Anti-CD73 antibody

Abstract

This invention relates to anti-CD73 antibodies and methods of using them to treat diseases and conditions related to CD73 activity, such as cancer.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 896,908, filed September 6, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. An electronic copy of the sequence list was created on September 1, 2020, and is named 022675_WO055_SL.txt, with a size of 30,355 bytes. Background of the Invention

[0006] Tumor cells are surrounded by a complex microenvironment (tumor microenvironment or TME) that influences every step of tumorigenesis. Within the TME, increased concentrations of immunomodulatory factors such as adenosine help tumor cells overcome the host's antitumor immune response.

[0007] Adenosine binds to four different receptors (ARs) expressed on various immune cells, such as CD4+ and CD8+ T cells and natural killer (NK) cells. These purinergic G protein-coupled receptors, A1R, A2AR, A2BR, and A3R, each exhibit unique characteristics and cellular and tissue distribution. Adenosine binding to ARs inhibits the antitumor responses of T cells and NK cells and also promotes the development and activation of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), thereby promoting cancer progression. In addition to the immunomodulatory effects of adenosine receptors, their signaling may also directly influence the survival and proliferation of cancer cells.

[0008] The accumulation of adenosine in the tumor mesenchymal exchange (TME) is mediated by cell surface enzymes CD73 and CD39, which play a role in the pathway of converting extracellular ATP into adenosine. CD73, also known as 5'-NT, is an extracellular enzyme composed of two 65kD subunits linked by an α-helical linker to form a homodimer. Many cancer cells overexpress CD73, especially under tumor hypoxic conditions, where CD73 mediates the hydrolysis of 5'-AMP to adenosine. Studies have shown that high expression of CD73 leads to poor prognosis in various cancers, including triple-negative breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, and melanoma. Inhibition of CD73 may be an effective cancer treatment. Invention Overview

[0010] This disclosure relates to novel recombinant antibodies targeting CD73, pharmaceutical compositions comprising one or more of these antibodies, and the use of said antibodies and pharmaceutical compositions for the treatment of cancer. Compared to currently available treatments for such cancers (including antibody therapies), the antibodies and compositions described herein are expected to provide superior clinical responses, either alone or in combination with another cancer therapeutic agent.

[0011] In some aspects, this disclosure provides an anti-CD73 antibody or its antigen-binding portion, wherein the antibody binds to the same epitope of antibody-binding human CD73 comprising the following:

[0012] a) Heavy chain (HC) containing the amino acid sequences of SEQ ID NO: 9 and 41 and light chain (LC) containing the amino acid sequences of SEQ ID NO: 13 and 42;

[0013] b) HC containing the amino acid sequences of SEQ ID NO:10 and 41 and LC containing the amino acid sequences of SEQ ID NO:14 and 42;

[0014] c) HC containing the amino acid sequences of SEQ ID NO:11 and 41 and LC containing the amino acid sequences of SEQ ID NO:15 and 42; or

[0015] d) HC containing the amino acid sequences of SEQ ID NO:12 and 41 and LC containing the amino acid sequences of SEQ ID NO:16 and 42.

[0016] In some embodiments, the heavy chain of the antibody comprises:

[0017] i) Heavy chain complementarity-determining regions (H-CDRs)-1-3 containing the amino acid sequences of SEQ ID NO:17-19 respectively;

[0018] ii) A heavy chain variable domain (VH) containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:9;

[0019] iii) VH containing the amino acid sequence of SEQ ID NO:9; or

[0020] iv) Heavy chain (HC) containing the amino acid sequences of SEQ ID NO: 9 and 41;

[0021] And the light chain of the antibody comprises:

[0022] i) Light chain complementarity-determining regions (L-CDRs)-1-3, each containing an amino acid sequence of SEQ ID NO:20-22;

[0023] 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;

[0024] iii) A VL containing the amino acid sequence of SEQ ID NO:13; or

[0025] iv) Light chains (LC) containing the amino acid sequences of SEQ ID NO:13 and 42.

[0026] In some embodiments, the heavy chain of the antibody comprises:

[0027] i) Heavy chain complementarity-determining regions (H-CDRs)-1-3 containing the amino acid sequences of SEQ ID NO:23-25 ​​respectively;

[0028] ii) A heavy chain variable domain (VH) comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:10;

[0029] iii) VH containing the amino acid sequence of SEQ ID NO:10; or

[0030] iv) Heavy chains (HC) containing the amino acid sequences of SEQ ID NO:10 and 41;

[0031] And the light chain of the antibody comprises:

[0032] i) Light chain complementarity-determining regions (L-CDRs)-1-3, each containing an amino acid sequence of SEQ ID NO:26-28;

[0033] 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:14;

[0034] iii) A VL containing the amino acid sequence of SEQ ID NO:14; or

[0035] iv) Light chains (LC) containing the amino acid sequences of SEQ ID NO:14 and 42.

[0036] In some embodiments, the heavy chain of the antibody comprises:

[0037] i) Heavy chain complementarity-determining regions (H-CDR)-1-3 respectively containing the amino acid sequences of SEQ ID NO:29-31;

[0038] ii) A heavy chain variable domain (VH) containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:11;

[0039] iii) VH containing the amino acid sequence of SEQ ID NO:11; or

[0040] iv) Heavy chains (HC) containing the amino acid sequences of SEQ ID NO:11 and 41;

[0041] And the light chain of the antibody comprises:

[0042] i) Light chain complementarity-determining regions (L-CDRs)-1-3, each containing an amino acid sequence of SEQ ID NO:32-34;

[0043] 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:15;

[0044] iii) A VL containing the amino acid sequence of SEQ ID NO:15; or

[0045] iv) Light chains (LC) containing the amino acid sequences of SEQ ID NO:15 and 42.

[0046] In some embodiments, the heavy chain of the antibody comprises:

[0047] i) Heavy chain complementarity-determining regions (H-CDRs)-1-3 containing the amino acid sequences of SEQ ID NO:35-37 respectively;

[0048] ii) A heavy chain variable domain (VH) comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:12;

[0049] iii) VH containing the amino acid sequence of SEQ ID NO:12; or

[0050] iv) Heavy chains (HC) containing the amino acid sequences of SEQ ID NO:12 and 41;

[0051] And the light chain of the antibody comprises:

[0052] i) Light chain complementarity-determining regions (L-CDRs)-1-3, each containing an amino acid sequence of SEQ ID NO:38-40;

[0053] 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:16;

[0054] iii) A VL containing the amino acid sequence of SEQ ID NO:16; or

[0055] iv) Light chains (LC) containing the amino acid sequences of SEQ ID NO:16 and 42.

[0056] In some embodiments, this disclosure provides an anti-CD73 antibody or its antigen-binding portion, wherein said antibody comprises the following H-CDR1-3 and L-CDR1-3 amino acid sequences:

[0057] a) These are SEQ ID NO: 17 to 22 respectively;

[0058] b) These are SEQ ID NO: 23 to 28 respectively;

[0059] c) are SEQ ID NO: 29 to 34 respectively; or

[0060] d) These are SEQ ID NO:35 to 40 respectively.

[0061] In some embodiments, this disclosure provides an anti-CD73 antibody or its antigen-binding portion, wherein said antibody comprises 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 following amino acid sequence:

[0062] a) These are SEQ ID NO:9 and 13, respectively;

[0063] b) These are SEQ ID NO: 10 and 14, respectively;

[0064] c) are SEQ ID NO:11 and 15 respectively; or

[0065] d) are SEQ ID NO:12 and 16 respectively.

[0066] In some embodiments, this disclosure provides an anti-CD73 antibody or its antigen-binding portion, wherein said antibody comprises a heavy chain variable domain and a light chain variable domain containing the following amino acid sequence:

[0067] a) These are SEQ ID NO:9 and 13, respectively;

[0068] b) These are SEQ ID NO: 10 and 14, respectively;

[0069] c) are SEQ ID NO:11 and 15 respectively; or

[0070] d) are SEQ ID NO:12 and 16 respectively.

[0071] In some embodiments, this disclosure provides an anti-CD73 antibody comprising the following:

[0072] a) Heavy chain (HC) containing the amino acid sequences of SEQ ID NO: 9 and 41 and light chain (LC) containing the amino acid sequences of SEQ ID NO: 13 and 42;

[0073] b) HC containing the amino acid sequences of SEQ ID NO:10 and 41 and LC containing the amino acid sequences of SEQ ID NO:14 and 42;

[0074] c) HC containing the amino acid sequences of SEQ ID NO:11 and 41 and LC containing the amino acid sequences of SEQ ID NO:15 and 42; or

[0075] d) HC containing the amino acid sequences of SEQ ID NO:12 and 41 and LC containing the amino acid sequences of SEQ ID NO:16 and 42.

[0076] In some embodiments, this disclosure provides an anti-CD73 antibody or its antigen-binding portion thereof that binds to an epitope on human CD73, said epitope comprising:

[0077] a) Amino acid residues R73, R109, and D168 of SEQ ID NO:43;

[0078] b) Amino acid residue R109 of SEQ ID NO:43; or

[0079] c) Amino acid residues I301, S302 and H304 of SEQ ID NO:43.

[0080] In some embodiments, this disclosure provides an anti-CD73 antibody or its antigen-binding portion thereof that binds to an epitope on human CD73, said epitope comprising:

[0081] a) Amino acid residues 27 to 31, 61 to 75 and 161 to 170 of SEQ ID NO:43;

[0082] b) Amino acid residues 61 to 70 and 161 to 170 of SEQ ID NO:43; or

[0083] c) Amino acid residues 27 to 31, 266 to 270 and 291 to 305 of SEQ ID NO:43.

[0084] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein has at least one property selected from the following:

[0085] a) In vitro inhibition of soluble CD73 activity;

[0086] b) Inhibit CD73 activity on Calu-6 cells in vitro;

[0087] c) Inhibit CD73 activity on H292 cells in vitro;

[0088] d) Specifically binds to human and cynomolgus monkey CD73 expressed on CHO-S cells;

[0089] e) ECD binding to human CD73, K measured by SPR D 1 nM or less;

[0090] f) ECD bound to CD73 in cynomolgus monkeys, K measured by SPR D 0.7 nM or less;

[0091] g) Does not bind to the same epitope of CD73 as oleclumab, CPX006 and / or 11E1;

[0092] h) binds to epitopes on CD73 homodimers in a manner that generates a 1:1 complex;

[0093] i) It inhibits soluble CD73 activity more effectively in vitro than olemumab;

[0094] j) In vitro inhibition of CD73 activity in Calu-6, H292 and Cynom-K1 cells;

[0095] k) In vitro inhibition of CD73 activity in Calu-6, NCI-H1775, KYSE-30 and Capan-2 cells;

[0096] l) In vitro inhibition of the survival and / or proliferation of MDA-MB-231 and MDA-MB-468 cells;

[0097] m) In vitro inhibition of primary CD4 + and CD8 + T cells and CD19 + CD73 activity on B cells;

[0098] n) In vitro CD4 recovery + T cell proliferation;

[0099] o) In vitro activated CD4 + and CD8 + T cells;

[0100] p) Combined with anti-PD-1 antibody, T cell proliferation was restored in the presence of AMP in a single-channel mixed lymphocyte response (MLR);

[0101] q) When combined with an anti-PD-1 antibody, T cell activation is enhanced in the presence of AMP in a single-channel MLR.

[0102] r) It does not stimulate B cell activation in vitro;

[0103] s) It does not reduce CD73 levels in H292 cells by more than 25% in vitro;

[0104] t) Inhibit CD73 activity in tumors harvested from PBMC humanized mice transplanted with A375 cells;

[0105] u) In vivo inhibition of tumor growth in NOD-scid mice transplanted with MDA-MB-231 cells;

[0106] v) In vivo inhibition of tumor growth in PBMC humanized mice transplanted with Calu-6 cells; and

[0107] w) In vivo inhibition of tumor growth in PBMC humanized mice transplanted with A375 cells.

[0108] In a specific implementation, 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 aforementioned characteristics.

[0109] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein is IgG, such as IgG1. This antibody can be used in F... C The region contains at least one mutation. For example, the antibody could be IgG1 and contain mutations at one or more heavy chain amino acid positions 234 and 235, wherein the heavy chain amino acid positions are determined according to... Numbering scheme number. In the specific implementation, one or two amino acid residues at positions 234 and 235 are mutated from Leu to Ala.

[0110] In some embodiments, this disclosure provides a pharmaceutical composition comprising the anti-CD73 antibody or its antigen-binding portion described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition may further comprise one or more of an immunostimulant, vaccine, chemotherapeutic agent, antitumor agent, antiangiogenic agent, tyrosine kinase inhibitor, and CD73 pathway inhibitor.

[0111] In some embodiments, this disclosure provides isolated nucleic acid molecules comprising a heavy chain or antigen-binding moiety thereof encoding the anti-CD73 antibody or antigen-binding moiety described herein, or a light chain or antigen-binding moiety thereof encoding the anti-CD73 antibody or antigen-binding moiety described herein, or both. In some embodiments, the nucleic acid molecule may comprise a nucleotide sequence of any one of SEQ ID NO: 1 to 8.

[0112] In some embodiments, this disclosure provides a vector containing the isolated nucleic acid molecule described herein, wherein the vector further contains an expression control sequence.

[0113] In some embodiments, this disclosure provides a host cell comprising a heavy chain or antigen-binding moiety thereof encoding the anti-CD73 antibody or antigen-binding moiety described herein, and a light chain or antigen-binding moiety thereof encoding the anti-CD73 antibody or antigen-binding moiety described herein, and a nucleotide sequence thereof.

[0114] In some embodiments, this disclosure provides a method for generating an anti-CD73 antibody or an antigen-binding portion thereof, comprising providing a host cell as described herein, culturing the host cell under conditions suitable for expressing the antibody or portion, and isolating the resulting antibody or portion.

[0115] In some embodiments, this disclosure provides bispecific binding molecules comprising one or two different antigen-binding moieties of the anti-CD73 antibodies described herein.

[0116] In some embodiments, this disclosure provides a method for reducing CD73 activity in a patient in need, comprising administering to the patient a therapeutically effective amount of the anti-CD73 antibody or antigen-binding moiety, pharmaceutical composition or bispecific binding molecule described herein.

[0117] In some implementations, this disclosure provides for increasing CD4 levels in patients in need. + Methods for T cell proliferation include administering a therapeutically effective amount of the antiCD73 antibody or antigen-binding moiety, pharmaceutical composition or bispecific binding molecule described herein to the patient.

[0118] In some embodiments, this disclosure provides a method for stimulating the immune system of a patient in need, comprising administering to the patient a therapeutically effective amount of the antiCD73 antibody or antigen-binding moiety described herein, a pharmaceutical composition, or a bispecific binding molecule.

[0119] In some embodiments, this disclosure provides a method for treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of the anti-CD73 antibody or antigen-binding moiety described herein, a pharmaceutical composition, or a bispecific binding molecule. In some embodiments, the cancer originates from a tissue selected from: 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 some 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.

[0120] In some implementations, the treatment described herein further includes administering to the patient an immunostimulant, vaccine, chemotherapeutic agent, antitumor agent, antiangiogenic agent, tyrosine kinase inhibitor, CD73 pathway inhibitor, or radiotherapy.

[0121] In some aspects, this disclosure provides the use of the anti-CD73 antibody or antigen-binding moiety, pharmaceutical composition, or bispecific binding molecule described herein for the preparation of a medicament for use in:

[0122] a) Reduces CD73 activity in patients;

[0123] b) Increase patients' CD4 + T cell proliferation;

[0124] c) Stimulate the patient's immune system; or

[0125] d) Treating the patient's cancer.

[0126] In some aspects, this disclosure provides the anti-CD73 antibody or antigen-binding moiety, pharmaceutical composition or bispecific binding molecule described herein for use in:

[0127] a) Reduces CD73 activity in patients;

[0128] b) Increase patients' CD4 + T cell proliferation;

[0129] c) Stimulate the patient's immune system; or

[0130] d) Treating the patient's cancer.

[0131] Other features, objects, and advantages of the invention will become apparent in the detailed description below. However, it should be understood that while this detailed description describes embodiments and aspects of the invention, it is given by way of illustration only and not by way of limitation. Those skilled in the art will understand from this detailed description various changes and modifications within the scope of the invention. Brief description of the attached diagram

[0133] Figure 1 This is a graph showing the activity of soluble CD73 after treatment with the indicated anti-CD73 antibody. Data are normalized to the untreated control and expressed as mean ± SEM.

[0134] Figure 2 These are a pair of graphs showing the activity of CD73 expressed on Calu-6 cells (top) or H292 cells (bottom) after treatment with the anti-CD73 antibody shown. Data are normalized to the untreated control and expressed as mean ± SEM.

[0135] Figure 3 This is a set of graphs showing the binding of the anti-CD73 antibody and a reference antibody (eurylmab) analog to human and cynomolgus CD73 expressed on CHO-S cells. A simulated transfected CHO-S cell line was used as a negative control. The “Control Ab” is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Data are presented as mean ± SEM.

[0136] Figure 4 This is a schematic diagram showing the competition patterns and epitope bins identified against the anti-CD73 antibodies. Connected black lines indicate cross-blocking activity. Connected dashed lines indicate antibodies that block in only one direction. Antibodies are grouped with other anti-CD73 antibodies according to their competition patterns.

[0137] Figure 5 Describes the binding epitopes of antibodies 21127 (A), 21163 (B), 21046 (C), 11E1 analog (D), eurymumab analog (E), and CPX006 analog (F) located on crystal structures of the CD73 dimer (4H2G) in an open state. The structures are shown in surface representation, with the N-terminal domain in light gray and the C-terminal domain in gray. Adenosine is shown as white rods (indicated by arrows). Linear epitopes are shown in dark gray, and contact residues are shown in black.

[0138] Figure 6A and 6B This is a series of SEC-MALS plots of antibodies 21127, 21163, 21046, and 11E1, eurymumab, and CPX006 analogs mixed with CD73 at ratios mAb:CD73 1:1 (black line), 0.5:1 (dark gray line), 0.1:1 (light gray line), 0:1 (gray dashed line), and 1:0 (black dashed line). The calculated sizes of the different peaks are shown in Table 10.

[0139] Figure 7This graph shows the activity of soluble CD73 after treatment with the indicated anti-CD73 antibody and a reference antibody (eucommia ulmosiderin) analog. The "Control Ab" is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Data are normalized to untreated controls and expressed as mean ± SEM. The vertical dashed line indicates antibody concentrations equimolar to soluble recombinant CD73.

[0140] Figure 8 This is a set of graphs showing the activity of CD73 expressed on Calu-6 cells (top), H292 cells (middle), and Cynom-K1 cells (bottom) after treatment with the indicated anti-CD73 antibody and a reference antibody (eucommia ulmosiderin) analogue. The "Control Ab" is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Data are normalized to untreated controls and expressed as mean ± SEM.

[0141] Figure 9 This is a set of graphs showing the activity of CD73 expressed on H292 cells after treatment with the shown anti-CD73 antibody and a reference antibody analog (eurylumab) for 3 hours (top), 6 hours (middle), or 24 hours (bottom). Data are normalized to untreated controls and expressed as mean ± SEM.

[0142] Figure 10 This graph shows the activity of CD73 expressed by 20 different cancer cell lines in the presence of the indicated antibodies, as measured by the CellTiter-Glo assay. The "Control Ab" is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Each data point represents the average of three technical replicates.

[0143] Figure 11 This is a pair of graphs showing the effect of blocking CD73 activity on the survival of two triple-negative breast cancer cell lines, MBA-MB-231 (top) and MBA-MB-468 (bottom), grown in the presence of 300 μM AMP. The “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.

[0144] Figure 12 This shows, as measured by the CellTiter-Glo assay, the presence of the indicated antibody, that primary CD4 from healthy human donors... + and CD8 + T cells and CD19 +A set of graphs showing the activity of CD73 expressed on B cells. Activity was normalized to untreated cells (100%). “Control Ab” is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Data are presented as mean ± SEM of three technical replicates.

[0145] Figure 13 This shows the treatment of CD4 with anti-CD3 / CD28 beads, AMP, and the indicated anti-CD73 antibody and reference antibody (Olelimumab) analogue. + A graph of T cell proliferation. Data were normalized to untreated controls and expressed as mean ± SEM.

[0146] Figure 14 This indicates that CD4 has been treated with anti-CD3 / CD28 beads, AMP, and 21127 or a reference antibody (Olelimumab) analogue. + (Above image) and CD8 + (Below) A pair of graphs illustrating T cell activation. The “Control Ab” is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Data are presented as mean ± SEM.

[0147] Figure 15 This is a graph showing T cell proliferation in a one-way mixed lymphocyte response (MLR) after culture with anti-PD-1 antibody (12819), AMP, and the indicated anti-CD73 antibody or reference antibody (eutromulinab) analogue. "Control Ab" is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Data are normalized to untreated controls and expressed as mean ± SEM.

[0148] Figure 16 These are a pair of graphs showing T cell proliferation in a single-channel MLR after culture with or without AMP (bottom) and with the indicated concentrations of anti-PD-1 and / or anti-CD73 antibodies. The 12819+21127 combination is a 1:1 mixture of the two antibodies, and the concentrations shown indicate the total concentration of the mixture. The "Control Ab" is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Data are normalized to untreated controls and expressed as mean ± SEM.

[0149] Figure 17A and 17B Describes B cell activation in PBMCs from healthy donors stimulated overnight with the antibody (10 μg / mL) and CD40 ligand (0.5 μg / mL). Figure 17A This shows that CD69 is present in B cells (CD20). + A series of horizontal figures on the ). Figure 17BThis is a series of graphs showing the mean fluorescence intensity (MFI) of antibody staining for B cell activation markers CD25, CD69, and CD83. The “IgG1-LALA control” is a non-CD73-specific FcγR-deficient IgG1-LALA isotype antibody. Data are two data points from a single donor and are presented as mean ± SEM.

[0150] Figure 18 This is a graph showing the CD73 expression levels in H292 cells 24 hours after treatment with the indicated anti-CD73 antibody or a reference antibody (eucommia ulmosiderin). Data are normalized to untreated controls and expressed as mean ± SEM.

[0151] Figure 19 These are two graphs showing CD73 activity in the human xenograft melanoma model A375 after treatment with the antibodies shown. Top: PBMC humanized mice transplanted with A375 received different doses of 21127 three times a week for two weeks. Tumors were harvested one day after the last dose for CD73 activity analysis. Bottom: NOD-scid mice subcutaneously transplanted with A375 were treated three times a week with the antibodies or combinations shown for one week. Tumors were harvested on days 3, 7, 10, 17, and 29 after the last treatment for CD73 activity analysis. Data are normalized to untreated controls and expressed as mean ± SEM.

[0152] Figure 20 This figure shows tumor growth in NOD-scid mice subcutaneously transplanted with the human triple-negative breast cancer cell line MDA-MB-231. The mice were treated twice weekly with either antibody 21127 or a reference antibody (eutromulumab) analogue. The gray area represents the treatment period. Data are presented as mean ± SEM.

[0153] Figure 21 This is a pair of graphs showing the effect of treatment with anti-CD73 antibody 21127 or a vehicle on tumor growth in PBMC humanized mice transplanted with Calu-6 or A375 tumor cells. The gray area represents the treatment period. Data are expressed as mean ± SEM. *P < 0.05. Invention Details

[0155] This invention provides a novel anti-human CD73 antibody that can be used to inhibit CD73 activity in patients (e.g., cancer patients). Unless otherwise stated, as used herein, "CD73" refers to human CD73. The human CD73 polypeptide sequence is available under UniProt accession number P21589(5NTD_HUMAN) (SEQ ID NO:43), as shown below:

[0156]

[0157] As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetramer comprising two heavy (H) chains (approximately 50 to 70 kDa) and two light (L) chains (approximately 25 kDa) linked together by disulfide bonds. Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain contains 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 contains three CDRs (H-CDRs refer to those from the heavy chain; and L-CDRs refer to those from the light chain) and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid numbering and the allocation of FR and CDR regions in the heavy or light chain can be determined according to... Definitions (EU number; Lefranc et al., Dev Comp Immunol 27(1):55-77(2003)); or Kabat's definition, 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 Plückthun, J. Mol. Biol. 309(3):657-70 (2001).

[0158] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line containing a nucleotide sequence encoding an antibody, wherein the nucleotide sequence is not naturally associated with that cell.

[0159] The terms “isolated protein,” “isolated polypeptide,” or “isolated antibody” refer to a protein, polypeptide, or antibody that, due to its origin or source, (1) is not naturally associated with the naturally associated component that accompanies it in its natural state, (2) does not contain other proteins from the same species, (3) is expressed by cells from a different species, and / or (4) is not present in nature. Therefore, polypeptides chemically synthesized or synthesized in cellular systems different from those from cells of their natural origin will be “isolated” from their naturally associated components. Proteins can also be isolated by using protein purification techniques well known in the art, thus presenting a composition substantially free of naturally associated components.

[0160] The term "affinity" refers to a measure of the attractive force between an antigen and an antibody. The intrinsic attraction of an antibody to an antigen is typically expressed as the binding affinity balance constant (K0) of a specific antibody-antigen interaction. D When K D When the concentration is ≤1mM, preferably ≤100nM, it is considered that the antibody specifically binds to the antigen. D The affinity constant can be obtained, for example, through surface plasmon resonance (BIAcore). TM Or biofilm layer interferometry, such as using the IBIS MX96 SPR system from IBIS Technologies or the Octet from ForteBio. TM System measurement.

[0161] As used herein, the term "epitope" refers to a portion (determinant) of an antigen that specifically binds to an antibody or related molecule (e.g., a bispecific binding molecule). Epitope determinants generally consist of chemically active surface groups of a molecule (e.g., an amino acid or sugar or a sugar side chain) and typically possess specific three-dimensional structural characteristics and specific charge properties. Epitopes can be "linear" or "conformal." In a linear epitope, all interaction sites between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, interaction sites occur across amino acid residues that are separated from each other in the primary amino acid sequence of the protein. Once a target epitope on an antigen is identified, an antibody for that epitope can be generated using techniques well known in the art. For example, an antibody for a linear epitope can be generated, for example, by immunizing an animal with a peptide containing the amino acid residues of the linear epitope. An antibody for a conformational epitope can be generated, for example, by immunizing an animal with a mini-domain containing the relevant amino acid residues of the conformational epitope. Antibodies targeting specific epitopes can also be generated, for example, by immunizing animals with a target molecule (e.g., CD73) or a related portion thereof, and then screening for binding to that epitope.

[0162] Whether an antibody binds to the same epitope or competitively binds to the anti-CD73 antibody of the present invention can be determined using methods known in the art, including but not limited to competitive assays, epitope binning, and alanine scanning. In some embodiments, the anti-CD73 antibody of the present invention is allowed to bind to CD73 under saturation 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 a reference anti-CD73 antibody, then the test antibody binds to a different epitope compared to the reference anti-CD73 antibody. However, if the test antibody cannot bind to CD73 simultaneously, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope close to the epitope bound by the anti-CD73 antibody of the present invention. This experiment can be performed using, for example, ELISA, RIA, BIACORE. TM SPR, biofilm interferometry, or flow cytometry can be used to test whether one anti-CD73 antibody cross-competes with another. The competition methods described above can be used in two directions: determining 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 Octet. TM The system is running.

[0163] The term "human antibody" refers to an antibody in which the variable domain and constant region sequences are derived from human sequences. This term includes antibodies having sequences derived from human genes but modified, for example, to reduce immunogenicity, increase affinity, and / or increase stability. Furthermore, this term includes antibodies recombinantly generated in non-human cells that can confer atypical glycosylations to human cells. This term also includes transgenic non-human organisms possessing human antibody genes (e.g., [examples not provided]). Antibodies produced in rats.

[0164] As used herein, the term “antigen-binding moiety” (or simply “antibody moiety”) of an antibody 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). Certain fragments of full-length antibodies have been shown to perform the antigen-binding function of the antibody. Examples of binding fragments included in the term “antigen-binding moiety” include (i) Fab fragments: monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments: bivalent fragments consisting of two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of VH domains; and (vi) separation complementarity-determining regions (CDRs) that can specifically bind to antigens. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using a recombination method, which allows them to be prepared as a single protein chain in which the VL and VH domains pair to form a monovalent molecule (called a single-chain Fv (scFv)). Antigen-binding molecules containing VH and / or VL are also within the scope of this invention. In the case of VH, the molecule may also contain one or more of the CH1, hinge, CH2, or CH3 regions. Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of the antibody. Other forms of single-chain antibodies are also included (e.g., diabodies). Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but linked using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites.

[0165] Antibody moieties (e.g., Fab and F(ab')2 fragments) can be prepared from whole antibodies using conventional techniques (e.g., papain or pepsin digestion of the whole antibody). Furthermore, antibodies, antibody moieties, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques (e.g., techniques described herein).

[0166] The class (isotype) and subclass of anti-CD73 antibodies can be determined by any method known in the art. Generally, the class and subclass of an antibody can be determined using antibodies specific to that particular class and subclass. Such antibodies are commercially available. The class and subclass can be determined by ELISA or Western blotting and other techniques. Alternatively, the class and subclass can be determined by sequencing all or a portion of the constant regions of the heavy and / or light chains of the antibody, comparing their amino acid sequences with known amino acid sequences of various classes and subclasses of immunoglobulins.

[0167] Unless otherwise indicated herein, all antibody amino acid residue numbers mentioned herein are based on... Numbering scheme (EU numbering)

[0168] Anti-CD73 antibody

[0169] This disclosure provides an antibody against CD73 and its antigen-binding portion. In one specific embodiment, the antibody disclosed herein is a human antibody produced from a transgenic animal (e.g., a rat) capable of producing antibodies encoded by rearranged human antibody genes. In some embodiments, the human antibody may contain certain mutations, for example, to alter primer-derived mutations to mutate them back to germline sequences (see, for example, “Symplex-corrected” variant sequences in Table 1).

[0170] In some embodiments, the anti-CD73 antibody of the present invention has a “LALA” mutation (L234A / L235A) in the Fc region. These mutations weaken the antibody’s binding to human FcγR (Fcγ 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.

[0171] In some implementations, the anti-CD73 antibody or antigen-binding moiety competes or cross-competes with antibodies containing the following to bind to human CD73, or to the same epitope of human CD73:

[0172] a) HC having the amino acid sequences of SEQ ID NO: 9 and 41 and LC having the amino acid sequences of SEQ ID NO: 13 and 42;

[0173] b) HC having the amino acid sequences of SEQ ID NO:10 and 41 and LC having the amino acid sequences of SEQ ID NO:14 and 42;

[0174] c) HC having the amino acid sequences of SEQ ID NO: 11 and 41 and LC having the amino acid sequences of SEQ ID NO: 15 and 42; or

[0175] d) HC having the amino acid sequences of SEQ ID NO:12 and 41 and LC having the amino acid sequences of SEQ ID NO:16 and 42.

[0176] In some embodiments, the anti-CD73 antibody or antigen-binding moiety has the heavy chain CDR3 (H-CDR3) amino acid sequence of SEQ ID NO:19, 25, 31 or 37.

[0177] In some embodiments, the anti-CD73 antibody or antigen-binding moiety has a heavy chain CDR1-3 (H-CDR1-3) comprising the amino acid sequences of SEQ ID NO:17-19, 23-25, 29-31 or 35-37, respectively.

[0178] In some embodiments, the anti-CD73 antibody or antigen-binding moiety 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 in any of SEQ ID NO:9 to 12.

[0179] In some embodiments, the anti-CD73 antibody or antigen-binding portion has a VH comprising an amino acid sequence of any one of SEQ ID NO:9 to 12.

[0180] 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 of SEQ ID NO: 9 to 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.

[0181] In some embodiments, the anti-CD73 antibody comprises the VH amino acid sequence of any one of SEQ ID NO:9 to 12 and the heavy chain constant region amino acid sequence of SEQ ID NO:41.

[0182] In some embodiments, the anti-CD73 antibody or antigen-binding moiety has a light chain CDR3 (L-CDR3) amino acid sequence of SEQ ID NO:22, 28, 34 or 40.

[0183] In some embodiments, the anti-CD73 antibody or antigen-binding moiety has a light chain CDR1-3 (L-CDR1-3) comprising the amino acid sequence of SEQ ID NO:20-22, 26-28, 32-34, or 38-40, respectively.

[0184] In some embodiments, the anti-CD73 antibody or antigen-binding moiety 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 of SEQ ID NO:13 to 16.

[0185] In some embodiments, the anti-CD73 antibody or antigen-binding portion has a VL containing an amino acid sequence of any one of SEQ ID NO:13 to 16.

[0186] 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 of SEQ ID NO:13 to 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.

[0187] In some embodiments, the anti-CD73 antibody comprises the VL amino acid sequence of any one of SEQ ID NO:13 to 16 and the light chain constant region amino acid sequence of SEQ ID NO:42.

[0188] In some embodiments, the anti-CD73 antibody comprises either of the heavy chains and either of the light chains described above.

[0189] In some embodiments, the anti-CD73 antibody or antigen-binding moiety of the present invention comprises the following H-CDR1-3 and L-CDR1-3 amino acid sequences:

[0190] a) These are SEQ ID NO: 17 to 22 respectively;

[0191] b) These are SEQ ID NO: 23 to 28 respectively;

[0192] c) are SEQ ID NO: 29 to 34 respectively; or

[0193] d) These are SEQ ID NO:35 to 40 respectively.

[0194] In some embodiments, the anti-CD73 antibody or antigen-binding moiety of the present invention comprises VH and VL that are identical to the following amino acid sequences in 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% proportions:

[0195] a) These are SEQ ID NO:9 and 13, respectively;

[0196] b) These are SEQ ID NO: 10 and 14, respectively;

[0197] c) are SEQ ID NO:11 and 15 respectively; or

[0198] d) are SEQ ID NO:12 and 16 respectively.

[0199] In some embodiments, the anti-CD73 antibody or antigen-binding moiety of the present invention comprises VH and VL containing the following amino acid sequence:

[0200] a) These are SEQ ID NO:9 and 13, respectively;

[0201] b) These are SEQ ID NO: 10 and 14, respectively;

[0202] c) are SEQ ID NO:11 and 15 respectively; or

[0203] d) are SEQ ID NO:12 and 16 respectively.

[0204] In some embodiments, the anti-CD73 antibody of the present invention comprises:

[0205] a) HC having the amino acid sequences of SEQ ID NO: 9 and 41 and LC having the amino acid sequences of SEQ ID NO: 13 and 42;

[0206] b) HC having the amino acid sequences of SEQ ID NO:10 and 41 and LC having the amino acid sequences of SEQ ID NO:14 and 42;

[0207] c) HC having the amino acid sequences of SEQ ID NO: 11 and 41 and LC having the amino acid sequences of SEQ ID NO: 15 and 42; or

[0208] d) HC having the amino acid sequences of SEQ ID NO:12 and 41 and LC having the amino acid sequences of SEQ ID NO:16 and 42.

[0209] The present invention also provides anti-CD73 antibodies or their antigen-binding portions that compete or cross-compete with antibodies 21028, 21046, 21127 or 21163, or bind to the same epitope.

[0210] In some embodiments, the anti-CD73 antibody or antigen-binding portion of the present invention comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 21028, 21046, 21127 or 21163.

[0211] In some embodiments, the anti-CD73 antibody or antigen-binding portion of the present invention comprises VH and VL whose amino acid sequences are at least 90% identical to those of VH and VL of antibodies 21028, 21046, 21127 or 21163, respectively.

[0212] In some embodiments, the anti-CD73 antibody or antigen-binding portion of the present invention comprises VH and VL of antibodies 21028, 21046, 21127 or 21163, respectively.

[0213] In some embodiments, the anti-CD73 antibody of the present invention is antibody 21028, 21046, 21127 or 21163, or an antibody having the same amino acid sequence as said antibody.

[0214] The class of anti-CD73 antibodies obtained by the methods described herein can be changed or converted to another class or subclass. In some embodiments of the invention, the nucleic acid molecules encoding VL or VH are isolated using methods well known in the art such that they do not include nucleic acid sequences encoding CL or CH, respectively. The nucleic acid molecules encoding VL or VH are then efficiently linked to nucleic acid sequences encoding CL or CH, respectively, from different classes of immunoglobulin molecules. As described above, this can be achieved using vectors or nucleic acid molecules containing CL or CH sequences. For example, an anti-CD73 antibody initially IgM can be class-converted to IgG. Furthermore, this class conversion can be used to convert one IgG subclass to another, for example, from IgG1 to IgG2. The κ light chain constant region can be changed, for example, to the λ light chain constant region, or vice versa. An exemplary method for generating antibodies of the present invention having the desired Ig isotype includes the following steps: 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 a variable domain of the heavy chain, linking the coding sequence of the variable domain of the heavy chain to the coding sequence of a constant region of the heavy chain having the desired isotype, expressing the light chain and heavy chain encoded by the linking sequence in cells, and collecting anti-CD73 antibodies having the desired isotype.

[0215] The anti-CD73 antibody of the present invention may be an IgG, IgM, IgE, IgA, or IgD molecule, but is typically an IgG isotype, such as an antibody against IgG subtypes IgG1, IgG2a, IgG2b, IgG3, or IgG4. In some embodiments, the antibody is an antibody against isotype subtype IgG1. In some embodiments, the antibody is an antibody against isotype subtype IgG2.

[0216] In some implementations, the anti-CD73 antibody may contain at least one mutation in the Fc region. Many different Fc mutations are known, where these mutations alter the effector function of the antibody. For example, in many cases, it is desirable to reduce or eliminate effector function, such as when ligand / receptor interactions are undesirable or in the case of antibody-drug conjugates.

[0217] In some embodiments, the anti-CD73 antibody contains at least one mutation in the Fc region that reduces effector function, for example, a mutation at one or more of positions 228, 233, 234, and 235, wherein the amino acid positions are determined according to... Numbering scheme number.

[0218] In some implementations, for example, in the case of antibodies that are IgG1 subclass antibodies, 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 the IgG1 antibody. The amino acid positions are determined according to... Numbering scheme number.

[0219] In some implementations, for example, in the case of an antibody that is an IgG4 subclass antibody, it may contain a mutant S228P, wherein the amino acid position is determined according to... Numbering scheme number. This mutation is known to reduce unwanted Fab arm swaps.

[0220] In some embodiments, the anti-CD73 antibody of the present invention, or its antigen-binding portion, binds to an epitope of CD73, comprising 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 some embodiments, the antibody or antigen-binding portion binds to an epitope of CD73 comprising residues R73, R109, and D168 (e.g., antibody 21127) or consisting of residues R73, R109, and D168 (e.g., antibody 21127). In some embodiments, the antibody or antigen-binding portion binds to an epitope of CD73 comprising residue R109 (e.g., antibody 21163) or consisting of residue R109 (e.g., antibody 21163). In some embodiments, the antibody or antigen-binding moiety binds to an epitope of CD73, which comprises residues I301, S302, and H304 (e.g., antibody 21046) or is composed of residues I301, S302, and H304 (e.g., antibody 21046).

[0221] In some embodiments, the anti-CD73 antibody of the present invention, or its antigen-binding portion, binds to an epitope of CD73, comprising residues 27 to 31, 61 to 70, 61 to 75, 161 to 170, 266 to 270, and / or 291 to 305 of SEQ ID NO:43. In some embodiments, the antibody or antigen-binding portion binds to an epitope of CD73, comprising residues 27 to 31, 61 to 75, and 161 to 170 (e.g., antibody 21127) or consisting of residues 27 to 31, 61 to 75, and 161 to 170 (e.g., antibody 21127). In some embodiments, the antibody or antigen-binding portion binds to an epitope of CD73, comprising residues 61 to 70 and 161 to 170 (e.g., antibody 21163) or consisting of residues 61 to 70 and 161 to 170 (e.g., antibody 21163). In some embodiments, the antibody or antigen-binding moiety binds to an epitope of CD73 comprising residues 27 to 31, 266 to 270, and 291 to 305 (e.g., antibody 21046) or consisting of residues 27 to 31, 266 to 270, and 291 to 305 (e.g., antibody 21046).

[0222] In some embodiments, the antibody or a portion thereof binds to an epitope containing residues 27 to 31 (or fragments thereof, such as fragments of 1, 2, 3, or 4 residues) of SEQ ID NO: 43 (e.g., antibodies 21127 and 21046). In some embodiments, the antibody or a portion thereof binds to an epitope containing residues 61 to 75 (or fragments thereof, such as fragments of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residues, such as residues 61 to 70) of SEQ ID NO: 43 (e.g., antibodies 21127 and 21163). In some embodiments, the antibody or a portion thereof binds to an epitope containing residues 161 to 170 (or fragments thereof, such as fragments of 1, 2, 3, 4, 5, 6, 7, 8, or 9 residues) of SEQ ID NO: 43 (e.g., antibodies 21127 and 21163). In some embodiments, the antibody or a portion thereof binds to an epitope containing residues 266 to 270 (or fragments thereof, such as fragments of 1, 2, 3, or 4 residues) of SEQ ID NO: 43 (e.g., antibody 21046). In some embodiments, the antibody or a portion thereof binds to an epitope containing residues 291 to 305 (or fragments thereof, such as fragments of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 residues) of SEQ ID NO: 43 (e.g., antibody 21046).

[0223] Epitopes with any combination of residues above or shown in Table 9 were also considered.

[0224] In some embodiments, the amino acid sequence containing the CD73 epitope described herein can be used as an immunogen (e.g., administered to an animal or used as an antigen to screen an antibody library) to generate or identify anti-CD73 antibodies or antigen-binding moieties bound to the epitope.

[0225] In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein inhibits 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 concentrations of 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 50, or 100 μg / mL, the anti-CD73 antibody or antigen-binding moiety described herein inhibits the activity of soluble CD73 by at least 90%.

[0226] In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein inhibits the activity of CD73 on Calu-6 cells by at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 75%).

[0227] In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein inhibits the activity of CD73 on H292 cells by at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 80%).

[0228] In some implementations, the anti-CD73 antibody or antigen-binding portion described herein specifically binds to human CD73, cynomolgus monkey CD73, or both expressed on CHO-S cells.

[0229] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein is in the form of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 nM or less (e.g., 1 nM or less). D Binding to the extracellular domain (ECD) of human CD73, as measured by surface plasmon resonance (SPR).

[0230] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein is in the form of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 nM or less (e.g., 0.7 nM or less). D The extracellular domain (ECD) of CD73 in cynomolgus monkeys is bound, as measured by surface plasmon resonance (SPR).

[0231] In some implementations, the anti-CD73 antibody or antigen-binding moiety described herein does not bind to the same epitope of CD73 as ovalbumin, CPX006, 11E1, or any combination thereof.

[0232] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein binds to the epitope on the CD73 homodimer in a manner that generates a 1:1 complex. In some embodiments, the antibody / CD73 binding generates only a 1:1 complex. In some embodiments, the antibody / CD73 binding primarily generates a 1:1 complex. In a specific embodiment, the binding of the antibody or antigen-binding moiety to CD73 generates a 1:1 complex independent of the CD73 concentration.

[0233] In some embodiments, for example, at concentrations of 1, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg / mL or less (e.g., at 10 μg / mL or less), the anti-CD73 antibody or antigen-binding moiety described herein inhibits soluble CD73 activity. In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibody or antigen-binding moiety inhibits CD73 activity more effectively than olemulombab. In specific embodiments (e.g., at one of the concentrations listed above), CD73 activity is inhibited by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%. For example, at a concentration of 3 μg / mL, the antibody or antigen-binding moiety can inhibit CD73 activity by 100%.

[0234] In some embodiments, for example, at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, or 100 μg / mL or less (e.g., at 3 μg / mL or less), the anti-CD73 antibody or antigen-binding moiety described herein inhibits CD73 activity on Calu-6, H292, Cynom-K1 cells, or any combination thereof. In some embodiments, the anti-CD73 antibody or antigen-binding moiety is more effective at inhibiting CD73 activity than olemulombab. In some embodiments, the anti-CD73 antibody or antigen-binding moiety remains more effective than olemulombab after an additional 3, 6, or 24 hours of culture. In specific embodiments (e.g., at one of the concentrations listed above for the antibody or antigen-binding moiety), at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% inhibition of CD73 activity is achieved.

[0235] In some embodiments (e.g., at a concentration of 25 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein inhibits CD73 activity on any cell line or combination of cell lines shown in Table 11. In some embodiments, the anti-CD73 antibody or antigen-binding moiety inhibits CD73 activity on Calu-6, NCI-H1775, KYSE-30, Capan-2 cells, or any combination thereof. In some embodiments, the anti-CD73 antibody or antigen-binding moiety inhibits CD73 activity more effectively than eurymumab.

[0236] 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 (e.g., at 0.01 μg / mL or less), the anti-CD73 antibody or antigen-binding moiety described herein inhibits the survival and / or proliferation of MDA-MB-231 cells, MDA-MB-468 cells, or both in vitro. In some embodiments (e.g., at concentrations of 0.5 μg / mL or greater), the anti-CD73 antibody or antigen-binding moiety is more effective than eurythromycin in inhibiting this survival / proliferation. In specific embodiments (e.g., at one of the concentrations listed above for the antibody or antigen-binding moiety), the number of viable cells is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0237] 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 (e.g., at 0.1 μg / mL or less), the anti-CD73 antibody or antigen-binding moiety described herein inhibits primary CD4. + Cells, CD8+ T cells, CD19 + CD73 activity on B cells or any combination thereof. In specific embodiments (e.g., when the antibody or antigen-binding moiety is at one of the concentrations listed above), CD73 activity is inhibited, for example, by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0238] In some embodiments, for example, at concentrations of 0.05, 0.1, 0.5, or 1 μg / mL or less (e.g., at 0.01 μg / mL or less), the anti-CD73 antibody or antigen-binding fraction described herein partially restores CD4. + T cell proliferation. In some embodiments, for example, at concentrations of 0.1, 0.5, 1, 10, 15, 20, 25, 30, 40, 50, or 100 μg / mL or less (e.g., at 25 μg / mL or less), the anti-CD73 antibody or antigen-binding moiety described herein will bind CD4. + T cell proliferation was restored to 100%. In some formulations (e.g., at concentrations of 0.1 μg / mL or higher), the anti-CD73 antibody or antigen-binding moiety was more effective than elemol in restoring CD4+. + T cell proliferation. In certain embodiments (e.g., when the antibody or antigen-binding portion is at one of the concentrations listed above), T cell proliferation is restored to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.

[0239] In some embodiments, for example, at concentrations of 0.001, 0.005, 0.01, 0.05, 1, 5, 10, 15, or 25 μg / mL or less (e.g., at 1 μg / mL or less), the anti-CD73 antibody or antigen-binding moiety described herein activates CD4. + and CD8 + T cells. In some implementations (e.g., at concentrations of 0.1 μg / mL or higher), this anti-CD73 antibody or antigen-binding moiety activates CD4 more effectively than olemumab. + and CD8 + T cells.

[0240] In some embodiments, for example, at concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μg / mL or less (e.g., at 0.01 μg / mL or less), the combination of the anti-CD73 antibody or antigen-binding moiety described herein with the anti-PD-1 antibody restores T cell proliferation in the presence of AMP during a one-way mixed lymphocyte reaction (MLR). In some embodiments (e.g., at concentrations of 1 μg / mL or less), the combination of the anti-CD73 antibody or antigen-binding moiety with the anti-PD-1 antibody completely restores T cell proliferation. In some embodiments (e.g., at concentrations of 0.1 μg / mL or greater), the combination of the anti-CD73 antibody or antigen-binding moiety with the anti-PD-1 antibody restores T cell proliferation more effectively than eurymumab. In specific implementations (e.g., when the antibody or antigen-binding portion is at one of the concentrations listed above, 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%.

[0241] In some embodiments, for example, at concentrations of 0.5, 1, 5, 10, 15, or 25 μg / mL or less (e.g., at 1 μg / mL or less), the anti-CD73 antibody or antigen-binding moiety described herein enhances T cell activation in a one-way MLR in the presence of AMP. In some embodiments, the combination of the anti-CD73 antibody or antigen-binding moiety with an anti-PD-1 antibody (e.g., 12819) enhances T cell activation in a one-way MLR in the presence of AMP.

[0242] In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein does not stimulate B cell activation in vitro. In some embodiments (e.g., at a concentration of 10 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein stimulates B cell activation in vitro. B cell activation can be determined by observing biomarkers (e.g., CD25, CD69, and / or CD83).

[0243] In some embodiments (e.g., at a concentration of 25 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein does not reduce CD73 levels in H292 cells in vitro. In some embodiments (e.g., at a concentration of 25 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein reduces CD73 levels in H292 cells in vitro by, for example, up to no more than 40%, 50%, 60%, 70%, 80%, or 90% compared to an untreated control. In some embodiments (e.g., at a concentration of 25 μg / mL), the anti-CD73 antibody or antigen-binding moiety described herein only moderately reduces CD73 levels in H292 cells in vitro. For example, in some embodiments, the reduction in CD73 levels is no more than, for example, 5%, 10%, 15%, 20%, 25%, 30%, or 35% (e.g., 25%).

[0244] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein inhibits CD73 activity in tumors harvested from PBMC humanized mice transplanted with A375 cells (e.g., the mice are administered the antibody or antigen-binding moiety at 5 mg / kg, 20 mg / kg, or 50 mg / kg three times a week for one or two weeks). In some embodiments, the 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.

[0245] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein inhibits tumor growth in vivo in NOD-scid mice transplanted with MDA-MB-231 cells (e.g., the mice are treated with the antibody or antigen-binding moiety at 10 mg / kg twice weekly for a total of 16 treatments). In some embodiments, at the end of treatment, the tumor growth inhibition is characterized by a limited increase in tumor volume (e.g., less than 10, 20, 30, 40, or 50 mm). 3 Maintain for at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 days (e.g., at least 60 days).

[0246] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein inhibits tumor growth in PBMC humanized mice transplanted with Calu-6 or A375 cells (e.g., the mice are treated with the antibody or antigen-binding moiety at 10 mg / kg three times a week for a total of six treatments).

[0247] The present invention also considers any combination of anti-CD73 antibodies or antigen-binding moieties having the properties described above.

[0248] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein has at least one of the following properties (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 properties):

[0249] a) In vitro inhibition of soluble CD73 activity;

[0250] b) Inhibit CD73 activity on Calu-6 cells in vitro;

[0251] c) Inhibit CD73 activity on H292 cells in vitro;

[0252] d) Specifically binds to human and cynomolgus monkey CD73 expressed on CHO-S cells;

[0253] e) with 1 nM or less K D ECD associated with human CD73, as measured by SPR;

[0254] f) with K 0.7 nM or less D ECD associated with CD73 in cynomolgus monkeys, as measured by SPR;

[0255] g) Does not bind to the same epitope of CD73 as ollilumab, CPX006 and / or 11E1;

[0256] h) binds to epitopes on CD73 homodimers in a manner that generates a 1:1 complex;

[0257] i) It inhibits soluble CD73 activity more effectively than olemumab in vitro;

[0258] j) In vitro inhibition of CD73 activity in Calu-6, H292 and Cynom-K1 cells;

[0259] k) In vitro inhibition of CD73 activity in Calu-6, NCI-H1775, KYSE-30 and Capan-2 cells;

[0260] l) In vitro inhibition of the survival and / or proliferation of MDA-MB-231 and MDA-MB-468 cells;

[0261] m) In vitro inhibition of primary CD4 + and CD8 + T cells and CD19 + CD73 activity on B cells;

[0262] n) In vitro CD4 recovery+ T cell proliferation;

[0263] o) In vitro activated CD4 + and CD8 + T cells;

[0264] p) Combined with anti-PD-1 antibody, T cell proliferation was restored in the presence of AMP in a single-channel mixed lymphocyte response (MLR);

[0265] q) When combined with an anti-PD-1 antibody, T cell activation is enhanced in the presence of AMP in a single-channel MLR.

[0266] r) It does not stimulate B cell activation in vitro;

[0267] s) It does not reduce CD73 levels in H292 cells by more than 25% in vitro;

[0268] t) Inhibit CD73 activity in tumors harvested from PBMC humanized mice transplanted with A375 cells;

[0269] u) In vivo inhibition of tumor growth in NOD-scid mice transplanted with MDA-MB-231 cells;

[0270] v) In vivo inhibition of tumor growth in PBMC humanized mice transplanted with Calu-6 cells; and

[0271] w) In vivo inhibition of tumor growth in PBMC humanized mice transplanted with A375 cells.

[0272] In some embodiments, the anti-CD73 antibody or antigen-binding moiety has all of the properties a) to w). In some embodiments, the anti-CD73 antibody or antigen-binding moiety has at least properties a) to k), n), and s). In some embodiments, the anti-CD73 antibody or antigen-binding moiety has at least properties a) to g), i) to k), and n). In some embodiments, the anti-CD73 antibody or antigen-binding moiety has at least properties a) to d), i) to k), n), and s).

[0273] In some implementations, the anti-CD73 antibody or antigen-binding moiety described herein may inhibit tumor growth in vivo and / or induce tumor regression, slow or reverse metastasis in cancer patients, and / or prolong the survival of cancer patients. Any combination of the properties described above is also considered.

[0274] In some embodiments, the anti-CD73 antibody or antigen-binding moiety described herein is more effective than anti-CD73 antibodies currently in clinical trials in increasing T cell proliferation and / or reducing CD73 activity. For example, in some embodiments, the anti-CD73 antibody or antigen-binding moiety has at least one of the following properties:

[0275] - At a concentration of 0.1 μg / mL, it increased the activity of activated CD4 cells cultured at 100 μM AMP by more than antibodies containing the following HC and LC amino acid sequences. + T cell proliferation:

[0276] - US Patent Publication 2016 / 0129108 contains SEQ ID NO: 17 and 19 respectively;

[0277] - US Patent Publication 2018 / 0009899 contains SEQ ID NO: 14 and 13 respectively; or

[0278] - US Patent Publication 2018 / 0030144 contains SEQ ID NO:3 and 4 respectively;

[0279] and / or

[0280] - At concentrations of 10 μg / mL or higher, after 24 hours of incubation, antibodies reduced CD73 activity in H292 cells more than those containing the following HC and LC amino acid sequences:

[0281] - US Patent Publication 2016 / 0129108 contains SEQ ID NO: 17 and 19 respectively;

[0282] - US Patent Publication 2018 / 0009899 contains SEQ ID NO: 14 and 13 respectively; or

[0283] - US Patent Publication 2018 / 0030144 contains SEQ ID NO:3 and 4, respectively.

[0284] In some embodiments, the antibody or its antigen-binding portion of the present invention may be part of a larger immunoadhesion molecule formed by covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of a streptavidin core region to prepare 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 prepare divalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Other examples include immunoadhesins in which one or more CDRs from an antibody are covalently or non-covalently incorporated into the molecule to make it specifically bind to a target antigen. In such embodiments, the CDR may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be non-covalently incorporated.

[0285] In some embodiments, fusion antibodies or immunoadhesives comprising all or a portion of the anti-CD73 antibody of the present invention linked to another polypeptide can be prepared. In some embodiments, only the variable domain of the anti-CD73 antibody is linked to the polypeptide. In some embodiments, the VH domain of the anti-CD73 antibody is linked to the first polypeptide, while the VL domain of the anti-CD73 antibody is linked to a second polypeptide that binds to the first polypeptide in such a manner 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, thereby allowing the VH and VL domains to interact with each other (e.g., single-chain antibodies). The VH-linker-VL antibody is then linked to the target polypeptide. Alternatively, fusion antibodies in which two (or more) single-chain antibodies are linked to each other can be generated. This is useful if the plan is to generate bivalent or multivalent antibodies on a single polypeptide chain or to generate bispecific antibodies.

[0286] To generate a single-chain antibody (scFv), a DNA fragment encoding VH and VL is efficiently linked to another fragment encoding a flexible adapter (e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:44)), such that the VH and VL sequences can be expressed as a continuous single-chain protein having the VL and VH domains linked by the flexible adapter. See, for example, 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). If only a single VH and VL are used, the single-chain antibody can be monovalent; if two VH and VL are used, it is divalent; or if more than two VH and VL are used, it is polyvalent. For example, bispecific or multivalent antibodies that specifically bind to human CD73 and another molecule can be generated.

[0287] In other embodiments, other modified antibodies can be prepared using nucleic acid molecules encoding anti-CD73 antibodies. For example, “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 “Janusin” (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Supplement) 7:51-52 (1992)) can be prepared using standard molecular biotechnology following the teachings of this specification.

[0288] The anti-CD73 antibody or antigen-binding moiety of the present invention may be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or a portion thereof is derivatized such that CD73 binding is not adversely affected by the derivatization or labeling. Therefore, the antibodies and antibody moiety of the present invention are intended to include both the complete and modified forms of the human anti-CD73 antibody described herein. For example, the antibody or antibody moiety of the present invention (by chemical conjugation, gene fusion, non-covalent binding, or otherwise) may be functionally linked to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a bimeric antibody), a detection agent, a drug, and / or a protein or peptide, which may mediate the binding of the antibody or antibody moiety to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0289] One type of antibody is the derivatized antibody produced by crosslinking two or more antibodies (of the same or different types, for example, to generate bispecific antibodies). Suitable crosslinking agents include those that are heterobifunctional, having two distinctly different reactive groups separated by suitable spacer groups (e.g., m-maleimide benzoyl-N-hydroxysuccinimide) or homobifunctional reactive groups (e.g., disuccinimide octanoate). Such linkers are available, for example, from Pierce Chemical, Rockford, IL.

[0290] Anti-CD73 antibodies or antigen-binding moieties can also be derived using chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, for example, to increase serum half-life.

[0291] The antibody or antigen-binding moiety according to the invention may also be labeled. As used herein, the terms "labeled" or "labeled" refer to the incorporation of another molecule into the antibody. In some embodiments, the label is a detectable marker, such as a polypeptide incorporated with a radiolabeled amino acid or bound to a biotinylated moiety, which can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optical or colorimetric methods). In some embodiments, the label or marker may be a therapeutic agent, such as a pharmaceutical conjugate or a toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of peptide labeling include, but are not limited to, the following: 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, biotin groups, predetermined peptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags), magnetic agents (e.g., gadolinium chelates), toxins (e.g., pertussis toxin), taxanes. Taxol, pinocembrin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogues or homologs. In some embodiments, the markers are connected by spacer arms of various lengths to reduce potential steric hindrance.

[0292] In some embodiments, the antibody or antigen-binding portion according to the invention may bind to a cytotoxic agent to form an immunoconjugate. In some embodiments, the antibody or antigen-binding portion according to the invention may conjugate to a radioactive isotope.

[0293] In some embodiments, the antibody of the present invention may exist in a neutral form (including zwitterionic form) or as a positively or negatively charged substance. In some embodiments, the antibody may complex with an anti-charged ion to form a pharmaceutically acceptable salt.

[0294] The term "medicinal salt" refers to a complex comprising one or more antibodies and one or more counter-charged ions, wherein the counter-charged ions are derived from medicinal inorganic and organic acids and bases.

[0295] Anti-CD73 antibody composition

[0296] The present invention also provides combination therapies (e.g., compositions) comprising one, two, three, four, or more anti-CD73 antibodies or antigen-binding moieties described herein. In some embodiments, the combination therapy (e.g., composition) comprises two anti-CD73 antibodies or antigen-binding moieties. The combination therapy may take the form of a treatment method, for example, using said antibodies or antigen-binding moieties or a pharmaceutical composition comprising said antibodies or antigen-binding moieties.

[0297] In some embodiments, this disclosure provides a composition comprising a first anti-CD73 antibody or an antigen-binding portion thereof and a second anti-CD73 antibody or an antigen-binding portion thereof, wherein: the first and second antibodies:

[0298] - These are antibodies 21028 and 21046, respectively;

[0299] - These are antibodies 21028 and 21127, respectively;

[0300] - These are antibodies 21028 and 21163, respectively;

[0301] - These are antibodies 21046 and 21127, respectively;

[0302] - These are antibodies 21046 and 21163, respectively; or

[0303] - These are antibodies 21127 and 21163, respectively.

[0304] In some embodiments, the composition comprises an antibody or its antigen-binding moiety that binds to the same epitope as the first and second antibodies or competes with the first and second antibodies for binding.

[0305] In some embodiments, the composition comprises an antibody or antigen-binding portion thereof containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of the first antibody, and an antibody or antigen-binding portion thereof containing the H-CDR1-3 and L-CDR1-3 amino acid sequences of the second antibody.

[0306] In some embodiments, the composition comprises an antibody or antigen-binding portion thereof containing VH and VL amino acid sequences identical to the respective VH and VL amino acid sequences of the first antibody in at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequences of the second antibody in at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequences of the respective VH and VL amino acid sequences of the second antibody.

[0307] In some embodiments, the composition comprises an antibody containing the VH and VL amino acid sequences of the first antibody or an antigen-binding portion thereof, and an antibody containing the VH and VL amino acid sequences of the second antibody or an antigen-binding portion thereof.

[0308] In some embodiments, the composition comprises an antibody containing the HC and LC amino acid sequences of the first antibody or an antigen-binding portion thereof, and an antibody containing the HC and LC amino acid sequences of the second antibody or an antigen-binding portion thereof.

[0309] In some embodiments, the composition may comprise one, two, or more antibodies or antigen-binding moieties selected from the following:

[0310] a) An antibody containing H-CDR1-3 with an amino acid sequence containing SEQ ID NO:17-19, 23-25, 29-31 or 35-57 respectively;

[0311] b) An antibody whose VH sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 9, 10, 11 or 12;

[0312] c) VH contains an antibody containing the amino acid sequence of SEQ ID NO: 9, 10, 11 or 12;

[0313] d) HC antibodies containing the amino acid sequences of SEQ ID NO: 9 and 41, 10 and 41, 11 and 41, or 12 and 41;

[0314] e) An antibody containing L-CDR1-3 containing the amino acid sequences SEQ ID NO:20-22, 26-28, 32-34, or 38-40 respectively;

[0315] f) An antibody whose VL sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16;

[0316] g) VL contains an antibody containing the amino acid sequence of SEQ ID NO: 13, 14, 15 or 16;

[0317] h) LC antibodies containing the amino acid sequences of SEQ ID NO: 13 and 42, 14 and 42, 15 and 42, or 16 and 42;

[0318] i) H-CDR1-3 and L-CDR1-3 contain antibodies containing the amino acid sequences of SEQ ID NO: 17 to 22, 23 to 28, 29 to 34 or 35 to 40, respectively;

[0319] j) Contains antibodies containing VH and VL whose amino acid sequences are at least 90% identical to those of SEQ ID NO:9 and 13, 10 and 14, 11 and 15, or 12 and 16, respectively;

[0320] k) Antibodies comprising VH and VL containing the amino acid sequences SEQ ID NO: 9 and 13, 10 and 14, 11 and 15, or 12 and 16, respectively; and

[0321] l) Antibodies containing HC and LC containing the amino acid sequences of SEQ ID NO: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.

[0322] In some embodiments, the anti-CD73 antibody compositions described herein can inhibit tumor growth and / or induce tumor regression in vivo, can slow or reverse metastasis in cancer patients, and / or can prolong the survival of cancer patients. Any combination of the properties described above is also considered.

[0323] The present invention also provides a method for generating the anti-CD73 antibody composition described herein, comprising providing a first anti-CD73 antibody or antigen-binding portion and a second anti-CD73 antibody or antigen-binding portion, and mixing the two antibodies or portions.

[0324] Bispecific binding molecules

[0325] This invention also provides bispecific binding molecules having the binding specificity of the anti-CD73 antibody described herein (e.g., comprising an antigen-binding moiety, such as six CDRs or VH and VL). In some embodiments, the bispecific binding molecule additionally has binding specificity to another different anti-CD73 antibody (e.g., another anti-CD73 antibody described herein) or an antibody targeting a different protein (e.g., a cancer antigen or another cell surface molecule whose activity mediates a disease symptom such as cancer). Such bispecific binding molecules are known in the art, and examples of different types of bispecific binding molecules are given elsewhere herein. In some embodiments, the bispecific binding molecule may bind to CD73 and PD-1, CD73 and PD-L1, or CD73 and CTLA-4.

[0326] Nucleic acid molecules and vectors

[0327] This invention also provides nucleic acid molecules and sequences encoding the anti-CD73 antibody or its antigen-binding moiety described herein. In some embodiments, different nucleic acid molecules encode the heavy and light chain amino acid sequences of the anti-CD73 antibody or antigen-binding moiety. In other embodiments, the same nucleic acid molecule encodes the heavy and light chain amino acid sequences of the anti-CD73 antibody or antigen-binding moiety.

[0328] Unless otherwise specified herein, references to nucleotide sequences include their complements. Therefore, it should be understood that references to nucleic acids having a specific sequence include their complementary strands, having their complementary sequences. As used herein, the term "polynucleotide" means a polymeric form of nucleotides of at least 10 bases in length, a modified form of a nucleotide type of ribonucleotide or deoxynucleotide or both. This term includes both single-stranded and double-stranded forms.

[0329] In some embodiments, this disclosure provides nucleic acid molecules comprising a heavy chain or antigen-binding moiety encoding the anti-CD73 antibody described herein or its antigen-binding moiety thereof, or a light chain or antigen-binding moiety encoding the anti-CD73 antibody described herein or its antigen-binding moiety thereof, or both.

[0330] This invention 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 described herein, for example, nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to 8, or nucleotide sequences encoding amino acid sequences selected from the group consisting of SEQ ID NO: 9 to 16. In the context of nucleic acid sequences, the term “sequence identity percentage” refers to the number of identical residues when two sequences are compared for maximum correspondence. The length of a sequence identity comparison can be more than a segment of at least about nine nucleotides, typically at least about 18 nucleotides, more typically 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. Many different algorithms are known in the art for measuring nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, a program from the Wisconsin Software Package version 10.0, Genetic Calculator Group (GCG), Madison, Wisconsin. FASTA, including procedures such as FASTA2 and FASTA3, provides alignments and percentage sequence identity between the best overlapping regions of the query and search sequences (see, for example, 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); all incorporated herein by reference). Unless otherwise specified herein, default parameters for a particular procedure or algorithm are used. For example, the percentage sequence identity between nucleic acid sequences can be determined using FASTA and its default parameters (word length 6 and NOPAM factor of the scoring matrix) or using Gap and its default parameters as provided in GCG version 6.1, which is incorporated herein by reference.

[0331] In some embodiments, this disclosure provides nucleic acid molecules comprising nucleotide sequences selected from the group consisting of SEQ ID NO:1 to 8. In some embodiments, the nucleic acid molecule comprises nucleotide sequences of SEQ ID NO:1 and 5, SEQ ID NO:2 and 6, SEQ ID NO:3 and 7, or SEQ ID NO:4 and 8.

[0332] In any of the embodiments described above, the nucleic acid molecule may be isolated. A nucleic acid molecule referred to herein as “isolated” or “purified” is (1) a nucleic acid isolated from the genomic DNA or cellular RNA of its origin; and / or (2) a nucleic acid not naturally occurring.

[0333] In some embodiments, this disclosure provides vectors suitable for expressing one or both of the chains of antibodies or their antigen-binding portions as described herein. As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid molecule already linked to that other nucleic acid. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded DNA in which an additional DNA segment can be linked. In some embodiments, the vector is a viral vector in which the additional DNA segment can be linked into a viral genome. In some embodiments, the vector is capable of autonomous replication within the host cell into which it is incorporated (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). In other embodiments, the vector (e.g., a non-free mammalian vector) integrates into the genome of the host cell upon introduction and thereby replicates along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are effectively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0334] This disclosure provides vectors comprising a heavy chain, a light chain, or both a heavy chain and a light chain encoding a nucleic acid molecule encoding an anti-CD73 antibody as described herein or its antigen-binding moiety. In some embodiments, the vectors of the present invention comprise the nucleic acid molecules described herein. The present invention further provides vectors comprising nucleic acid molecules encoding a fusion protein, a modified antibody, an antibody fragment, and a probe thereof. The vector may further comprise an expression control sequence.

[0335] As used herein, the term "expression control sequence" refers to a polynucleotide sequence essential for the expression and processing of the coding sequence influencing its connection. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; and, if necessary, sequences enhancing protein secretion. The nature of these control sequences varies depending on the host organism; in prokaryotes, these control sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, generally, these control sequences include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components necessary for expression and processing, and may also include additional components that are advantageous, such as leader sequences and fusion chaperone sequences.

[0336] Nucleic acid molecules encoding the heavy and / or light chains of an anti-CD73 antibody or its antigen-binding moiety as described herein can be isolated from any source from which this antibody or moiety is produced. In some embodiments, the nucleic acid molecule is isolated from B cells expressing an anti-CD73 antibody isolated from an animal immunized with a human CD73 antigen, or from immortalized cells derived 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 polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In some embodiments, nucleic acid molecules as described herein can be synthesized instead of isolated.

[0337] In some embodiments, nucleic acid molecules as described herein comprise a nucleotide sequence encoding a VH domain from an anti-CD73 antibody or antigen-binding moiety as described herein, with an intrareading frame linked to a nucleotide sequence encoding a heavy chain constant region from any source. Similarly, nucleic acid molecules as described herein may comprise a nucleotide sequence encoding a VL domain from an anti-CD73 antibody or antigen-binding moiety as described herein, with an intrareading frame linked to a nucleotide sequence encoding a light chain constant region from any source.

[0338] In some embodiments of the present invention, nucleic acid molecules encoding VH and / or VL can be "converted" into full-length antibody genes. In some embodiments, nucleic acid molecules encoding VH or VL domains are converted into full-length antibody genes by insertion into expression vectors that respectively encode heavy chain constant (CH) or light chain constant (CL) regions, such that the VH segment is effectively linked to the CH segment within the vector and / or the VL segment is effectively linked to the CL segment within 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., ligate) the nucleic acid molecules encoding VH and / or VL domains to nucleic acid molecules encoding CH and / or CL regions using standard molecular biotechnology. The nucleic acid molecules encoding the full-length heavy chain and / or light chain can then be expressed in cells from which the nucleic acid molecules have been introduced, and anti-CD73 antibodies can be isolated.

[0339] Nucleic acid molecules can be used for recombinant expression of large quantities of anti-CD73 antibodies. As described in this article, these nucleic acid molecules can also be used to generate chimeric antibodies, bispecific antibodies, single-chain antibodies, immunoadhesives, bisomal antibodies, mutant antibodies, and antibody derivatives.

[0340] In some embodiments, the nucleic acid molecule of the present invention is used as a probe for a specific antibody sequence or as a PCR primer. For example, the nucleic acid can be used as a probe in diagnostic methods or as a PCR primer to amplify DNA regions of additional nucleic acid molecules that can be used, in particular, to isolate variable domains encoding anti-CD73 antibodies. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the oligonucleotide is derived from highly variable domains of the heavy and light chains of the target antibody. In some embodiments, the oligonucleotide encodes all or a portion of one or more of the CDRs of the anti-CD73 antibody of the present invention as described herein or its antigen-binding portion.

[0341] In some implementations, nucleic acid molecules and vectors can be used to prepare mutated anti-CD73 antibodies. The antibody can be mutated in variable domains of the heavy and / or light chains, for example, to alter the antibody's binding properties. For instance, mutations can be made in one or more of the CDRs to increase or decrease the K+ of the anti-CD73 antibody. D Increase or decrease k off This may alter the binding specificity of the antibody. In some embodiments, one or more mutations are made at amino acid residues that are known to vary compared to strains in monoclonal antibodies of the present invention. The mutations may be made in the CDR or framework region of the variable domain or in the constant region. In some embodiments, the mutations are made in the variable domain. In a specific embodiment, one or more mutations are made at amino acid residues that are known to vary compared to strains in the CDR or framework region of the variable domain of the antibody or its antigen-binding moiety of the present invention.

[0342] In some embodiments, the framework region is mutated so that the resulting framework region has the corresponding germline amino acid sequence. The mutation can be made in the framework region or the constant region, for example, to increase the half-life of the anti-CD73 antibody. See, for example, PCT disclosure WO 00 / 09560. Mutations in the framework region or the constant region 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 invention, the antibody can have mutations in any one or more of the variable domain's CDR or the framework region, or in the constant region.

[0343] In some embodiments, the anti-CD73 antibody or its antigen-binding portion thereof of the present invention is expressed by inserting the coding portion or full-length light and heavy chain DNA obtained as described above into an expression vector, thereby efficiently linking the gene to necessary expression control sequences (e.g., transcription and translation control sequences). Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses (e.g., cauliflower mosaic virus, tobacco mosaic virus), granules, YACs, EBV-derived episomes, etc. The antibody coding sequence can be linked into the vector such that the transcription and translation control sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody coding sequence. The expression vector and expression control sequences can be selected to be compatible with the expression host cells used. The antibody light chain coding sequence and the antibody heavy chain coding sequence can be inserted into the same or different vectors and can be efficiently linked to the same or different expression control sequences (e.g., promoters). In some implementations, both coding sequences are inserted into the same expression vector and can be efficiently ligated to the same expression control sequence (e.g., a common promoter), efficiently ligated to separate identical expression control sequences (e.g., promoters), or efficiently ligated to different expression control sequences (e.g., promoters). The antibody coding sequence can be inserted into the expression vector using standard methods (e.g., ligation of the antibody gene fragment and a complementary restriction site on the vector, or blunt-end ligation if no restriction site is present).

[0344] As described above, convenient vector systems encode fully functional human CH or CL immunoglobulin sequences, with appropriate restriction sites modified to allow easy insertion and expression of any VH or VL sequence. The genes encoding HC and LC in these vectors can contain intron sequences that lead to enhanced overall antibody protein yield by stabilizing the associated mRNA. These intron sequences are flanked by splice donor and acceptor sites, determining where RNA splicing will occur. When multiple introns are used, the intron sequences can be located in the variable or constant regions of the antibody chain, or both. Polyadenylation and transcription termination can occur at native chromosomal sites downstream of the coding region. Recombinant expression vectors can also encode signal peptides that promote antibody chain secretion from host cells. The antibody chain gene can be cloned into the vector to link the signal peptide reading frame to the N-terminus of the immunoglobulin chain. This signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0345] In addition to the antibody chain gene, the recombinant expression vector of the present invention may carry a regulatory sequence that controls the expression of the antibody chain gene in host cells. Those skilled in the art will recognize that the design of the expression vector (including the selection of the regulatory sequence) can depend on factors such as the selection of the host cell to be transformed, the desired protein expression level, etc. Preferred regulatory sequences for expression in mammalian host cells include viral components that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from: retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), polyomaviruses, and strong mammalian promoters (e.g., innate immunoglobulin and actin promoters). For further description of viral regulatory components and their sequences, see, for example, U.S. Patents 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants, including descriptions of promoters and vectors and plant transformation, are known in the art. See, for example, U.S. Patent 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells (e.g., yeast cells) are also well known in the art.

[0346] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the present invention may also carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells in which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, typically, selection marker genes can confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) in host cells in which the vector has been introduced. For example, selection marker genes include, for instance, a dihydrofolate reductase (DHFR) gene (for DHFR-host cells with methotrexate selection / amplification), a neo gene (for G418 selection), and a glutamate synthase gene.

[0347] Host cells and methods for producing antibodies and antibody compositions

[0348] This invention also provides methods for generating the antibody compositions and antibodies and their antigen-binding moieties described herein. In some embodiments, the invention relates to a method for generating anti-CD73 antibodies or antigen-binding moieties as described herein, comprising providing a recombinant host cell containing a heavy chain or antigen-binding moiety encoding the heavy chain or antigen-binding moiety described herein, and a light chain or antigen-binding moiety encoding the light chain or antigen-binding moiety described herein; culturing the host cell under conditions suitable for expressing the antibody or antigen-binding moiety; and isolating the resulting antibody or antigen-binding moiety. The antibody or antigen-binding moieties thus expressed in such recombinant host cells are referred to herein as “recombinant” antibodies or antigen-binding moieties. The invention also provides progeny cells of such host cells and antibodies or antigen-binding moieties generated therefrom.

[0349] As used herein, the term "recombinant host cell" (or simply "host cell") means a cell in which a recombinant expression vector has been introduced. By definition, recombinant host cells do not exist naturally. This disclosure provides host cells that may contain, for example, vectors as described herein. The invention also provides host cells that contain, for example, a heavy chain or an antigen-binding moiety thereof encoding an anti-CD73 antibody or an antigen-binding moiety thereof as described herein, a light chain or an antigen-binding moiety thereof encoding an anti-CD73 antibody or an antigen-binding moiety thereof, or both. It should be understood that "recombinant host cell" and "host cell" refer not only to the specific subject cell but also to its offspring. Due to mutations or environmental influences, certain modifications may appear in the offspring, and thus the offspring may be substantially different from the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0350] Nucleic acid molecules encoding anti-CD73 antibodies and their antigen-binding moieties, and vectors containing these nucleic acid molecules, can be used to transfect suitable mammalian, plant, bacterial, or yeast host cells. Transformation can be performed 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 cell nucleus. Alternatively, 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. Patents 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, bio-projectile transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0351] Available mammalian cell lines for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, in particular, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, young hamster kidney (BHK) cells, African green monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and many other cell lines. Particularly preferred cell lines are selected by identifying which cell lines exhibit high expression levels. Other cell lines that can be used are insect cell lines (e.g., 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 sufficient to allow expression of the antibody in the host cell, or more preferably, by secreting the antibody into the culture medium in which the host cell grows. The antibody can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, species of tobacco (Nicotiana), Arabidopsis, duckweed, corn, wheat, and potato. Bacterial host cells include species of *Escherichia coli* and *Streptomycetes*. Yeast host cells include *Schizosaccharomyces pombe*, *Saccharomyces cerevisiae*, and *Pichia pastoris*.

[0352] Furthermore, the expression of the antibody or its antigen-binding moiety from the cell line of the present invention can be enhanced using many known techniques. For example, the glutamine synthase gene expression system (GS system) is a commonly used method for enhancing expression under certain conditions. EP patents 0 216 846, 0 256 055, 0 323 997, and 0 338 841 discuss the GS system in whole or in part.

[0353] Antibodies expressed in different cell lines or in transgenic animals will likely have different glycosylation patterns from one another. However, all antibodies encoded by nucleic acid molecules provided herein or containing amino acid sequences provided herein are part of this invention, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modifications.

[0354] Pharmaceutical Composition

[0355] Another aspect of the invention is a pharmaceutical composition comprising, as an active ingredient (or as the sole active ingredient), the anti-CD73 antibody of the invention or its antigen-binding moiety, a bispecific binding molecule, or an antibody composition. The pharmaceutical composition may additionally comprise pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is intended for the improvement, prevention, and / or treatment of cancer (e.g., the cancers described herein). In some embodiments, the cancer is in tissues such as the skin, lungs, intestines, colon, ovaries, brain, prostate, kidneys, soft tissues, hematopoietic system, head and neck, liver, bones, bladder, breast, stomach, uterus, cervix, and pancreas. In some 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, bile duct cancer, thyroid cancer, or testicular cancer.

[0356] The pharmaceutical compositions of the present invention will comprise one or more of the anti-CD73 antibodies, antigen-binding moieties, antibody compositions, or bispecific binding molecules of the present invention, for example, one or two anti-CD73 antibodies, antigen-binding moieties, or bispecific binding molecules. In some embodiments, the composition comprises a single anti-CD73 antibody of the present invention or its antigen-binding moiety. In some embodiments, the composition comprises two different anti-CD73 antibodies of the present invention or their antigen-binding moieties.

[0357] In some embodiments, the pharmaceutical composition may comprise at least one anti-CD73 antibody of the present invention or its antigen-binding portion, for example, an anti-CD73 antibody or portion, and one or more additional antibodies targeting one or more associated cell surface receptors (e.g., one or more cancer-associated receptors).

[0358] Generally, the antibodies, antigen-binding moieties, and bispecific binding molecules of the present invention are suitable for administration as formulations in combination with one or more pharmaceutically acceptable excipients (e.g., excipients described below).

[0359] The term "excipient" is used herein to describe any component other than the compounds of this invention. The selection of excipients will depend to a great extent on factors such as the specific administration method, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutical-grade excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, and physiologically compatible analogs. Some examples of pharmaceutical-grade excipients are water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride, are preferably included in the composition. Additional examples of pharmaceutically-grade substances are wetting agents or small amounts of adjuvants such as wetting agents or emulsifiers, preservatives, or buffers that enhance the shelf life or efficacy of the antibody.

[0360] The pharmaceutical compositions and their preparation methods of the present invention will be readily apparent to those skilled in the art. Such compositions and their preparation methods can be found, for example, in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing, 1995). The pharmaceutical compositions are preferably prepared under GMP (Good Manufacturing Practice) conditions.

[0361] The pharmaceutical compositions of the present invention may 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 a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to a subject or a convenient portion of that dose (e.g., half or one-third of the dose).

[0362] Formulations of pharmaceutical compositions intended for parenteral administration typically comprise a combination of an active ingredient and a pharmaceutically acceptable carrier (e.g., sterile water or sterile isotonic saline). These formulations can be prepared, packaged, or sold in bolus administration or continuous administration. Injectable formulations can be prepared, packaged, or sold in unit dosage forms (e.g., in ampoules or in multi-dose containers containing preservatives). Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous carriers, pastes, etc. These formulations may further comprise one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In some embodiments of formulations for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water), and then the reconstituted composition is administered parenterally. Parenteral formulations also include aqueous solutions (preferably with a pH of 3 to 9) that may contain excipients (e.g., salts, sugars, and buffers), but for some applications, they may be more suitable as sterile non-aqueous solutions or as dry forms to be used in combination with suitable carriers (e.g., sterile, pyrogen-free water). Exemplary parenteral formulations include solutions or suspensions in sterile aqueous solutions (e.g., propylene glycol or dextran aqueous solutions). Such dosage forms may be appropriately buffered as needed. Other useful parenteral formulations include those containing active ingredients in microcrystalline form or as liposome preparations. Formulations for parenteral administration can be formulated for immediate and / or modified release. Modified release formulations include delayed, sustained, pulsatile, controlled, targeted, and programmed release.

[0363] In some embodiments, a sterile injectable solution can be prepared by incorporating the desired amount of anti-CD73 antibody, its antigen-binding moiety, bispecific binding molecule, or antibody composition into a suitable solvent having one or more of the components listed above, as needed, followed by filtration and sterilization. Generally, dispersants are prepared by incorporating the active compound into a sterile carrier containing an alkaline dispersion medium and other desired components listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying, which produce powders of the active ingredient plus any additional target components from their pre-sterilely filtered solutions. Appropriate flowability of the solution can be maintained, for example, by using a coating (e.g., lecithin), in the case of a dispersant, by maintaining the desired particle size, and by using a surfactant. Extended absorption of the injectable composition can be achieved by including a substance with delayed absorption (e.g., monostearate or gelatin) in the composition, and / or by using a modified release coating (e.g., a sustained-release coating).

[0364] The antibodies of the present invention can also be administered intranasally or by inhalation, typically in the form of dry powder from a dry powder inhaler (alone, as a mixture, or as a mixed component particle, for example, mixed with a suitable pharmaceutically acceptable excipient); as an aerosol spray from a pressurized container, pump, sprayer, nebulizer (preferably using electrohydraulic mechanics to produce a fine mist) or nebulizer, with or without a suitable propellant; or as nasal drops.

[0365] The antibodies and antibody portions of the present invention can also be formulated for oral administration. Oral administration may involve swallowing, allowing the compound to enter the gastrointestinal tract, and / or administration via the buccal, tongue, or sublingual route, through which the compound enters the bloodstream directly from the oral cavity. Formulations suitable for oral administration include solid, semi-solid, and liquid systems such as tablets; soft or hard capsules containing multiple particles or nanoparticles, liquids, or powders; lozenges (including liquid-filled lozenges); chewables; gels; rapidly dispersible dosage forms; films; ovoid bodies; sprays; and buccal / mucosal adhesive patches. Liquid formulations include suspensions, solutions, syrups, and elixirs. Such formulations can be used as fillers in soft or hard capsules (e.g., made from gelatin or hydroxypropyl methylcellulose) and typically contain carriers, such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or suitable oils, and one or more emulsifiers and / or suspending agents. Liquid formulations can also be prepared, for example, by reconstructing solids from sachets.

[0366] Therapeutic uses of the antibodies and compositions of the present invention

[0367] In some embodiments, the anti-CD73 antibody of the present invention and its antigen-binding moiety, anti-CD73 composition, and bispecific binding molecule are used to reduce CD73 activity in desired mammals (e.g., humans). For example, a physician may promote antitumor activity in a patient by administering the anti-CD73 antibody of the present invention alone or in combination with other therapeutic agents (sequentially or simultaneously). The anti-CD73 antibody reduces CD73 activity, thereby inhibiting pathways that suppress the patient's antitumor response.

[0368] In some embodiments, the antibody or its antigen-binding moiety, composition, or bispecific binding molecule is used to treat CD73-positive cancers. The cancer can occur in one or more tissues such as skin, lungs, intestines, colon, ovaries, brain, prostate, kidneys, soft tissues, hematopoietic system, head and neck, liver, bones, bladder, breast, stomach, uterus, cervix, and pancreas.

[0369] In some embodiments, cancers treated by the anti-CD73 antibody, antigen-binding moiety, bispecific binding molecule, and / or antibody composition of the present invention may include, for example, melanoma (e.g., advanced or metastatic melanoma), basal cell carcinoma of the skin, glioblastoma, glioma, gliosarcoma, astrocytoma, meningioma, neuroblastoma, adrenocortical carcinoma, head and neck squamous cell carcinoma, oral cancer, salivary gland carcinoma, nasopharyngeal carcinoma, 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, bile duct cancer, colon cancer, colorectal cancer, and ovarian cancer. Ovarian cancer, fallopian tube cancer, bladder cancer, upper urinary tract cancer, urothelial carcinoma, renal cell carcinoma, kidney cancer, urogenital system cancers, 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 myeloid leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, mast cell leukemia, small lymphocytic lymphoma, Burkitt's lymphoma. This includes lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, monocytic lymphoma, HTLV-associated T-cell leukemia / lymphoma, mesothelioma, and solid tumors. The cancer may be in, for example, early, intermediate, advanced, locally advanced, or metastatic stages, and may be relapsed or refractory to other treatments (e.g., other anti-CD73 agents) or there may be no available standard therapy.

[0370] In some embodiments, cancers treated by the anti-CD73 antibody, antigen-binding moiety, composition, and / or bispecific binding molecule of the present invention 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, bile duct cancer, thyroid cancer, and testicular cancer.

[0371] "Treatment" refers to any method of alleviating or eliminating at least one of a biological disease and / or its accompanying symptoms. As used herein, "alleviation" of a disease, ailment, or condition means reducing the severity and / or frequency of the symptoms of that disease, ailment, or condition. Furthermore, references to "treatment" herein include references to curative, palliative, and preventative treatments.

[0372] "Therapeutic effective dose" refers to the amount of a therapeutic agent administered that will alleviate one or more of the symptoms of a disease under treatment to a certain extent. Therapeutic effective doses of anticancer agents may, for example, lead to delayed tumor growth, tumor shrinkage, increased survival, elimination of cancer cells, slowing or reducing disease progression, reversal of metastasis, or other clinical endpoints desired by healthcare professionals.

[0373] The anti-CD73 antibodies or their antigen-binding moieties, antibody compositions, or bispecific binding molecules described herein may be administered alone or in combination with one or more other drugs or antibodies (or as any combination thereof). Therefore, as detailed below, the pharmaceutical compositions, methods, and uses described herein also include embodiments in combination with other active agents (co-administration).

[0374] As used herein, the terms “co-administered,” “after co-administration,” and “combined” with one or more other therapeutic agents relating to the anti-CD73 antibodies and their antigen-binding moieties, antibody compositions, and bispecific binding molecules of the present invention are intended to refer to and specifically refer to, and include, the following:

[0375] a) When such components are formulated into a single dosage form that releases the components substantially simultaneously to a patient in need, this combination of the antibody / antigen-binding moiety / antibody composition / bispecific binding molecule and therapeutic agent of the present invention is administered to the patient simultaneously.

[0376] b) When these components are formulated separately into separate dosage forms that are intended for simultaneous administration to a patient, thereby releasing the components substantially simultaneously to the patient, this combination of the antibody / antigen-binding moiety / antibody composition / bispecific binding molecule and therapeutic agent of the present invention is administered substantially simultaneously to the patient.

[0377] c) When these components are formulated separately into separate dosage forms for a patient in need to take continuously with significant time intervals between administrations, such that the components are released to the patient at substantially different times, this combination of the antibody / antigen-binding moiety / antibody composition / bispecific binding molecule and therapeutic agent of the present invention is administered to the patient sequentially; and

[0378] d) When such components are formulated together to release the components in a controlled manner, such that they are released simultaneously, sequentially and / or overlappingly to patients in need at the same and / or different times, wherein each part can be administered via the same or different routes in a single dosage form, this combination of the antibody / antigen binding portion / antibody composition / bispecific binding molecule and therapeutic agent of the present invention is sequentially administered to the patient.

[0379] The anti-CD73 antibody of the present invention, or its antigen-binding portion, antibody composition, or bispecific binding molecule, can be administered without additional therapeutic treatment, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment using the anti-CD73 antibody of the present invention, or its antigen-binding portion, antibody composition, or bispecific binding molecule, may include at least one additional therapeutic treatment (combination therapy), such as an immunostimulant, an anticancer agent (e.g., a chemotherapeutic agent, an antitumor drug, an antiangiogenic agent, or a tyrosine kinase inhibitor), or a vaccine (e.g., a tumor vaccine).

[0380] In some embodiments, the antibody or its antigen-binding portion, antibody composition, or bispecific binding molecule may be co-administered or formulated with another agent / drug to treat cancer. This additional therapeutic treatment may include, for example, immunostimulants, vaccines, chemotherapeutic agents, antitumor drugs, anti-angiogenic agents, tyrosine kinase inhibitors, and / or radiation therapy. In some embodiments, this additional therapeutic treatment may comprise different anticancer antibodies.

[0381] Pharmaceuticals comprising the anti-CD73 antibody or its antigen-binding moiety described herein, antibody compositions or bispecific binding molecules, and at least one other agent (e.g., a chemotherapeutic agent, an antitumor agent, or an antiangiogenic agent) may be used as combination therapy for simultaneous, separate, or sequential administration in cancer therapy. The other agent may be any agent suitable for treating the specific cancer being treated, for example, selected from: alkylating agents, such as platinum derivatives, such as cisplatin, carboplatin, and / or oxaliplatin; plant alkaloids, such as paclitaxel, docetaxel, and / or irinotecan; antitumor antibiotics, such as adriamycin, donomycin, epirubicin, idarubicin, and mitoxantrone. Dactinomycin, bleomycin, actinomycin, luteomycin, and / or mitomycin; topoisomerase inhibitors, such as topotecan; antimetabolites, such as fluorouracil and / or other fluoropyrimidines; FOLFOX; osimertinib; cyclophosphamide; anthracycline; dacarbazine; gemcitabine; or any combination thereof. In some embodiments, the anti-CD73 antibody described herein, or its antigen-binding moiety, antibody composition, or bispecific binding molecule, reconstitutes reactivity to another agent.

[0382] The anti-CD73 antibody or its antigen-binding moiety, antibody composition, or bispecific binding molecule of the present invention can also be used in combination with other anticancer therapies such as vaccines, cytokines, enzyme inhibitors, immunostimulatory compounds, and T-cell therapies. In the case of a vaccine, it can be, for example, a protein, peptide, or DNA vaccine containing one or more antigens associated with the cancer being treated, or a vaccine containing dendritic cells along with the antigen. Suitable cytokines include, for example, IL-2, IFN-γ, and GM-CSF. Examples of enzyme inhibitors with anticancer activity are indoleamine-2,3-dioxygenase (IDO) inhibitors, such as 1-methyl-D-tryptophan (1-D-MT). Adoptive T-cell therapy refers to various immunotherapeutic techniques involving the expansion or engineering of a patient's own T cells to recognize and attack their tumors.

[0383] It is also contemplated that the anti-CD73 antibody of the present invention, or its antigen-binding moiety, antibody composition, or bispecific binding molecule, can bind to tyrosine kinase inhibitors for use in adjuvant therapy. These are synthetic, primarily quinazoline-derived, low molecular weight molecules that, for example, interact with the intracellular tyrosine kinase domain of the receptor and inhibit ligand-induced receptor phosphorylation by competing for intracellular Mg-ATP binding sites.

[0384] In some embodiments, antibodies or their antigen-binding moieties, antibody compositions, or bispecific binding molecules may be used in combination with agents / pharmaceuticals that mediate immune system activation, including but not limited to those modulating 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, An agent that expresses or activates CHK2, CTLA-3, CEACAM (e.g., CEACAM-1 and / or 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 the expression of CD39, NKG2A, LAG-3, TIM-3, or TNFR2. In some embodiments, the agent is a small molecule inhibitor. In some embodiments, the agent is an antibody or its antigen-binding fragment that binds to one of the above molecules. In specific embodiments, the agent is an anti-PD-L1 antibody (e.g., duruvalumab or atozolizumab), an anti-PD-1 antibody, or an anti-CTLA-4 antibody. It is also contemplated that the anti-CD73 antibody of the present invention, or its antigen-binding moiety, antibody composition, or bispecific binding molecule, may be used in combination with cytokines (e.g., IL-1, IL-2, IL-12, IL-15, or IL-21), EGFR inhibitors, VEGF inhibitors, etc.

[0385] In some embodiments, the antibody and antigen binding moiety, antibody composition, or bispecific binding molecule of the present invention may be administered in combination with another inhibitor of the adenosine pathway, which may target, for example, CD73, CD39, or CD38, or an adenosine receptor selected from A1R, A2AR, A2BR, and / or A3R. These 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 these inhibitors include other anti-CD73 antibodies and anti-CD39 and anti-CD38 antibodies. In some embodiments, the anti-CD73 antibody or its antigen-binding portion, bispecific antibody or antibody composition of the present invention may be administered in combination with A001421, APCP, eurylumab, CPX-006 / CPI-006, CPX-016, NZV-930, BMS-986179, IPH53, PT199, duruvazumab, atomicumab, dalatumab or ixartuximab.

[0386] The present invention also considers the use of the anti-CD73 antibody or antigen-binding sequence described herein (e.g., six CDR or VH and VL sequences) to prepare chimeric antigen receptors that can be used in CAR-T technology.

[0387] It should be understood that the antibodies and their antigen-binding portions, antibody compositions, and bispecific binding molecules of the present invention can be used in the treatment methods described herein, in the treatments described herein, and / or in the preparation of medicaments for the treatments described herein. The present invention also provides kits and articles comprising the antibodies and their antigen-binding portions, antibody compositions, and bispecific binding molecules described herein.

[0388] Dosage and route of administration

[0389] The antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present invention can be administered in an effective amount to treat the treated condition, i.e., administered at the dose and for the duration necessary to achieve the desired outcome. The effective therapeutic amount may vary depending on factors such as the specific condition being treated, the patient's age, sex, and weight, and whether the antibody is administered as a standalone treatment or in combination with one or more additional anticancer therapies.

[0390] Dosing regimens can be adjusted to provide the optimal desired response. For example, a single bolus injection can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased according to the urgency of the treatment situation. It is particularly advantageous to formulate the parenteral composition into dose units to facilitate dosage administration and uniformity. As used herein, dose units refer to physically discrete units suitable as unit doses for use in the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect associated with the desired pharmaceutical carrier. The specification of the dose units of the present invention is generally defined by and directly dependent on the following: (a) the unique characteristics of the therapeutic agent and the specific therapeutic or preventive effect to be achieved, and (b) the inherent limitations in the field of sensitivities of the individual to which this active compound is combined for treatment.

[0391] Therefore, those skilled in the art will recognize that, based on the disclosure provided herein, dosage and administration regimens can be adjusted according to methods well known in the therapeutic field. That is, the maximum tolerable dose can be readily established, and the effective amount to provide a detectable therapeutic benefit to the patient can be determined, as can the time required for administering each drug to provide a detectable therapeutic benefit to the patient. Therefore, although certain dosages and administration regimens are illustrated herein, such examples do not in any way limit the dosages and administration regimens that can be provided to patients to practice the invention.

[0392] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated, and may include single or multiple doses. It should be further understood that, for any particular subject, the specific dosage regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition. The dosage ranges listed herein are merely exemplary and are not intended to limit the scope or practice of the specific compositions. Furthermore, dosage regimens using the compositions of the present invention may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific antibody used. Therefore, the dosage regimen can vary widely but can be routinely determined using standard methods. For example, the dosage may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Therefore, the present invention includes intra-patient dose escalation as determined by those skilled in the art. The determination of appropriate dosages and regimens is well known in the relevant art and will be understood by those skilled in the art upon the provision of the teachings disclosed herein.

[0393] Effective doses for cancer therapy can be measured by their ability to stabilize disease progression and / or improve symptoms in patients, and preferably reverse disease progression (e.g., by reducing tumor size). The ability of the antibodies, antigen-binding moieties, antibody compositions, or bispecific binding molecules of the present invention to inhibit cancer can be evaluated by in vitro assays (e.g., as described in the examples) and by suitable animal models for predicting efficacy in human tumors. Appropriate dosing regimens will be selected to provide an optimized therapeutic response in each specific situation, e.g., as a single bolus or as a continuous infusion, and the dose can be adjusted according to the urgency indicated by each situation.

[0394] The antibodies or antigen-binding portions thereof, antibody compositions, or bispecific binding molecules of the present invention can be administered by any method acceptable in the art for administering peptides, proteins, or antibodies, and are generally suitable for parenteral administration. As used herein, “parenteral administration” includes any route of administration characterized by physical destruction of the subject’s tissue and administration through an opening in that tissue, thus generally resulting in direct administration into the bloodstream, muscle, or internal organs. Therefore, parenteral administration includes, but is not limited to, administration by injection, administration through a surgical incision, administration through a tissue-penetrating non-surgical wound, etc. In particular, parenteral administration is contemplated including, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intracisional, intravenous, intraarterial, intrathecal, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion. Specific embodiments include intravenous and subcutaneous routes.

[0395] Diagnostic uses and compositions

[0396] The antibody-antigen binding portion of the present invention is also used in diagnostic methods (e.g., in vitro, ex vivo). For example, the antibody-antigen binding portion can be used to detect and / or measure CD73 levels in samples from patients (e.g., tissue samples or bodily fluid samples, 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 immunohistochemistry. The present invention further covers kits (e.g., diagnostic kits) comprising the antibody-antigen binding portion described herein.

[0397] Products and medicine boxes

[0398] This invention also provides articles, such as kits, comprising one or more containers (e.g., single-use or multiple-use containers) of a pharmaceutical composition containing an anti-CD73 antibody or its antigen-binding moiety, composition, or bispecific binding molecule as described herein, optionally additional bioactive molecules (e.g., another therapeutic agent), and instructions for use. The antibody or antigen-binding moiety, composition, or bispecific binding molecule, and optional additional bioactive molecules may be individually packaged in suitable packaging, such as vials or ampoules made of non-reactive glass or plastic. In some embodiments, the vial or ampoule contains a concentrated stock solution (e.g., 2x, 5x, 10x, or greater) of the antibody or antigen-binding moiety, composition, or bispecific binding molecule and optionally bioactive molecules. In some embodiments, the articles, such as kits, include a medical device (e.g., syringe and needle) for administering the antibody or antigen-binding moiety, composition, or bispecific binding molecule and / or bioactive molecules; and / or a suitable diluent (e.g., sterile water and saline). This invention also includes methods for preparing said articles.

[0399] Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials described herein may also be used in the practice or testing of this invention, exemplary methods and materials are described below. In case of conflict, this specification (including definitions) shall prevail.

[0400] Generally, the nomenclature and techniques used in conjunction with those described herein in cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, and protein and nucleic acid chemistry and hybridization are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly practiced in the art or as described herein.

[0401] Furthermore, unless the context otherwise requires, singular terms should include plurals and plural terms should include singulars. Throughout the specification and embodiments, the words “have” and “comprise” or variations thereof, such as “has,” “having,” “comprises,” or “comprising,” should be understood to imply inclusion of the integers or groups of integers mentioned, but do not exclude any other integers or groups of integers.

[0402] All publications and other references mentioned herein are incorporated herein by reference in their entirety. While this document cites some references, such citations do not constitute an acknowledgment that any of such references constitutes part of the general common knowledge in the field.

[0403] To better understand the present invention, the following embodiments are described. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. Example

[0404] Example 1: Cloning of anti-CD73 antibody from rat B cells

[0405] Materials and methods

[0406] since Anti-human CD73 antibodies were isolated from a rat-derived antibody library (Osborn et al., J Immunol. 190(4):1481-90(2013)). The rats are a transgenic rat strain from Ligand Pharmaceuticals Inc. that produces antibodies with a completely human idiotype. Antibody genes derived from antibody-secreting B-cell (ASC) clone rats sorted from single cells were obtained via Symplex. TM Antibody discovery technology was developed (Meijer et al., J Mol Biol 358(3):764-72(2006)).

[0407] An antibody library construct encoding complete human immunoglobulins in the form of IgG1-LALA (see below) was transfected into HEK293 cells. Flow cytometry was used to screen cell supernatants for binding to CD73 expressed on the surface of CHO cells in a high-throughput manner. CD73-reactive clones were analyzed by DNA sequencing, and DNA sequences encoding antibodies were extracted. Selected antibody clones were expressed and functionally tested as described below.

[0408] The antibody will be passed through a Symplex of cDNA fragments encoding the antibody. TM Missense mutations at the N-terminus of the heavy and light chains introduced using degenerate primers in cloning were corrected back to the germline sequences. Table 1 shows the nucleotide sequences of the variable domains of the heavy and light chains of germline antibodies named 21028, 21046, 21127, and 21163. The correction methods involved germline N-terminal sequence correction and codon usage optimization. Targets matching human germline sequences were identified by blast homology searching of the variable regions of the heavy and light chains.

[0409] 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.

[0410] result

[0411] Table 1 shows the nucleotide sequences encoding the variable domains of antibodies 21028, 21046, 21127, and 21163.

[0412] Table 1: Variable domain nucleotide sequences of antibodies 21028, 21046, 21127, and 21163:

[0413]

[0414]

[0415]

[0416] Table 2 shows the deduced amino acid sequences of antibodies 21028, 21046, 21127, and 21163. CDRs are in bold / underlined.

[0417] Table 2: Variable domain amino acid sequences of 21028, 21046, 21127, and 21163:

[0418]

[0419] Table 3 shows the amino acid sequences of the constant regions of the heavy and light chains (CH and CL, respectively). “IgG1LALA” refers to the presence of the “LALA” mutation (L234A / L235A, numbered according to the Kabat numbering scheme) in the heavy chain, which is known to reduce the effector function of the Fc region of the IgG1 antibody (Hezareh et al., J Virol. 75(24):12161-68(2001); Hescell et al., Nature 449(7158):101-04(2007)).

[0420] Table 3: Constant region amino acid sequences of antibodies 21028, 21046, 21127, and 21163:

[0421]

[0422] Table 4 shows the heavy and light chain CDR amino acid sequences of antibodies 21028, 21046, 21127 and 21163, wherein the CDRs are defined according to the IMGT system.

[0423] Table 4: CDR amino acid sequences of antibodies 21028, 21046, 21127 and 21163

[0424]

[0425] SID:SEQ ID NO:

[0426] Table 5 shows the SEQ ID NO information for antibodies 21028, 21046, 21127, and 21163. Unless otherwise specified herein, the sequences are amino acid sequences.

[0427] Table 5: SEQ ID NO of antibodies 21028, 21046, 21127 and 21163

[0428]

[0429] nt: nucleotide

[0430] aa: amino acids

[0431] Example 2: Screening for anti-CD73 antibodies using a soluble CD73 activity assay

[0432] The ability of a group of anti-CD73 antibodies to inhibit the enzymatic activity of soluble recombinant CD73 was evaluated. Anti-CD73 antibodies at a concentration of 10 μg / mL were incubated with recombinant CD73 (SinoBiological Incorporated), AMP, and ATP for 30 minutes at 37°C. [The text then abruptly shifts to a different topic:] ...by using... 2.0 (Promega) AMP inhibition studies of CD73 activity by measuring ATP, as described by Sachsenmeier et al., Journal of Biomolecular Screening 17(7):993-998 (2012).

[0433] Inhibition of CD73 enzyme activity after treatment with anti-CD73 antibody (see [link to relevant documentation]). Figure 1 Clearly, the CD73 activity varied greatly after treatment with different anti-CD73 antibodies, indicating that some antibodies were non-functional in this assay, while others strongly inhibited CD73 activity.

[0434] Example 3: Screening for anti-CD73 antibodies using a cell-based CD73 activity assay

[0435] The inhibitory effect of a group of anti-CD73 antibodies on the activity of CD73 expressed in cancer cell lines was evaluated. Anti-CD73 antibodies at a concentration of 10 μg / mL were incubated with CD73-expressing cell lines at 37°C for 30 minutes, followed by the addition of the CD73 substrate AMP, and incubation at 37°C for another 3 hours. When investigating CD73 activity, ATP was added to the supernatant, and... 2.0 (Promega) Measure AMP inhibition by ATP detection, as described by Sachsenmeier et al. (ibid.).

[0436] After treatment with anti-CD73 antibody, the activity of CD73 expressed on two different cancer cell lines was shown to be... Figure 2Clearly, CD73 activity varied considerably after treatment with different anti-CD73 antibodies; some antibodies were non-functional in this assay, while others strongly inhibited CD73 activity. In particular, the same ten antibodies showed maximum activity in both cell lines.

[0437] Example 4: Cloning anti-CD73 reference antibody analogs

[0438] Materials and methods

[0439] The amino acid sequences encoding the heavy and light chain variable domains of antibody analogs, as listed in Table 6, were obtained from the patents or patent applications cited herein. The protein sequences were reverse-translated into DNA sequences using human codons. The corresponding DNA sequences were genetically synthesized and cloned into expression vectors containing the human heavy or light chain constant regions, resulting in the expression of the full-length antibody chain. The selected human antibody isotypes for expression, along with any additional mutations introduced into the Fc region as needed, are listed in the antibody form column. CHO cells were transfected with the resulting expression plasmid using a standard protein expression system. The corresponding antibody supernatant was purified using standard protein A purification column chromatography.

[0440] Table 6: List of genetically synthesized antibody analogs and corresponding antibody forms

[0441]

[0442] Example 5: Direct binding of anti-CD73 antibody to CHO-S cells transfected with human or cynomolgus monkey CD73 protein

[0443] The binding of anti-CD73 antibodies 21028, 21046, 21127, and 21163 to human or cynomolgus monkey CD73 protein expressed on CHO-S cells was evaluated and compared with binding to eurymumab analogues.

[0444] The anti-CD73 antibody was cultured for 30 minutes at 4°C with a hamster CHO-S cell line transiently expressing human or cynomolgus macaque CD73. The cells were washed twice and then cultured for another 20 minutes with a secondary anti-human IgG (H+L) antibody conjugated with AF647. After the washing step, antibody binding was detected by measuring the geometric mean of the AF647 signal in each well using high-throughput flow cytometry with an iQue Screener PLUS (Sartorius). Triple analysis was performed for each concentration and for each antibody to generate a 12-point titration curve.

[0445] The binding curves of the antibody to human or cynomolgus monkey CD73 expressed on cells are shown in the figure. Figure 3The analyzed antibodies bound to human and cynomolgus monkey CD73 proteins displayed on cells at varying titers and potencies. Specifically, mAb 21127 bound to human and cynomolgus monkey CD73 at the highest titer of the tested antibody.

[0446] Example 6: Measurement of affinity of antibody and Fab fragment for human and cynomolgus monkey CD73 extracellular domain (ECD)

[0447] This example demonstrates the binding of anti-CD73Fab fragments and antibodies to the extracellular domain (ECD) of CD73 in recombinant humans and cynomolgus monkeys, as measured by surface plasmon resonance (SPR).

[0448] Materials and methods

[0449] Kinetic binding analysis of anti-CD73 mAb and Fab fragments was performed using surface plasmon resonance (SPR) with a combination of a continuous flow micro-spotting instrument (CFM, Wasatch Microfluidics, Salt Lake City, US) and an IBIS MX96 SPR instrument (IBIS Technologies, The Netherlands). CD73 cDNA encoding the extracellular domains of human and cynomolgus monkey CD73 was synthesized and cloned into vectors containing a CMV promoter and a C-terminal 6x histidine sequence (SEQ ID NO: 45) for a His tag on the CD73 ECD, or a human IgG1 Fc sequence (AA P101-K330), resulting in C-terminal fusion of the IgG1 Fc into the cloned CD73 ECD. ExpiCHO was used... TM The expression system transiently expresses His-tagged constructs and Fc fusion constructs, respectively, and passes them through standard Ni-NTA chromatography or standard MabSelect chromatography. TM Sure TM Purification was performed. The anti-CD73Fab fragment was generated by digesting the full-length IgG1 antibody with GingisKHAN enzyme using a kit provided by Genovis (Sweden). The Fc-tagged antigen was captured in Ga-hu-IgG Fc using CFM. The affinity of the Fab fragment was measured 15 minutes later. After spotting, Antigens were placed in an IBIS MX96 biosensor and immobilized using the SensEye FixIt kit. Kinetic analysis was performed by injecting monomeric Fab fragments at concentrations increasing from 0.8 nM to 300 nM. After each Fab fragment injection cycle, the surface was regenerated with 10 mM glycine (pH 3) and 10% glycerol. Fab binding and antigen dissociation were performed for 15 minutes each. mAbs were captured onto Ga-hu-IgG Fc using a continuous flow micro-spotting system (CFM, Wasatch Microfluidics, Salt Lake City, US). Affinity of full-length monoclonal antibody (mAb) was measured at 15 minutes on a SensEye (Ssens BV, The Netherlands) instrument. After spotting, the SensEye was stopped in an IBIS MX96 and the spotted mAb was fixed using the SensEye FixIt kit (Ssens BV, The Netherlands). Kinetic analysis was performed by administering a series of kinetic titrations at 2-fold dilutions, injected with His-tagged antigen at concentrations increasing from 0.16 nM to 10 nM. The surface was regenerated with 100 mM H3PO4 at pH 3 before the next cycle of cynomolgus monkey antigen injection. MAb binding was performed for 15 minutes and antigen dissociation for 45 minutes. The recorded binding reactions were fitted to a simple Langmuir 1:1 binding model, and the binding rate (kb / g) was calculated using Scrubber 2.0 software. on or k a ), dissociation rate (k off or k d ) and affinity (K D The constant is the average of six independent measurement points, which is combined with the dynamic parameters.

[0450] result

[0451] The binding affinity and kinetic parameters for the anti-CD73 Fab fragment and full-length antibodies are shown in Tables 7 and 8, respectively. The full-length antibody binds to human CD73 with high affinity in the sub-nanomolar range. All antibodies recognize human and cynomolgus CD73 with binding kinetics comparable to reference antibodies (11E1, eurymumab, and analogs of CPX006). All antibodies are characterized by very slow dissociation rate constants, which facilitate the measurement of high affinity. The monovalent binding affinity of the Fab fragment is 20 to 245 times weaker than that of the corresponding full-length antibodies. The dissociation rate (kd) is strongly influenced by changes in antibody form, with the Fab fragment showing much faster dissociation from CD73 than the full-length antibody.

[0452] The difference in binding kinetics between the full-length antibody and the Fab fragment suggests that the antibody binds to the CD73 homodimer, and that the high affinity measured against the full-length antibody is due to an affinity effect. CD73 is known to exist as a non-covalent homodimer (Knapp et al., Structure 20(12):2161-2173(2012)), and the binding of 11E1 and eurymumab analogues has been previously found to depend on bivalent binding to the CD73 homodimer, which has similarly different binding kinetics to the Fab fragment as described herein (Perrot, Cell Reports 27(8):2411-2425(2019)).

[0453] SPR analysis of the anti-CD73 antibody and the Fab fragment showed that the anti-CD73 antibody depends on the divalent binding to the CD73 homodimer, and that the binding affinity of the full-length antibody illustrated in this paper is comparable to that of the reference mAb when binding to the CD73 homodimer.

[0454] Table 7: Binding kinetics of anti-CD73Fab fragment to human (Hs) and cynomolgus monkey (Cy) CD73 ECD, as measured by SPR.

[0455]

[0456]

[0457] Table 8: Binding kinetics of anti-CD73 antibody to human (Hs) and cynomolgus monkey (Cy) CD73 ECD, as measured by SPR.

[0458]

[0459] Example 7: Epitope binning of anti-CD73 antibody

[0460] This embodiment describes grouping anti-CD73 antibodies into epitope loci based on a pairwise competition model. Antibodies belonging to different epitope loci recognize different epitopes on the ECD of CD73.

[0461] Materials and methods

[0462] The study of pairwise antibody competition was performed using an IBIS-MX96 instrument (IBIS, Netherlands) via SPR. Anti-CD73 antibody was diluted to 3 μg / mL in PBS and captured for 15 minutes using a continuous flow micropipe sampler before being spotted onto Ga-hu-IgG Fc. The remaining binding sites were then blocked with Herceptin (trastuzumab) and chemically cross-linked using the SensEye FixIt kit (IBIS, Netherlands). After sensor preparation, antibody competition analysis was performed using a classic sandwich assay. The CD73-His ECD antigen was diluted in PBS, 0.05% Tween 20, and 200 nM Herceptin running buffer, then injected at a concentration of 10 nM and captured by a conjugated array of anti-CD73 antibodies. Subsequently, individual injections of each CD73 antibody, diluted to 100 nM in running buffer, were performed to establish antibody competition. Data were analyzed using epitope compartment 2.0 (Wasatch, USA).

[0463] result

[0464] The competitive pattern of 16 anti-CD73 antibodies was shown in Figure 4 The anti-CD73 antibodies tested were grouped into four overlapping major epitope bins: 1, 2, 3, and 4. Bind 1 comprised olimulumab and an 11E1 analog, which cross-blocked each other. Olimulumab and 11E1 antibodies have been shown to bind overlapping epitopes at the top of the N-terminal domain and opposite 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. Bind 2 comprised antibodies 21385, 21127, and 21163, which showed comparable competitive patterns, indicating that the antibodies bound similar epitopes. The antibodies in bin 2 cross-blocked all tested antibodies except those in bin 4. The antibodies in bin 4 were divided into two sub-bins: bins 4a and 4b, which competed with one or more antibodies in bin 3. Antibody 21046 (Channel 4a) and CPX006 analog (Channel 4b) compete with different antibodies in Channel 3, indicating that the two antibodies have closely related but different binding epitopes.

[0465] In summary, antibodies 21127 and 21163 (Channel 2) bind to a unique epitope of CD73, which overlaps somewhat with the epitopes of 11E1 and eurylumab analogs (Channel 1). The epitopes of antibodies 21127 and 21163 differ from the epitope of the CPX006 analog (Channel 4). Antibody 21046 (Channel 4a) binds to an epitope similar to that of the CPX006 analog (Channel 4b), but different from the epitopes of antibodies 21127 and 21163 (Channel 2) and the epitopes of 11E1 and eurylumab analogs (Channel 1).

[0466] Example 8: Epitope localization of anti-CD73 antibody induced by CD73 mutagenesis

[0467] This example illustrates how binding epitopes of antibodies 21127, 21163, and 21046 can be classified into linear epitopes and contact residues by measuring binding affinity and kinetics to 94 different CD73 mutants.

[0468] Materials and methods

[0469] The protein sequences of human and rat (Rattus norvegicus) CD73 were downloaded from UniProt (accession numbers P21589 and P21588, respectively). The 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). Available CD73 structural PDBs 4H2F (open), 4H2G (open), and 4H2I (closed) were disclosed for locating surface-exposed amino acid residues. The different surface-exposed residue positions between human and rat CD73 were mutated to alanine. To locate linear antibody epitopes, a CD73 chimeric protein was generated, in which 10 amino acids from the human CD73 ECD sequence were sequentially interchanged with overlapping 5-amino acid segments to form the chicken sequence.

[0470] The cDNA encoding the extracellular domain of human CD73 was synthesized and cloned into a vector containing the CMV promoter and the human Ig Fc sequence (residues P101 to K330), resulting in the C-terminus fusion of the Ig Fc into the cloned CD73ECD. Wild-type (wt) and mutant human CD73Fc fusion constructs were produced using standard gene synthesis techniques, and the proteins were prepared using ExpiCHO. TM The expression system was transiently expressed in 2 mL of culture.

[0471] After harvesting the human CD73Fc fusion construct, the binding of the supernatant to anti-CD73 Fab was tested by surface plasmon resonance (SPR). The culture supernatant containing the CD73 fusion protein was immobilized on Ga-hu-lgG Fc using a continuous flow micro-spotting system (CFM, Wasatch Microfluidics, Salt Lake City, US). (Ssens BV, The Netherlands) 15 minutes. After sampling, The captured proteins were placed in an IBIS MX96 biosensor and immobilized on the surface using a FixIT kit (Ssens BV, The Netherlands). Kinetic analysis was performed by applying a series of kinetic titrations, in which the monomeric Fab fragment of the antibody of the present invention was injected at concentrations increasing from 0.8 nM to 500 nM. After each cycle of Fab fragment injection, the surface was regenerated with 10 mM glycine at pH 3 and 10% glycerol. Fab binding and antigen dissociation were performed for 15 minutes. The recorded binding reactions were fitted to a simple Langmuir 1:1 binding model, and the binding rate (kJ / kb) was calculated using Scrubber 2 software. on or k a ), dissociation rate (k off or k d ) and affinity (K D )constant.

[0472] result

[0473] Chimeric receptor constructs were used to target the CD73 epitopes recognized by antibodies 21127, 21163, and 21046 and 11E1, eurymumab, and CPX006 analogues, wherein 10 amino acid segments of the human CD73 sequence were replaced with chicken sequences; or by alanine scanning, wherein surface-exposed amino acids that differ between human and rat CD73 were mutated to alanine. The binding affinity of antibodies to wild-type CD73 and mutants was measured by SPR, and a cutoff value of at least 5-fold decrease in affinity compared to wt or deviation from the 1:1 binding model was used to identify constructs with significant loss of binding to anti-CD73 antibodies (Table 9).

[0474] Table 9: Summary of binding specificity of antibodies 21127, 21163, 21046, 11E1, oliguria, and CPX006 analogs

[0475]

[0476]

[0477] The identified linear epitopes and contact residues were located in their open state on the crystal structure of the CD73 homodimer. Figure 5 The epitopes recognized by antibodies 21127, 21163, 21046, and 11E1, as well as olivazoli and CPX006 analogues, were found to be located on the N-terminal domain of CD73. Figure 5 The epitopes of antibodies 21127, 21163, and 11E1, and eurylomarazole analogs, were found to be located on surfaces perpendicular to the dimerization interface of the CD73 homodimer and on opposite sides relative to the catalytic center. Figure 5(Figures A, B, D, and E). The epitopes of the four antibodies differ in that they bind to different contact residues and linear epitopes. Antibodies binding to the top surface of CD73 can block enzyme activity by locking the enzyme in an open, inactive conformation, preventing it from transitioning to a closed, active state (Perrot et al., Cell Reports 27:2411-2425 (2019)).

[0478] The epitopes of antibody 21046 and CPX006 analogs were found to be located before the N-terminal domain, directly above the catalytic center of CD73. Figure 5 (See sub-figures C and F). These two antibodies differ in terms of the linear epitopes they recognize and the contact residues they contact.

[0479] In summary, epitope localization at single-amino acid resolution revealed that the example anti-CD73 antibodies each bind to distinct epitopes. Antibodies 21127 and 21163 bind to similar epitopes at the top of the CD73 N-terminal domain; these epitopes differ from those of 11E1, eurymumab, and CPX006 analogs. Antibodies 21046 and CPX006 analogs bind to overlapping but still distinct epitopes preceding the CD73 N-terminal domain.

[0480] Example 9: Stoichiometry of the antibody / CD73 complex formed in solution

[0481] This example describes the size of the antibody / CD73 ECD complex formed in solution, as measured by SEC-MALS.

[0482] Materials and methods

[0483] The size of the complex formed between the CD73 homodimer and the anti-CD73 antibody was analyzed at different ratios and separately. Samples were prepared by mixing 900 pmol CD73-His with antibodies diluted in PBS at pH 7.4 at 900, 450, 90, or 0 pmol. Samples were incubated at room temperature for 30 min, followed by separation using a UHPLC-UltiMate 3000 (Thermo Scientific) and an SEC X-Bridge column (Waters) at a flow rate of 1.2 mL / min. The sample run 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 graphs were generated using GraphPad Prism.

[0484] result

[0485] Divalent binding of the antibody to the CD73 homodimer locks the enzyme into 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 can be through cross-linking of the CD73 homodimer or through binding of a single CD73 dimer in the inactive state. To characterize the stoichiometry of the antibody / CD73 complex formed in solution, the size of the protein complex was determined by SEC-MALS. The size of the 1:1 antibody / CD73 homodimer complex was in the range of 270 to 280 kDa, calculated from the molecular weights of the antibody and the CD73 homodimer (approximately 150 and 125 kDa, respectively). Figure 6A and 6B Table 10 shows the SEC-MALS plots and sizes of the complexes formed by antibodies 21127, 21163, and 21046 and 11E1, eurymumab, and CPX006 analogs binding to the CD73 homodimer at various molecular ratios. Separate runs of CD73 and each antibody showed monodisperse peaks matching the expected protein size and confirmed the absence of aggregates. Figure 6A and 6B (dashed line). The SEC-MALS plot of antibody 21127 bound to CD73 shows a main peak (peak 2) with an average size of 276 kDa, corresponding to the predicted size of the 1:1 21127:CD73 homodimer complex, and a smaller peak (peak 1), corresponding to excess 21127 or CD73 dimer. Antibodies 21163 and 11E1 and CPX006 analogs mainly form 1:1 complexes, but tend to form higher-order complexes at the highest antibody:CD73 ratio (1:1). For CPX006 and 11E1 analogs, polydisperse higher-order complexes are most prominent. Antibodies 21046 and eurylumab analogs form large complexes, indicating that these antibodies mainly crosslink the respective CD73 dimers, which has also been shown for eurylumab (MEDI9447) (Geoghegan et al., MABS 8(3):454-467(2016)).

[0486] Table 10: Molecular weight (MW, kDa) of complexes formed when CD73 and anti-CD73 antibodies are mixed at various ratios, as measured by SEC-MALS.

[0487]

[0488]

[0489] If counted from right to left, the peaks in Table 10 correspond to... Figure 6A and 6B Peaks 1 to 4 are visible on the chromatogram. Peak 1 corresponds to unbound / excess CD73 homodimer (~125 kDa) or unbound / excess antibody (~150 kDa). Peaks 2 to 4 correspond to mAb:CD73 complexes of various sizes and stoichiometry, where the complex size of ~275 kDa represents a 1:1 stoichiometry.

[0490] As shown in Table 10 and Figure 6A and 6B The results showed that antibody 21127 binds to the epitope on the CD73 homodimer in a manner independent of CD73 concentration, forming a 1:1 complex. Antibodies 21163 and 11E1 and CPX006 analogs primarily bind to this CD73 dimer to form 1:1 complexes, but also showed a tendency to form higher-order complexes to varying degrees. Antibodies 21046 and eurylomarone analogs bind only to the CD73 homodimer to form oligomeric complexes, indicating cross-linking of multiple CD73 dimers.

[0491] Example 10: Functional role of anti-CD73 antibody in soluble CD73 activity assay

[0492] The ability of anti-CD73 antibodies 21028, 21046, 21127, and 21163 to inhibit the enzymatic activity of soluble recombinant CD73 was evaluated in more detail using a soluble CD73 activity assay and compared with eurymumab analogues. The anti-CD73 antibodies were incubated with recombinant CD73, AMP, and ATP at 37°C for 2 hours. [Further details regarding the assay are needed for accurate translation.] 2.0 (Promega) AMP inhibition was measured by ATP assay to study CD73 activity, as described by Sachsenmeier et al. (ibid.).

[0493] Inhibition of CD73 enzyme activity after treatment with indicated concentrations of anti-CD73 antibody or eurymumab analogue was shown in Figure 7 Clearly, the inhibitory function of anti-CD73 antibodies is concentration-dependent, and all anti-CD73 antibodies inhibit CD73 activity despite differences in titer and efficacy. Furthermore, this eurymarumab analogue is less effective at inhibiting CD73 enzyme activity at higher antibody concentrations. MedImmune has published similar results for eurymarumab (formerly MEDI9447) (Geoghegan et al., MABS8(3):454-467(2016)). Compared to intermediate concentrations, antibodies 21028 and 21046 also showed slightly less inhibition of CD73 enzyme activity at higher concentrations. Conversely, antibodies 21127 and 21163 showed maximum inhibition of CD73 enzyme activity at all concentrations above approximately 3 μg / mL.

[0494] Example 11: Functional role of anti-CD73 antibody in cell-based CD73 activity assay

[0495] 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 with eurymumab analogues. Anti-CD73 antibodies were incubated at 37°C for 30 min with CD73-expressing human cell lines (Calu-6 and H292) or CD73-expressing cynomolgus monkey cell line (Cynom-K1), followed by addition of the CD73 substrate AMP and incubation at 37°C for another 3 h. ATP was added to the supernatant and... 2.0 (Promega) AMP inhibition was measured by ATP assay to study CD73 activity, as described by Sachsenmeier et al. (ibid.).

[0496] Inhibition of CD73 activity after treatment with different anti-CD73 antibodies was shown in Figure 8 Clearly, the inhibitory function of anti-CD73 antibodies is concentration-dependent, and despite differences in titer and efficacy, all antibodies inhibited CD73 activity in both humans and cynomolgus monkeys. Antibodies 21127 and 21163 showed the highest efficacy in both human cell lines, Calu-6 and H292, but in all three cell lines, all four tested antibodies were significantly superior to eurymumab analogues.

[0497] Example 12: Functional role of anti-CD73 antibody in long-term cell-based CD73 activity assay

[0498] 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 using a long-term cell-based assay and compared with eurymumab analogues. The anti-CD73 antibodies were incubated with the CD73-expressing human cell line H292 at 37°C for 30 min, followed by the addition of the CD73 substrate AMP and incubation at 37°C for 3, 6, or 24 hours. The activity was assessed by adding ATP to the supernatant and using… 2.0 (Promega) AMP inhibition was measured by ATP assay to study CD73 activity, as described by Sachsenmeier et al. (ibid.).

[0499] Inhibition of CD73 activity after treatment with different anti-CD73 antibodies was shown in Figure 9Clearly, the inhibitory function of anti-CD73 antibodies is concentration-dependent, and all antibodies inhibit CD73 activity despite differences in titer and efficacy. Specifically, incubating cells with antibodies for up to 24 hours further differentiated antibodies 21127 and 21163 from eurymumab analogs (and two other anti-CD73 antibodies tested).

[0500] Example 13: Efficacy of anti-CD73 antibody against cancer cell lines

[0501] To evaluate the ability of anti-CD73 antibodies 21127, 21046, and 21163 to inhibit the activity of CD73 expressed on a large group of cancer cell lines representing a broad range of CD73 expression and activity levels. As described in Table 11, this cell group spans several cancer indications across multiple tissues of origin.

[0502] Materials and methods

[0503] Anti-CD73 antibody was incubated at 25 μg / mL with CD73-expressing cell lines at 37°C for 30 minutes, followed by incubation at 37°C for another 3 hours with the CD73 substrate AMP. ATP was added to the supernatant and then... 2.0 (Promega) AMP inhibition was measured using ATP assays to assess cellular CD73 activity, as described by Sachsenmeier et al. (ibid.). CD73 activity was detected by luminescence.

[0504] result

[0505] The effect of anti-CD73 antibody on the activity of CD73 expressed on 20 different cancer cell lines was shown in Figure 10 Clearly, CD73 activity varies after treatment with different anti-CD73 antibodies. In cell lines such as Calu-6, NCI-H1775, KYSE-30, and Capan-2, some antibodies (21046 and eurymumab analogs) showed some ability to inhibit CD73 at lower cellular CD73 activity levels, but failed 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.

[0506] Table 11: List of cell lines and tissues of origin

[0507]

[0508]

[0509] Example 14: In vitro assay of anti-CD73 antibody activity using a cell viability assay

[0510] This example describes the in vitro functional characterization of antibody 21127 using two cancer cell lines grown in the presence of 300 μM AMP in a survival assay.

[0511] Materials and methods

[0512] The ability of the antibody to directly inhibit the survival (survival and / or proliferation) of triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 was assessed in vitro. Cells were seeded at 1000 cells / well in 384-well plates in RPMI 1640 Glutamax (MDA-MB-231) or DMEM (MDA-MB-468) supplemented with 2% FBS and 1% P / S, and incubated for four days at 37°C in a humidified incubator with antibody titrated from 25 μg / mL in the presence of 300 μM AMP (adenosine monophosphate, Sigma-Aldrich). Cell viability was quantified using the WST-1 cell proliferation reagent (Roche) according to the manufacturer's instructions.

[0513] result

[0514] Clearly, the functional readouts for 21127 and the olemumab analogue are different. Figure 11 Antibody 21127 showed significant effects on both cell lines, reducing viable cell counts to approximately 60% in MBA-MB-231 and to 0% in MBA-MB-468 compared to untreated cells (i.e., no measurable viable cells relative to the culture background). Olemimarab analogues did not show significant effects on the viability of the MBA-MB-231 cell line, while MBA-MB-468 was affected to some extent, with viable cell counts reduced to approximately 70-80% compared to untreated cells.

[0515] Similar effects can be expected in cancer cell lines derived from other tissues.

[0516] Example 15: Inhibition of primary CD4 expression from healthy human donors by anti-CD73 antibody + and CD8 + T cells and CD19 + CD73 on B cells

[0517] Evaluation of anti-CD73 antibody inhibition of primary CD4 expression from healthy human donors + and CD8 + T cells and CD19 + The ability of CD73 to function on B cells.

[0518] Materials and methods

[0519] According to the manufacturer's instructions (Miltenyi Biotec), primary CD4+ cells were isolated from PBMCs (peripheral blood mononuclear cells) in the erythrocyte sedimentation rate (ESR) layer using magnetically specific (MACS) beads. + and CD8 + T cells and CD19 + B cell line. Primary cells were incubated at 37°C for 30 minutes with a four-fold dilution of anti-CD73 antibody at concentrations of 50 to 0.003 μg / mL, followed by incubation at 37°C for another 20 hours with the CD73 substrate AMP. (CD19) + B cells) or 40 hours (CD4) + and CD8 + T cells). By adding ATP to the supernatant and using 2.0 (Promega) AMP inhibition was measured by ATP assay to assess CD73 activity, as described by Sachsenmeier et al. (ibid.).

[0520] result

[0521] After treatment with anti-CD73 antibody, it was expressed on primary CD4 cells. + and CD8 + T cells and CD19 + CD73 activity on B cells is shown to Figure 12 Clearly, there was a significant difference in CD73 activity after treatment with antibody 21127 and eurylumab analogue. Higher concentrations of antibody 21127 showed almost complete inhibition of CD73 activity in all primary cell types, while eurylumab analogue showed inhibition of CD19... + B cells and CD8 + Very limited inhibition of CD73 activity in T cells and CD4 + Only partial inhibition occurs in T cells. The ability of oliguria analogs to inhibit CD73 activity is correlated with the level of CD73 expression, because peripheral blood CD4 from healthy donors... + T cells, on average, have limited CD73 expression, while CD19... + B cells express high levels of CD73 and CD8 on average. + T cells express moderate levels of CD73 on average (Allard et al., Immunol Rev 276(1):121-144(2017)).

[0522] Example 16: Functional role of anti-CD73 antibody in T cell proliferation assay

[0523] In an in vitro assay, the inhibitory effects of anti-CD73 antibodies 21028, 21046, 21127, and 21163 on CD4 expression were evaluated. + The ability of CD73 to activate on T cells was compared with that of eurymumab analogues. CD43 isolated from healthy donors... + T cells were activated for 48 hours using anti-CD3 / CD28 beads (Thermo Fisher Scientific), AMP, and anti-CD73 antibody, followed by the addition of 3H-thymidine (PerkinElmer) for another 24 hours. T cell proliferation was measured as a control with 3H-thymidine incorporation and normalized to the untreated control.

[0524] T cell proliferation after treatment with different anti-CD73 antibodies was shown to be... Figure 13 Clearly, the stimulatory effect of anti-CD73 antibodies on T cell proliferation is concentration-dependent. Although their titers and efficacy differ, all antibodies stimulate T cell proliferation. Except for the eurylumab analogue, all antibodies completely restored T cell proliferation, with antibody 21127 exhibiting the highest titer.

[0525] Example 17: Functional role of anti-CD73 antibody in T cell activation assay

[0526] In an in vitro assay, the inhibition of CD73 antibody expression on CD4 was evaluated. + and CD8 + The ability of CD73 to activate on T cells was compared with that of eurymumab analogues.

[0527] CD4 isolated from healthy donors + and CD8 + T cells were activated for 72 hours using anti-CD3 / CD28 beads (Thermo Fisher Scientific), AMP, and anti-CD73 antibody, followed by harvesting of the supernatant. T cell activation was measured as the IFN-γ level in the supernatant using an ELISA (Thermo Fisher Scientific).

[0528] T cell activation after treatment with different anti-CD73 antibodies was shown to be Figure 14 Clearly, the stimulatory effect of antibody 21127 on T cell activation is concentration-dependent, and 21127 leads to higher IFN-γ levels than eurymumab analogues.

[0529] Example 18: Functionality of anti-CD73 antibody in single-phase MLR assay

[0530] The ability of anti-CD73 antibodies to inhibit CD73 activity in a single-channel mixed lymphocyte response (MLR) was evaluated in an in vitro assay and compared with eurymumab analogues.

[0531] Dendritic cells (DCs) and CD4 cells isolated from two different healthy donors + T cell co-culture was used to induce an allogeneic antigen-specific response, leading to cytokine production and T cell activation and / or proliferation. Dendritic cells (DCs) were cultured for 7 days with 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng / mL interleukin-4 (IL-4) from CD14 cells. + Monocyte differentiation, and the combination of dendritic cells (DCs) with CD4+ from peripheral blood mononuclear cells (PBMCs) from healthy donor material. + T cells were mixed at a 1:10 ratio. One-way MLR was performed by incubating with 25 μg / mL anti-PD-1 antibody (12819; see PCT patent publication WO 2017 / 055547), 50 μM AMP, and the indicated concentration of anti-CD73 antibody for 48 hours, followed by the addition of 3H-thymidine (PerkinElmer) for another 24 hours. Proliferation was measured as a control with 3H-thymidine incorporation and normalized to untreated AMP.

[0532] T cell proliferation after treatment with different anti-CD73 antibodies was shown to be... Figure 15 Clearly, the proliferative stimulant effect of anti-CD73 antibodies is concentration-dependent, and both antibody 21127 and euryloabinumab analogues stimulate T cell proliferation. However, only antibody 21127 completely restored proliferation.

[0533] Example 19: Combination of anti-CD73 antibody and anti-PD-1 antibody in a single-phase MLR assay

[0534] This example examines the ability of the combination of anti-PD-1 antibody (12819) and anti-CD73 antibody (21127) to enhance T cell activation in a one-way mixed lymphocyte response (MLR).

[0535] Dendritic cells (DCs) and CD4 cells isolated from two different healthy donors + T cell co-culture was used to induce allogeneic antigen-specific responses, leading to cytokine production and T cell activation and / or proliferation. T cells were cultured for 7 days with 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng / mL interleukin-4 (IL-4) from CD14 cells. + Monocytes differentiated into dendritic cells (DCs) by combining DCs with CD4+ cells from peripheral blood mononuclear cells (PBMCs) from healthy donor materials. +T cells were mixed at a 1:10 ratio. One-way MLR was performed with or without 50 μM AMP and incubated for 72 hours with anti-CD73 antibody 21127 and / or anti-PD-1 antibody (12819; see PCT patent publication WO 2017 / 055547), followed by harvesting the supernatant. The combination of anti-CD73 antibody 21127 and anti-PD-1 antibody 12819 was added at a 1:1 ratio of the two antibodies. T cell activation was measured by quantifying IFN-γ levels in the supernatant using an ELISA (Thermo Fisher Scientific) and normalized to an untreated control.

[0536] T cell activation in unidirectional MLRs, measured by IFN-γ levels, after treatment with antibodies 12819 and / or 21127 was observed. Figure 16 In the figure above, anti-PD-1 antibody 12819 strongly activated unidirectional MLRs in the absence of AMP and in both the presence and absence of co-incubation with antibody 21127 (top figure). When AMP was added to the unidirectional MLRs, the activation of unidirectional MLRs by the anti-PD-1 antibody was significantly reduced (bottom figure). However, the combination of this anti-PD-1 antibody and anti-CD73 antibody 21127 at least partially restored strong T cell activation, highlighting the benefits of this anti-PD-1 antibody / anti-CD73 antibody combination.

[0537] Example 20: Functional role of anti-CD73 antibody in B cell activation assay

[0538] The ability of antibodies 21046 and 21127 to stimulate B cell activation was evaluated in an in vitro B cell activation assay. PBMCs from healthy donors were stimulated overnight with 21046, 21127, or an oliguria analogue (10 μg / mL) and CD40 ligand (0.5 μg / mL). Flow cytometry analysis was performed on B cells (CD40... + Gating is implemented to assess B cell activation as the upregulation of B cell activation markers CD25, CD69, and CD83.

[0539] like Figure 17A and 17B The study showed that antibody 21046 strongly upregulated B cell activation markers CD25, CD69, and CD83, while antibody 21127 and eurymumab analogues had limited effects on B cell activation.

[0540] Example 21: Decrease in CD73 levels in H292 cells induced by anti-CD73 antibody

[0541] The ability of anti-CD73 antibodies 21127, 21028, 21046, and 21163 to regulate CD73 levels in cell lines was evaluated and compared with eurymumab analogues. The anti-CD73 antibodies were incubated with the human cell line H292 at 37°C for 24 hours at 25 μg / mL, followed by cell lysis and assessment of CD73 levels using Simple Western blotting (ProteinSimple).

[0542] Figure 18 The figures show CD73 levels after treatment with antibodies 21127, 21028, 21046, or 21163 or eurylmumaab analogues. Incubation with anti-CD73 antibody 21046 or eurylmumaab analogues resulted in some downregulation of CD73, while other anti-CD73 antibodies had (if any) a moderate effect on CD73 levels.

[0543] Example 22: Functional role of anti-CD73 antibody in human tumor xenograft model

[0544] 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.

[0545] Materials and methods

[0546] The human melanoma cell line A375 was subcutaneously injected into the ventral inner side of 6- to 8-week-old female NOG or NOD-scid mice. In one example ( Figure 19 (See the image above) Human PBMCs were injected intraperitoneally one day after tumor cell inoculation. The tumor was measured three times a week in two dimensions using calipers, and the tumor volume (in mm) was calculated according to the following formula. 3 (Width) 2 x length x 0.5. Mice were treated three times a week by intraperitoneal injection of load buffer or (1) antibody 21127, (2) eurymumab analog or (3) a combination of antibody 21127 and anti-mouse CD73 antibody TY / 23 (BioXcell). Unlike the eurymumab analog, antibody 21127 is not cross-reactive to mouse CD73. Therefore, to compare the inhibition of CD73 activity in tumor masses containing mouse cells (endothelial cells and stromal cells) expressing CD73, antibody 21127 was combined with anti-mouse CD73 antibody TY / 23. The antibody was administered at 5 mg / kg, 20 mg / kg or 50 mg / kg. At different time points after treatment cessation, tumors were harvested and dissociated using a tumor dissociation kit and a gentleMACS Octo dissociator (Miltenyi). The resulting cell suspension was incubated with CD73 substrate AMP at 37°C for 3 hours. The cells were then separated by adding ATP to the supernatant and passing through the tumor dissociation device. 2.0 (Promega) AMP inhibition was measured by ATP assay to study CD73 activity, as described by Sachsenmeier et al. (ibid.).

[0547] result

[0548] After two weeks of repeated administration, antibody 21127 showed dose-dependent inhibition of CD73 activity in tumors harvested from PBMC humanized mice with A375 human melanoma cell line. Figure 19 (See the figure above). It was also found that the effect on CD73 enzyme activity persisted for a prolonged period after treatment was discontinued.

[0549] like Figure 19 As shown in the figure below, eurylumab analogues inhibited CD73 activity for 16 days after treatment, and enzyme activity was 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 cessation.

[0550] Example 23: In vivo efficacy of anti-CD73 antibody in a human xenograft tumor model without immune cells

[0551] This example demonstrates the ability of anti-CD73 antibodies to inhibit tumor growth in a human triple-negative breast cancer xenograft model.

[0552] Human MDA-MB-231 triple-negative breast cancer cells were subcutaneously inoculated into the ventral medial aspect of 6- to 8-week-old female NOD-scid mice. Tumor volume was measured three times weekly in two dimensions using calipers, and the tumor volume (in mm) was calculated according to the following formula. 3 (Width) 2 x length x 0.5. Mice were treated twice weekly for a total of 16 treatments, followed by an observation period, by intraperitoneal injection of load buffer, antibody 21127, or an olemulsification analogue. The antibody was administered at a dose of 10 mg / kg. Tumor volume at each time point was compared between treatment groups using a two-way ANOVA with Bonferroni's multiple comparison test. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad software, Inc.).

[0553] During treatment, antibody 21127 and oliguria analogues showed significant tumor-suppressive effects. Figure 20 After treatment was discontinued, regrowth of tumors treated with the eurylumab analogue was observed. Conversely, during treatment, mice treated with antibody 21127 effectively controlled tumor growth, with limited increase in tumor size 60 days post-treatment.

[0554] Example 24: In vivo efficacy of anti-CD73 antibody in a human xenograft tumor model in mice reconstructed with human PBMCs

[0555] This example demonstrates the in vivo efficacy of anti-CD73 antibody 21127 in PBMC humanized mice transplanted with human lung cancer cells or human melanoma cells.

[0556] One day prior to intraperitoneal injection of human PBMCs, cells from the human lung cancer cell line Calu-6 or the human A375 melanoma cell line were subcutaneously implanted into NOG mice. Treatment was initiated on the day of PBMC injection by intraperitoneal injection of either load buffer or anti-CD73 antibody 21127 at a dose of 10 mg / kg (n = 10 / group), three times a week for a total of six treatments. Tumor volume was measured three times a week in two dimensions using calipers, and the tumor volume (in mm) was calculated according to the following formula. 3 (Width) 2 x length x0.5. Two-way ANOVA with Bonferroni multiple comparison test was used to compare tumor volume between treatment groups at each time point. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad software, Inc.).

[0557] like Figure 21 The results showed that in mice reconstructed with human PBMCs, treatment with antibody 21127 resulted in significant tumor growth delay in both human tumor xenograft models (Calu-6 and A375) (P < 0.05 compared to the load control). Figure 21 Each figure in the diagram represents a human PBMC donor.

Claims

1. An anti-CD73 antibody comprising a heavy chain HC and a light chain LC, wherein the variable domain amino acid sequence of the HC is SEQ ID NO:11, and the constant region amino acid sequence of the HC is SEQ ID NO:41; the variable domain amino acid sequence of the LC is SEQ ID NO:15, and the constant region amino acid sequence of the LC is SEQ ID NO:

42.

2. The anti-CD73 antibody according to claim 1, wherein the antibody has at least one property selected from the group consisting of: a) Inhibits the activity of soluble CD73 in vitro; b) Inhibits CD73 activity on Calu-6 cells in vitro; c) Inhibits CD73 activity on H292 cells in vitro; d) Specifically binds to human and cynomolgus monkey CD73 expressed on CHO-S cells; e) When measured by SPR, use K0 at 1 nM or less. D Combined with human CD73's ECD; f) When measured by SPR, use a K0.7 or smaller. D ECD associated with CD73 in cynomolgus monkeys; g) Does not bind to the same epitope of CD73 as ollilumab, CPX006 and / or 11E1; h) binds to epitopes on CD73 homodimers in a manner that generates a 1:1 complex; i) It inhibits soluble CD73 activity more effectively than olemumab in vitro; j) In vitro inhibition of CD73 activity in Calu-6, H292 and Cynom-K1 cells; k) inhibited CD73 activity in Calu-6, NCI-H1775, KYSE-30 and Capan-2 cells in vitro; l) Inhibits the proliferation of MDA-MB-231 and MDA-MB-468 cells in vitro; m) inhibits primary CD4 in vitro + and CD8 + T cells and CD19 + CD73 activity on B cells; n) In vitro recovery of CD4 + T cell proliferation; o) In vitro activation of CD4 + and CD8 + T cells; p) Combined with anti-PD-1 antibody, T cell proliferation was restored in the presence of AMP in a single-channel mixed lymphocyte response (MLR); q) When combined with an anti-PD-1 antibody, T cell activation is enhanced in the presence of AMP in a single-channel MLR. r) It does not stimulate B cell activation in vitro; s) It does not reduce CD73 levels in H292 cells by more than 25% in vitro; t) Inhibit CD73 activity in tumors harvested from PBMC humanized mice transplanted with A375 cells; u) In vivo inhibition of tumor growth in NOD-scid mice transplanted with MDA-MB-231 cells; v) In vivo inhibition of tumor growth in PBMC humanized mice transplanted with Calu-6 cells; and w) In vivo inhibition of tumor growth in PBMC humanized mice transplanted with A375 cells.

3. The anti-CD73 antibody according to claim 2, wherein the antibody has 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 described properties.

4. The anti-CD73 antibody according to claim 1, wherein the antibody is IgG.

5. The anti-CD73 antibody according to claim 4, wherein the antibody is IgG1.

6. The anti-CD73 antibody according to claim 1, wherein the antibody is in F C The region contains at least one mutation.

7. The anti-CD73 antibody according to claim 5, wherein the antibody is IgG1 and contains a mutation at one or more of the heavy chain amino acid positions 234 and 235, which is numbered according to the Eu numbering scheme.

8. The anti-CD73 antibody according to claim 7, wherein one or both of the amino acid residues at positions 234 and 235 are mutated from Leu to Ala.

9. A pharmaceutical composition comprising an antiCD73 antibody according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 9, further comprising an immunostimulant, a vaccine, a chemotherapeutic agent, an antitumor drug, an antiangiogenic agent, a tyrosine kinase inhibitor, or a CD73 pathway inhibitor.

11. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of the anti-CD73 antibody of any one of claims 1 to 8 or the antigen-binding portion thereof, and a nucleotide sequence encoding the light chain of the anti-CD73 antibody of any one of claims 1 to 8 or the antigen-binding portion thereof.

12. The isolated nucleic acid molecule according to claim 11, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:

7.

13. A vector comprising the isolated nucleic acid molecule of claim 11 or 12, wherein the vector further comprises an expression control sequence.

14. A host cell comprising a nucleotide sequence encoding the heavy chain of the anti-CD73 antibody of any one of claims 1 to 8 or the antigen-binding portion thereof, and a nucleotide sequence encoding the light chain of the anti-CD73 antibody of any one of claims 1 to 8 or the antigen-binding portion thereof.

15. A method for producing an anti-CD73 antibody, comprising providing a host cell as described in claim 14, culturing the host cell under conditions suitable for expressing the antibody or a portion thereof, and isolating the resulting antibody or a portion thereof.

16. A bispecific binding molecule comprising an antigen-binding portion of an anti-CD73 antibody according to any one of claims 1 to 8 and an antigen-binding portion of another different antibody.

17. Use of the antiCD73 antibody according to any one of claims 1 to 8 for the preparation of a medicament for treating a patient’s cancer, wherein the cancer is melanoma, head and neck cancer, triple-negative breast cancer, bladder cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, or colorectal cancer.

18. Use of the pharmaceutical composition according to claim 9 or 10 for preparing a medicament for treating a patient’s cancer, wherein the cancer is melanoma, head and neck cancer, triple-negative breast cancer, bladder cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, or colorectal cancer.

19. Use of the bispecific binding molecule according to claim 16 for preparing a medicament for treating a patient’s cancer, wherein the cancer is melanoma, head and neck cancer, triple-negative breast cancer, bladder cancer, non-small cell lung cancer, pancreatic cancer, ovarian cancer, renal cell carcinoma, prostate cancer, or colorectal cancer.

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