-231 and MDA-MB-468 cells in vitro; m) In vitro primary CD4 + and CD8 + T cells and CD19 + Inhibits CD73 activity on B cells; n) In vitro CD4 + restores T-cell proliferation; o) In vitro CD4 + and CD8 + Activating T cells; p) in combination with anti-PD-1 antibodies, restores T cell proliferation in a one-way mixed lymphocyte reaction (MLR) in the presence of AMPs; q) in combination with anti-PD-1 antibodies in a unidirectional MLR to enhance T cell activation in the presence of AMPs; r) does not stimulate B cell activation in vitro; s) does not reduce the levels of CD73 in H292 cells in vitro by more than 25%; t) inhibit CD73 activity in tumors harvested from PBMC-humanized mice implanted with A375 cells; u) inhibiting in vivo tumor growth in NOD-scid mice implanted with MDA-MB-231 cells; v) inhibiting in vivo tumor growth in PBMC-humanized mice engrafted with Calu-6 cells; and w) Inhibits in vivo tumor growth in PBMC-humanized mice implanted with A375 cells In one embodiment, the compound has at least one of the following structures (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all 23). In some embodiments, the anti-CD73 antibody or antigen-binding portion has all of properties a) through w). In some embodiments, the anti-CD73 antibody or antigen-binding portion has at least properties a) through k), n), and s). In some embodiments, the anti-CD73 antibody or antigen-binding portion has at least properties a) through g), i) through k), and n). In some embodiments, the anti-CD73 antibody or antigen-binding portion has at least properties a) through d), i) through k), n), and s).

[0106] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein may inhibit tumor growth and / or induce tumor growth regression in vivo, slow or reverse metastasis in cancer patients, and / or prolong survival of cancer patients. Any combination of the above properties is also contemplated.

[0107] In some embodiments, the anti-CD73 antibodies or antigen-binding portions described herein increase T cell proliferation and / or reduce CD73 activity more effectively than anti-CD73 antibodies currently in clinical trials. For example, in certain embodiments, the anti-CD73 antibodies or antigen-binding portions have the following properties: - At a concentration of 0.1 μg / mL, - SEQ ID NOs: 17 and 19, respectively, of US Patent Application Publication No. 2016 / 0129108; -SEQ ID NOs: 14 and 13, respectively, of U.S. Patent Application Publication No. 2018 / 0009899; or -SEQ ID NOs: 3 and 4, respectively, of US Patent Application Publication No. 2018 / 0030144 Antibodies containing the HC and LC amino acid sequences containing the HC and LC amino acid sequences inhibited the activation of CD4 + Increases T-cell proliferation; and / or -At concentrations of 10 μg / mL or more, - SEQ ID NOs: 17 and 19, respectively, of US Patent Application Publication No. 2016 / 0129108; -SEQ ID NOs: 14 and 13, respectively, of U.S. Patent Application Publication No. 2018 / 0009899; or -SEQ ID NOs: 3 and 4, respectively, of US Patent Application Publication No. 2018 / 0030144 Reduces CD73 activity in H292 cells after 24 hours of incubation more than antibodies containing the HC and LC amino acid sequences The present invention has at least one of the following:

[0108] In certain embodiments, the antibodies or antigen-binding portions thereof of the present disclosure may be part of a larger immunoadhesin molecule formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesin molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Another example includes the incorporation, either covalently or non-covalently, of one or more CDRs from an antibody into a molecule, making it an immunoadhesin that specifically binds to an antigen of interest. In such embodiments, the CDRs may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be incorporated non-covalently.

[0109] In some embodiments, fusion antibodies or immunoadhesins can be made that include all or a portion of an anti-CD73 antibody of the present disclosure linked to another polypeptide. In certain embodiments, only the variable domain of the anti-CD73 antibody is linked to the polypeptide. In certain embodiments, the VH domain of the anti-CD73 antibody is linked to a first polypeptide and the VL domain of the anti-CD73 antibody is linked to a second polypeptide that associates with the first polypeptide such that the VH and VL domains can interact with each other to form an antigen binding site. In some embodiments, the VH domain is separated from the VL domain by a linker (e.g., a single chain antibody) such that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to a polypeptide of interest. Additionally, fusion antibodies can be made in which two (or more) single chain antibodies are linked to each other. This is useful when one wants to create a bivalent or multivalent antibody on a single polypeptide chain or when one wants to create a bispecific antibody.

[0110] To make a single chain antibody (scFv), a DNA fragment encoding the VH and VL is operably linked to another fragment encoding a flexible linker, e.g., the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:44), such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains linked by the flexible linker. See, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). Single chain antibodies can be monovalent, when only a single VH and VL are used; bivalent, when two VHs and VLs are used; or multivalent, when three or more VHs and VLs are used. For example, bispecific or multivalent antibodies can be generated that specifically bind to human CD73 and to another molecule.

[0111] In other embodiments, nucleic acid molecules encoding anti-CD73 antibodies can be used to prepare other modified antibodies, such as "kappa bodies" (Ill et al., Protein Eng. 10:949-57 (1997)), "minibodies" (Martin et al., EMBO J. 13:5303-9 (1994)), "diabodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or "Janusins" (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)), which can be prepared using standard molecular biology techniques following the teachings herein.

[0112] The anti-CD73 antibodies or antigen-binding portions of the present disclosure may be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibodies or portions thereof are derivatized such that CD73 binding is not adversely affected by the derivatization or labeling. Thus, the antibodies and antibody portions of the present disclosure are intended to include both intact and modified forms of the human anti-CD73 antibodies described herein. For example, the antibodies or antibody portions of the present disclosure may be functionally linked (by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0113] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate), having two distinct reactive groups separated by a suitable spacer. Such linkers are available, for example, from Pierce Chemical Company, Rockford, Ill.

[0114] An anti-CD73 antibody or antigen-binding portion thereof can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups can be useful to improve the biological characteristics of the antibody, for example to increase serum half-life.

[0115] The antibody or antigen-binding portion according to the present disclosure may be labeled. As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into the antibody. In some embodiments, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid, or the attachment of a biotinyl moiety to the polypeptide that can be detected by a marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). In some embodiments, the label or marker can be a therapeutic agent, such as a drug conjugate or a toxin. A variety of methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, etc.). , metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogues or homologues thereof. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0116] In some embodiments, an antibody or antigen-binding portion according to the present disclosure may be conjugated to a cytotoxic agent to form an immunoconjugate. In some embodiments, an antibody or antigen-binding portion according to the present disclosure may be conjugated to a radioisotope.

[0117] In certain embodiments, the antibodies of the present disclosure may exist in neutral form (including zwitterionic forms) or as positively or negatively charged species. In some embodiments, the antibodies may be complexed with a counterion to form a pharma- ceutically acceptable salt.

[0118] The term "pharmaceutically acceptable salts" refers to a complex comprising one or more antibodies and one or more counterions derived from pharmaceutically acceptable inorganic and organic acids and bases.

[0119] Anti-CD73 antibody composition The present disclosure also provides combination therapies (e.g., compositions) comprising one, two, three, four, or more of the anti-CD73 antibodies or antigen-binding portions thereof described herein. In certain embodiments, the combination therapy (e.g., composition) comprises two anti-CD73 antibodies or antigen-binding portions. The combination therapy may take the form of, for example, a method of treatment using the antibodies or antigen-binding portions or a pharmaceutical composition comprising the antibodies or antigen-binding portions.

[0120] In some embodiments, the present disclosure provides a method for the treatment of a pulmonary artery disease, comprising administering to a subject the method of the present disclosure a first and second antibody comprising: - antibodies 21028 and 21046, respectively; - antibodies 21028 and 21127, respectively; - antibodies 21028 and 21163, respectively; - antibodies 21046 and 21127, respectively; - antibodies 21046 and 21163, respectively; or - Antibodies 21127 and 21163, respectively The present invention provides a composition comprising a first anti-CD73 antibody, or antigen-binding portion thereof, and a second anti-CD73 antibody, or antigen-binding portion thereof, wherein

[0121] In some embodiments, the composition comprises an antibody or antigen-binding portion thereof that binds to the same epitope as, or competes for binding with, said first and second antibodies.

[0122] In some embodiments, the composition comprises an antibody or antigen-binding portion thereof comprising the amino acid sequences of H-CDR1-3 and L-CDR1-3 of the first antibody, and an antibody or antigen-binding portion thereof comprising the amino acid sequences of H-CDR1-3 and L-CDR1-3 of the second antibody.

[0123] In some embodiments, the composition comprises an antibody, or antigen-binding portion thereof, comprising a VH and a VL that comprise amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VH and VL amino acid sequences, respectively, of the first antibody, and an antibody, or antigen-binding portion thereof, comprising a VH and a VL that comprise amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VH and VL amino acid sequences, respectively, of the second antibody.

[0124] In some embodiments, the composition comprises an antibody, or antigen-binding portion thereof, that comprises the VH and VL amino acid sequences of the first antibody, and an antibody, or antigen-binding portion thereof, that comprises the VH and VL amino acid sequences of the second antibody.

[0125] In some embodiments, the composition comprises an antibody, or antigen-binding portion thereof, that comprises the HC and LC amino acid sequences of the first antibody, and an antibody, or antigen-binding portion thereof, that comprises the HC and LC amino acid sequences of the second antibody.

[0126] In certain embodiments, the composition comprises: a) an antibody comprising H-CDR1 to 3 containing the amino acid sequences of SEQ ID NOs: 17 to 19, 23 to 25, 29 to 31, or 35 to 57, respectively; b) an antibody whose VH has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 9, 10, 11, or 12; c) an antibody whose VH comprises the amino acid sequence of SEQ ID NO: 9, 10, 11, or 12; d) an antibody whose HC comprises the amino acid sequence of SEQ ID NOs: 9 and 41, 10 and 41, 11 and 41, or 12 and 41; e) an antibody comprising L-CDR1 to 3 each comprising the amino acid sequence of SEQ ID NO: 20 to 22, 26 to 28, 32 to 34, or 38 to 40; f) an antibody whose VL has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; g) an antibody whose VL comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16; h) an antibody whose LC comprises the amino acid sequence of SEQ ID NOs: 13 and 42, 14 and 42, 15 and 42, or 16 and 42; i) an antibody in which H-CDR1 to 3 and L-CDR1 to 3 comprise the amino acid sequences of SEQ ID NOs: 17 to 22, 23 to 28, 29 to 34, or 35 to 40, respectively; j) an antibody comprising a VH and a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NOs: 9 and 13, 10 and 14, 11 and 15, or 12 and 16, respectively; k) an antibody comprising a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 9 and 13, 10 and 14, 11 and 15, or 12 and 16, respectively; and l) an antibody comprising an HC and an LC comprising the amino acid sequences of SEQ ID NOs: 9 and 41, and 13 and 42; 10 and 41, and 14 and 42; 11 and 41, and 15 and 42; or 12 and 41, and 16 and 42, respectively. The antibody may comprise one, two or more antibodies or antigen-binding portions thereof selected from the group consisting of:

[0127] In some embodiments, the anti-CD73 antibody compositions described herein may inhibit tumor growth and / or induce tumor growth regression in vivo, slow or reverse metastasis in cancer patients, and / or prolong survival of cancer patients. Any combination of the above properties is also contemplated.

[0128] The disclosure also provides a method of making an anti-CD73 antibody composition described herein, the method comprising providing a first anti-CD73 antibody or antigen-binding portion and a second anti-CD73 antibody or antigen-binding portion, and combining the two antibodies or portions.

[0129] bispecific binding molecules The present disclosure also provides bispecific binding molecules (e.g., comprising six CDRs or antigen binding portions such as a VH and a VL) that have the binding specificity of an anti-CD73 antibody described herein. In some embodiments, the bispecific binding molecule further has the binding specificity of another, different anti-CD73 antibody (e.g., another anti-CD73 antibody described herein), or an antibody whose activity targets a different protein, such as a cancer antigen or another cell surface molecule that mediates a disease state, such as cancer. Such bispecific binding molecules are known in the art, and examples of different types of bispecific binding molecules are provided elsewhere herein. In certain embodiments, the bispecific binding molecule may bind to CD73 and PD-1, CD73 and PD-L1, or CD73 and CTLA-4.

[0130] Nucleic Acid Molecules and Vectors The present disclosure also provides nucleic acid molecules and sequences encoding the anti-CD73 antibodies or antigen-binding portions thereof described herein. In some embodiments, different nucleic acid molecules encode the heavy and light chain amino acid sequences of the anti-CD73 antibodies or antigen-binding portions. In other embodiments, the same nucleic acid molecule encodes the heavy and light chain amino acid sequences of the anti-CD73 antibodies or antigen-binding portions.

[0131] Reference to a nucleotide sequence includes its complement unless otherwise specified. Thus, reference to a nucleic acid having a specific sequence should be understood to include its complementary strand, including its complementary sequence. The term "polynucleotide" referred to herein means a polymeric form of nucleotides, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide, of at least 10 bases in length. This term includes single-stranded and double-stranded forms.

[0132] In some embodiments, the disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, or a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, or both, of an anti-CD73 antibody, or antigen-binding portion thereof, described herein.

[0133] The present disclosure also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to one or more nucleotide sequences listed herein, such as a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-8, or a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16. The term "percent sequence identity" in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison can span a stretch of at least about 9 nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are several different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, FASTA, Gap, or Bestfit, programs of the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin, can be used to compare polynucleotide sequences. FASTA, including, for example, the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (see, e.g., Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); and Pearson, J. Mol. Biol. 276:71-84 (1998), which are incorporated herein by reference). Unless otherwise specified, default parameters of a particular program or algorithm are used.For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (word size 6 and NOPAM factor of the scoring matrix), or using Gap with its default parameters as provided in GCG version 6.1, which are incorporated herein by reference.

[0134] In some embodiments, the disclosure provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-8. In particular embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NOs: 1 and 5, SEQ ID NOs: 2 and 6, SEQ ID NOs: 3 and 7, or SEQ ID NOs: 4 and 8.

[0135] In any of the above embodiments, the nucleic acid molecule may be isolated. Nucleic acid molecules referred to herein as "isolated" or "purified" are nucleic acids that (1) have been separated from the genomic DNA or cellular RNA nucleic acids from which they originate; and / or (2) are non-naturally occurring.

[0136] In some embodiments, the disclosure provides vectors suitable for expressing one or both chains of an antibody or antigen-binding portion thereof described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector into which additional DNA segments can be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vector (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby be replicated along with the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0137] The present disclosure provides vectors comprising nucleic acid molecules encoding the heavy chain, light chain, or both the heavy and light chains of an anti-CD73 antibody or antigen-binding portion thereof described herein. In certain embodiments, the vectors of the present disclosure comprise the nucleic acid molecules described herein. The present disclosure further provides vectors comprising nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof. The vectors may further comprise expression control sequences.

[0138] The term "expression control sequences" as used herein refers to polynucleotide sequences necessary for the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion, if desired. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequences" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0139] Nucleic acid molecules encoding the heavy and / or light chains of the anti-CD73 antibodies or antigen-binding portions thereof described herein can be isolated from any source that produces such antibodies or portions. In some embodiments, the nucleic acid molecules are isolated from B cells expressing anti-CD73 antibodies isolated from animals immunized with human CD73 antigen or from immortalized cells made from such B cells. Methods for isolating nucleic acids encoding antibodies are well known in the art. mRNA can be isolated and used to generate cDNA for use in polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In certain embodiments, the nucleic acid molecules described herein can be synthesized rather than isolated.

[0140] In some embodiments, the nucleic acid molecules described herein comprise a nucleotide sequence encoding a VH domain from an anti-CD73 antibody or antigen-binding portion described herein linked in-frame to a nucleotide sequence encoding a heavy chain constant region from any source. Similarly, the nucleic acid molecules described herein can comprise a nucleotide sequence encoding a VL domain from an anti-CD73 antibody or antigen-binding portion described herein linked in-frame to a nucleotide sequence encoding a light chain constant region from any source.

[0141] In some embodiments of the present disclosure, nucleic acid molecules encoding VH and / or VL may be "converted" into full-length antibody genes. In certain embodiments, nucleic acid molecules encoding VH or VL domains are converted into full-length antibody genes by inserting them into an expression vector already encoding a heavy chain constant (CH) or light chain constant (CL) region, respectively, such that the VH segment is operably linked to a CH segment in the vector and / or the VL segment is operably linked to a CL segment in the vector. In some embodiments, nucleic acid molecules encoding VH and / or VL domains are converted into full-length antibody genes by linking, e.g., ligating, the nucleic acid molecules encoding the VH and / or VL domains to nucleic acid molecules encoding the CH and / or CL regions using standard molecular biology techniques. The nucleic acid molecules encoding the full-length heavy and / or light chains may then be expressed from a cell into which they have been introduced and the anti-CD73 antibody may be isolated.

[0142] The nucleic acid molecules can be used to recombinantly express large amounts of anti-CD73 antibodies. The nucleic acid molecules can also be used to generate chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies, and antibody derivatives described herein.

[0143] In some embodiments, the nucleic acid molecules of the present disclosure are used as probes or PCR primers for specific antibody sequences. For example, the nucleic acids can be used as probes in diagnostic methods or as PCR primers to amplify regions of DNA that can be used to isolate additional nucleic acid molecules that encode, inter alia, variable domains of anti-CD73 antibodies. In some embodiments, the nucleic acid molecules are oligonucleotides. In some embodiments, the oligonucleotides are derived from the hypervariable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotides encompass all or a portion of one or more of the CDRs of the anti-CD73 antibodies of the present disclosure or antigen-binding portions thereof described herein.

[0144] In some embodiments, the nucleic acid molecules and vectors can be used to generate mutated anti-CD73 antibodies. The antibodies can be mutated in the variable domains of the heavy and / or light chains, for example to alter the binding characteristics of the antibody. For example, the K D To increase or decrease k off Mutations may be made in one or more of the CDRs to increase or decrease the affinity or to change the binding specificity of the antibody. In some embodiments, one or more mutations are made at amino acid residues known to vary compared to germline in the monoclonal antibodies of the present disclosure. Mutations may be made in the CDRs or framework regions of the variable domain, or in the constant region. In certain embodiments, mutations are made in the variable domain. In specific embodiments, one or more mutations are made at amino acid residues known to vary compared to germline in the CDRs or framework regions of the variable domain of the antibody or antigen-binding portion thereof of the present disclosure.

[0145] In some embodiments, the framework regions are mutated so that the resulting framework regions have the amino acid sequence of the corresponding germline gene. For example, mutations can be made in the framework regions or constant regions to increase the half-life of the anti-CD73 antibody. See, for example, WO 00 / 09560. Mutations in the framework regions or constant regions can also be made to alter the immunogenicity of the antibody and / or to provide a site for covalent or non-covalent binding to another molecule. According to the present disclosure, the antibody can have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant region.

[0146] In some embodiments, the anti-CD73 antibodies or antigen-binding portions thereof of the present disclosure are expressed by inserting DNA encoding the partial or full-length light and heavy chains obtained as described above into an expression vector such that the genes are operably linked to the necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The antibody coding sequence may be ligated into a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody coding sequence. The expression vector and expression control sequences may be selected to be compatible with the expression host cell used. The antibody light chain coding sequence and the antibody heavy chain coding sequence may be inserted into the same or separate vectors and operably linked to the same or different expression control sequences (e.g., promoters). In some embodiments, both coding sequences are inserted into the same expression vector and may be operably linked to the same expression control sequence (e.g., a common promoter), separate identical expression control sequences (e.g., promoters), or different expression control sequences (e.g., promoters). The antibody coding sequence may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0147] A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed as described above. The HC and LC coding genes in such vectors may contain intron sequences that stabilize the associated mRNA, thereby providing overall antibody protein yield enhancement. The intron sequences are flanked by splice donor and splice acceptor sites that determine where RNA splicing occurs. The location of the intron sequence may be in either the variable or constant region of the antibody chain, or in both the variable and constant regions if multiple introns are used. Polyadenylation and transcription termination may occur at native chromosomal sites downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0148] In addition to the antibody chain genes, the recombinant expression vectors of the disclosure may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Those of skill in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of the protein desired, and the like. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian promoters such as the native immunoglobulin and actin promoters. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants, including promoters and vectors, as well as plant transformation instructions, are known in the art. See, for example, U.S. Patent No. 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0149] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on the host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0150] Host cells and methods for producing antibodies and antibody compositions The present disclosure also provides antibody compositions and methods of making the antibodies and antigen-binding portions thereof described herein. In some embodiments, the present disclosure relates to a method of making an anti-CD73 antibody or antigen-binding portion thereof described herein, comprising providing a recombinant host cell comprising a nucleotide sequence encoding a heavy chain or antigen-binding portion thereof, and a nucleotide sequence encoding a light chain or antigen-binding portion thereof, of an anti-CD73 antibody or antigen-binding portion thereof described herein; culturing the host cell under conditions suitable for expression of the antibody or antigen-binding portion; and isolating the resulting antibody or antigen-binding portion. The antibody or antigen-binding portion produced by such expression in such a recombinant host cell is referred to herein as a "recombinant" antibody or antigen-binding portion. The present disclosure also provides progeny of such host cells, and the antibody or antigen-binding portion produced by the same.

[0151] The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which a recombinant expression vector has been introduced. By definition, a recombinant host cell does not exist in nature. The present disclosure provides host cells that may contain, for example, a vector described herein. The present disclosure also provides host cells that contain, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portion thereof, a nucleotide sequence encoding a light chain or antigen-binding portion thereof, or both, of an anti-CD73 antibody or antigen-binding portion thereof described herein. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0152] Nucleic acid molecules encoding anti-CD73 antibodies and antigen-binding portions thereof, as well as vectors containing these nucleic acid molecules, can be used to transfect suitable mammalian, plant, bacterial, or yeast host cells. Transformation can be by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistec transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0153] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and several other cell lines. A particularly preferred cell line is selected by determining which cell line has a high expression level. Other cell lines that may be used are insect cell lines such as Sf9 or Sf21 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0154] Furthermore, expression of the antibodies or antigen-binding portions thereof of the present disclosure from production cell lines can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or in part in connection with European Patent Nos. 0216846, 0256055, 0323997 and 0338841.

[0155] Antibodies expressed by different cell lines or transgenic animals may have different glycosylation patterns from each other, however, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the disclosure, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modifications.

[0156] Pharmaceutical Compositions Another aspect of the present disclosure is a pharmaceutical composition comprising an anti-CD73 antibody or antigen-binding portion thereof, bispecific binding molecule, or antibody composition of the present disclosure as an active ingredient (or as the only active ingredient). The pharmaceutical composition may further comprise a pharma-ceutically acceptable excipient. In some embodiments, the pharmaceutical composition is intended for the amelioration, prevention, and / or treatment of cancer, such as the cancers described herein. In certain embodiments, the cancer is in tissues such as skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas. In certain embodiments, the cancer is melanoma, head and neck cancer, breast cancer (e.g., triple-negative breast cancer), bladder cancer, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), ovarian cancer, renal cell carcinoma, prostate cancer, colorectal cancer, cholangiocarcinoma, thyroid cancer, or testicular cancer.

[0157] A pharmaceutical composition of the disclosure comprises one or more anti-CD73 antibodies, antigen-binding portions, antibody compositions, or bispecific binding molecules of the disclosure, e.g., one or two anti-CD73 antibodies, antigen-binding portions, or bispecific binding molecules. In some embodiments, a composition comprises a single anti-CD73 antibody or antigen-binding portion thereof of the disclosure. In some embodiments, a composition comprises two separate anti-CD73 antibodies or antigen-binding portions thereof of the disclosure.

[0158] In some embodiments, a pharmaceutical composition may comprise at least one anti-CD73 antibody or antigen-binding portion thereof of the present disclosure, e.g., an anti-CD73 antibody or portion, and one or more additional antibodies that target one or more associated cell surface receptors, e.g., one or more cancer-associated receptors.

[0159] Generally, the antibodies, antigen-binding portions, and bispecific binding molecules of the disclosure are suitable for administration as a formulation in combination with one or more pharma- ceutically acceptable excipients, e.g., as described below.

[0160] The term "excipient" is used herein to describe any ingredient other than the compound of the present disclosure. The choice of excipient largely depends on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferable to include an isotonicity agent, such as a sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride, in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting agents or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody.

[0161] The pharmaceutical compositions of the present disclosure and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). Pharmaceutical compositions are preferably manufactured under GMP (Good Manufacturing Practice) conditions.

[0162] The pharmaceutical composition of the present disclosure can be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of active ingredient that would be administered to a subject, or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

[0163] A formulation of a pharmaceutical composition suitable for parenteral administration typically comprises the active ingredient combined with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In some embodiments of formulations for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffers (preferably up to pH 3-9), although for some applications they may be more suitably formulated as sterile nonaqueous solutions or as dry forms used in combination with an appropriate vehicle such as sterile pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, for example aqueous propylene glycol or dextrose. Such dosage forms may be suitably buffered if desired. Other parenterally administrable formulations that are useful include those which contain the active ingredient in microcrystalline form or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release.

[0164] In some embodiments, sterile injectable solutions can be prepared by incorporating the anti-CD73 antibody, its antigen-binding portion, bispecific binding molecule, or antibody composition in the required amount in an appropriate solvent containing one or a combination of ingredients listed above, as needed, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable composition can be brought about by including an agent that delays absorption, such as monostearate salts or gelatin, in the composition, and / or by using modified release coatings (e.g., delayed release coatings).

[0165] Antibodies of the disclosure may also be administered intranasally or by inhalation, typically in the form of a dry powder (alone, in a mixture, or as mixed component particles mixed, e.g., with suitable pharma- ceutically acceptable excipients) from a dry powder inhaler; as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer that uses electrohydrodynamics to produce a fine mist), or nebulizer, with or without a suitable propellant; or as nasal drops.

[0166] The antibodies and antibody portions of the present disclosure may also be formulated for oral route administration. Oral administration may involve swallowing so that the compound enters the digestive tract, and / or buccal, lingual, or sublingual administration, where the compound enters the bloodstream directly from the mouth. Formulations suitable for oral administration include solid, semi-solid, and liquid systems such as tablets; soft or hard capsules containing multiparticulates or nanoparticles, liquids, or powders; lozenges (including liquid-filled); chewable tablets; gels; fast dispersing dosage forms; films; obules; sprays; and buccal / mucoadhesive patches. Liquid formulations include suspensions, solutions, syrups, and elixirs. Such formulations may be used as fillers in soft or hard capsules (e.g., made from gelatin or hydroxypropylmethylcellulose), typically containing a carrier such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying and / or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.

[0167] Therapeutic Uses of the Antibodies and Compositions of the Disclosure In some embodiments, the anti-CD73 antibodies and antigen-binding portions thereof, anti-CD73 compositions, and bispecific binding molecules of the present disclosure are used to reduce CD73 activity in a mammal (e.g., a human) in need thereof. For example, a physician can enhance anti-tumor activity in a patient by administering an anti-CD73 antibody of the present disclosure alone or in combination (sequentially or simultaneously) with other therapeutic agents. The anti-CD73 antibody reduces the activity of CD73 and inhibits pathways that suppress anti-tumor responses in a patient.

[0168] In certain embodiments, the antibody or antigen-binding portion thereof, composition, or bispecific binding molecule is for use in treating a CD73-positive cancer, which may be in one or more tissues, such as skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas.

[0169] In some embodiments, the cancer treated by the anti-CD73 antibodies, antigen-binding portions, bispecific binding molecules, and / or antibody compositions of the disclosure is, for example, melanoma (e.g., advanced or metastatic melanoma), basal cell carcinoma of the skin, glioblastoma, glioma, gliosarcoma, astrocytoma, meningioma, neuroblastoma, adrenocortical carcinoma, squamous cell carcinoma of the head and neck, oral cancer, salivary gland cancer, nasopharyngeal cancer, breast cancer, thyroid cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and squamous cell lung cancer), esophageal cancer, gastroesophageal junction cancer, gastric cancer, gastrointestinal cancer, primary peritoneal cancer, liver cancer, hepatocellular carcinoma, biliary tract cancer, cholangiocarcinoma, colon cancer, colorectal cancer, ovarian cancer, fallopian tube cancer, bladder cancer, upper urinary tract cancer, urothelial cancer, The cancers may include, but are not limited to, renal cell carcinoma, kidney cancer, genitourinary cancer, cervical cancer, prostate cancer, testicular cancer, fibrosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, histiocytoma, pancreatic cancer, endometrial cancer, appendix cancer, advanced Merkel cell carcinoma, multiple myeloma, sarcoma, choriocarcinoma, erythroleukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, mast cell leukemia, small lymphocytic lymphoma, Burkitt lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, monocytic lymphoma, HTLV-associated T-cell leukemia / lymphoma, mesothelioma, and solid tumors. The cancer may be, for example, at an early, intermediate, late, locally advanced, or metastatic stage, may be relapsed or refractory to other therapeutics (e.g., other anti-CD73 therapeutics), or may have no standard treatment available.

[0170] In some embodiments, cancers treated by the anti-CD73 antibodies, antigen-binding portions, compositions, and / or bispecific binding molecules of the disclosure may include, for example, melanoma, head and neck cancer, breast cancer (e.g., triple-negative breast cancer), bladder cancer, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), ovarian cancer, renal cell carcinoma, prostate cancer, colorectal cancer, cholangiocarcinoma, thyroid cancer, and testicular cancer.

[0171] "Treat", "treating" and "treatment" refer to a method of alleviating or arresting a biological disorder and / or at least one of the symptoms associated therewith. As used herein, "alleviating" a disease, disorder or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder or condition. Furthermore, references herein to "treatment" include references to curative, palliative and prophylactic treatment.

[0172] "Therapeutically effective amount" refers to the amount of therapeutic agent being administered that relieves to some extent one or more of the symptoms of the disorder being treated. A therapeutically effective amount of an anti-cancer therapeutic agent may result in, for example, delayed tumor growth, tumor shrinkage, increased survival, elimination of cancer cells, delayed or reduced disease progression, reversal of metastasis, or other clinical endpoint desired by a medical professional.

[0173] The anti-CD73 antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules described herein may be administered alone or in combination with one or more other drugs or antibodies (or in any combination thereof). Thus, the pharmaceutical compositions, methods and uses described herein also encompass embodiments of combination (co-administration) with other active agents, as detailed below.

[0174] As used herein, the terms "co-administration," "co-administered," and "in combination with," referring to the anti-CD73 antibodies and antigen-binding portions thereof, antibody compositions, and bispecific binding molecules of the disclosure and one or more therapeutic agents, are intended to mean, refer to, and include: a) simultaneous administration of such combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent to a patient in need of treatment, where such components are formulated together in a single dosage form that releases said components to said patient substantially simultaneously; b) substantially simultaneous administration of such combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent to a patient in need of treatment, where such components are formulated separately from one another in separate dosage forms taken by said patient at substantially the same time, whereby said components are released to said patient at substantially the same time; c) sequential administration of such combination of an antibody / antigen-binding portion / antibody composition / bispecific binding molecule of the present disclosure and a therapeutic agent to a patient in need of treatment, where such components are formulated separately from one another in separate dosage forms taken by said patient at successive times with a significant time interval between each administration, whereby said components are released to said patient at substantially different times; and d) Sequential administration of such combinations of antibodies / antigen-binding portions / antibody compositions / bispecific binding molecules of the present disclosure and therapeutic agents to a patient in need of treatment, where such components are formulated together in a single dosage form that releases said components in a controlled manner, where they are released to said patient at the same and / or different times, simultaneously, sequentially and / or overlappingly, and where each portion can be administered by the same or different routes.

[0175] The anti-CD73 antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be administered without additional therapeutic treatments, i.e., as stand-alone therapy (monotherapy). Alternatively, treatment with the anti-CD73 antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may include at least one additional therapeutic treatment (combination therapy), such as an immunostimulant, an anti-cancer agent (e.g., a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, or a tyrosine kinase inhibitor), or a vaccine (e.g., a tumor vaccine).

[0176] In some embodiments, the antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule may be co-administered or formulated with another medicament / drug for treating cancer. The additional therapeutic treatment may include, for example, an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, and / or radiation therapy. In some embodiments, the additional therapeutic treatment may include a different anti-cancer antibody.

[0177] Medicaments comprising an anti-CD73 antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule described herein and at least one other agent (e.g., a chemotherapeutic agent, an anti-neoplastic agent, or an anti-angiogenic agent) may be used as a combination treatment for simultaneous, separate or sequential administration in the treatment of cancer. The other agent may be any agent suitable for treating the particular cancer in question, such as an agent selected from the group consisting of alkylating agents, e.g., platinum derivatives such as cisplatin, carboplatin, and / or oxaliplatin; plant alkaloids, e.g., paclitaxel, docetaxel, and / or irinotecan; antitumor antibiotics, e.g., doxorubicin (adriamycin), daunorubicin, epirubicin, idarubicin, mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and / or mitomycin; topoisomerase inhibitors, such as topotecan; antimetabolites, e.g., fluorouracil and / or other fluoropyrimidines; FOLFOX; osimertinib; cyclophosphamide; anthracyclines; dacarbazine; gemcitabine; or any combination thereof. In some embodiments, the anti-CD73 antibodies, or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules described herein re-establish responsiveness to other agents.

[0178] The anti-CD73 antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may also be used in combination with other anti-cancer therapies, such as vaccines, cytokines, enzyme inhibitors, immunostimulatory compounds, and T cell therapy. In the case of a vaccine, this may be, for example, a protein, peptide, or DNA vaccine containing one or more antigens associated with the cancer being treated, or a vaccine that includes dendritic cells with antigens. Suitable cytokines include, for example, IL-2, IFN-γ, and GM-CSF. One example of a class of enzyme inhibitors with anti-cancer activity is an indoleamine-2,3-dioxygenase (IDO) inhibitor, such as 1-methyl-D-tryptophan (1-D-MT). Adoptive T cell therapy refers to a variety of immunotherapy techniques that involve expanding or engineering a patient's own T cells to recognize and attack tumors.

[0179] It is also contemplated that the anti-CD73 antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be used in adjunctive therapy in conjunction with tyrosine kinase inhibitors, which are synthetic, primarily quinazoline-derived, small molecular weight molecules that interact with the intracellular tyrosine kinase domain of the receptor and inhibit ligand-induced receptor phosphorylation, e.g., by competing for the intracellular Mg-ATP binding site.

[0180] In some embodiments, the antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule is selected from the group consisting of A2AR, A1AR, A2BR, A3AR, ADA, ALP, BTLA, B7-H3, B7-H4, CTLA-4, CD27, CD28, CD39, CD40, CD47, CD55, CD122, CD137, CD160, CGEN-15049, CHK1, CHK2, CTLA-3, CEACAM (e.g., CEACAM-1 and / or may be used in combination with medicines / drugs that mediate immune system activation, including, but not limited to, agents that modulate expression or activity of CEACAM-5), GAL9, GITR, HVEM, LAG-3, LY108, LAIR1, ICOS, IDO, KIR, NKG2A, PAP, PD-1 / PD-L1 / PD-L2, OX40, TIGIT, TIM-3, TGFR-β, TNFR2, VISTA, LILRB2, CMTM6, and / or 2B4. In some embodiments, the agent modulates expression of CD39, NKG2A, LAG-3, TIM-3, or TNFR2. In certain embodiments, the agent is a small molecule inhibitor. In certain embodiments, the agent is an antibody or antigen-binding fragment thereof that binds to one of the above molecules. In specific embodiments, the agent is an anti-PD-L1 antibody (e.g., durvalumab or atezolizumab), an anti-PD-1 antibody, or an anti-CTLA-4 antibody. It is also contemplated that the anti-CD73 antibodies, or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be used in combination with cytokines (e.g., IL-1, IL-2, IL-12, IL-15, or IL-21), EGFR inhibitors, VEGF inhibitors, and the like.

[0181] In certain embodiments, the antibodies and antigen-binding portions, antibody compositions, or bispecific binding molecules of the disclosure may be administered in combination with another inhibitor of the adenosinergic pathway, which may target, for example, CD73, CD39, or CD38, or an adenosine receptor selected from A1R, A2AR, A2BR, and / or A3R. Such inhibitors include, but are not limited to, AR1 inhibitors (e.g., DPCPX, SCH58261, and FSPTP), A2AR inhibitors (e.g., PBF-509, CPI-444, AZD4635, ZM241385, AB928, istradefylline, SYN-115, ANR94, PSB1115, PSB603, and NIR178), A2B inhibitors (e.g., miR-128b, ATL801, aminophylline, MRS1754, IPDX, and PBF-1129), and A3R inhibitors (e.g., MRS1191, MRS1220, MRS1523, and CF-102). Other examples of such inhibitors include other anti-CD73 antibodies, as well as anti-CD39 and anti-CD38 antibodies. In some embodiments, an anti-CD73 antibody or antigen-binding portion thereof, bispecific antibody, or antibody composition of the disclosure may be administered in combination with A001421, APCP, oleculab, CPX-006 / CPI-006, CPX-016, NZV-930, BMS-986179, IPH53, PT199, durvalumab, atezolizumab, daratumumab, or isatuximab.

[0182] The present disclosure also contemplates the use of the sequences of the anti-CD73 antibodies or antigen binding portions described herein (e.g., the six CDRs or VH and VL sequences) in the preparation of chimeric antigen receptors, which may be for use in CAR-T technology.

[0183] It is understood that the antibodies and antigen-binding portions thereof, antibody compositions, and bispecific binding molecules of the present disclosure can be used in the methods of treatment described herein, can be used in the treatments described herein, and / or can be for use in the manufacture of a medicament for the treatments described herein. The present disclosure also provides kits and articles of manufacture that include the antibodies and antigen-binding portions thereof, antibody compositions, and bispecific binding molecules described herein.

[0184] Dose and Route of Administration The antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present disclosure may be administered in an amount effective to treat the condition in question, i.e., at dosages and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether the antibody is administered as a stand-alone treatment or in combination with one or more additional anti-cancer treatments.

[0185] Dosage regimens can be adjusted to provide the optimum desired response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for dosage unit forms of the present disclosure are generally dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds to treat susceptibility in an individual.

[0186] Therefore, based on the disclosure provided herein, those skilled in the art will understand that dosage and administration regimen will be adjusted according to the method well known in the therapeutic field. That is, the maximum tolerated dosage can be easily established, and the effective amount that provides a patient with a detectable therapeutic benefit can also be determined, as can the time requirement for administering each agent to provide a patient with a detectable therapeutic benefit. Thus, although specific dosage and administration regimen are exemplified herein, these examples in no way limit the dosage and administration regimen that can be provided to a patient when implementing the present disclosure.

[0187] It should be noted that dosage values ​​vary with the type and severity of the condition to be alleviated and may include single or multiple administrations. For any particular subject, a particular dosage regimen should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and it should be further understood that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the embodied compositions. Furthermore, dosage regimens using the compositions of the present disclosure may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, medical condition, the severity of the condition, the route of administration, and the specific antibody used. Thus, dosage regimens may vary widely, but may be routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or experimental values. Thus, the present disclosure encompasses intrapatient dose escalation as determined by one of ordinary skill in the art. It will be understood that the determination of appropriate dosages and regimens is well known in the relevant art and is encompassed by one of ordinary skill in the art once provided with the teachings disclosed herein.

[0188] An effective amount of tumor treatment may be measured by its ability to stabilize disease progression and / or improve symptoms in a patient, preferably by its ability to reverse disease progression, for example by reducing tumor size. The ability of an antibody, antigen-binding portion, antibody composition, or bispecific binding molecule of the present disclosure to inhibit cancer may be evaluated by in vitro assays, for example as described in the Examples, as well as in appropriate animal models predicting efficacy in human tumors. An appropriate dosing regimen will be selected to provide the optimal therapeutic response in each specific situation, and may be administered, for example, as a single bolus or as a continuous infusion, with possible adjustment of dosage indicated by the exigencies of each case.

[0189] The antibody or antigen-binding portion thereof, antibody composition, or bispecific binding molecule of the present disclosure may be administered by any method accepted in the art for administering peptides, proteins, or antibodies, and is typically suitable for parenteral administration. As used herein, "parenteral administration" includes any route of administration characterized by physical disruption of the tissue of interest and administration through tissue disruption, thus generally resulting in direct administration into the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administration by injection, application through a surgical incision, application through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intracisternal, intravenous, intraarterial, intrathecal, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion. Particular embodiments include intravenous and subcutaneous routes.

[0190] Diagnostic Uses and Compositions The antibodies and antigen-binding portions of the present disclosure are also useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the antibodies and antigen-binding portions can be used to detect and / or measure levels of CD73 in a sample from a patient (e.g., a tissue sample or a bodily fluid sample such as inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistology. The present disclosure further encompasses kits (e.g., diagnostic kits) that include the antibodies and antigen-binding portions described herein.

[0191] Products and Kits The present disclosure also provides articles of manufacture, e.g., kits, that include one or more containers (e.g., single-use or multi-use containers) that contain a pharmaceutical composition of an anti-CD73 antibody or antigen-binding portion thereof, composition, or bispecific binding molecule described herein, optionally an additional biologically active molecule (e.g., another therapeutic agent), and instructions for use. The antibody or antigen-binding portion, composition, or bispecific binding molecule, and any additional biologically active molecule may be packaged separately in suitable packing, such as a vial or ampoule made from non-reactive glass or plastic. In certain embodiments, the vial or ampoule holds a concentrated stock (e.g., 2x, 5x, 10x or more) of the antibody or antigen-binding portion, composition, or bispecific binding molecule, and optionally the biologically active molecule. In certain embodiments, an article of manufacture, such as a kit, includes a medical device for administering the antibody or antigen-binding portion, composition, or bispecific binding molecule and / or the biologically active molecule (e.g., a syringe and needle); and / or a suitable diluent (e.g., sterile water and saline). The present disclosure also includes methods of making said articles of manufacture.

[0192] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, will control.

[0193] Generally, the nomenclatures used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein.

[0194] Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprise" or "comprising", are understood to mean the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0195] All publications and other references mentioned herein are incorporated herein by reference in their entirety. Although certain documents are cited in this specification, the citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0196] In order that this disclosure may be better understood, the following examples are set forth. These examples are for illustrative purposes only, and are not to be construed in any way as limiting the scope of the disclosure. EXAMPLES

[0197] Example 1. Cloning of anti-CD73 antibodies from rat B cells Materials and Methods Antibodies against human CD73 were isolated from the antibody repertoire derived from OmniRat® rats (Osborn et al., J Immunol. 190(4):1481-90(2013)), a transgenic rat line from Ligand Pharmaceuticals Inc. that produces antibodies with fully human idiotypes. Cloning of rat-derived antibody genes from single-cell sorted antibody-secreting B cells (ASC) was performed by the Simplex™ antibody discovery technology (Meijer et al., J Mol Biol 358(3):764-72(2006)).

[0198] Antibody repertoire constructs encoding fully human immunoglobulins in IgG1-LALA format (see below) were transfected into HEK293 cells. Cell supernatants were screened for binding to CD73 expressed on the surface of CHO cells using flow cytometry in a high-throughput format. CD73-reactive clones were analyzed by DNA sequencing and DNA sequences encoding the antibodies were extracted. Selected antibody clones were expressed and functionally tested as described below.

[0199] The missense mutations in the amino termini of the heavy and light chains introduced by the use of degenerate primers in the Simplex™ cloning of the antibody-encoding cDNA fragment were corrected back to germline sequences. Table 1 shows the heavy and light chain variable domain nucleotide sequences of the germline antibodies designated 21028, 21046, 21127, and 21163. The correction process involved amino-terminal sequence correction to germline and codon usage optimization. Targets for matching with human germline sequences were identified by blast homology searches for the heavy and light chain variable regions.

[0200] The protein sequences of the variable domains, constant regions and complementarity determining regions (CDRs) of antibodies 21028, 21046, 21127 and 21163 are shown in Tables 2, 3 and 4, respectively.

[0201] result Table 1 shows the nucleotide sequences encoding the variable domains of antibodies 21028, 21046, 21127 and 21163. [Table 1] TIFF0007662617000002.tif246169 TIFF0007662617000003.tif26169

[0202] Table 2 shows the deduced amino acid sequences of antibodies 21028, 21046, 21127 and 21163. The CDRs are in bold / underlined. [Table 2]

[0203] Table 3 shows the heavy chain constant region amino acid sequence and the light chain constant region amino acid sequence (CH and CL, respectively). "IgG1LALA" refers to the presence of "LALA" mutations (L234A / L235A, numbered according to the Kabat numbering scheme) in the heavy chain, which are known to reduce the effector function of the Fc region of IgG1 antibodies (Hezareh et al., J Virol. 75(24):12161-68(2001); Hessell et al., Nature 449(7158):101-04(2007)). [Table 3]

[0204] Table 4 shows the heavy and light chain CDR amino acid sequences of antibodies 21028, 21046, 21127, and 21163, where the CDRs are defined according to the IMGT system. [Table 4]

[0205] Table 5 shows sequence information for antibodies 21028, 21046, 21127 and 21163. Unless otherwise stated, sequences are amino acid sequences. [Table 5]

[0206] Example 2. Screening of anti-CD73 antibodies in a soluble CD73 activity assay A panel of anti-CD73 antibodies was evaluated for their ability to inhibit the enzymatic activity of soluble recombinant CD73. Anti-CD73 antibodies were incubated with recombinant CD73 (SinoBiological Incorporated), AMP, and ATP at a concentration of 10 μg / mL for 30 minutes at 37° C. CD73 activity was investigated by measuring AMP inhibition of ATP detection using CellTiter-Glo® 2.0 (Promega Corporation) as described in Sachsenmeier et al., Journal of Biomolecular Screening 17(7):993-998 (2012).

[0207] Inhibition of CD73 enzymatic activity after treatment with anti-CD73 antibodies is shown in Figure 1. Strong variations in CD73 activity after treatment with different anti-CD73 antibodies are evident, indicating that some antibodies were not functional in this assay, whereas others strongly inhibited CD73 activity.

[0208] Example 3. Screening of anti-CD73 antibodies in a cell-based CD73 activity assay A panel of anti-CD73 antibodies was evaluated for their ability to inhibit the activity of CD73 expressed on cancer cell lines. Anti-CD73 antibodies were incubated with CD73-expressing cell lines at a concentration of 10 μg / mL for 30 minutes at 37° C., followed by addition of the CD73 substrate, AMP, and further incubation for 3 hours at 37° C. CD73 activity was investigated by adding ATP to the supernatant and measuring AMP inhibition of ATP detection using CellTiter-Glo® 2.0 (Promega Corporation) as described in Sachsenmeier et al., supra.

[0209] The activity of CD73 expressed on two different cancer cell lines after treatment with anti-CD73 antibodies is shown in Figure 2. It is clear that CD73 activity after treatment with different anti-CD73 antibodies strongly changed; some antibodies were not functional in this assay, while others strongly inhibited CD73 activity. Notably, the same ten antibodies showed the highest activity in both cell lines.

[0210] Example 4. Cloning of anti-CD73 reference antibody analogs Materials and Methods The amino acid sequences encoding the heavy and light chain variable domains of the antibody analogs in Table 6 were obtained from the listed patents or patent applications. The protein sequences were reverse translated into DNA sequences with human codon usage. The corresponding DNA sequences were gene synthesized and cloned into expression vectors containing human heavy or light chain constant regions to obtain expression of full-length antibody chains. The human antibody isotypes selected for expression are listed in the antibody format column along with additional mutations introduced into the Fc region, if applicable. CHO cells were transfected with the resulting expression plasmids using a standard protein expression system. The corresponding antibody supernatants were purified using standard Protein A purification column chromatography. [Table 6]

[0211] Example 5. Direct binding of anti-CD73 antibodies to CHO-S cells transfected with human or cynomolgus CD73 protein The binding of anti-CD73 antibodies 21028, 21046, 21127, and 21163 to human or cynomolgus CD73 protein expressed on CHO-S cells was assessed and compared to the binding of the oleculab analog.

[0212] Anti-CD73 antibodies were incubated with hamster CHO-S cell lines transiently expressing human or cynomolgus CD73 for 30 min at 4°C. Cells were washed twice and then incubated with AF647-conjugated secondary anti-human IgG(H+L) antibody for an additional 20 min. After the washing step, antibody binding was detected using a high-throughput flow cytometer iQue Screener PLUS (Sartorius), which measures the GeoMean of the AF647 signal in each well. All concentrations were assayed in triplicate and a 12-point titration curve was generated for each antibody.

[0213] The binding curves of the antibodies to human or cynomolgus CD73 expressed on cells are shown in Figure 3. The antibodies assayed bind to cell-presented human and cynomolgus CD73 proteins with different potencies and efficacies. In particular, mAb21127 binds to human and cynomolgus CD73 with the highest potency among the antibodies tested.

[0214] Example 6. Measurement of antibody and FAB fragment affinity to human and cynomolgus monkey CD73 extracellular domain (ECD) This example demonstrates binding of anti-CD73 Fab fragments and antibodies to recombinant human and cynomolgus CD73 extracellular domain (ECD) as measured by surface plasmon resonance (SPR).

[0215] Materials and Methods Kinetic binding analysis of anti-CD73 mAb and Fab fragments was performed by surface plasmon resonance (SPR) using a Continuous Flow Microspotter (CFM, Wasatch Microfluidics, Salt Lake City, USA) coupled with an IBIS MX96 SPR instrument (IBIS Technologies, The Netherlands). CD73 cDNA encoding the extracellular domain of human and cynomolgus CD73 was synthesized and cloned into vectors containing a CMV promoter and either a C-terminal 6x histidine sequence of His-tagged CD73 ECD (SEQ ID NO: 45) or a human IgG1 Fc sequence (AA P101-K330), respectively, fusing IgG1 Fc C-terminally to the cloned CD73 ECD. His-tagged and Fc-fusion constructs were transiently expressed using the ExpiCHO™ expression system and purified by standard Ni-NTA chromatography or standard MabSelect™ SuRe™ procedures, respectively. Anti-CD73 Fab fragments were generated by digesting full-length IgG1 antibody with GingisKHAN enzyme using a kit provided by Genovis (Sweden). The affinity of the Fab fragments was measured by capturing Fc-tagged antigen on a Ga-hu-IgG Fc SensEye® for 15 min using CFM. After spotting, the SensEye® was placed on an IBIS MX96 biosensor and the immobilized antigen was fixed by the SensEye FixIt kit. Kinetic analysis was performed by injecting monomeric Fab fragments at increasing concentrations from 0.8 nM to 300 nM. After each cycle of Fab fragment injection, the surface was regenerated with 10 mM glycine pH 3, 10% glycerol. Fab association was performed for 15 min and antigen dissociation was performed for 15 min. The affinity of full-length monoclonal antibodies (mAbs) was measured by capturing the mAbs on a Ga-hu-IgG Fc SensEye® (Ssens BV, The Netherlands) for 15 min using a Continuous Flow Microspotter (CFM, Wasatch Microfluidics, Salt Lake City, USA).After spotting, the SensEye was docked to an IBIS MX96 and the spotted mAbs were fixed by SensEye FixIt kit (Ssens BV, The Netherlands). Kinetic analysis was performed by applying a kinetic titration series injecting increasing concentrations of His-tagged antigen from 0.16 nM to 10 nM as 2-fold dilutions. The surface was regenerated with 100 mM H3PO4, pH 3, before the next cycle of cynomolgus antigen injection. mAb association was performed for 15 min and antigen dissociation was performed for 45 min. The recorded binding responses were expressed as association rates (k. on or k a ), dissociation rate (k off or k d ) and affinity (K D To calculate the ΔΨ constants, a simple Langmuir 1:1 binding model was fitted using Scrubber 2.0 software. Binding kinetic parameters were determined as the average of six independent measurement points.

[0216] result The binding affinity and kinetic parameters of anti-CD73 Fab fragments and full-length antibodies are shown in Tables 7 and 8, respectively. The full-length antibodies bound to human CD73 with high affinity in the sub-nanomolar range. All antibodies recognized human and cynomolgus CD73 with binding kinetics comparable to reference antibodies (11E1, oleclumab, and CPX006 analogs). All antibodies were characterized by very slow dissociation rate constants that contribute to the high affinities measured. The monovalent binding affinities of the Fab fragments were 20-245 times weaker than those of the corresponding full-length antibodies. The dissociation rates (kd) were strongly affected by the change in format, with Fab fragments showing much faster dissociation from CD73 than the intact antibody.

[0217] The difference in binding rates of the intact antibody and the Fab fragment suggests that the antibody binds to a CD73 homodimer and that the high affinity measured for the full-length antibody is due to an avidity effect. CD73 is known to exist as a non-covalent homodimer (Knapp et al., Structure 20(12):2161-2173 (2012)), and binding of 11E1 analogs and oleculab analogs was previously found to depend on bivalent binding to the CD73 homodimer, with similar differences in binding kinetics between the full-length antibody and the Fab fragment described here (Perrot, Cell Reports 27(8):2411-2425 (2019)).

[0218] SPR analysis of anti-CD73 antibodies and Fab fragments demonstrated that the anti-CD73 antibodies rely on bivalent binding to dimeric CD73, and that the binding affinity of the full-length antibodies exemplified herein is comparable to that of the reference mAb when bound to CD73 homodimers. [Table 7] [Table 8]

[0219] Example 7. Epitope binning of anti-CD73 antibodies This example describes the grouping of anti-CD73 antibodies into epitope bins based on pairwise competition patterns. Antibodies belonging to different epitope bins recognize different epitopes on the ECD of CD73.

[0220] Materials and Methods Paired antibody competition studies were performed by SPR using an IBIS-MX96 instrument (IBIS, The Netherlands). Anti-CD73 antibodies were diluted to 3 μg / mL in PBS and spotted onto a Ga-hu-IgG Fc SensEye® by capturing for 15 min using a Continuous Flow Microspotter, followed by blocking of remaining binding sites with Herceptin (Trastuzumab) and chemical cross-linking with the SensEye FixIt kit (IBIS, The Netherlands). After sensor preparation, antibody competition analysis was performed using a classical sandwich assay. CD73-His ECD antigen was diluted in PBS, 0.05% Tween20, and 200 nM Herceptin running buffer, then injected at a concentration of 10 nM and captured by the conjugate array of anti-CD73 antibodies. Then, individual injections of each of the CD73 antibodies diluted to 100 nM in running buffer were performed to establish the antibody competition pattern. Data were analyzed by Epitope Binning 2.0 (Wasatch, USA).

[0221] result The competition patterns of the 16 anti-CD73 antibodies are shown in Figure 4. The anti-CD73 antibody group tested is in four overlapping major epitope bins: 1, 2, 3, and 4. Bin 1 contained the oleclumab and 11E1 analogs, which cross-blocked each other. The oleclumab and 11E1 antibodies have been shown to bind overlapping epitopes at the top of the N-terminal domain and on opposite sides of the catalytic site of CD73 (Geoghegan et al., MABS 8(3):454-467 (2016), and Perrot et al., Cell Reports 27:2411-2425 (2019)). All antibodies in bin 1 cross-blocked the antibodies grouped in bin 2. Bin 2 contained antibodies 21385, 21127, and 21163, which showed comparable competition patterns, suggesting that the antibodies bind similar epitopes. Antibodies in bin 2 cross-blocked all tested antibodies except those in bin 4. The bin 4 antibodies could be divided into two subbins: bin 4a and 4b, which competed with one or more antibodies in bin 3. Antibody 21046 (bin 4a) and the CPX006 analog (bin 4b) competed with different antibodies in bin 3, indicating that the two antibodies have closely related, but not identical, binding epitopes.

[0222] In conclusion, antibodies 21127 and 21163 (bin 2) bind to a unique epitope of CD73 with some overlap with the epitopes of the 11E1 analog and oleculab analog (bin 1). The epitope of antibodies 21127 and 21163 is distinct from that of the CPX006 analog (bin 4). Antibody 21046 (bin 4a) binds to an epitope similar to the CPX006 analog epitope (bin 4b) but distinct from the epitopes of antibodies 21127 and 21163 (bin 2) and the 11E1 analog and oleculab analog (bin 1).

[0223] Example 8. Epitope mapping of anti-CD73 antibodies by CD73 mutagenesis This example illustrates how the binding epitopes of antibodies 21127, 21163, and 21046 can be divided into linear epitopes and individual contact residues by measuring the binding affinities and kinetics to 94 different CD73 variants.

[0224] Materials and Methods Protein sequences of human and rat (Rattus norvegicus) CD73 were downloaded from UniProt (accession numbers P21589 and P21588, respectively). Full-length protein sequences of cynomolgus monkey (Macaca fascicularis) and chicken (Gallus gallus) CD73 were downloaded from NCBI (XP_005552488.1 and XP_004940453.1, respectively). Surface-exposed amino acid residues were mapped using the publicly available CD73 structures PDB 4H2F (open), 4H2G (open), and 4H2I (closed). Surface-exposed residue positions that differed between human and rat CD73 were mutated to alanine. To map linear antibody epitopes, a CD73 chimeric protein was generated in which 10 amino acids in the human CD73 ECD sequence were sequentially exchanged with the chicken sequence in overlapping segments of 5 amino acids.

[0225] cDNA encoding the extracellular domain of human CD73 was synthesized and cloned into a vector containing a CMV promoter and human Ig Fc sequence (residues P101-K330) to fuse the Ig Fc C-terminally to the cloned CD73 ECD. Wild-type (wt) and mutant human CD73 Fc fusion constructs were made by standard gene synthesis techniques and proteins were expressed transiently in 2 mL cultures using the ExpiCHO™ Expression System.

[0226] After harvesting the human CD73 Fc fusion constructs, the supernatants were tested for binding to anti-CD73 Fab by surface plasmon resonance (SPR). Culture supernatants containing CD73 fusion proteins were immobilized on Ga-hu-lgG Fc SensEye® (Ssens BV, The Netherlands) for 15 min using a Continuous Flow Microspotter (CFM, Wasatch Microfluidics, Salt Lake City, USA). After spotting, the SensEye® was placed on an IBIS MX96 biosensor and the captured proteins were immobilized on the surface using the FixIT kit (Ssens BV, The Netherlands). Kinetic analysis was performed by applying a kinetic titration series in which monomeric Fab fragments of the antibodies of the invention were injected at increasing concentrations from 0.8 nM to 500 nM. After each cycle of Fab fragment injection, the surface was regenerated with 10 mM glycine pH 3, 10% glycerol. Fab association was performed for 15 min and antigen dissociation was performed for 15 min. The recorded binding response was calculated as the association rate (k on or k a ), dissociation rate (k off or k d ) and affinity (K D ) constants were fitted to a simple Langmuir 1:1 binding model using Scrubber2 software.

[0227] result The epitopes of CD73 recognized by antibodies 21127, 21163 and 21046, as well as 11E1, oleculab, and CPX006 analogs, were mapped using chimeric receptor constructs in which a 10 amino acid segment of the human CD73 sequence was replaced with the chicken sequence; or by alanine scanning, in which surface exposed amino acids that differ between human and rat CD73 were mutated to alanine. Binding affinities of antibodies to wild-type CD73 and mutants were measured by SPR, and a cutoff of at least a 5-fold affinity reduction compared to wt, or deviation from the 1:1 binding model, was used to find constructs with significant loss of binding to anti-CD73 antibodies (Table 9). [Table 9]

[0228] The identified linear epitopes and contact residues were mapped onto the crystal structure of the open state CD73 homodimer (Figure 5). The epitopes recognized by antibodies 21127, 21163, and 21046, as well as the 11E1, oleclumab, and CPX006 analogs, were all found to be on the N-terminal domain of CD73 (Figure 5). The epitopes of antibodies 21127 and 21163, as well as the 11E1 and oleclumab analogs, were found to be located on a surface perpendicular to the dimerization interface of the CD73 homodimer and on the opposite side to the catalytic center (Figure 5, panels A, B, D, and E). The epitopes of the four antibodies were found to differ by binding to different contact residues and linear epitopes. Antibodies that bind to the upper surface of CD73 can block enzyme activity by locking the enzyme in an open, inactive conformation and preventing it from transitioning to the closed, active state (Perrot et al., Cell Reports 27:2411-2425(2019)).

[0229] The epitopes of antibody 21046 and the CPX006 analog were found to be located in front of the N-terminal domain just above the catalytic center of CD73 (Figure 5, panels C and F). The two antibodies differ with respect to the linear epitope recognized and the contact residues.

[0230] In summary, epitope mapping at single amino acid resolution showed that the exemplified anti-CD73 antibodies each have distinct binding epitopes. Antibodies 21127 and 21163 bind to similar epitopes at the top of the N-terminal domain of CD73; these epitopes are distinct from those of 11E1, oleclumab, and the CPX006 analog. Antibodies 21046 and the CPX006 analog bind to overlapping, yet distinct, epitopes at the front of the N-terminal domain of CD73.

[0231] Example 9. Stoichiometry of antibody / CD73 complexes formed in solution This example describes the size of antibody / CD73 ECD complexes formed in solution as measured by SEC-MALS.

[0232] Materials and Methods Anti-CD73 antibody and His-tagged CD73 were analyzed in different ratios as well as individually to analyze the size of the complex formed between CD73 homodimer and anti-CD73 antibody. Samples were prepared by mixing 900 pmol of CD73-His with 900, 450, 90 or 0 pmol of antibody diluted in PBS, pH 7.4. Samples were incubated for 30 min at room temperature and subsequently separated using a UHPLC UltiMate 3000 (Thermo Scientific) and a SEC X-Bridge column (Waters) at a flow rate of 1.2 mL / min. Sample running buffer was 0.01 M citrate, 250 mM L-arginine·HCl, pH 6.0. After HPLC separation, all samples were analyzed using a MiniDAWN TREOS MALS detector (Wyatt) and an Optilab T-rEX refractive index detector (Wyatt). Data plots were generated using GraphPad Prism.

[0233] result Bivalent binding of antibodies to CD73 homodimers may lock the enzyme in an inactive conformation (Geoghegan et al., MABS 8(3):454-467 (2016) and Perrot et al., Cell Reports 27:2411-2425 (2019)). The blocking mechanism could be by cross-linking of CD73 homodimers or by binding to single CD73 dimers in the inactive state. To characterize the stoichiometry of the antibody / CD73 complexes formed in solution, the size of the protein complexes was determined by SEC-MALS. The size of the 1:1 antibody / CD73 homodimer complexes is in the range of 270-280 kDa, calculated from the individual molecular weights of the antibody and CD73 homodimer (approximately 150 and 125 kDa, respectively). Figures 6A and 6B and Table 10 show the SEC-MALS profiles and size of the complexes formed for antibodies 21127, 21163, and 21046, as well as 11E1, oleculab, and CPX006 analogs, which bind to CD73 homodimers at various molecular ratios. Individual runs of each of CD73 and antibody show monodisperse peaks consistent with the expected size of the protein, confirming the absence of aggregates (Figures 6A and 6B, dashed lines). The SEC-MALS profile of antibody 21127 binding to CD73 shows one major peak (peak 2) with an average size of 276 kDa, corresponding to the size of the predicted 1:1 21127:CD73 homodimer complex, and one smaller peak (peak 1) corresponding to an excess of either 21127 or CD73 dimers. Antibody 21163, as well as the 11E1 and CPX006 analogs, formed primarily 1:1 complexes, but tended to form higher order complexes at the highest antibody:CD73 ratios (1:1). Polydisperse higher order complexes were most prominent with the CPX006 and 11E1 analogs. Antibody 21046 and the oleculab analog formed large complexes, indicating that these antibodies primarily crosslink distinct CD73 dimers, which has also been shown for oleculab (MEDI9447) (Geoghegan et al, MABS 8(3):454-467(2016)). [Table 10]

[0234] The peaks in Table 10 correspond to peaks 1-4 seen from right to left in the chromatograms of Figures 6A and 6B. Peak 1 corresponds to either unbound / excess CD73 homodimer (-125 kDa) or unbound / excess antibody (-150 kDa). Peaks 2-4 correspond to mAb:CD73 complexes of various sizes and stoichiometries, with a complex size of -275 kDa representing a 1:1 stoichiometry.

[0235] As shown in Table 10 and Figures 6A and 6B, antibody 21127 binds to an epitope on the CD73 homodimer in a manner that results in a 1:1 complex independent of CD73 concentration. Antibodies 21163 and the 11E1 and CPX006 analogs bind CD73 dimers primarily as 1:1 complexes, but also show a tendency to form higher order complexes to various degrees. Antibody 21046 and the oleculab analog bind CD73 homodimers only as oligomeric complexes, showing cross-linking of multiple CD73 dimers.

[0236] Example 10. Functionality of anti-CD73 antibodies in soluble CD73 activity assays The ability of anti-CD73 antibodies 21028, 21046, 21127, and 21163 to inhibit the enzymatic activity of soluble recombinant CD73 in a soluble CD73 activity assay was evaluated in more detail and compared to the oleculab analog. Anti-CD73 antibodies were incubated with recombinant CD73, AMP, and ATP for 2 hours at 37° C. CD73 activity was investigated by measuring AMP inhibition of ATP detection using CellTiter-Glo® 2.0 (Promega Corporation) as described in Sachsenmeier et al., supra.

[0237] Inhibition of CD73 enzyme activity after treatment with the indicated concentrations of anti-CD73 antibodies or oleculab analogs is shown in FIG. 7. It is clear that the inhibitory function of anti-CD73 antibodies is concentration-dependent, and all of the anti-CD73 antibodies inhibit CD73 activity, albeit with different potencies and efficacies. Furthermore, oleculab analogs inhibit CD73 enzyme activity less effectively at higher antibody concentrations. Similar results have been published for oleculab (formerly MEDI9447) by MedImmune (Geoghegan et al., MABS 8(3):454-467(2016)). Antibodies 21028 and 21046 also inhibit CD73 enzyme activity slightly less at higher concentrations compared to intermediate concentrations. In contrast, antibodies 21127 and 21163 show maximal inhibition of CD73 enzyme activity at all concentrations above approximately 3 μg / mL.

[0238] Example 11. Functionality of anti-CD73 antibodies in a cell-based CD73 activity assay The ability of anti-CD73 antibodies 21028, 21046, 21127, and 21163 to inhibit the activity of CD73 expressed on cells was evaluated in more detail and compared to the oleclumab analog. Anti-CD73 antibodies were incubated with CD73-expressing human cell lines (Calu-6 and H292) or CD73-expressing cynomolgus monkey cell line (Cynom-K1) for 30 minutes at 37° C., followed by addition of the CD73 substrate, AMP, and further incubation for 3 hours at 37° C. CD73 activity was investigated by adding ATP to the supernatant and measuring AMP inhibition of ATP detection using CellTiter-Glo® 2.0 (Promega Corporation) as described in Sachsenmeier et al., supra.

[0239] Inhibition of CD73 activity after treatment with different anti-CD73 antibodies is shown in Figure 8. It is clear that the inhibitory function of the anti-CD73 antibodies is concentration dependent and that all of the antibodies inhibit both human and cynomolgus CD73 activity, albeit with different potencies and efficacies. Antibodies 21127 and 21163 show the highest efficacy in the two human cell lines, Calu-6 and H292, but all four antibodies tested are clearly superior to the oleculab analog in all three cell lines.

[0240] Example 12. Functionality of anti-CD73 antibodies in long-term cell-based CD73 activity assays The ability of anti-CD73 antibodies 21028, 21046, 21127, and 21163 to inhibit the activity of CD73 expressed on cell lines was evaluated in more detail and compared to the oleclumab analog in a long-term cell-based assay. Anti-CD73 antibodies were incubated with the CD73-expressing human cell line H292 for 30 minutes at 37° C., followed by addition of the CD73 substrate, AMP, and further incubation for 3, 6, or 24 hours at 37° C. CD73 activity was investigated by adding ATP to the supernatant and measuring AMP inhibition of ATP detection using CellTiter-Glo® 2.0 (Promega Corporation) as described in Sachsenmeier et al., supra.

[0241] Inhibition of CD73 activity following treatment with different anti-CD73 antibodies is shown in Figure 9. It is clear that the inhibitory function of the anti-CD73 antibodies is concentration dependent, and that all of the antibodies inhibit CD73 activity, albeit with different potencies and efficacies. Notably, antibodies 21127 and 21163 are further differentiated from the oleclumab analog (and the other two anti-CD73 antibodies tested) when cells are incubated with the antibodies for up to 24 hours.

[0242] Example 13. Efficacy of anti-CD73 antibodies against cancer cell lines Anti-CD73 antibodies 21127, 21046, 21163 were evaluated for their ability to inhibit the activity of CD73 expressed in a large panel of cancer cell lines representing a wide range of CD73 expression and activity levels. The cell panel spans several cancer indications across multiple tissues of origin as listed in Table 11.

[0243] Materials and Methods Anti-CD73 antibodies were incubated with CD73-expressing cell lines at a concentration of 25 μg / mL for 30 min at 37° C., followed by addition of the CD73 substrate, AMP, and further incubation for 3 h at 37° C. CellTiter-Glo® 2.0 (Promega Corporation) was used to assess cellular CD73 activity by adding ATP to the supernatant and measuring AMP inhibition of ATP detection. CD73 activity is detected via luminescence.

[0244] result The effect of anti-CD73 antibodies on the activity of CD73 expressed on 20 different cancer cell lines is shown in Figure 10. It is clear that CD73 activity was altered after treatment with different anti-CD73 antibodies. Some antibodies (21046 and oleclumab analogues) showed some ability to inhibit CD73 at lower cellular CD73 activity levels but were unable to inhibit the enzyme at the highest activity levels, while other antibodies (21127 and 21163) strongly inhibited CD73 activity even at the highest activity levels observed, for example, in Calu-6, NCI-H1775, KYSE-30 and Capan-2 cell lines. [Table 11]

[0245] Example 14. In vitro testing of anti-CD73 antibody activity in a cell-based viability assay This example describes the in vitro functional characterization of antibody 21127 in a viability assay using two cancer cell lines grown in the presence of 300 μM AMP.

[0246] Materials and Methods Antibodies were evaluated in vitro for their ability to directly inhibit the viability (survival and / or proliferation) of triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468. Cells were seeded at 1000 cells / well in 384-well plates in RPMI 1640 Glutamax (MDA-MB-231) or DMEM (MDA-MB-468) medium supplemented with 2% FBS and 1% P / S and incubated for 4 days in a humidified incubator at 37° C. with antibodies titrated from 25 μg / mL in the presence of 300 μM AMP (adenosine monophosphate, Sigma-Aldrich). Cell viability was quantified using WST-1 cell proliferation reagent (Roche) according to the manufacturer's instructions.

[0247] result It is clear that 21127 and the oleculab analogues differ with respect to the functional readout (Figure 11). Antibody 21127 showed a significant effect on both cell lines, reducing MBA-MB-231 to approximately 60% viable cells compared to untreated cells and MBA-MB-468 to 0% viable cells (i.e., no measurable viable cells in the medium background). The oleculab analogue showed no significant effect on the viability of the MBA-MB-231 cell line, while MBA-MB-468 was affected to some extent, reducing the number of viable cells to approximately 70-80% compared to untreated cells.

[0248] Similar effects may be expected in cancer cell lines derived from other tissues.

[0249] Example 15. Primary CD4 from healthy human donors with anti-CD73 antibodies + and CD8 + T cells and CD19 + Inhibition of CD73 expressed on B cells Primary CD4 from healthy human donors + and CD8 + T cells and CD19 + Anti-CD73 antibodies were evaluated for their ability to inhibit the activity of CD73 expressed on B cells.

[0250] Materials and Methods Primary CD4 IgE was isolated from buffy coat PBMCs (peripheral blood mononuclear cells) using magnetic (MACS) beads with the relevant specificity according to the manufacturer's instructions (Miltenyi Biotec). + and CD8 + T cells and CD19 + B cells were isolated. A 4-fold dilution titration of anti-CD73 antibodies at concentrations ranging from 50 to 0.003 μg / mL was incubated with the isolated primary cells for 30 min at 37 °C, followed by addition of the CD73 substrate, AMP, and incubation at 37 °C for 20 h (CD19 + B cells) or 40 hours (CD4 + and CD8 + T cells) were further incubated. CD73 activity was assessed using CellTiter-Glo® 2.0 (Promega Corporation) by adding ATP to the supernatant and measuring AMP inhibition of ATP detection.

[0251] result Primary CD4 after treatment with anti-CD73 antibody + and CD8 + T cells and CD19 + The activity of CD73 expressed on B cells is shown in Figure 12. It is clear that CD73 activity after treatment with antibody 21127 and the olecurab analog was strongly different. Higher concentrations of antibody 21127 demonstrated almost complete inhibition of CD73 activity in all primary cell types, whereas the olecurab analog showed no significant inhibition of CD19. + B cells and CD8 + Very limited inhibition of CD73 activity in T cells, as well as CD4 + Only partial inhibition was observed in peripheral blood CD4 T cells from healthy donors. + T cells have on average restricted CD73 expression, but CD19 + B cells express, on average, high levels of CD73 and CD8 +The ability of oleculab analogs to inhibit CD73 activity correlates with CD73 expression levels, as T cells express intermediate levels on average (Allard et al., Immunol Rev 276(1):121-144(2017)).

[0252] Example 16. Functionality of anti-CD73 antibodies in T cell proliferation assays Anti-CD73 antibodies 21028, 21046, 21127, and 21163 were identified as CD4 + Its ability to inhibit the activity of CD73 expressed on T cells was assessed in an in vitro assay and compared to the oleculab analog. + T cells were isolated from healthy donors and activated with anti-CD3 / CD28 beads (Thermo Fisher Scientific), AMPs and anti-CD73 antibodies for 48 h, followed by the addition of 3H-thymidine (PerkinElmer Corporation) for an additional 24 h. T cell proliferation was measured as 3H-thymidine incorporation and normalized to controls not treated with AMPs.

[0253] T cell proliferation after treatment with different anti-CD73 antibodies is shown in Figure 13. It is clear that the stimulatory function of anti-CD73 antibodies on T cell proliferation is concentration dependent and all of the antibodies stimulate T cell proliferation, albeit with different potencies and efficacies. All antibodies, except for the oleculab analog, fully restored T cell proliferation, with antibody 21127 doing so with the highest potency.

[0254] Example 17. Functionality of anti-CD73 antibodies in T cell activation assays Anti-CD73 antibody is CD4 + and CD8 + Its ability to inhibit the activity of CD73 expressed on T cells was assessed in an in vitro assay and compared to an oleculab analog.

[0255] CD4 + and CD8 +T cells were isolated from healthy donors and activated with anti-CD3 / CD28 beads (Thermo Fisher Scientific), AMP and anti-CD73 antibodies for 72 h, followed by harvesting of supernatants. T cell activation was measured as IFN-γ levels in the supernatants using ELISA (Thermo Fisher Scientific).

[0256] T cell activation after treatment with different anti-CD73 antibodies is shown in Figure 14. It is evident that the stimulatory function of antibody 21127 on T cell activation is concentration dependent, with 21127 resulting in higher IFN-γ levels than the oleclumab analog.

[0257] Example 18. Functionality of anti-CD73 antibodies in a one-way MLR assay The ability of anti-CD73 antibodies to inhibit the activity of CD73 in a one-way mixed lymphocyte reaction (MLR) was assessed in an in vitro assay and compared with an oleculab analog.

[0258] Dendritic cells (DCs) and CD4 cells isolated from two different healthy donors + T cells were co-cultured to induce alloantigen-specific responses leading to cytokine production and T cell activation and / or proliferation. DCs were cultured with 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng / mL interleukin-4 (IL-4) for 7 days to induce CD14 + CD4 cells differentiated from monocytes and isolated from peripheral blood mononuclear cells (PBMCs) derived from healthy donor material + T cells were mixed at a ratio of 1:10. One-way MLRs were incubated with 25 μg / mL anti-PD-1 antibody (12819; see WO 2017 / 055547), 50 μM AMP, and the indicated concentrations of anti-CD73 antibody for 48 h, followed by the addition of 3H-thymidine (PerkinElmer Corporation) for an additional 24 h. Proliferation was measured as 3H-thymidine incorporation and normalized to controls not treated with AMP.

[0259] T cell proliferation after treatment with different anti-CD73 antibodies is shown in Figure 15. It is evident that the stimulatory function of anti-CD73 antibodies on proliferation was concentration-dependent, with both antibody 21127 and the oleculab analogue stimulating T cell proliferation. However, only antibody 21127 fully restored proliferation.

[0260] Example 19. Combination of anti-CD73 and anti-PD-1 antibodies in a one-way MLR assay This example examines the ability of a combination of an anti-PD-1 antibody (12819) and an anti-CD73 antibody (21127) to enhance T cell activation in a one-way mixed lymphocyte reaction (MLR).

[0261] Dendritic cells (DCs) and CD4 cells isolated from two different healthy donors + T cells were co-cultured to induce alloantigen-specific responses leading to cytokine production and T cell activation and / or proliferation. DCs were cultured with 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng / mL interleukin-4 (IL-4) for 7 days to induce CD14 + CD4 cells differentiated from monocytes and isolated from peripheral blood mononuclear cells (PBMCs) derived from healthy donor material + T cells were mixed at a ratio of 1:10. One-way MLRs were incubated with anti-CD73 antibody 21127 and / or anti-PD-1 antibody (12819; see WO 2017 / 055547) with or without 50 μM AMP for 72 hours, and the supernatants were subsequently harvested. The combination of anti-CD73 antibody 21127 and anti-PD-1 antibody 12819 was combined at a 1:1 ratio of the two antibodies. T cell activation was measured by quantifying IFN-γ levels in the supernatants using ELISA (Thermo Fisher Scientific) and normalized to controls not treated with AMP.

[0262] T cell activation in the unidirectional MLR, as measured by IFN-γ levels after treatment with antibodies 12819 and / or 21127, is shown in Figure 16. It is clear that the anti-PD-1 antibody 12819 robustly activated the unidirectional MLR both without AMP and with and without co-incubation with antibody 21127 (top panel). Activation of the unidirectional MLR by the anti-PD-1 antibody was much less when AMP was added to the unidirectional MLR (bottom panel). However, combining the anti-PD-1 antibody with the anti-CD73 antibody 21127 at least partially restored robust T cell activation, highlighting the benefit of the anti-PD-1 antibody / anti-CD73 antibody combination.

[0263] Example 20. Functionality of anti-CD73 antibodies in B cell activation assays The ability of antibodies 21046 and 21127 to stimulate B cell activation was assessed in an in vitro B cell activation assay. PBMCs from healthy donors were stimulated overnight with 21046, 21127, or oleculab analog (10 μg / mL) and CD40 ligand (0.5 μg / mL). Flow cytometry analysis was performed to determine the activation of B cells (CD20 + ), and B cell activation was assessed as upregulation of B cell activation markers CD25, CD69, and CD83.

[0264] As shown in Figures 17A and 17B, antibody 21046 stimulates robust upregulation of B cell activation markers CD25, CD69 and CD83, whereas antibody 21127 and the oleculab analog have limited effects on B cell activation.

[0265] Example 21. Anti-CD73 antibody-induced reduction of CD73 levels in H292 cells The ability of anti-CD73 antibodies 21127, 21028, 21046, and 21163 to modulate CD73 levels in cell lines was evaluated and compared to the oleculab analog. Anti-CD73 antibodies were incubated at 25 μg / mL with the human cell line H292 for 24 hours at 37° C., followed by cell lysis and assessment of CD73 levels using Simple Western technology (ProteinSimple).

[0266] Figure 18 shows CD73 levels after treatment with antibodies 21127, 21028, 21046, or 21163, or an oleculab analog. Incubation with anti-CD73 antibody 21046 or an oleculab analog resulted in some CD73 downregulation, while other anti-CD73 antibodies had modest effects, if any, on CD73 levels.

[0267] Example 22. Functionality of anti-CD73 antibodies in human tumor xenograft models This example demonstrates the ability of anti-CD73 antibody 21127 to inhibit the enzymatic activity of CD73 expressed on cells isolated from human tumor xenografts.

[0268] Materials and Methods The human melanoma cell line A375 was inoculated subcutaneously into the flank of 6-8 week old female NOG or NOD-scid mice. In one example (Figure 19, top panel), human PBMCs were injected intraperitoneally 1 day after tumor cell inoculation. Tumors were measured in two dimensions by calipers three times a week, and tumor volumes (mm 3 ) to the formula: (width) 2Calculations were made according to x length x 0.5. Mice were treated three times a week by intraperitoneal injection of vehicle buffer or (1) antibody 21127, (2) olecurumab analog, or (3) antibody 21127 combined with anti-mouse CD73 antibody TY / 23 (BioXcell). Unlike olecurumab analog, antibody 21127 is not cross-reactive to mouse CD73. Therefore, antibody 21127 was combined with anti-mouse CD73 antibody TY / 23 to compare inhibition of CD73 activity in tumor masses that also contain mouse cells expressing CD73 (endothelial and stromal cells). Antibodies were administered at 5 mg / kg, 20 mg / kg, or 50 mg / kg. At different time points after cessation of treatment, tumors were harvested and dissociated using a tumor dissociation kit and a gentleMACS Octo Dissociator (Miltenyi). The resulting cell suspension was incubated with the CD73 substrate AMP for 3 hours at 37°C. CD73 activity was examined by adding ATP to the supernatants and measuring AMP inhibition of ATP detection by CellTiter-Glo® 2.0 (Promega Corporation) as described in Sachsenmeier et al., supra.

[0269] result Antibody 21127 demonstrated dose-dependent inhibition of CD73 activity in tumors harvested from PBMC-humanized mice implanted with the A375 human melanoma cell line after repeated dosing over a 2-week period (Figure 19, top panel). The effect on CD73 enzyme activity was found to persist long-term after treatment was discontinued.

[0270] As shown in Figure 19, bottom panel, the oleculab analog inhibited CD73 activity for 16 days after treatment, with enzyme activity fully restored by day 28 after treatment. In contrast, antibody 21127, alone or in combination with TY / 23, showed sustained inhibition of CD73 activity for 28 days after treatment was discontinued.

[0271] Example 23. In vivo efficacy of anti-CD73 antibodies in immune cell-free human xenograft tumor models This example demonstrates the ability of anti-CD73 antibodies to inhibit tumor growth in a human triple-negative breast cancer xenograft model.

[0272] Human MDA-MB-231 triple-negative breast cancer cells were inoculated subcutaneously into the flank of 6- to 8-week-old female NOD-scid mice. Tumors were measured in two dimensions by calipers three times a week, and tumor volumes (mm 3 ) to the formula: (width) 2 Calculations were made according to x length x 0.5. Mice were treated twice weekly with a total of 16 treatments by intraperitoneal injection of vehicle buffer, antibody 21127, or oleculab analog, followed by an observation period. Antibodies were administered at 10 mg / kg. Two-way ANOVA with Bonferroni's multiple comparison test was applied to compare tumor volumes at each time point between treatment groups. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

[0273] Antibody 21127 and olecurab analogs showed significant tumor inhibitory effects during treatment (Figure 20). Regrowth of tumors treated with olecurab analogs was observed after treatment was discontinued. In contrast, mice treated with antibody 21127 effectively controlled tumor growth during treatment, with limited increase in tumor size over the 60 days following treatment.

[0274] Example 24. In vivo efficacy of anti-CD73 antibodies in human xenograft tumor models in mice reconstituted with human PBMCs This example demonstrates the in vivo efficacy of anti-CD73 antibody 21127 in PBMC-humanized mice engrafted with human lung cancer cells or human melanoma cells.

[0275] Cells from the human lung cancer cell line Calu-6 or the human A375 melanoma cell line were implanted subcutaneously into NOG mice one day prior to intraperitoneal injection of human PBMCs. Treatment began on the day of PBMC injection and mice were treated with intraperitoneal injection of 10 mg / kg vehicle buffer or anti-CD73 antibody 21127 three times per week for a total of six treatments (n=10 / group). Tumors were measured three times per week in two dimensions by calipers and tumor volumes (mm 3 ) to the formula: (width) 2 The mean tumor volume was calculated by ×length×0.5. Two-way ANOVA with Bonferroni's multiple comparison test was applied to compare tumor volumes at each time point between treatment groups. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc.).

[0276] As shown in Figure 21, treatment with antibody 21127 resulted in significant tumor growth delay (P<0.05 vs. vehicle control) in two human tumor xenograft models (Calu-6 and A375) in mice reconstituted with human PBMCs. Each graph in Figure 21 represents one human PBMC donor.

Claims

1. a) SEQ ID NOs: 17-22, respectively; b) SEQ ID NOs: 23-28, respectively; c) SEQ ID NOs: 29-34, respectively; or d) SEQ ID NOs: 35 to 40, respectively An anti-CD73 antibody or an antigen-binding portion thereof, comprising the H-CDR1-3 and L-CDR1-3 amino acid sequences of:

2. a) SEQ ID NOs: 9 and 13, respectively; b) SEQ ID NOs: 10 and 14, respectively; c) SEQ ID NOs: 11 and 15, respectively; or d) SEQ ID NOs: 12 and 16, respectively 2. The anti-CD73 antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain variable domain amino acid sequence and a light chain variable domain amino acid sequence that are at least 90% identical to the amino acid sequence of

3. a) SEQ ID NOs: 9 and 13, respectively; b) SEQ ID NOs: 10 and 14, respectively; c) SEQ ID NOs: 11 and 15, respectively; or d) SEQ ID NOs: 12 and 16, respectively 2. The anti-CD73 antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain variable domain and a light chain variable domain comprising the amino acid sequence of:

4. a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 9 and 41 and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 13 and 42; b) a HC comprising the amino acid sequences of SEQ ID NOs: 10 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 14 and 42; c) a HC comprising the amino acid sequences of SEQ ID NOs: 11 and 41 and a LC comprising the amino acid sequences of SEQ ID NOs: 15 and 42; or d) HC comprising the amino acid sequences of SEQ ID NOs: 12 and 41 and LC comprising the amino acid sequences of SEQ ID NOs: 16 and 42 4. An anti-CD73 antibody comprising:

5. a) inhibiting the activity of soluble CD73 in vitro; b) inhibiting the activity of CD73 on Calu-6 cells in vitro; c) inhibiting the activity of CD73 on H292 cells in vitro; d) specifically binds to human and cynomolgus CD73 expressed on CHO-S cells; e) A K of 1 nM or less as measured by SPR Ｄ binds to the ECD of human CD73 at f) a K of 0.7 nM or less as measured by SPR Ｄ binds to the ECD of cynomolgus monkey CD73 at g) does not bind to the same epitope on CD73 as oleclumab, CPX006, and / or 11E1; h) binds to an epitope on the CD73 homodimer in a manner that produces a 1:1 complex; i) inhibits soluble CD73 activity more effectively than oleculab in vitro; j) inhibits CD73 activity on Calu-6, H292, and Cynom-K1 cells in vitro; k) inhibits CD73 activity on Calu-6, NCI-H1775, KYSE-30, and Capan-2 cells in vitro; l) inhibiting the proliferation of MDA-MB-231 and MDA-MB-468 cells in vitro; m) In vitro primary CD4 ＋ and CD8 ＋ T cells and CD19 ＋ Inhibits CD73 activity on B cells; n) In vitro CD4 ＋ restores T cell proliferation; o) In vitro CD4 ＋ and CD8 ＋ Activates T cells; p) in combination with anti-PD-1 antibodies, restores T cell proliferation in a one-way mixed lymphocyte reaction (MLR) in the presence of AMP; q) in combination with anti-PD-1 antibodies in a one-way MLR to enhance T cell activation in the presence of AMP; r) does not stimulate B cell activation in vitro; s) does not reduce the levels of CD73 in H292 cells in vitro by more than 25%; t) inhibiting CD73 activity in tumors harvested from PBMC-humanized mice implanted with A375 cells; u) inhibiting tumor growth in vivo in NOD-scid mice implanted with MDA-MB-231 cells; v) inhibiting in vivo tumor growth in PBMC-humanized mice implanted with Calu-6 cells; and w) inhibiting in vivo tumor growth in PBMC-humanized mice implanted with A375 cells The anti-CD73 antibody or antigen-binding portion of any one of claims 1 to 4, having at least one property selected from the group consisting of:

6. The anti-CD73 antibody of any one of claims 1 to 4, which is an IgG.

7. IgG １ The anti-CD73 antibody of claim 6,

8. F Ｃ The anti-CD73 antibody of any one of claims 1 to 7, comprising at least one mutation in the region.

9. IgG １ and comprising a mutation at one or more of heavy chain amino acid positions 234 and 235, numbered according to the EU numbering scheme.

10. The anti-CD73 antibody of claim 9, wherein one or both of the amino acid residues at positions 234 and 235 are mutated from Leu to Ala.

11. A pharmaceutical composition comprising the anti-CD73 antibody or antigen-binding portion thereof according to any one of claims 1 to 10, and a pharma- ceutically acceptable excipient.

12. 12. The pharmaceutical composition of claim 11, further comprising an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, or a CD73 pathway inhibitor.

13. 11. An isolated recombinant nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain, or an antigen-binding portion thereof, and a nucleotide sequence encoding the light chain, or an antigen-binding portion thereof, of an anti-CD73 antibody according to any one of claims 1 to 10.

14. 14. The isolated recombinant nucleic acid molecule of claim 13, comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 8.

15. 15. A vector comprising the isolated nucleic acid molecule of claim 13 or 14, further comprising an expression control sequence.

16. A host cell comprising a nucleotide sequence encoding the heavy chain or an antigen-binding portion thereof, and a nucleotide sequence encoding the light chain or an antigen-binding portion thereof, of an anti-CD73 antibody according to any one of claims 1 to 10.

17. 20. A method for producing an anti-CD73 antibody or antigen-binding portion thereof, comprising providing a host cell as described in claim 16, culturing the host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion.

18. A bispecific binding molecule comprising an antigen-binding portion of an anti-CD73 antibody according to any one of claims 1 to 10 and an antigen-binding portion of another, different antibody.

19. a) reducing CD73 activity in a patient; b) The patient's CD4 ＋ Increases T-cell proliferation; c) stimulating the patient's immune system; or d) Treating cancer in a patient 19. A pharmaceutical composition for treating a patient suffering from atopic dermatitis, comprising an effective amount of an anti-CD73 antibody or antigen-binding portion thereof according to any one of claims 1 to 10, or a bispecific binding molecule according to claim 18.

20. The pharmaceutical composition of claim 19, wherein the patient has cancer originating in a tissue selected from the group consisting of skin, lung, intestine, colon, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bone, bladder, breast, stomach, uterus, cervix, and pancreas.

21. The pharmaceutical composition of claim 19, wherein the patient has melanoma, head and neck cancer, breast cancer, bladder cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, colorectal cancer, cholangiocarcinoma, thyroid cancer, or testicular cancer.

22. The pharmaceutical composition of claim 19, wherein the treatment further comprises an immunostimulant, a vaccine, a chemotherapeutic agent, an anti-neoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, a CD73 pathway inhibitor, or radiation therapy.

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