Binding molecules specific for CD73 and uses thereof

Abstract

The present invention relates to binding molecules specific for CD73 and their uses. The present disclosure provides anti-CD73 binding molecules, such as antibodies and antigen-binding fragments thereof. Also provided are pharmaceutical formulations comprising the disclosed compositions and methods for diagnosing and treating diseases associated with CD73 expression, such as cancer. Such diseases can be treated, for example, by direct therapy with the anti-CD73 binding molecules disclosed herein (e.g., naked antibodies or antibody drug conjugates that bind to CD73), by adjuvant therapy with other antigen-binding anticancer agents such as immune checkpoint inhibitors (e.g., anti-CTLA-4 and anti-PD-1 monoclonal antibodies), and / or by combination therapy in which these anti-CD73 molecules are administered before, after, or simultaneously with chemotherapy.

Description

[0001] This application is a divisional application of Chinese patent application No. 201580060993.4, filed on November 9, 2015, entitled "Binding molecules specific for CD73 and uses thereof." Technical Field

[0002] The present invention relates to anti-CD73 binding molecules, such as antibodies and antigen-binding fragments thereof. Pharmaceutical formulations comprising the disclosed compositions and methods for diagnosing and treating diseases associated with CD73 expression, such as cancer, are also provided. Background Art

[0003] CD73, or ecto-5′-nucleotidase (5′-NT), is ubiquitously expressed in many tissues. This protein is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) bond, has exoenzymatic activity, and plays a role in signal transduction. The primary function of CD73 is to convert extracellular nucleotides (e.g., 5′-AMP), which are normally impermeable to cells, into their corresponding nucleotides (e.g., adenosine), which can readily enter most cells. The production of adenosine by CD73 through dephosphorylation of AMP has been shown to regulate adenosine receptor engagement in many tissues, suggesting that adenosine plays a role in cytoprotection, cell growth, angiogenesis, and immunosuppression, and also plays a role in tumorigenesis.

[0004] CD73 expression on tumor cells has been reported in several types of cancer, including colorectal cancer, pancreatic cancer, bladder cancer, leukemia, lymphoma, glioma, glioblastoma, melanoma, ovarian cancer, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer. Increased CD73 expression is associated with tumor invasion and metastasis, and reduced patient survival. CD73 creates an immunosuppressive environment, characterized by increased adenosine levels, which promotes cancer development and progression. Notably, CD73 expression is associated with a pro-metastatic phenotype in melanoma and breast cancer.

[0005] Immune checkpoint inhibitors hold great potential as cancer therapies. However, the clinical benefits of immune checkpoint inhibition have been modest. One possible explanation is that tumors employ non-overlapping immunosuppressive mechanisms to promote immune evasion. Therefore, improved compositions and methods for reducing tumor-mediated immunosuppression are urgently needed. Summary of the Invention

[0006] The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73. In some aspects, such CD73 binding molecules are, for example, antibodies or antigen-binding fragments thereof. In specific embodiments, the anti-CD73 antibodies of the present invention (e.g., MEDI9447) are useful for reducing tumor-mediated immunosuppression. Therefore, the present invention also provides therapeutic combinations featuring anti-CD73 antibodies (e.g., MEDI9447) and other agents targeting additional aspects of the cancer immune cycle (i.e., anti-PD-1 or anti-PD-L1 antibodies; anti-CTLA4 antibodies, A2aR antagonists, STAT-3 inhibitors) and methods of using such combinations to reduce tumor-mediated immunosuppression.

[0007] In one aspect, the present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to a CD73 epitope, wherein the binding molecule specifically binds to a heavy chain variable region (V H ) and light chain variable region (V L ) or an antigen-binding fragment thereof that binds to the same CD73 epitope as the epitope: CD730002, CD730003, CD730004, CD730008, CD730010, CD730011, CD730021, CD730042, CD730046, CD730047, or CD730058.

[0008] In another aspect, the present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73 and competitively inhibits the expression of V33 by V33 comprising CD730002, CD730003, CD730004, CD730008, CD730010, CD730011, CD730021, CD730042, CD730046, CD730047, or CD730058. H and V L An antibody or antigen-binding fragment thereof binds to CD73.

[0009] In another aspect, the present invention provides a method comprising: L An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V L Having the amino acid sequence:

[0010] [FW1]SGLSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4]

[0011] Where [FW1], [FW2], [FW3] and [FW4] represent VL Framework region, and wherein X1 represents the amino acid residue proline (P), glutamic acid (E) or aspartic acid (D); X2 represents the amino acid residue asparagine (N) or aspartic acid (D);

[0012] X3 represents the amino acid residue glutamine (Q) or leucine (L);

[0013] X4 represents the amino acid residue leucine (L) or proline (P);

[0014] X5 represents the amino acid residue glycine (G) or serine (S);

[0015] X6 represents the amino acid residue leucine (L) or histidine (H);

[0016] X7 represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N); and

[0017] X8 represents the amino acid residue leucine (L) or threonine (T).

[0018] In various embodiments of any aspect delineated herein, the isolated binding molecule or antigen-binding fragment thereof of item 6, wherein FW1 comprises SEQ ID NO: 25 or 26, FW2 comprises SEQ ID NO: 27 or 28, FW3 comprises SEQ ID NO: 29, and FW4 comprises SEQ ID NO: 30.

[0019] In another aspect, the present invention provides a method comprising: H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V H Having the amino acid sequence:

[0020] [FW5]SYAX9S[FW6]X 10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8]

[0021] wherein [FW5], [FW6], [FW7] and [FW8] represent VH framework regions, and wherein

[0022] X9 represents the amino acid residue methionine (M) or tyrosine (Y);

[0023] X 10represents the amino acid residue leucine (L) or alanine (A);

[0024] X 11 represents the amino acid residue tryptophan (W) or serine (S);

[0025] X 12 represents the amino acid residue tryptophan (W) or glycine (G);

[0026] X 13 represents the amino acid residue serine (S) or arginine (R);

[0027] X 14 represents the amino acid residue glycine (G) or serine (S);

[0028] X 15 represents the amino acid residue arginine (R) or threonine (T);

[0029] X 16 represents the amino acid residue valine (V) or isoleucine (I); and

[0030] X 17 Indicates the amino acid residues tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0031] In another aspect, the present invention provides a method comprising: L and antibody V H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V L Includes amino acid sequence:

[0032] [FW1]SGLSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4]

[0033] Where [FW1], [FW2], [FW3] and [FW4] represent V L framework area, and wherein

[0034] X1 represents the amino acid residue proline (P), glutamic acid (E) or aspartic acid (D);

[0035] X2 represents the amino acid residue asparagine (N) or aspartic acid (D);

[0036] X3 represents the amino acid residue glutamine (Q) or leucine (L);

[0037] X4 represents the amino acid residue leucine (L) or proline (P);

[0038] X5 represents the amino acid residue glycine (G) or serine (S);

[0039] X6 represents the amino acid residue leucine (L) or histidine (H);

[0040] X7 represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N); and

[0041] X8 represents the amino acid residue leucine (L) or threonine (T);

[0042] And where the V H Includes amino acid sequence:

[0043] [FW5]SYAX9S[FW6]X 10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8]

[0044] wherein [FW5], [FW6], [FW7] and [FW8] represent VH framework regions, and wherein

[0045] X9 represents the amino acid residue methionine (M) or tyrosine (Y);

[0046] X 10 represents the amino acid residue leucine (L) or alanine (A);

[0047] X 11 represents the amino acid residue tryptophan (W) or serine (S);

[0048] X 12 represents the amino acid residue tryptophan (W) or glycine (G);

[0049] X 13 represents the amino acid residue serine (S) or arginine (R);

[0050] X 14 represents the amino acid residue glycine (G) or serine (S);

[0051] X 15 represents the amino acid residue arginine (R) or threonine (T);

[0052] X 16 represents the amino acid residue valine (V) or isoleucine (I); and

[0053] X17 Indicates the amino acid residues tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0054] In another aspect, the present invention provides a method for producing an antibody having V L An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V L With V L A complementarity determining region-2 (VL-CDR2) amino acid sequence that is identical to, or identical to, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 52 except for four, three, two or one amino acid substitutions.

[0055] In another aspect, the present invention provides a method for producing an antibody having V L An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V L having a complementarity determining region-3 (VL-CDR3) amino acid sequence identical to, or identical to, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56 except for four, three, two, or one amino acid substitution.

[0056] In another aspect, the present invention provides a method for producing an antibody having V H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V H The polypeptide has a complementarity determining region-1 (VH-CDR1) amino acid sequence identical to or identical to SEQ ID NO: 35 or SEQ ID NO: 36 except for four, three, two or one amino acid substitutions.

[0057] In another aspect, the present invention provides a method for producing an antibody having V H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V H having a complementarity determining region-2 (VH-CDR2) amino acid sequence identical to, or identical to, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40 except for four, three, two, or one amino acid substitution.

[0058] In another aspect, the present invention provides a method for producing an antibody having V HAn isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V H having a complementarity determining region-3 (VH-CDR3) amino acid sequence identical to, or identical to, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45 except for four, three, two, or one amino acid substitutions.

[0059] In another aspect, the present invention provides a method for producing an antibody having V L An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V L having VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequences that are identical to, or identical to, SEQ ID NOs: 46, 49 and 53; SEQ ID NOs: 47, 49 and 53; SEQ ID NOs: 47, 49 and 54; SEQ ID NOs: 46, 50 and 54; SEQ ID NOs: 46, 51 and 55; SEQ ID NOs: 48, 52 and 54; SEQ ID NOs: 46, 49 and 56; SEQ ID NOs: 47, 49 and 56; SEQ ID NOs: 46, 50 and 56; SEQ ID NOs: 46, 51 and 56; or SEQ ID NOs: 48, 52 and 56, respectively.

[0060] In another aspect, the present invention provides a method for producing an antibody having V H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V H having VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequences that are identical to, or identical to, SEQ ID NOs: 35, 37 and 41; SEQ ID NOs: 36, 37 and 42; SEQ ID NOs: 36, 38 and 43; SEQ ID NOs: 36, 39 and 44; SEQ ID NOs: 36, 40 and 44; SEQ ID NOs: 35, 37 and 45; SEQ ID NOs: 36, 37 and 45; SEQ ID NOs: 36, 38 and 45; SEQ ID NOs: 36, 39 and 45; or SEQ ID NOs: 36, 40 and 45, respectively, or except for four, three, two or one amino acid substitutions in one or more of these VH-CDRs.

[0061] In another aspect, the present invention provides a method having V L and V H An isolated antibody or antigen-binding fragment thereof that specifically binds to CD73, the V L and V H having VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequences identical to or identical to, except for four, three, two or one amino acid substitutions in one or more CDRs, SEQ ID NOs: 46, 49, 53, 35, 37 and 41; SEQ ID NOs: 47, 49, 53, 35, 37 and 41; SEQ ID NOs: 47, 49, 54, 36, 37 and 42; SEQ ID NOs: 46, 50, 54, 36, 38 and 43; SEQ ID NOs: 46, 51, 55, 36, 39 and 44; SEQ ID NOs: 48, 52, 54, 36, 40 and 44; SEQ ID NOs: 46, 49, 56, 35, 37 and 41; SEQ ID NOs: 47, 49, 54, 36, 37 and 42; NO: 46, 49, 53, 35, 37 and 45; SEQ ID NO: 47, 49, 56, 36, 37 and 45; SEQ ID NO: 46, 50, 56, 36, 38 and 45; SEQ ID NO: 46, 51, 56, 36, 39 and 45; SEQ ID NO: 48, 52, 56, 36, 40 and 45; or SEQ ID NO: 46, 49, 56, 35, 37 and 45.

[0062] In another aspect, the present invention provides a method for producing an antibody having V L and antibody V H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V L having an amino acid sequence that is at least about 90% to about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70.

[0063] In another aspect, the present invention provides a method for producing an antibody having V L and antibody V HAn isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V H having an amino acid sequence that is at least about 90% to about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.

[0064] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD73, wherein the antibody or antigen-binding fragment has a V L , the V L having a sequence at least about 90% to about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70, and wherein the antibody or antigen-binding fragment has a V H , the V H Having a sequence that is at least about 90% to about 100% identical to a reference amino acid sequence selected from SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.

[0065] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof having a V sequence consisting essentially of SEQ ID NO: 57. L and V consisting essentially of SEQ ID NO: 71 H .

[0066] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof having a V sequence consisting essentially of SEQ ID NO: 68. L and V consisting essentially of SEQ ID NO: 82 H .

[0067] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof having a V sequence consisting of SEQ ID NO: 57. L and V consisting of SEQ ID NO: 71 H .

[0068] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof having a V sequence consisting of SEQ ID NO: 68. L and V consisting of SEQ ID NO: 82 H .

[0069] In another aspect, the present invention provides a composition comprising an isolated antibody or antigen-binding fragment thereof according to the present invention and a carrier.

[0070] In another aspect, the invention provides a nucleic acid having a sequence encoding an isolated antibody or antigen-binding fragment thereof according to the invention.

[0071] In another aspect, the invention provides a composition comprising a nucleic acid according to the invention.

[0072] In another aspect, the invention provides a vector comprising a nucleic acid according to the invention.

[0073] In another aspect, the present invention provides a host cell comprising a nucleic acid sequence, composition, or vector according to the present invention.

[0074] In another aspect, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof according to the present invention, which method involves culturing cells comprising a nucleic acid sequence, composition, or vector according to the present invention; and isolating the antibody or antigen-binding fragment thereof.

[0075] In another aspect, the present invention provides a diagnostic reagent comprising a labeled isolated antibody or antigen-binding fragment according to the present invention.

[0076] In another aspect, the invention provides a kit comprising the isolated antibody or antigen-binding fragment thereof, composition, or diagnostic reagent according to the invention.

[0077] In another aspect, the present invention provides a method of inhibiting the growth of a cell expressing CD73, the method involving contacting the cell with an antibody or antigen-binding fragment thereof according to the present invention.

[0078] In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, the method involving administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the present invention.

[0079] In another aspect, the invention provides a method of treating cancer in a subject, the method involving administering to the subject a therapeutically effective amount of a first agent (the first agent being an antibody or antigen-binding fragment according to the invention) in combination with a therapeutically effective amount of a second agent (the second agent being an anti-cancer agent other than the first agent).

[0080] In another aspect, the invention provides a therapeutic method involving administering an anti-CD73 antibody or antigen-binding fragment thereof to a subject identified as having a tumor with increased CD73 expression relative to a reference.

[0081] In another aspect, the present invention provides a therapeutic method involving administering an anti-CD73 antibody, or an antigen-binding fragment thereof, and anti-PD-1, anti-PD-L1, or anti-CTLA4, or an antigen-binding fragment thereof, to a subject identified as having a tumor with increased CD73 expression compared to a reference.

[0082] In another aspect, the present invention provides methods involving administering MEDI9447 or Phen0203 hIgG1 or an antigen-binding fragment thereof, and pembrolizumab to a subject identified as having a tumor with increased CD73 expression compared to a reference. or nivolumab or antigen-binding fragments thereof.

[0083] In another aspect, the invention provides a therapeutic method involving administering MEDI9447 or Phen0203 hIgG1, or an antigen-binding fragment thereof, and MEDI4736, or an antigen-binding fragment thereof, to a subject identified as having a tumor with increased CD73 expression compared to a reference.

[0084] In another aspect, the present invention provides a treatment method involving administering MEDI9447 or Phen0203 hIgG1, or an antigen-binding fragment thereof, and tremelimumab, or an antigen-binding fragment thereof, to a subject identified as having a tumor with increased CD73 expression compared to a reference.

[0085] In another aspect, the present invention provides a method of identifying a subject having a cancer responsive to anti-CD73 therapy, the method involving detecting an increased level of CD73 expression or activity in tumor cells or blood cells of the subject relative to a reference, thereby identifying the cancer responsive to anti-CD73 therapy.

[0086] In another aspect, the present invention provides a method of identifying a subject having a cancer that is responsive to anti-CD73 therapy in combination with one or more of anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy, the method involving detecting an increased level of CD73 expression or activity in tumor cells or blood cells of the subject relative to a reference, thereby identifying the cancer that is responsive to anti-CD73 therapy in combination with one or more of anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

[0087] In another aspect, the present invention provides a method of identifying a subject having a cancer responsive to anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy, the method involving detecting a reduced level of CD73 expression or activity in tumor cells or blood cells of the subject relative to a reference, thereby identifying the cancer responsive to anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

[0088] In another aspect, the present invention provides a method of inhibiting tumor growth in a subject, which involves administering to a subject in need thereof an anti-CD73 antibody or an antigen-binding fragment thereof, and one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof.

[0089] In another aspect, the present invention provides a method of increasing an anti-tumor immune response in a subject, the method involving administering an anti-CD73 antibody or an antigen-binding fragment thereof to a subject in need thereof, and administering to the subject one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof.

[0090] In another aspect, the present invention provides a method of treating a tumor in a subject, which involves administering to a subject in need thereof an anti-CD73 antibody or an antigen-binding fragment thereof, and one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof.

[0091] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of an anti-CD73 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0092] In another aspect, the present invention provides a method comprising an effective amount of MEDI9447 or an antigen-binding fragment thereof and pembrolizumab. or a pharmaceutical formulation of an antigen-binding fragment thereof.

[0093] In another aspect, the present invention provides a method comprising an effective amount of MEDI9447 or an antigen-binding fragment thereof and nivolumab. or a pharmaceutical formulation of an antigen-binding fragment thereof.

[0094] In another aspect, the present invention provides a method comprising an effective amount of Phen0203 hIgG1 or an antigen-binding fragment thereof and pembrolizumab. or a pharmaceutical formulation of an antigen-binding fragment thereof.

[0095] In another aspect, the present invention provides a method comprising an effective amount of Phen0203 hIgG1 or an antigen-binding fragment thereof, nivolumab, or a pharmaceutical formulation of an antigen-binding fragment thereof.

[0096] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of an anti-CD73 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0097] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of MEDI9447 or an antigen-binding fragment thereof and MEDI4736 or an antigen-binding fragment thereof.

[0098] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of Phen0203 hIgG1 or an antigen-binding fragment thereof and MEDI4736 or an antigen-binding fragment thereof.

[0099] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of an anti-CD73 antibody or an antigen-binding fragment thereof and an anti-CTLA4 antibody or an antigen-binding fragment thereof.

[0100] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of MEDI9447 or an antigen-binding fragment thereof and tremelimumab or an antigen-binding fragment thereof.

[0101] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of MEDI9447 or an antigen-binding fragment thereof and ipilimumab or an antigen-binding fragment thereof.

[0102] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of Phen0203 hIgG1 or an antigen-binding fragment thereof and Tremelimumab or an antigen-binding fragment thereof.

[0103] In another aspect, the present invention provides a pharmaceutical formulation comprising an effective amount of Phen0203 hIgG1 or an antigen-binding fragment thereof and ipilimumab or an antigen-binding fragment thereof.

[0104] In another aspect, the present invention provides a kit for increasing anti-tumor activity, which comprises an anti-CD73 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0105] In another aspect, the present invention provides a kit for increasing anti-tumor activity, which comprises an anti-CD73 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0106] In another aspect, the present invention provides a kit for increasing anti-tumor activity, which comprises an anti-CD73 antibody or an antigen-binding fragment thereof and an anti-CTLA4 antibody or an antigen-binding fragment thereof.

[0107] In various embodiments of any aspect delineated herein, the VL and VH of CD730002 are or comprise SEQ ID NOs: 1 and 2, respectively, and the VL and VH of CD730010 are or comprise SEQ ID NOs: 3 and 4, respectively.

[0108] In various embodiments of any aspect delineated herein, the isolated binding molecule or antigen-binding fragment thereof comprises an antibody or antigen-binding fragment thereof.

[0109] In various embodiments of any aspect delineated herein, the binding molecule is affinity matured.

[0110] In various embodiments of any aspect delineated herein, FW5 is or includes SEQ ID NO:31, FW6 is or includes SEQ ID NO:32, FW7 is or includes SEQ ID NO:33, and FW8 is or includes SEQ ID NO:34.

[0111] In various embodiments of any aspect delineated herein, FW1 is or includes SEQ ID NO: 25 or 26, FW2 is or includes SEQ ID NO: 27 or 28, FW3 is or includes SEQ ID NO: 29, FW4 is or includes SEQ ID NO: 30, FW5 is or includes SEQ ID NO: 31, FW6 is or includes SEQ ID NO: 32, FW7 is or includes SEQ ID NO: 33, and FW8 is or includes SEQ ID NO: 34.

[0112] In various embodiments of any aspect delineated herein, the VL comprises a VL complementarity determining region-1 (VL-CDR1) amino acid sequence identical to, or identical except for four, three, two, or one amino acid substitutions to, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.

[0113] In various embodiments of any aspect delineated herein, the isolated antibody or antigen-binding fragment thereof has a VL having SEQ ID NO:57 and a VH having SEQ ID NO:71.

[0114] In various embodiments of any aspect delineated herein, the isolated antibody or antigen-binding fragment thereof has a VL having SEQ ID NO:68 and a VH having SEQ ID NO:82.

[0115] In various embodiments of any aspect delineated herein, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain constant region or fragment thereof.

[0116] In various embodiments, the heavy chain constant region or fragment thereof is an IgG constant region, including, for example, an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region.

[0117] In various embodiments of any aspect delineated herein, the isolated antibody or antigen-binding fragment thereof comprises a light chain constant region selected from a human kappa constant region and a human lambda constant region.

[0118] In various embodiments of any aspect delineated herein, the IgG constant region has one or more amino acid substitutions relative to a wild-type IgG constant region, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type IgG constant region.

[0119] In various embodiments of any aspect delineated herein, the IgG constant region has one or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, wherein numbering is according to the EU index as set forth by Kabat.

[0120] In various embodiments of any aspect delineated herein, at least one IgG constant region amino acid substitution is selected from:

[0121] (a) the amino acid at position 252 is substituted with tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T);

[0122] (b) the amino acid at position 254 is substituted with threonine (T);

[0123] (c) the amino acid at position 256 is substituted with serine (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), or threonine (T);

[0124] (d) the amino acid at position 257 is substituted with leucine (L);

[0125] (e) the amino acid at position 309 is substituted with proline (P);

[0126] (f) the amino acid at position 311 is substituted with serine (S);

[0127] (g) the amino acid at position 428 is substituted with threonine (T), leucine (L), phenylalanine (F), or serine (S);

[0128] (h) the amino acid at position 433 is substituted with arginine (R), serine (S), isoleucine (I), proline (P), or glutamine (Q);

[0129] (i) the amino acid at position 434 is substituted with tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F), or tyrosine; and

[0130] (j) a combination of two or more of the above substitutions,

[0131] The numbering is based on the EU index proposed by Kabat.

[0132] In various embodiments of any aspect delineated herein, the human IgG constant region has amino acid substitutions at positions 252, 254, and 256 relative to a wild-type human IgG constant region, wherein

[0133] (a) the amino acid at position 252 is substituted with tyrosine (Y),

[0134] (b) the amino acid at position 254 is substituted with threonine (T), and

[0135] (c) the amino acid at position 256 is substituted with glutamic acid (E),

[0136] The numbering is based on the EU index proposed by Kabat.

[0137] In various embodiments of any aspect delineated herein, the amino acid at position 434 is substituted with an amino acid selected from the group consisting of tryptophan (W), methionine (M), tyrosine (Y), and serine (S), and wherein numbering is according to the EU index as set forth by Kabat.

[0138] In various embodiments of any aspect delineated herein, the amino acid at position 428 is substituted with an amino acid selected from the group consisting of threonine (T), leucine (L), phenylalanine (F), and serine (S), and wherein numbering is according to the EU index as set forth by Kabat.

[0139] In various embodiments of any aspect delineated herein, the amino acid at position 257 is substituted with leucine (L), and the amino acid at position 434 by Kabat is substituted with tyrosine (Y), and wherein numbering is according to the EU index as set forth by Kabat.

[0140] In various embodiments of any aspect delineated herein, the amino acid at Kabat position 428 is substituted with leucine (L), and the amino acid at Kabat position 434 is substituted with serine (S).

[0141] In various embodiments of any aspect delineated herein, the human IgG constant region has amino acid substitutions at positions 252, 254, and 256 relative to a wild-type human IgG constant region, where numbering is according to the EU index as set forth by Kabat, and wherein

[0142] (a) the amino acid at position 252 is substituted with tyrosine (Y),

[0143] (b) the amino acid at position 254 is substituted with threonine (T), and

[0144] (c) The amino acid at position 256 was substituted with glutamic acid (E).

[0145] In various embodiments of any aspect delineated herein, the antibody is a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a multispecific antibody, or an antigen-binding fragment thereof.

[0146] In various embodiments of any aspect delineated herein, the antigen binding fragment is Fv, Fab, F(ab')2, Fab', dsFv, scFv, or sc(Fv)2.

[0147] In various embodiments of any aspect delineated herein, the isolated antibody or antigen-binding fragment thereof is conjugated to at least one xenologous agent, including, for example, an anti-cancer agent.

[0148] In various embodiments of any aspect delineated herein, the composition according to the present invention further comprises an anti-cancer agent.

[0149] In various embodiments of any aspect delineated herein, the isolated antibody or antigen-binding fragment thereof does not induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0150] In various embodiments of any aspect delineated herein, the isolated antibody or antigen-binding fragment thereof is an antagonist of CD73.

[0151] In various embodiments, the isolated antibody or antigen-binding fragment thereof is an antagonist of CD73 in a cell selected from MB-MDA-231, 4T1, MK1, or a combination of two or more of said cells.

[0152] In various embodiments of any aspect delineated herein, the CD73 is human CD73.

[0153] In various embodiments of any aspect delineated herein, binding of the antibody or antigen-binding fragment to CD73 reduces cell proliferation.

[0154] In various embodiments of any aspect delineated herein, the antibody or antigen-binding fragment against CD73 can bind to human CD73, cynomolgus monkey CD73, and mouse CD73.

[0155] In various embodiments of any aspect delineated herein, the cancer is selected from colorectal cancer, pancreatic cancer, bladder cancer, leukemia, lymphoma, glioma, glioblastoma, melanoma, ovarian cancer, thyroid cancer, esophageal cancer, prostate cancer, and breast cancer.

[0156] In various embodiments of any aspect delineated herein, the cancer has a pro-metastatic phenotype, including melanoma or breast cancer

[0157] In various embodiments of any aspect delineated herein, the subject is a human.

[0158] In various embodiments of any aspect delineated herein, the combination of the first and second agents has superior anti-tumor activity; either additive or synergistic.

[0159] In various embodiments of any aspect delineated herein, the second agent is an antibody or antigen-binding fragment thereof.

[0160] In various embodiments, the second agent specifically binds to PD-1 (programmed death 1 protein), PD-L1 (programmed death 1 protein ligand 1), PD-L2 (programmed death 1 protein ligand 2), or CTLA-4 (cytotoxic T lymphocyte antigen 4 protein).

[0161] In various embodiments of any aspect delineated herein, the second agent is an anti-CTLA-4 antibody or antigen-binding fragment thereof, including, for example, ipilimumab, tremelimumab (tesitumumab, CP-675,206), or an antigen-binding fragment thereof.

[0162] In various embodiments of any aspect delineated herein, the second agent is an anti-PD-1 antibody or antigen-binding fragment thereof, including, for example, pembrolizumab ( lambrolizumab (MK-3475), nivolumab ( BMS-936558, MDX-1106, ONO-4538), AMP-224 or an antigen-binding fragment thereof.

[0163] In various embodiments of any aspect delineated herein, the second agent is an anti-PD-L1 antibody or an antigen-binding fragment thereof, including, for example, MEDI4736, BMS-936559, MPDL3280A, or an antigen-binding fragment thereof.

[0164] In various embodiments of any aspect delineated herein, the anti-CD73 antibody is MEDI9447, Phen0203hIgG1, or an antigen-binding fragment thereof.

[0165] In various embodiments of any aspect delineated herein, the subject is undergoing, has undergone, or will undergo anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

[0166] In various embodiments of any aspect delineated herein, the anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy involves administering an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or antigen-binding fragment thereof, respectively.

[0167] In various embodiments, the anti-PD-1 antibody is pembrolizumab ( LambrOlizumab (MK-3475), Nivolumab ( BMS-936558, MDX-1106, ONO-4538), AMP-224 or an antigen-binding fragment thereof.

[0168] In various embodiments, the anti-PD-L1 antibody is MEDI4736, BMS-936559, MPDL3280A, or an antigen-binding fragment thereof.

[0169] In various embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab (tesitumumab, CP-675,206), or an antigen-binding fragment thereof.

[0170] In various embodiments of any aspect delineated herein, the tumor is colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Burkitt's lymphoma, ovarian cancer, breast cancer, head and neck cancer, or pancreatic cancer.

[0171] In various embodiments of any aspect delineated herein, CD73 expression or activity is detected in a tumor sample, a blood sample, or a lymph sample.

[0172] In various embodiments of any aspect delineated herein, CD73 expression is detected in tumor cells or peripheral blood cells, including one or more lymphoid or myeloid cell subsets, i.e., B lymphocytes, CD4+, FoxP3+ lymphocytes, or myeloid-derived suppressor cells (MDSCs).

[0173] In various embodiments of any aspect delineated herein, CD73 expression is detected by flow cytometry, immunohistochemistry (IHC), or CD73 enzymatic activity or soluble CD73 levels in a sample.

[0174] In various embodiments of any aspect delineated herein, the anti-CD73 antibody or antigen-binding fragment thereof and the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or antigen-binding fragment thereof are administered simultaneously.

[0175] In various embodiments of any aspect delineated herein, the method induces or increases a tumor-specific immune response.

[0176] In various embodiments of any aspect delineated herein, the method reduces the immunosuppressive effects of the AMP / CD73 / adenosine pathway.

[0177] In various embodiments of any aspect delineated herein, the tumor is a CD73-overexpressing tumor.

[0178] In another aspect, the present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73 having an antibody VL and an antibody VH, wherein the isolated binding molecule or antigen-binding fragment thereof specifically binds to a CD73 protein epitope having one or more amino acids corresponding to Val144, Lys180, and Asn185.

[0179] In various embodiments of any aspect delineated herein, the isolated binding molecule or antigen binding protein further comprises one or more amino acids corresponding to Tyrl35, Lysl36, and Asnl87.

[0180] In various embodiments of any aspect delineated herein, the isolated binding molecule or antigen-binding fragment thereof comprises amino acids corresponding to Tyrl35, Lysl36, and Asnl87.

[0181] In various embodiments of any aspect delineated herein, the isolated binding molecule or antigen-binding fragment thereof comprises amino acids corresponding to Tyrl35, Lysl36, Asnl87, Tyrl35, Lysl36, and Asnl87.

[0182] In various embodiments of any aspect delineated herein, the isolated binding molecule or antigen-binding fragment thereof binds to an epitope within one or more of the following regions of the CD73 protein: Tyr132-Val144 and / or Lys180-Asn187.

[0183] In various embodiments of any aspect delineated herein, the isolated binding molecule or antigen-binding fragment thereof comprises or is within the amino acid sequence at Tyr132-Val144 and / or Lys180-Asn187.

[0184] In another aspect, the present invention provides a conformational epitope on the surface of a CD73 protein having one or more amino acids corresponding to Val144, Lys180, and Asn185, wherein the CD73 protein comprising the epitope can be specifically bound by the monoclonal antibody MEDI9447 or an antigen-binding fragment, variant, analog or derivative thereof.

[0185] In various embodiments of any aspect delineated herein, the conformational epitope further comprises one or more amino acids corresponding to Tyrl35, Lysl36, and Asnl8.

[0186] In various embodiments of any aspect delineated herein, the conformational epitope comprises amino acids corresponding to Tyrl35, Lysl36, and Asnl87.

[0187] In various embodiments of any aspect delineated herein, the conformational epitope comprises amino acids corresponding to Tyrl35, Lysl36, Asnl87, Tyrl35, Lysl36, and Asnl87.

[0188] In various embodiments of any aspect delineated herein, the conformational epitope is within one or more of the following regions of the CD73 protein: Tyr132-Val144 and / or Lys180-Asn187.

[0189] In various embodiments of any aspect delineated herein, the conformational epitope comprises or is within the amino acid sequence at Tyr132-Val144 and / or Lys180-Asn187.

[0190] In various embodiments of any aspect delineated herein, MEDI9447 binds to CD73 protein in an inactive or catalytically active state, or in an open or closed state.

[0191] In various embodiments of any aspect delineated herein, the CD73 protein is human CD73.

[0192] In various embodiments of any aspect delineated herein, the isolated binding molecule or antigen-binding fragment thereof, wherein the VL and VH are the VL and VH of MED19447.

[0193] Specifically, the present invention relates to the following:

[0194] 1. A method comprising antibody V L An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V L Includes amino acid sequence:

[0195] [FW1]SGLSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4]

[0196] Where [FW1], [FW2], [FW3] and [FW4] represent V L Framework region, and wherein X1 represents the amino acid residue proline (P), glutamic acid (E) or aspartic acid (D); X2 represents the amino acid residue asparagine (N) or aspartic acid (D);

[0197] X3 represents the amino acid residue glutamine (Q) or leucine (L);

[0198] X4 represents the amino acid residue leucine (L) or proline (P);

[0199] X5 represents the amino acid residue glycine (G) or serine (S);

[0200] X6 represents the amino acid residue leucine (L) or histidine (H);

[0201] X7 represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N); and

[0202] X8 represents the amino acid residue leucine (L) or threonine (T).

[0203] 2. The isolated binding molecule or antigen-binding fragment thereof according to item 1, wherein FW1 comprises SEQ ID NO: 25 or 26, FW2 comprises SEQ ID NO: 27 or 28, FW3 comprises SEQ ID NO: 29, and FW4 comprises SEQ ID NO: 30.

[0204] 3. An isolated binding molecule comprising an antibody VH that specifically binds to CD73 or an antigen-binding fragment thereof, wherein the V H Includes amino acid sequence:

[0205] [FW5]SYAX9S[FW6]X10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8]

[0206] wherein [FW5], [FW6], [FW7] and [FW8] represent VH framework regions, and wherein

[0207] X9 represents the amino acid residue methionine (M) or tyrosine (Y);

[0208] X 10 represents the amino acid residue leucine (L) or alanine (A);

[0209] X 11 represents the amino acid residue tryptophan (W) or serine (S);

[0210] X 12 represents the amino acid residue tryptophan (W) or glycine (G);

[0211] X 13 represents the amino acid residue serine (S) or arginine (R);

[0212] X 14 represents the amino acid residue glycine (G) or serine (S);

[0213] X 15 represents the amino acid residue arginine (R) or threonine (T);

[0214] X 16 represents the amino acid residue valine (V) or isoleucine (I); and

[0215] X 17 Indicates the amino acid residues tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0216] 4. The isolated binding molecule or antigen-binding fragment thereof according to item 3, wherein FW5 comprises SEQ ID NO: 31, FW6 comprises SEQ ID NO: 32, FW7 comprises SEQ ID NO: 33, and FW8 comprises SEQ ID NO: 34.

[0217] 5. A method comprising antibody V L and antibody V H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V LIncludes amino acid sequence:

[0218] [FW1]SGLSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4]

[0219] Where [FW1], [FW2], [FW3] and [FW4] represent V L framework area, and wherein

[0220] X1 represents the amino acid residue proline (P), glutamic acid (E), or aspartic acid (D);

[0221] X2 represents the amino acid residue asparagine (N) or aspartic acid (D);

[0222] X3 represents the amino acid residue glutamine (Q) or leucine (L);

[0223] X4 represents the amino acid residue leucine (L) or proline (P);

[0224] X5 represents the amino acid residue glycine (G) or serine (S);

[0225] X6 represents the amino acid residue leucine (L) or histidine (H);

[0226] X7 represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N); and

[0227] X8 represents the amino acid residue leucine (L) or threonine (T);

[0228] And where the V H Includes amino acid sequence:

[0229] [FW5]SYAX9S[FW6]X 10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8]

[0230] wherein [FW5], [FW6], [FW7] and [FW8] represent VH framework regions, and wherein

[0231] X9 represents the amino acid residue methionine (M) or tyrosine (Y);

[0232] X 10 represents the amino acid residue leucine (L) or alanine (A);

[0233] X 11 represents the amino acid residue tryptophan (W) or serine (S);

[0234] X 12 represents the amino acid residue tryptophan (W) or glycine (G);

[0235] X 13 represents the amino acid residue serine (S) or arginine (R);

[0236] X 14 represents the amino acid residue glycine (G) or serine (S);

[0237] X 15 represents the amino acid residue arginine (R) or threonine (T);

[0238] X 16 represents the amino acid residue valine (V) or isoleucine (I); and

[0239] X 17 Indicates the amino acid residues tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0240] 6. The isolated binding molecule or antigen-binding fragment thereof of claim 5, wherein FW1 comprises SEQ ID NO: 25 or 26, FW2 comprises SEQ ID NO: 27 or 28, FW3 comprises SEQ ID NO: 29, FW4 comprises SEQ ID NO: 30, FW5 comprises SEQ ID NO: 31, FW6 comprises SEQ ID NO: 32, FW7 comprises SEQ ID NO: 33, and FW8 comprises SEQ ID NO: 34.

[0241] 7. The isolated binding molecule or antigen-binding fragment thereof according to any one of items 1 to 6, including an antibody or antigen-binding fragment thereof.

[0242] 8. A method comprising antibody V L An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V LIncluded are VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequences that are identical to, or identical to, SEQ ID NOs: 46, 49 and 53; SEQ ID NOs: 47, 49 and 53; SEQ ID NOs: 47, 49 and 54; SEQ ID NOs: 46, 50 and 54; SEQ ID NOs: 46, 51 and 55; SEQ ID NOs: 48, 52 and 54; SEQ ID NOs: 46, 49 and 56; SEQ ID NOs: 47, 49 and 56; SEQ ID NOs: 46, 50 and 56; SEQ ID NOs: 46, 51 and 56; or SEQ ID NOs: 48, 52 and 56, respectively.

[0243] 9. A method comprising antibody V H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the V H Included are VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequences that are identical to, or identical to, SEQ ID NOs: 35, 37 and 41; SEQ ID NOs: 36, 37 and 42; SEQ ID NOs: 36, 38 and 43; SEQ ID NOs: 36, 39 and 44; SEQ ID NOs: 36, 40 and 44; SEQ ID NOs: 35, 37 and 45; SEQ ID NOs: 36, 37 and 45; SEQ ID NOs: 36, 38 and 45; SEQ ID NOs: 36, 39 and 45; or SEQ ID NOs: 36, 40 and 45, respectively.

[0244] 10. The isolated binding molecule or antigen-binding fragment thereof according to item 8 or 9, including an antibody or antigen-binding fragment thereof.

[0245] 11. A method comprising: L and V H An isolated antibody or antigen-binding fragment thereof that specifically binds to CD73, the V L and V HSEQ ID NOs: 46, 51, 55, 36, 39, and 44; SEQ ID NOs: 48, 52, 54, 36, 40, and 44; SEQ ID NOs: 46, 49, 56, 35, 37, and 41; SEQ ID NOs: 47, 49, 53, 35, 37, and 41; SEQ ID NOs: 47, 49, 54, 36, 37, and 42; SEQ ID NOs: 46, 50, 54, 36, 38, and 43; SEQ ID NOs: 46, 51, 55, 36, 39, and 44; SEQ ID NOs: 48, 52, 54, 36, 40, and 44; SEQ ID NOs: 46, 49, 56, 35, 37, and 41; SEQ ID NOs: 47, 49, 54, 36, 37, and 42; NO: 46, 49, 53, 35, 37 and 45; SEQ ID NO: 47, 49, 56, 36, 37 and 45; SEQ ID NO: 46, 50, 56, 36, 38 and 45; SEQ ID NO: 46, 51, 56, 36, 39 and 45; SEQ ID NO: 48, 52, 56, 36, 40 and 45; or SEQ ID NO: 46, 49, 56, 35, 37 and 45.

[0246] 12. The isolated antibody or antigen-binding fragment thereof according to item 11, comprising an antibody or antigen-binding fragment thereof.

[0247] 13. An isolated antibody or antigen-binding fragment thereof comprising a V sequence comprising SEQ ID NO: 68 L and V comprising SEQ ID NO: 82 H .

[0248] 14. An isolated antibody or antigen-binding fragment thereof comprising V consisting essentially of SEQ ID NO: 68 L and V consisting essentially of SEQ ID NO: 82 H .

[0249] 15. An isolated antibody or antigen-binding fragment thereof comprising V consisting of SEQ ID NO: 68 L and V consisting of SEQ ID NO: 82 H .

[0250] 16. The isolated binding molecule or antigen-binding fragment thereof according to item 7, comprising a heavy chain constant region or a fragment thereof.

[0251] 17. An isolated binding molecule or antigen-binding fragment thereof according to item 10, comprising a heavy chain constant region or a fragment thereof.

[0252] 18. The isolated antibody or antigen-binding fragment thereof according to any one of items 11 to 15, comprising a heavy chain constant region or a fragment thereof.

[0253] 19. A nucleic acid comprising a sequence encoding the isolated antibody or antigen-binding fragment thereof according to any one of items 7 and 10 to 18.

[0254] 20. A host cell comprising the nucleic acid sequence according to item 19.

[0255] 21. A method for preparing the antibody or antigen-binding fragment thereof according to any one of items 7 and 10 to 18, the method comprising (a) culturing the cell according to item 20; and (b) isolating the antibody or antigen-binding fragment thereof.

[0256] 22. The isolated binding molecule or antigen-binding fragment thereof according to item 7, which is an antagonist of CD73.

[0257] 23. The isolated binding molecule or antigen-binding fragment thereof according to item 10, which is an antagonist of CD73.

[0258] 24. The isolated antibody or antigen-binding fragment thereof according to any one of items 11 to 15, which is an antagonist of CD73.

[0259] 25. The isolated binding molecule or antigen-binding fragment thereof of claim 22, wherein the CD73 is human CD73.

[0260] 26. The isolated binding molecule or antigen-binding fragment thereof of claim 23, wherein the CD73 is human CD73.

[0261] 27. The isolated antibody or antigen-binding fragment thereof of claim 24, wherein the CD73 is human CD73.

[0262] 28. Use of the antibody or antigen-binding fragment thereof according to any one of items 7 to 27 in the manufacture of a medicament for treating cancer.

[0263] 29. The use according to item 28, wherein the cancer is selected from the group consisting of colorectal cancer, pancreatic cancer, bladder cancer, leukemia, lymphoma, glioma, glioblastoma, melanoma, ovarian cancer, thyroid cancer, esophageal cancer, prostate cancer and breast cancer.

[0264] 30. Use of an anti-CD73 antibody or antigen-binding fragment thereof in the manufacture of a medicament for treating a subject identified as having a tumor with increased CD73 expression relative to a reference.

[0265] 31. The use of claim 30, wherein the anti-CD73 antibody is MEDI9447 or Phen0203 hIgG1.

[0266] 32. The use of claim 30 or 31, wherein the subject is undergoing, has undergone, or will undergo anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

[0267] 33. The use of claim 32, wherein the anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy comprises administering an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or an antigen-binding fragment thereof.

[0268] 34. The use of item 33, wherein the anti-PD-1 antibody is pembrolizumab ( Lambrolizumab, MK-3475), nivolumab ( BMS-936558, MDX-1106, ONO-4538), AMP-224 or an antigen-binding fragment thereof.

[0269] 35. The method of claim 33, wherein the anti-PD-L1 antibody is MEDI4736, BMS-936559, or MPDL3280A or an antigen-binding fragment thereof.

[0270] 36. The method of claim 33, wherein the anti-CTLA-4 antibody is ipilimumab, tremelimumab (tesitumumab, CP-675,206), or an antigen-binding fragment thereof.

[0271] 37. A pharmaceutical formulation comprising an effective amount of an anti-CD73 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof.

[0272] 38. The pharmaceutical formulation of claim 37, wherein the anti-CD73 antibody is MEDI9447, Phen0203 hIgG1, or an antigen-binding fragment thereof.

[0273] 39. The pharmaceutical formulation of claim 37 or 38, wherein the anti-PD-L1 antibody is MEDI4736, BMS-936559, or MPDL3280A, or an antigen-binding fragment thereof.

[0274] 40. A pharmaceutical formulation comprising an effective amount of an anti-CD73 antibody or an antigen-binding fragment thereof and an anti-CTLA4 antibody or an antigen-binding fragment thereof.

[0275] 41. The pharmaceutical formulation of claim 40, wherein the anti-CD73 antibody is MEDI9447, Phen0203 hIgG1, or an antigen-binding fragment thereof.

[0276] 42. The pharmaceutical formulation of claim 40 or 41, wherein the anti-CTLA4 antibody is ipilimumab or tremelimumab (tesitumumab, CP-675,206) or an antigen-binding fragment thereof.

[0277] 43.An antibody V L and antibody V H An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, wherein the isolated binding molecule or antigen-binding fragment thereof specifically binds to a CD73 protein epitope that includes one or more amino acids corresponding to Val144, Lys180, and Asn185.

[0278] 44. The isolated binding molecule or antigen-binding fragment thereof of claim 43, further comprising one or more amino acids corresponding to Tyr135, Lys136, and Asn187.

[0279] 45. The isolated binding molecule or antigen-binding fragment thereof of claim 43 or 44, comprising amino acids corresponding to Tyr135, Lys136, and Asn187.

[0280] 46. ​​The isolated binding molecule or antigen-binding fragment thereof of claim 43 or 44, comprising amino acids corresponding to Tyr135, Lys136, Asn185, Tyr135, Lys136, and Asn187.

[0281] 47. An isolated binding molecule or antigen-binding fragment thereof as described in claim 43 or 44, which binds to an epitope within one or more of the following regions of the CD73 protein: Tyr132-Val144 and / or Lys180-Asn187.

[0282] 48. An isolated binding molecule or antigen-binding fragment thereof as described in claim 43 or 44, comprising the amino acid sequence at Tyr132-Va1144 and / or Lys180-Asn187.

[0283] The compositions and articles defined by the present invention are made individually or otherwise in connection with the examples provided below. Other features and advantages of the present invention will appear in the detailed description and in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0284] Figure 1A The nucleotide sequence and amino acid translation of the MEDI9447 VH domain are shown, and the CDRs are shown based on the Kabat numbering convention.

[0285] Figure 1BThe nucleotide sequence and amino acid translation of the MEDI9447 VL domain are shown, and the CDRs are shown based on the Kabat numbering convention.

[0286] Figure 1C Alignment of MEDI9447 VH with the closest human VH and JH germline sequences is shown. CDRs are highlighted based on Kabat numbering convention and residues that differ from the germline sequence are boxed.

[0287] Figure 1D An alignment of MEDI9447 VL with the closest human VL and JL germline sequences is shown. The CDRs are highlighted based on the Kabat numbering convention and residues that differ from the germline sequence are boxed.

[0288] Figure 2 Two graphs showing antibody-mediated internalization of cytotoxic FabZAP reagents into MDA-MB-231 and 4T1 cells are provided, where the antibodies are MEDI9447 and control antibody R347.

[0289] Figure 3A is a graph showing the inhibition of 5' ectonucleotidase by the anti-CD73 antibody MEDI9447.

[0290] Figure 3B This is a graph showing the inhibition of AMP hydrolysis by the anti-CD73 antibody CD370010.

[0291] Figure 4 Figure 2 shows that MEDI9447 inhibits tumor growth in the CT26 syngeneic tumor model. Murine CT26 tumor cells were subcutaneously implanted into the right flank of female Balb / C mice. Tumors were allowed to grow for 3 days and treated with MEDI9447 or an isotype control twice weekly for two weeks. On day 16, tumors were harvested for flow cytometric analysis.

[0292] Figure 5 Figure 2 shows that MEDI9447 inhibits tumor-infiltrating myeloid-derived suppressor cells (MDSCs). CT26 tumor-bearing mice treated with MEDI9447 were sacrificed and tumors were harvested on study day 16. Tumors were dissociated into single cells, stained for CD45 and MDSC markers, and analyzed by flow cytometry.

[0293] Figure 6Included are six spider graphs showing the effect of MEDI9447 mIgG1, anti-PD-1, or the combination on tumor volume. Control antibodies include rIgG2a, a rat IgG2a control monoclonal rat antibody specific for E. coli β-galactosidase (β-Gal), and an isotype control mouse IgG1. Tumor volume for each group of animals was plotted for a single animal up to study day 40. All control group mice had tumors at the end of the 40-day study period. Anti-CD73 treatment alone resulted in 10% tumor-free animals at the end of the study. Anti-PD1 treatment alone also resulted in 10% tumor-free animals at the end of the study. Remarkably, the combination of anti-CD73 and anti-PD treatment resulted in 60% tumor-free mice. None of the control group mice were tumor-free at the end of the study.

[0294] Figure 7 is a graph showing the effect of MEDI9447 mIgG1, anti-PD1, or the combination on survival.

[0295] Figure 8 Figure 2 is a graph showing that the combination of MEDI9447 and anti-PD-1 significantly enhanced tumor growth inhibition in colorectal cancer tumors when compared to either agent alone (p < 0.05). Mice were subcutaneously injected with syngeneic MC38-OVA colorectal cancer cells and treated twice weekly with 10 mg / kg MEDI9447, 10 mg / kg anti-PD-1 antibody, or the combination of both antibodies. Tumor volume was measured twice weekly.

[0296] Figure 9 Figure 2 is a graph showing that anti-PD-1 induces a CD73-rich tumor microenvironment, as measured by CD73 expression on tumor cells isolated from tumor-bearing mice. Mice (n=4) were injected subcutaneously with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg anti-PD-1 or an irrelevant isotype control antibody. One day after the first treatment, tumors were isolated, cells were dissociated, and surface phenotypes were analyzed by flow cytometry.

[0297] Figure 10 Figure 2 is a graph showing that anti-PD-1 induces a CD73-rich tumor microenvironment, as measured by CD73 expression on myeloid-derived suppressor cells (MDSCs) isolated from tumor-bearing mice. Mice (n=4) were injected subcutaneously with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg anti-PD-1 or an irrelevant isotype control antibody. One day after the first treatment, tumors were isolated, tumor cells were isolated, peripheral whole blood cells were harvested, and surface CD73 expression was analyzed by flow cytometry.

[0298] Figure 11Figure 2 shows that anti-PD-1 induces a CD73-enriched tumor microenvironment, as measured by CD4 T cells isolated from tumor-bearing mice. + 、FoxP3 + CD73 expression on lymphocytes was measured. Mice (n=4) were injected subcutaneously with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg anti-PD-1 or irrelevant isotype control antibody. Three days after the first treatment, tumors were isolated, peripheral whole blood cells were harvested, and surface CD73 expression was analyzed by flow cytometry.

[0299] Figure 12 Figure 2 is a graph showing that the combination of MEDI9447 and anti-PD-L1 significantly enhanced tumor growth inhibition in melanoma tumors when compared to either agent alone (p < 0.05). Mice were injected subcutaneously with syngeneic B16F10 melanoma cells and treated twice weekly with 10 mg / kg MEDI9447, 10 mg / kg anti-PD-L1 antibody, or a combination of both antibodies. Tumor volume was measured twice weekly.

[0300] Figure 13 Figure 2 is a graph showing that the combination of MEDI9447 and anti-PD-L1 significantly enhanced tumor growth inhibition in lymphoma tumors when compared to either agent alone (p < 0.01). Mice were injected subcutaneously with syngeneic EG7-OVA lymphoma cells and treated twice weekly with 10 mg / kg MEDI9447, 10 mg / kg anti-PD-L1 antibody, or a combination of both antibodies. Tumor volume was measured twice weekly.

[0301] Figure 14 Graph showing that anti-PD-L1 induces a CD73-rich tumor microenvironment as measured by surface expression of CD73 on draining lymph node B lymphocytes. Mice (n=4) were injected subcutaneously with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg anti-PD-L1 or an irrelevant isotype control antibody. One day after the first treatment, cells were isolated from draining lymph nodes and analyzed for surface phenotype by flow cytometry.

[0302] Figure 15 is shown by the presence of tumor-infiltrating CD4 + 、FoxP3 + Figure 3. Anti-PD-L1 induces a CD73-rich tumor microenvironment as measured by surface expression of CD73 on lymphocytes. Mice (n=4) were injected subcutaneously with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg anti-PD-L1 or an irrelevant isotype control antibody. Three days after the first treatment, tumors were isolated, cells were dissociated, and analyzed for surface phenotype by flow cytometry.

[0303] Figure 16A A and B are graphs showing that MEDI9447 alone or in combination with anti-PD-L1 reduces CD73 expression on tumor-infiltrating lymphocytes. Mice bearing colorectal CT26 syngeneic tumors were treated twice weekly (days 12 and 16) with 30 mg / kg MEDI9447 alone or 30 mg / kg anti-PD-L1 or a combination of both MEDI9447 and anti-PD-L1. On day 17, tumors were harvested and analyzed by flow cytometry for surface CD73 expression. CD73 expression on tumor-infiltrating (A) CD4+FoxP3+Treg and (B) CD8+T cells.

[0304] Figure 17A A and B are graphs showing that MEDI9447 alone or in combination with anti-PD-L1 reduces CD73 activity on (A) tumor cells and (B) peripheral whole blood cells. Mice bearing colorectal CT26 syngeneic tumors were treated twice weekly (days 12 and 16) with 30 mg / kg MEDI9447 alone or 30 mg / kg anti-PD-L1 or a combination of both MEDI9447 and anti-PD-L1. On day 17, tumors and peripheral whole blood cells were harvested and analyzed for surface CD73 expression using Cell-Titre Glo for enzymatic activity.

[0305] Figure 18 It is a set of graphs depicting the cytokine profiles of peripheral blood mononuclear cells treated with MEDI9447 and antibodies or fusion proteins specific for CTLA4, OX40, PD-1, and PD-L1. Primary human peripheral blood mononuclear cells were incubated for 72 hours in a mixed leukocyte reaction with MEDI9447 and / or antibodies or fusion proteins specific for the specified targets. Cytokines (IFN-γ, IL-1β, TNF-α) in duplicate supernatants were quantified by ELISA. The data shown represent the optimal dose combination of anti-CD73 antibodies with 4 different partner agents. The anti-PD-1 and anti-CD73 combination showed significant (p < 0.05) synergy, as determined by Bliss response surface methodology (Zhao et al.). The cytokine profiles indicate that both myeloid and lymphoid lineages are affected. More than 50 donor pairs were tested.

[0306] Figure 19A and 19B Depicted are the results of hydrogen deuterium exchange MS (HDX-MS) analysis of CD73 in complex with MEDI9447. Figure 19ADepicted is a heat map of hydrogen-deuterium exchange showing those CD73 regions (N- to C-terminus) that experience reduced deuterium uptake when bound to MEDI9447. The relative exchange between antibody-bound and unbound CD73 is depicted as a function of exposure time, with decreased exchange in red, increased exchange in blue, and no change to white. The N-terminal regions at positions 132-143 and 182-187 exhibit the highest degree of differential exchange. Figure 19B The crystal structure of the CD73 monomer is shown, depicting the location of the HDX-identified binding interface (cyan) within the N-terminal domain (yellow). The CD73 linker region and C-terminal domain are represented in orange and blue, respectively.

[0307] Figures 20A-20E Depicted are the results of hydrogen deuterium exchange MS (HDX-MS) analysis indicating regions of CD73 and MEDI9447 that undergo differential hydrogen exchange in free versus bound states. Figure 20A Depicted are graphs representing relative deuterium uptake (mass change in Daltons) as a function of deuterium exposure time within a peptide encompassing the 132-143 region. Figure 20B Depicted is a graph representing the relative deuterium uptake (mass change in Daltons) as a function of deuterium exposure time within a peptide encompassing the 182-187 region. Figure 20A and 20B In the figure, uptake for CD73 alone is shown in squares, while uptake for CD73 bound to MEDI9447 Fab is shown in red. Peptide sequence, position, and mass are indicated in the plot box. To narrow down the region containing sequences that exhibit hydrogen exchange changes and are predicted to form epitopes, the relative mass changes in overlapping peptides were compared. For example, the peptide spanning positions 173-186 exhibited differential exchange, while the peptide spanning 173-181 did not. Therefore, it was inferred that residues upstream of 182 were not differentially labeled. Figure 20C Depicted is a DynamX difference graph of the MEDI9447 Fab heavy chain. Figure 20D DynamX difference graph depicting the light chain of MEDI9447 Fab. Figure 20C and 20D , each data point indicates the difference in deuterium uptake between the CD73+Fab complex (positive values ​​on the y-axis) and the Fab alone (negative values ​​on the y-axis). The vertical bars represent the sum of the uptake differences between exposure time points. CDRs with relatively lower uptake when the Fab binds to CD73 are indicated. Figure 20EDepicted is a DynamX difference chart of CD73 alone (negative values ​​on the y-axis) and CD73 bound to Fab (positive values ​​on the y-axis). Regions E1 (aa 132-143) and E2 (aa 182-187) are indicated. The horizontal axis corresponds to the peptides analyzed from N- to C-terminus (left to right). The dotted line overlays the chart, showing the 1.6 Dalton, 98% confidence interval cutoff for statistically significant changes.

[0308] Figures 21A-21H Depicted are sensor chip data showing that the MEDI9447 epitope is located within the N-terminal domain of CD73. Figure 21A Figure 2 is a graph depicting sensor chip data for wild-type CD73 protein. Wild-type CD73 protein was immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by surface plasmon resonance (SPR). Figure 21B Figure 2 is a graph depicting sensor chip data for N-terminal domain-swapped CD73 protein. N-terminal domain-swapped CD73 protein was immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. When the N-terminal domain was switched, MEDI9447 did not bind to CD73. Figure 21C Figure 2 is a graph depicting sensor chip data for CD73 proteins with N- and C-terminal domains switched. CD73 proteins with N- and C-terminal domains switched were immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. MEDI9447 did not bind to CD73 when both the N- and C-terminal domains were switched. Figure 21D This graph depicts sensor chip data for linker-region-switched CD73 proteins. Linker-region-switched CD73 proteins were immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. Switching the linker region alone did not affect binding. Figure 21E Figure 2 is a graph depicting sensor chip data for CD73 proteins with C-terminal domain switching. CD73 proteins with C-terminal domain switching were immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. Switching the C-terminal domain alone did not affect binding. Figure 21F Figure 2 is a graph depicting sensor chip data for interface E1 (aa 132-143)-converted CD73 protein. Interface E1 (aa 132-143)-converted CD73 protein was immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. Figure 21GFigure 2 is a graph depicting sensor chip data for interfacial E2 (aa 182-187)-converted CD73 protein. Interfacial E2 (aa182-187)-converted CD73 protein was immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. Figure 21H Figure 1 is a graph depicting sensor chip data for CD73 protein converted by interface E1 (aa 132-143) and interface E2 (aa 182-187). CD73 protein converted by interface E1 (aa 132-143) and interface E2 (aa 182-187) was immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. Figures 21F-21H , convert HDX interface E1(aa 132-143)( Figure 21F ) had a slight effect on binding, compared to converting HDX interface E2 (aa182-187) alone ( Figure 21G ) or in combination with E1 ( Figure 21H ) on the contrary. Figures 21A-21H The sensorgrams and overlay fits are shown in matching colors. The kinetic measurements for each binding assay are shown in Table 16.

[0309] Figure 22 Depicted is an alignment of the human and chicken CD73 protein sequences. Only the mature protein sequence is shown. Non-conserved residues in the chicken sequence are highlighted. Regions that were converted between chicken and human to generate knockout variants are annotated (e.g., DS1a, DS1b, etc.).

[0310] Figure 23 Binding of MEDI9447 to CD73 variants is depicted. Figure 23 is a data table showing the binding of MEDI9447 to CD73 variants. The K values ​​of the variants highlighted in blue are D >2-fold change compared to WT or KO parental construct. *Kinetic measurements derived from a 2:1 heterologous ligand fit. **Numbers correspond to chicken sequences (129=133, 140=144 and 181=185 in human).

[0311] Figures 24A-24F The MEDI9447 epitope is depicted as being located at the apex of the N-terminal domain. Figure 24A showed that MEDI9447 interacts with a panel of CD73 variants (see Figure 22 and 23 Evaluation of the binding of the HDX protein revealed six positions that constitute the interaction site. Two of the three most influential residues (highlighted blocks) are located outside the HDX interface region (highlighted in blue). The three less important residues (pink blocks) are located within the HDX interface. Figure 24BThe results show that knocking in N185 and V144 (K180 is conserved) into a CD73 construct containing chicken N- and C-terminal domain sequences restored the expression of MEDI9447 to that of wild-type CD73 (from 5 nM to 0.3 nM dilution; and Figure 21B Compared with K D 20 times smaller binding. Figure 24C A close-up of the epitope residues located within the N-terminal domain of CD73 is depicted. The most important binding residues are highlighted, while less influential positions (Y135, K136, and N187) are in pink. The HDX interface is overlaid in blue. Figure 24D A surface representation showing that the epitopes form a nearly continuous binding surface is depicted. Figure 24E Depicted is the crystal structure of the open conformation of CD73, showing the location of the epitope on the apical surface of the N-terminal domain. Figure 24F The epitope is shown to be located away from the substrate binding site (adenosine depicted as a sphere) and the zinc ion (grey sphere) coordination site (cyan side chains). In all crystal structures, the CD73 N-terminal domain, linker region, and C-terminal domain are depicted in yellow, orange, and blue, respectively.

[0312] Figures 25A-25C MEDI9447 was shown to be a non-competitive inhibitor of CD73 hydrolysis of AMP. Figure 25A is a graph depicting the kinetics of CD73 phosphohydrolysis of AMP measured in the presence of MEDI9447 or an isotype-matched control mAb. Figure 25B is a graph showing that MEDI9447 acts as a noncompetitive inhibitor, in that it inhibits hydrolysis equally regardless of substrate concentration. In contrast, APCP, a known competitive inhibitor of CD73, increases K m , but without increasing V 最大 . Figure 25C Graph depicting the dose response of MEDI9447 IgG, Fab, or control IgG to inhibition of CD73 hydrolysis of AMP. MEDI9447 IgG achieved maximum inhibition at a 1:1 molar stoichiometry with CD73 dimers (arrows). At high concentrations with excess MEDI9447 IgG (>10 nM), a loss of inhibition or "hook effect" was observed. MEDI9447 Fab and control IgG did not inhibit CD73. All experiments were performed using the CellTiterGlo assay as described herein (RLU, relative light units).

[0313] Figures 26A-26C Anti-CD73 mAb (clone 0069) binding was shown to be dependent on CD73 N- and C-terminal domain residues. Figure 26Ais a graph showing sensor chip data for histidine-tagged CD73. Histidine-tagged CD73 was immobilized on a HIS2 biosensor and binding by mAb A was measured by biolayer interferometry (BLI). Binding of mAb A to WT CD7 (blue sensorgram), N-terminal domain-swapped knockout CD73 (KO_1-291, green sensorgram), and C-terminal domain-swapped knockout CD7 (KO_311-523, cyan sensorgram) shows that mAb binding is affected by residues in both the N- and C-terminal domains. Figure 26B Crystal structures of open and closed CD73 showing the locations of the highlighted mAb A binding hotspots (aa 114-134 and 153-170), which are located near the interface of the N- and C-terminal domains (N-terminal domain in yellow, linker in orange, and C-terminal domain in blue). Figure 26A and 26C The combined data. Figure 26C Binding sensorgrams of mAb A to different domain-switch knockout variants of CD73 are shown. Switching subdomains DS2c (aa 114-134) or DS3a (aa 153-170) knocked out binding. All binding analyses were performed on an Octet QK384 instrument as described herein.

[0314] Figures 27A-27C MEDI9447 was shown to inhibit the transition of CD73 to a conformationally active structure. Figure 27A Figure 2 is a graph showing biosensor data for wild-type CD73. Wild-type CD73 was immobilized on a HIS2 biosensor and the binding of MEDI9447 (blue sensorgram) and anti-CD73 mAb A (brown sensorgram) was measured by BLI on an OctetQK384. 2+ When pre-incubated with APCP, MEDI9447 retained binding (black sensorgram), but mAb A lost binding (orange sensorgram). Figure 27B It shows that although Zn 2+ Preincubation of CD73 with APCP resulted in loss of mAb A binding (orange sensorgram), and the addition of Zn 2+ Pre-incubation with MEDI9447 before APCP recovery (purple sensorgram). The blue and brown sensorgrams show the binding of mAb A to CD73 alone and to CD73 treated with MEDI9447 (but not Zn 2+ and APCP) pre-incubated with CD73. Figure 27C Figure 3 depicts how MEDI9447 blocks Zn uptake by CD73 2+Proposed model of the fully closed active conformation induced by APCP. MEDI9947 may restrict the transition to an intermediate state with lower affinity for mAb A.

[0315] Figure 28A and 28B Shown are the binding of MEDI9447 or mAb A to CD73 under different conditions measured by BLI, as described herein, unless otherwise indicated. Figure 28A Figure 2 is a graph depicting the binding of anti-CD73 mAb A to histidine-tagged wild-type CD73 immobilized on a HIS2 biosensor. After a 100-second baseline, captured CD73 was treated with Zn 2+ The biosensors were incubated with 30 nM mAb A for 600 seconds to measure binding. mAb A bound to CD73 (blue sensorgram), but not with Zn. 2+ CD73 pre-incubated with APCP (purple sensorgram). mAb A was maintained with APCP and EDTA (green sensorgram) or Zn 2+ , APCP and EDTA (gold sensorgram). The chelation effect of EDTA shows that when CD73 is treated with Zn 2+ Divalent cations are required for the loss of mAb A binding when incubated with APCP. Figure 28B It shows that MEDI9447 Fab or control IgG did not rescue mAb A with Zn 2+ Figure 3 shows the binding of CD73 pre-incubated with APCP. Figure 28B The determination was performed as shown. 2+ Before APCP, MEDI9447 Fab or isotype-matched control IgG was pre-incubated with CD73. mAb A immobilized on the biosensor binds to CD73 alone (blue sensorgram), CD73 pre-incubated with MEDI9447 Fab (light blue sensorgram) or control IgG (black sensorgram), but not with Zn 2+ CD73 incubated with APCP (brown sensorgram), or incubated with Zn 2+ Fab (gold sensorgram) or control IgG (purple sensorgram) pre-incubated with CD73 before APCP.

[0316] Figure 29A and 29B Anti-CD73 mAb B binding was shown to be dependent on residues in subregion DS2b (aa 92-134) or DS2c (aa 114-134). Figure 29A Is displayed corresponding to Figures 30A-30CTable of SEC-MALS data for CD73 and MEDI9447. For each mixture of CD73 and either MEDI9447 or mAb B, the corresponding SEC retention time, Mw, and polydispersity of the formed complex are shown. Figure 29B Determination of the binding hotspot of mAb B on CD73 is depicted. According to the methods herein, the binding of mAb B to CD73 variants immobilized on HIS2 biosensors was measured by BLI, as directed against Figures 26A-26C The binding sensorgrams showed that switching subregions DS2b (aa 92-134) or DS2c (aa 114-134) knocked out the binding of mAb B.

[0317] Figures 30A-30C MEDI9447 is shown to form interdimer bridges between soluble CD73 molecules. CD73 was incubated with varying amounts of MEDI9447 or anti-CD73 mAb B and analyzed by SEC-MALS. Shown are SEC UV chromatograms with protein retention time on the x-axis and molar mass determined by MALS on the y-axis. Figure 30A Figure 1 is a chromatogram showing that MEDI9447 forms a complex with CD73 of approximately 1.7 (λ) and approximately 6.6 (λ) megadaltons at a 1:1 molar ratio (green trace). Complexes of comparable size are formed at lower ratios of MEDI9447:CD73 (0.5:1 in blue and 0.1:1 in magenta). MEDI9447 and CD73 alone are represented by the black and red UV traces, respectively. Figure 30B Figure 2: A top-down view of the crystal structure of the CD73 dimer showing the mAb B binding hotspot (purple) and the MEDI9447 epitope (magenta and pink). mAb B binds to a site near the central groove between the dimers in the open conformation. Figure 30C Figure 2 is a chromatogram showing the formation of a single major complex of approximately 270-290 kD (peak at approximately 7.2 minutes) when CD73 binds to mAb B. The UV traces shown represent 1:1 mAb B:CD73 (red), 0.5:1 (blue), and 0.1:1 (green). mAb A and CD73 alone are magenta and black, respectively.

[0318] Figures 31A-31D Depicted is the inhibition of surface-bound CD73 by IgG and Fab forms of MEDI9447. Figure 31Ais a graph depicting inhibition of AMP hydrolysis by immobilized CD73 by MEDI9447 IgG, Fab, or a control antibody. CD73 was immobilized to nickel-coated microtiter plates via a C-terminal histidine tag, and inhibition of AMP hydrolysis by MEDI9447 IgG, Fab, or a control antibody was measured using the malachite green assay described herein. MEDI9447 IgG, but not control IgG, inhibited CD73 hydrolysis of AMP in a dose-dependent manner. MEDI9447 Fab also inhibited CD73 activity, but to a much lesser extent. Figure 31B Depicted is a complex comprising MEDI9447 Fab (green) bound to an anti-Fd antibody (xFd, red). When MEDI9447 Fab (green) was bound to one arm of the anti-Fd antibody (xFd, red) and the other arm was bound to a nonspecific polyclonal Fab (pFab, orange), inhibition increased to comparable to that of MEDI9447 IgG (Fab+xFd+pFab vs. MEDI9447 IgG and MEDI9447 IgG+xFd+pFab) (see Figure 31A ). Figure 31C Figure 2 is a graph depicting the inhibition of AMP hydrolysis by GPI-anchored CD73 by MEDI9447 IgG, Fab, or a control antibody. The enzymatic activity of endogenously expressed CD73 in MDA-MB-231 cells was determined by the CellTiterGlo assay. Similar to immobilized recombinant CD73, MEDI9447 IgG inhibited AMP hydrolysis to a greater extent than Fab, but increasing the effective size of MEDI9447 Fab by forming a complex with an anti-Fd antibody enhanced the inhibition. Figure 31D Graph depicting inhibition of AMP hydrolysis of soluble CD73 (sCD73) by MEDI9447 IgG, Fab, or control antibody. To test whether xFd+MEDI9447 could inhibit soluble CD73, AMP hydrolysis was measured using a malachite green assay. MEDI9447 Fab alone or bound to a single xFd arm did not inhibit soluble CD73 activity. In contrast, MEDI9947 Fab bound to two xFd arms (MEDI9447 Fab+xFd) imparted bivalency that led to CD73 inhibition.

[0319] Figure 32Figure 2 is a graph showing that MEDI94447 IgG and Fab inhibit CD73 hydrolysis of AMP. CD73 activity was measured using the malachite green assay in the presence of increasing concentrations of antibody, as described herein. MEDI9447 IgG inhibited CD73 hydrolysis activity in a dose-dependent manner, and no hook effect or loss of inhibition was observed. MEDI9447 Fab also inhibited CD73 function, but achieved lower levels of maximal inhibition. The experiment was performed twice with comparable results. Data from only one experiment are shown.

[0320] Figure 33 Depicted is a model showing that inhibition of CD73 hydrolysis activity by MEDI9447 occurs through a dual mechanism. MEDI9447 IgG (green) inhibits soluble CD73 by forming an inter-dimer bridge that prevents conformational transition to the closed state. Monovalently bound IgG or Fab do not inhibit soluble CD73. When CD73 is bound to the surface, inhibition can occur through bridging of adjacent CD73 dimers or steric blocking from monovalently bound IgG or Fab / xFd (red) complexes. DETAILED DESCRIPTION

[0321] The present invention provides isolated binding molecules or antigen-binding fragments thereof that specifically bind to CD73. In some aspects, such molecules are antibodies and antigen-binding fragments thereof that specifically bind to CD73. Also provided are related polynucleotides, vectors, and pharmaceutical compositions comprising anti-CD73 antibodies or antigen-binding fragments thereof. Also provided are methods for preparing and using the anti-CD73 antibodies and antigen-binding fragments disclosed herein, such as methods for diagnosing and treating cancer in subjects (as direct therapy, adjuvant therapy, or combination therapy). The present invention also provides antibody-drug conjugates derived from the CD73 binding molecules disclosed herein. In addition, the present invention provides therapeutic combinations characterized by anti-CD73 antibodies (e.g., MEDI9447) and one or more agents targeting other aspects of the cancer immune cycle, such as anti-PD-1 antibodies, anti-PD-L1 antibodies (e.g., MEDI4736), and anti-CTLA4 antibodies; and methods for using such combinations to reduce tumor-mediated immunosuppression.

[0322] In order to more readily understand the present disclosure, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0323] I. Definition

[0324] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or method steps, as these compositions or method steps may vary. As used in this specification and appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. The term "a" (or "an"), along with the terms "one or more," and "at least one," are used interchangeably herein.

[0325] Furthermore, when used herein, "and / or" is to be understood as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or," as used herein in phrases such as "A and / or B," is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or," as used herein in phrases such as "A, B, and / or C," is intended to encompass each of the following: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0326] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, 2000, Oxford University Press provide those of skill with general dictionary references to many of the terms used in this disclosure.

[0327] Units, prefixes and symbols are all expressed in their International System of Units (SI) accepted form. Numerical ranges include numbers that limit this range. Unless otherwise indicated, amino acid sequences are written from left to right in an amino to carboxyl orientation. The titles provided herein are not limitations on the various aspects, which can be obtained by reference to the specification sheets as a whole. Therefore, the terms directly defined below are more completely defined by reference to the specification sheets in their entirety.

[0328] It should be understood that when an aspect is described with the language "comprising," other similar aspects described with respect to "consisting of" and / or "consisting essentially of" are also provided.

[0329] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides are referred to by their universally recognized single-letter codes.

[0330] As used herein, the term "CD73 polypeptide" refers to the CD73 (cluster of differentiation 73) protein, also referred to in the literature as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase, encoded by the NT5E gene. See, for example, Misumi et al. Eur. J. Biochem. 191(3):563-9 (1990). The sequences corresponding to the human and murine forms of CD73 are available in the Uniprot database under accession numbers P21589 and Q61503, respectively. In defining any CD73 antibody epitope, the amino acid numbering used refers to the amino acid residues of the mature CD73 protein that does not include the signal sequence residues. Thus, an antibody that binds to amino acids Val144, Lys180, and Asn185, for example, refers to the amino acid position after the cleavage signal sequence, i.e., the amino acid in the mature protein.

[0331] An exemplary CD73 polypeptide is provided below:

[0332] >sp|P21589|5NTD_Human 5′-nucleotidase OS=Homo sapiens GN=NT5E PE=1 SV=1

[0333]

[0334] Soluble and membrane-bound forms of CD73 have been identified. See Klemens et al., Biochem. Biophys. Res. Commun. 172(3): 1371-7 (1990). In addition, several different isozymes have been identified. See Rosi et al. Life Sci. 62(25): 2257-66 (1998). The full-length CD73 protein comprises 574 amino acids. Mature CD73 protein is produced after removal of the signal sequence of the propeptide (positions 1 to 26) and the C-terminal region (positions 550-574). In addition, amino acids 404 to 453 are removed in isoform 2 of CD73 after alternative splicing. Natural variants are also known, for example, variant C358Y, variant T376A, and variant M379T. See Misumi et al., Eur. J. Biochem. 191:563-569 (1990); Otsuki et al., DNA Res. 12:117-126 (2005); Mungall et al., Nature 425:805-811 (2003); Hansen et al., Gene 167:307-312 (1995); Klemens et al., Gene 168:307-312 (1996). ens et al. Biochem. Biophys. Res. Commun. 172: 1371-1377 (1990); Knapp et al. Structure 20: 2161-2173 (2012); or St. Hilaire et al. N. Engl. J. Med. 364: 432-442 (2011), all of which are incorporated herein by reference in their entirety.

[0335] Typical diseases that lead to changes in the level of CD73 in the patient's tissue fluid, especially serum: tissue trauma; reperfusion injury caused by myocardial infarction or stroke, organ transplantation or other surgical procedures; cancer or cancer metastasis; or inflammatory conditions caused by the above-mentioned trauma or reperfusion injury or by chronic diseases (including allergic disorders, autoimmune diseases and inflammatory diseases). Examples of such chronic conditions can be arthritis, allergic disorders such as asthma, inflammatory disorders such as inflammatory bowel disease or skin inflammatory disorders, psoriasis, Parkinson's disease, Alzheimer's disease, autoimmune diseases, type I or type II diabetes, atherosclerosis, multiple sclerosis, Crohn's disease, or rejection caused by organ transplantation. Specifically, inflammatory diseases systemic inflammatory response syndrome (SIRS), acute lung injury (ALI), multiple organ failure (MOF), local ischemia reperfusion injury (IRI) and adverse drug reactions (ADRS) lead to changes in tissue fluid CD73 protein. Thus, the CD73 binding molecules disclosed herein can be used, for example, to treat or diagnose cancer (e.g., colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Burkitt's lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer). In addition, measuring the level of CD73 in a sample (e.g., tissue fluid) from a patient using the CD73 binding molecules disclosed herein can be used to monitor the progression of the above-mentioned diseases, to assess the efficacy of therapy, to select patients for specific therapy treatment, or to make medical decisions, such as starting, ending, interrupting, or modifying certain treatments.

[0336] The terms "inhibit," "block," "suppress," and grammatical variations thereof are used interchangeably herein and refer to any statistically significant decrease in a biological activity, including complete prevention of the activity. For example, "inhibition" can refer to a decrease in biological activity of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Thus, when the term "inhibition" or "suppression" is used to describe, for example, an effect on the enzymatic activity of CD73, the term refers to the ability of an anti-CD73 antibody or antigen-binding fragment thereof to statistically significantly reduce the 5'-nucleotidase activity of CD73 (hydrolyzing catabolized adenosine monophosphate AMP to adenosine) relative to the 5'-nucleotidase activity mediated by CD73 in untreated (control) cells. Cells expressing CD73 can be naturally occurring cells or cell lines (e.g., cancer cells) or can be recombinantly produced by introducing a nucleic acid encoding CD73 into a host cell. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof can statistically significantly reduce the 5'-nucleotidase activity of a soluble form of CD73 in a biological fluid. In one aspect, the anti-CD73 binding molecule, e.g., antibody, or antigen-binding fragment thereof, inhibits CD73-mediated 5'-nucleotidase activity by at least 10%, at least 15%, or at least 20%, at least 25%, or at least 30%, at least 35%, or at least 40%, at least 45%, or at least 50%, at least 55%, or at least 60%, at least 65%, or at least 70%, at least 75%, or at least 80%, at least 85%, or at least 90%, at least 95%, or about 100%, as determined, for example, by the methods described in the Examples below and / or by methods known in the art.

[0337] As used herein, the term "inhibit CD73 activity" refers to the ability of an anti-CD73 binding molecule, such as an antibody or antigen-binding fragment thereof, to statistically significantly reduce CD73-dependent 5'-nucleotidase activity in a cell expressing CD73 or a sample containing CD73. In some aspects, inhibition of CD73 activity can be a decrease of at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or about 100% when a cell or sample is contacted with an anti-CD73 binding molecule, such as an antibody or antigen-binding fragment thereof of the disclosure relative to CD73 activity measured in the absence of the anti-CD73 binding molecule, such as an antibody or antigen-binding fragment thereof (control conditions).

[0338] The terms "antibody" or "immunoglobulin" as used interchangeably herein include whole antibodies and any antigen-binding fragment or single chain thereof.

[0339] A typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH or VL). H ) and heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or V L ) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), with more conserved regions called framework regions (FWs) inserted between them. Each VH and VL is composed of three CDRs and four FWs, arranged in the following order from amino terminus to carboxyl terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or host factors, including different cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Exemplary antibodies of the present disclosure include anti-CD73 antibodies (original and germlined), affinity-optimized clones, optimized antibodies lacking ADCC, conjugated antibodies (e.g., ADCs), and other optimized antibodies (e.g., serum half-life optimized antibodies, including, for example, YTE mutations, see Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006) and U.S. Pat. No. 7,083,784, hereby incorporated by reference in their entireties).

[0340] The term "germlining" means that the amino acids at specific positions in an antibody are mutated back to those in the germline.

[0341] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above substances through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies (e.g., bispecific antibodies) produced from at least two complete antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigenic determinants of antibodies, and any other modified immunoglobulin molecules comprising antigen enzyme recognition sites, as long as these antibodies exhibit the desired biological activity.

[0342] Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are designated α, δ, ε, γ, and μ, respectively, based on the properties of their heavy chain constant regions. Different classes of immunoglobulins have distinct and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, such as toxins, radioisotopes, etc., to form ADCs.

[0343] "Blocking" antibodies or "antagonist" antibodies are antibodies that inhibit or reduce the biological activity of the antigen to which the antibody binds, such as CD73. In certain aspects, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. Ideally, the biological activity is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100%.

[0344] The terms "CD73 antibody," "antibody that binds to CD73," or "anti-CD73" refer to an antibody or antigen-binding fragment thereof that binds to CD73 with sufficient affinity to render the molecule useful as a therapeutic or diagnostic agent in targeting CD73. The extent of binding of the anti-CD73 antibody to unrelated, non-CD73 proteins is less than about 10% of the binding of the antibody to CD73, as determined, for example, by radioimmunoassay (RIA), BIACORE, or other methods. TM In certain aspects, the antibody binds to CD73 with a dissociation constant (K) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤10 pM, ≤1 pM, or ≤0.1 pM. D ). The term "anti-CD73" broadly also encompasses molecules comprising, for example, the CDRs of the antibodies disclosed herein bound to a scaffold. Thus, the phrase "isolated binding molecules or antigen-binding fragments thereof that specifically bind to CD73" will refer not only to antibodies and antigen-binding fragments thereof, but will also refer to molecules comprising, for example, one or more scaffolds (e.g., a fibronectin III domain from fibronectin or tenascin-3) to which the CDRs of the antibodies disclosed herein are bound. See, e.g., U.S. Patent Publication No. 20150098955, hereby incorporated by reference in its entirety.

[0345] In one embodiment, the anti-CD73 antibody is an IgG1-TM format antibody such that the IgG1 Fc domain includes mutations L234, L235E, and P331, binds to soluble and cell surface-displayed CD73, and inhibits CD73 enzymatic activity. Figures 1A-1D The nucleotide and amino acid sequences of the MEDI9447 VH and VL domains are provided.

[0346] By "CTLA4 polypeptide" is meant a polypeptide having at least 85% amino acid sequence identity to GenBank Accession No. AAL07473.1, or a fragment thereof having T cell inhibitory activity. The sequence of AAL07473.1 is provided below:

[0347] CTLA4 polypeptide sequence [Homo sapiens]

[0348]

[0349] By "CTLA4 nucleic acid molecule" is meant a polynucleotide encoding a CTLA4 polypeptide. An exemplary CTLA4 nucleic acid molecule sequence is provided as Genbank Accession No. AAL07473.

[0350] By "anti-CTLA4 antibody" is meant an antibody that selectively binds to a CTLA4 polypeptide. Exemplary anti-CTLA4 antibodies are described, for example, in U.S. Patent Nos. 6,682,736; 7,109,003; 7,123,281; 7,411,057; 7,824,679; 8,143,379; 7,807,797; and 8,491,895 (wherein tremelimumab is 11.2.1), which are incorporated herein by reference. Tremelimumab is an exemplary anti-CTLA4 antibody. The tremelimumab sequence (SEQ ID NOs: 130-137) is provided in the sequence listing herein below.

[0351] By "PD-1 polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_005009 and having PD-L1 and / or PD-L2 binding activity. The sequence of NP_005009 is provided below.

[0352] PD-1 peptide sequence

[0353] NCBI accession number NP_005009

[0354]

[0355] By "PD-1 nucleic acid molecule" is meant a polynucleotide encoding a PD-1 polypeptide. An exemplary PD-1 nucleic acid molecule sequence is provided as NCBI Accession No. NM_005018.

[0356] By "anti-PD-1 antibody" is meant an antibody or antigen-binding fragment thereof that selectively binds to a PD-1 polypeptide. Exemplary anti-PD-1 antibodies include, for example, pembrolizumab ( Lambrolizumab, MK-3475), nivolumab ( BMS-936558, MDX-1106, ONO-4538), or AMP-224.

[0357] By "PD-L1 polypeptide" is meant a polypeptide or fragment thereof having at least about 85%, 95%, or 100% amino acid identity to NCBI Accession No. NP_001254635 and having PD-1 and CD80 binding activity. The sequence of NP_001254635 is provided below.

[0358] PD-L1 peptide sequence

[0359] NCBI accession number NP_001254635

[0360]

[0361] By "PD-L1 nucleic acid molecule" is meant a polynucleotide encoding a PD-L1 polypeptide. An exemplary PD-L1 nucleic acid molecule sequence is provided as NCBI Accession No. NM_001267706.

[0362] By "anti-PD-L1 antibody" is meant an antibody or antigen-binding fragment thereof that selectively binds to a PD-L1 polypeptide. Exemplary anti-PD-L1 antibodies are described, for example, in US 20130034559 / US 8779108 and US 20140356353, incorporated herein by reference. MEDI4736 is an exemplary PD-L1 antibody. The sequence of MEDI4736 is provided in the sequence listing herein below (SEQ ID NOs: 138-145).

[0363] The term "antigen-binding fragment" refers to a molecule that comprises a portion of an intact antibody, and in particular, a molecule that comprises the antigen-determining variable region of an intact antibody. It is known in the art that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0364] "Monoclonal antibody" refers to a homogeneous antibody population that is capable of highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants.

[0365] The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain variable fragments (scFv), fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal antibody" refers to such antibodies prepared in any number of ways, including but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals (e.g., expressing human antibodies in transgenic mice).

[0366] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that has been engineered to contain minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody is a human immunoglobulin in which residues from CDRs are replaced with residues from non-human species (e.g., mouse, rat, rabbit, or hamster) with desired specificity, affinity, and ability. (Jones et al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature, 332: 323-327; Verhoeyen et al., 1988, Science, 239: 1534-1536) In some cases, the framework (FW) amino acid residues of a human immunoglobulin are replaced with corresponding residues from antibodies of non-human species with desired specificity, and / or affinity, and / or ability.

[0367] Humanized antibodies can also be further modified to improve and optimize antibody specificity, affinity and / or ability by replacing the extra residues in the non-human residue in the Fv framework region and / or replacement.Usually, humanized antibodies will comprise substantially all at least one (and typically two or three) variable domains, and this variable domains comprises all or substantially all CDR districts corresponding to non-human immunoglobulin, and all or substantially all FR districts are those of human immunoglobulin consensus sequences.Humanized antibodies can also comprise at least a portion of immunoglobulin constant region or domain (Fc), typically constant region or domain of human immunoglobulin.The example of the method for producing humanized antibodies is described in U.S. Patent number 5,225,539 or 5,639,641.

[0368] The "variable region" of an antibody refers to the variable region of an antibody light chain alone or the variable region of an antibody heavy chain, or a combination thereof. The variable regions of the heavy and light chains are each composed of four FW regions connected by three CDR regions. The CDRs in each chain are closely held together by the FW regions and, together with the CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site. There are at least two techniques for determining CDRs: (1) a method based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, MD); and (2) a method based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273: 927-948). Furthermore, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0369] The Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain).

[0370] The phrases "amino acid position numbering in Kabat," "Kabat position," and grammatical variants thereof refer to the numbering system used in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland (1991) for heavy chain variable domains or light chain variable domains of antibodies. Using this numbering system, the actual linear amino acid sequence may comprise fewer or additional amino acids corresponding to truncations or insertions of the FW or CDRs of the variable domains. For example, a heavy chain variable domain may comprise a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an insertion residue after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat).

[0371] Table 1

[0372]

[0373] The Kabat numbering of residues for a given antibody can be determined by aligning the antibody sequence with regions of homology to the "standard" Kabat numbering sequence. In contrast, Chothia refers to the position of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbering using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by the Oxford Molecular AbM antibody modeling software.

[0374] IMGT (Immunogenetics) also provides a numbering system for immunoglobulin variable regions (including CDRs). See, for example, Lefranc, MP et al., Dev. Comp. Immunol 27:55-77 (2003), which is incorporated herein by reference. The IMGT numbering system is based on alignments, structural data, and characterizations of more than 5,000 sequences of hypervariable loops and allows for easy comparison of variable and CDR regions across all species. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0375] EU index or EU numbering system is the sequence numbering of the first human IgG (EU antibody) based on sequencing.Because the most common quoting to this regulation is Kabat sequence manual (Kabat (Kabat) et al., 1991), EU index is sometimes mistakenly used synonymously with Kabat index.EU index does not provide insertion and deletion, and therefore in some cases, the comparison of the IgG position across IgG subclass and kind can be clear, particularly in hinge region. Nevertheless, in many Fc structure function studies, this regulation is enough to enable direct comparison between Fc district.Therefore, in this manual, the numbering scheme used for the replacement and insertion in the Fc region is the EU index as in Kabat.By contrast, the numbering scheme used in this manual for variable region (VH and VL) is conventional Kabat numbering.

[0376] As used throughout this specification, the VH CDR sequences correspond to the classical Kabat numbering positions, i.e., Kabat VH-CDR1 is at positions 31-35, VH-CDR2 is at positions 50-65, and VH-CDR3 is at positions 95- 102. VL-CDR1, VL-CDR2, and VL-CDR3 also correspond to the classical Kabat numbering positions, i.e., positions 24-34, 50-56, and 89-97, respectively.

[0377] As used herein, the Fc region includes a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include a J chain. For IgG, Fc comprises the hinge between immunoglobulin domains C gamma 2 and C gamma 3 (Cγ2 and Cγ3) and C gamma 1 (Cγ1) and C gamma 2 (Cγ2).

[0378] Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined as comprising residues C226 or P230 at its carboxyl terminus, where numbering is according to the EU index as described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Fc can refer to this region in isolation, or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of different Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index, and therefore slight differences may exist between the presented sequences and prior art sequences.

[0379] The term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human prepared using any technique known in the art (e.g., recombinant expression in cultured cells, or expression in transgenic animals). Thus, the term human antibody also encompasses antibodies having an amino acid sequence corresponding to an antibody originally produced by a human (or an engineered variant or derivative thereof) but expressed in a non-human system (e.g., produced by chemical synthesis; recombinantly expressed in microorganisms, mammals, or insect cells; or expressed in an animal subject). Thus, antibodies obtained from a human subject or from a human cell (e.g., a hybridoma or cell line expressing a recombinant antibody or fragment thereof) and subsequently expressed in an animal (e.g., a mouse) are considered human antibodies. This definition of a human antibody includes complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide, such as an antibody comprising a mouse light chain and a human heavy chain polypeptide.

[0380] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more animal species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies derived from one mammal (e.g., mouse, rat, rabbit, etc.) with the desired specificity, and / or affinity, and / or capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0381] As used herein, the term "epitope" refers to an antigenic protein determinant capable of binding to the CD73 antibodies or CD73 binding molecules disclosed herein. Epitopes are typically composed of chemically active surface groups of molecules such as amino acids or sugar side chains and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. The portion of the antibody or binding molecule that recognizes the epitope is called the paratope. Epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational epitopes are composed of discontinuous segments of the amino acid sequence of the antigen. These epitopes interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. In contrast, linear epitopes interact with the paratope based on its primary structure. Linear epitopes are formed by a continuous sequence of amino acids from the antigen.

[0382] The term "antibody binding site" refers to a region of an antigen (e.g., CD73) comprising a continuous or discontinuous site (i.e., an epitope) to which a complementary antibody specifically binds. Thus, the antibody binding site may comprise additional regions of the antigen outside of the epitope, and such additional regions may determine properties such as binding affinity and / or stability or influence properties such as antigen enzymatic activity or dimerization. Thus, even if two antibodies bind to the same epitope within an antigen, if the antibody molecules establish different intermolecular contacts with amino acids outside of the epitope, such antibodies are considered to bind to different antibody binding sites.

[0383] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) represents. Affinity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure.

[0384] Unless otherwise stated, "potency" is generally expressed as IC in nM. 50 IC 50 is the median inhibitory concentration of the antigen-binding molecule. In functional assays, IC 50 The concentration that reduces a biological response by 50% of its maximum value. In ligand binding studies, IC 50 IC is the concentration that reduces receptor binding by 50% of the maximum specific binding level. 50This can be calculated by any number of means known in the art.The improvement in potency can be determined by measuring, for example, against a parent antibody (eg, the parent antibody before germlining or the parent antibody before affinity optimization).

[0385] The fold improvement in potency for an antibody or polypeptide of the disclosure compared to the parent antibody can be at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, at least about 130-fold, at least about 140-fold, at least about 150-fold, at least about 160-fold, at least about 170-fold, or at least about 180-fold or more.

[0386] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells with cytotoxins. Specific high-affinity IgG antibodies directed against the surface of the target cells "arm" the cytotoxic cells and are absolutely required for this killing. Lysis of the target cells is extracellular, requires direct cell-to-cell contact, and does not involve complement. It is expected that, in addition to antibodies, other proteins containing an Fc region (specifically, an Fc fusion protein) that have the ability to specifically bind to target cells bearing antigen will be able to affect cell-mediated cytotoxicity. For simplicity, the cell-mediated cytotoxicity resulting from the activity of the Fc fusion protein is also referred to herein as ADCC activity.

[0387] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is one that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in a form found in nature. In some aspects, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0388] The term "subject" refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, etc. Typically, the terms "subject" and "patient" are used interchangeably herein with respect to human subjects.

[0389] The term "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient (e.g., an anti-CD73 binding molecule disclosed herein) to be effective and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such compositions can be sterile.

[0390] As disclosed herein, an "effective amount" of an anti-CD73 binding molecule is an amount sufficient to carry out the specifically recited purpose. For the recited purpose, an "effective amount" can be determined empirically and in a routine manner.

[0391] The term "therapeutically effective amount" refers to an amount of an anti-CD73 binding molecule or other drug disclosed herein sufficient to "treat" a disease or disorder in a subject or mammal.

[0392] The term "label" as used herein refers to a detectable compound or composition that is fused (e.g., genetically fused) or conjugated (e.g., chemically conjugated) directly or indirectly to an anti-CD73 binding molecule disclosed herein to produce a "labeled" anti-CD73 binding molecule. The label can be detectable by itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze chemical alteration of a detectable substrate compound or composition.

[0393] Terms such as "derivatizable group" and "derivatizable functional group" are used interchangeably and refer to a functional group capable of reacting to permit covalent bond formation between an anti-CD73 binding molecule disclosed herein (e.g., a CD73 antibody) and another substance. In some aspects, such substances are therapeutic agents (e.g., cytotoxins), detectable labels, polymers (e.g., PEG), etc. Exemplary derivatizable groups include sulfhydryl, hydroxyl, amino, carboxyl, and amide, as well as modified forms thereof such as activated or protected forms.

[0394] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "alleviate" refer to (1) therapeutic measures that result in a cure, slowdown, alleviation of symptoms, and / or halting of progression of a diagnosed pathological condition or disorder and (2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathological condition or disorder. Thus, those in need of treatment include those already having the disorder; those predisposed to having the disorder; and those in whom prevention of the disorder is desired. In certain aspects, a subject is successfully "treated" for cancer according to the methods of the present disclosure if the subject shows, for example, total, partial, or transient remission of a certain type of cancer.

[0395] The terms "cancer," "tumor," "cancerous," and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, epithelial cancers, including adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer (such as hepatocellular carcinoma and hepatoma), bladder cancer, breast cancer (including hormone-mediated breast cancer, see, e.g., Innes et al. (2006) Br. J. Cancer 94: 1057-1065), colon cancer, colorectal cancer, endometrial cancer, myeloma (such as multiple myeloma), salivary gland cancer, kidney cancer (such as renal cell carcinoma and Wilms' tumor), basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, various types of head and neck cancer, and cancers of mucinous origin (such as mucinous ovarian cancer), bile duct cancer (liver), and renal papillary carcinoma. In some aspects, the term cancer as used herein refers specifically to cancers that express CD73. In some specific aspects, the term cancer refers to a cancer that expresses low levels of CD73. In some aspects, the term cancer as used herein specifically refers to a cancer that expresses CD73 (e.g., colon cancer, breast cancer, lymphoma, non-small cell carcinoma).

[0396] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refers to polymers of nucleotides of any length, and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0397] The term "vector" means a construct that is capable of delivering one or more genes or sequences of interest in a host cell and, in some aspects, capable of expressing these genes or sequences. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (such as production cells).

[0398] The terms "polypeptide," "peptide," and "protein," used interchangeably herein, refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that because the polypeptides of the present disclosure are antibody-based, in certain aspects, the polypeptides may appear as single chains or related chains.

[0399] A "recombinant" polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in engineered host cells are considered isolated, as are native or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any suitable technique. The polypeptides disclosed herein can be recombinantly produced using methods known in the art. Alternatively, the proteins and peptides disclosed herein can be chemically synthesized.

[0400] The term "amino acid substitution" refers to replacing an amino acid residue present in the parent sequence with another amino acid residue. The amino acid in the parent sequence can be replaced, for example, via chemical peptide synthesis or by recombinant methods known in the art. Therefore, reference to "substitution at position X" or "substitution at position X" refers to replacing the amino acid present at position X with an alternative amino acid residue. In some aspects, the substitution type can be described according to the pattern AXY, where A is the single letter code corresponding to the amino acid naturally present at position X, and Y is the substituted amino acid residue. In other aspects, the substitution type can be described according to the pattern XY, where Y is the single letter code corresponding to the amino acid residue that replaces the amino acid naturally present at position X.

[0401] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In another aspect, a string of amino acids can be conservatively substituted with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0402] Non-conservative substitutions include those in which (i) a residue with an electropositive side chain (e.g., Arg, His, or Lys) is substituted for or replaced by an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for or replaced by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val), (iii) cysteine ​​or proline is substituted for or replaced by any other residue, or (iv) a residue with a large hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is substituted for or replaced by one with a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

[0403] Other substitutions can be readily identified by one of ordinary skill in the art. For example, for the amino acid alanine, substitutions can be made with any of D-alanine, glycine, β-alanine, L-cysteine, and D-cysteine. For lysine, substitutions can be made with any of D-lysine, arginine, D-arginine, homoarginine, methionine, D-methionine, ornithine, or D-ornithine. In general, substitutions in functionally important regions that can be expected to induce changes in the properties of the isolated polypeptide are those in which (i) a polar residue, such as serine or threonine, is substituted for a hydrophobic residue, such as leucine, isoleucine, phenylalanine, or alanine (or are substituted therewith); (ii) a cysteine ​​residue is substituted for any other residue (or are substituted therewith); (iii) a residue having an electropositive side chain, such as lysine, arginine, or histidine, is substituted for a residue having an electropositive side chain, such as glutamic acid or aspartic acid (or are substituted therewith); or (iv) a residue having a bulky side chain, such as phenylalanine, is substituted for one that does not have such a side chain, such as glycine (or are substituted therewith). The likelihood that one of the aforementioned non-conservative substitutions may alter the functional properties of the protein is also related to the position of the substitution with respect to the functionally important region of the protein: some non-conservative substitutions may therefore have little or no effect on biological properties.

[0404] The term "amino acid insertion" refers to the introduction of a new amino acid residue between two amino acid residues present in the parent sequence. An amino acid can, for example, be inserted into the parent sequence via chemical peptide synthesis or by recombinant methods known in the art. Therefore, as used herein, phrases "insertion between position X and Y" or "insertion between Kabat position X and Y" (wherein X and Y correspond to amino acid positions (e.g., insertion of the cysteine ​​amino acid between position 239 and 240)) refer to the insertion of the amino acid between X and Y positions, and also refer to the insertion in the nucleic acid sequence of the codons encoding amino acids between the codons encoding amino acids at positions X and Y. Insertion types can be described according to the pattern AXin, where A is the single letter code corresponding to the amino acid to be inserted, and X is the position between the insertion.

[0405] The term "percentage of sequence identity" between two polypeptide or polynucleotide sequences refers to the number of identical matching positions shared by these sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced in order to optimally align the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps that are present in the target sequence are not counted because gaps are not nucleotides or amino acids. Similarly, gaps that are present in the reference sequence are not counted because the target sequence nucleotides or amino acids are counted, while nucleotides or amino acids from the reference sequence are not counted.

[0406] The calculation of sequence identity percentage is by determining the number of positions that identical amino acid residues or nucleic acid bases appear in two sequences to produce matching position number, the matching position number is divided by the total number of positions in the comparison window and the result is multiplied by 100, thereby producing sequence identity percentage. The determination of the sequence identity percentage between the comparison of sequence and two sequences can be completed using software that is easy to obtain for online use and downloading. Suitable software programs can be obtained from different sources, and are used to compare proteins and nucleotide sequences. A kind of suitable program for determining sequence identity percentage is b12seq, which is a part of the BLAST program suite that can be obtained from the National Center for Biotechnology Information (National Centerfor Biotechnology Information) BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithm to perform comparison between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0407] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It should be noted that percent sequence identity values ​​are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that length values ​​will always be integers.

[0408] In certain aspects, the percent identity "X" of a first amino acid sequence to a second amino acid sequence is calculated as 100 x (Y / Z), where Y is the number of amino acid residues, scored as the number of identical matches when aligning the first and second sequences (as aligned by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0409] Those skilled in the art will appreciate that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence comparisons driven entirely by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program for generating multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com / muscle / . ClustalW2 and MUSCLE are alternatively available from, for example, EBI.

[0410] It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structure), functional data (e.g., mutation positions), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, which is available from www.tcoffee.org and alternatively available from, for example, the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated automatically or manually.

[0411] As used herein, the term "consensus sequence" for light chain (VL) and heavy chain (VH) variable regions refers to a composite or universalized VL or VH sequence, defined based on information that the amino acid residues within the VL or VH chain can undergo modification without impairing antigen binding. Thus, in the "consensus sequence" of a VL or VH chain, certain amino acid positions are occupied by one of the multiple possible amino acid residues at that position. For example, if arginine (R) or serine (S) occurs at a specific position, the specific position within the consensus sequence can be arginine or serine (R or S). The consensus sequence of a VH and VL chain can be defined, for example, by in vitro affinity maturation (e.g., randomizing each amino acid position in a certain CDR using degenerate encoding primers), scanning mutagenesis of amino acid residues within an antibody CDR (e.g., alanine scanning mutagenesis), or any other method known in the art, followed by evaluation of the binding of these mutants to the antigen to determine whether the mutated amino acid position affects antigen binding. In some aspects, mutations are introduced into the CDR region. In other aspects, mutations are introduced into the framework region. In some other aspects, mutations are introduced in the CDR and framework regions.

[0412] II. CD73 Binding Molecules

[0413] The present disclosure provides CD73 binding molecules, such as antibodies and antigen-binding fragments thereof, that specifically bind to CD73 (e.g., human CD73). The full-length amino acid (aa) and nucleotide (nt) sequences of CD73 are known in the art (see, e.g., UniProt accession number P21589 for human CD73, or UniProt accession number Q61503 for mouse CD73). In some aspects, the anti-CD73 binding molecule is a human antibody (e.g., clone 10.3 antibody, clone 2C5 antibody, MEDI9447). In certain aspects, the CD73 binding molecule is an antibody or an antigen-binding fragment thereof.

[0414] In some aspects, the CD73 binding molecules, such as antibodies or antigen-binding fragments thereof, include Fab, Fab′, F(ab′)2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgG CH2, minibody, F(ab′)3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv) 2, or scFv-Fc. In some aspects, the antibody is of the IgG class, for example, of the IgG1 class.

[0415] In some aspects, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment comprises a heavy chain constant region or a fragment thereof. In some specific aspects, the heavy chain constant region is an IgG constant region. The IgG constant region may include a light chain constant region selected from the group consisting of a kappa constant region and a lambda constant region.

[0416] In certain aspects, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein are modified compared to a parent antibody, such as CD730010 antibody or CD730002 antibody. In some aspects, the parent antibody is CD730010. In other aspects, the parent antibody is CD730002. In other aspects, the parent antibody is CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. These modifications can include mutations in the CDR regions and / or FW regions compared to a parent antibody, such as CD730010 or CD730002.

[0417] The phrase "CD730002 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO: 1 and two VH domains comprising the amino acid sequence of SEQ ID NO: 2.

[0418] The phrase "CD730004 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO: 104 and two VH domains comprising the amino acid sequence of SEQ ID NO: 103.

[0419] The phrase "CD730008 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO: 106 and two VH domains comprising the amino acid sequence of SEQ ID NO: 107.

[0420] The phrase "CD730010 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO:3 and two VH domains comprising the amino acid sequence of SEQ ID NO:4.

[0421] The phrase "CD730011 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO:5 and two VH domains comprising the amino acid sequence of SEQ ID NO:6.

[0422] The phrase "CD730021 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO:7 and two VH domains comprising the amino acid sequence of SEQ ID NO:8.

[0423] The phrase "CD730042 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO:9 and two VH domains comprising the amino acid sequence of SEQ ID NO:10.

[0424] The phrase "CD730046 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO:11 and two VH domains comprising the amino acid sequence of SEQ ID NO:12.

[0425] The phrase "CD730047 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO: 13 and two VH domains comprising the amino acid sequence of SEQ ID NO: 14.

[0426] The phrase "CD730068 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO: 108 and two VH domains comprising the amino acid sequence of SEQ ID NO: 107.

[0427] The phrase "CD730069 antibody" refers to an IgG1 comprising two VL domains comprising the amino acid sequence of SEQ ID NO:110 and two VH domains comprising the amino acid sequence of SEQ ID NO:109.

[0428] (i) CD730010-derived anti-CD73 antibodies

[0429] In certain aspects, the anti-CD73 antibodies of the present disclosure include modifications to the CDR1 and / or CDR2 and / or CDR3 of the light chain of the CD730010 antibody, including but not limited to:

[0430] 1) a light chain CDR1 comprising the consensus sequence SGSLSNIGRNX1VN, wherein X1 represents the amino acid residue proline (P), glutamic acid (E) or aspartic acid (D); and / or

[0431] 2) a light chain CDR2 comprising the consensus sequence LX2NX3RX4X5, wherein X2 represents the amino acid residue asparagine (N) or aspartic acid (D), X3 represents the amino acid residue glutamine (Q) or leucine (L), X4 represents the amino acid residue leucine (L) or proline (P), and X5 represents the amino acid residue glycine (G) or serine (S); and / or

[0432] 3) A light chain CDR3 comprising the consensus sequence ATWDDSX6X7GWX8, wherein X6 represents the amino acid residue leucine (L) or histidine (H), X7 represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N), and X8 represents the amino acid residue leucine (L) or threonine (T).

[0433] In certain aspects, the anti-CD73 antibodies of the present disclosure include modifications to the CDR1 and / or CDR2 and / or CDR3 of the heavy chain of the CD730010 antibody, including but not limited to:

[0434] 1) a heavy chain CDR1 comprising the consensus sequence SYAX9S, wherein X9 represents the amino acid residue methionine (M) or tyrosine (Y); and / or

[0435] 2) Including consensus sequence X 10 IX 11 GSX 12 GX 13 The heavy chain CDR2 of TYYADSVKG, where X 10 represents the amino acid residue leucine (L) or alanine (A), X 11 represents the amino acid residue tryptophan (W) or serine (S), X 12 represents the amino acid residue tryptophan (W) or glycine (G), and X13 represents the amino acid residue serine (S) or arginine (R); and / or

[0436] 3) including the consensus sequence LGYX 14 X 15 X 16 DX 17 The heavy chain CDR3 of 14 represents the amino acid residue glycine (G) or serine (S), X 15 represents the amino acid residue arginine (R) or threonine (T), X 16 represents the amino acid residue valine (V) or isoleucine (I), and X 17 Indicates the amino acid residues tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0437] In one aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL region comprising the consensus amino acid sequence:

[0438] [FW1]SGLSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4]

[0439] wherein [FW1], [FW2], [FW3], and [FW4] represent the amino acid residues of VL framework region 1 (SEQ ID NO: 25 or 26), VL framework region 2 (SEQ ID NO: 27 or 28), VL framework region 3 (SEQ ID NO: 29), and VL framework region 4 (SEQ ID NO: 30), respectively, and wherein

[0440] X1 represents the amino acid residue proline (P), glutamic acid (E), or aspartic acid (D);

[0441] X2 represents the amino acid residue asparagine (N) or aspartic acid (D);

[0442] X3 represents the amino acid residue glutamine (Q) or leucine (L);

[0443] X4 represents the amino acid residue leucine (L) or proline (P);

[0444] X5 represents the amino acid residue glycine (G) or serine (S);

[0445] X6 represents the amino acid residue leucine (L) or histidine (H);

[0446] X7 represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N); and

[0447] X8 represents the amino acid residue leucine (L) or threonine (T).

[0448] In one aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH region comprising the consensus amino acid sequence:

[0449] [FW5]SYAX9S[FW6]X 10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8]

[0450] wherein [FW5], [FW6], [FW7], and [FW8] represent the amino acid residues of VH framework region 1 (SEQ ID NO: 31), VH framework region 2 (SEQ ID NO: 32), VH framework region 3 (SEQ ID NO: 33), and VH framework region 4 (SEQ ID NO: 34), respectively, and wherein

[0451] X9 represents the amino acid residue methionine (M) or tyrosine (Y);

[0452] X 10 represents the amino acid residue leucine (L) or alanine (A);

[0453] X 11 represents the amino acid residue tryptophan (W) or serine (S);

[0454] X 12 represents the amino acid residue tryptophan (W) or glycine (G);

[0455] X 13 represents the amino acid residue serine (S) or arginine (R);

[0456] X 14 represents the amino acid residue glycine (G) or serine (S);

[0457] X 15 represents the amino acid residue arginine (R) or threonine (T);

[0458] X 16 Indicates the amino acid residue valine (V) or isoleucine (I)

[0459] X 17 Indicates the amino acid residues tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0460] In one aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL region comprising the consensus amino acid sequence:

[0461] [FW1]SGLSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4]

[0462] wherein [FW1], [FW2], [FW3], and [FW4] represent the amino acid residues of VL framework region 1 (SEQ ID NO: 25 or 26), VL framework region 2 (SEQ ID NO: 27 or 28), VL framework region 3 (SEQ ID NO: 29), and VL framework region 4 (SEQ ID NO: 30), respectively, and wherein

[0463] X1 represents the amino acid residue proline (P), glutamic acid (E), or aspartic acid (D);

[0464] X2 represents the amino acid residue asparagine (N) or aspartic acid (D);

[0465] X3 represents the amino acid residue glutamine (Q) or leucine (L);

[0466] X4 represents the amino acid residue leucine (L) or proline (P);

[0467] X5 represents the amino acid residue glycine (G) or serine (S);

[0468] X6 represents the amino acid residue leucine (L) or histidine (H);

[0469] X7 represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N); and

[0470] X8 represents the amino acid residue leucine (L) or threonine (T);

[0471] and wherein the anti-CD73 antibody or antigen-binding fragment thereof further comprises a VH region comprising the consensus amino acid sequence:

[0472] [FW5]SYAX9S[FW6]X 10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8]

[0473] wherein [FW5], [FW6], [FW7], and [FW8] represent the amino acid residues of VH framework region 1 (SEQ ID NO: 31), VH framework region 2 (SEQ ID NO: 32), VH framework region 3 (SEQ ID NO: 33), and VH framework region 4 (SEQ ID NO: 34), respectively, and wherein

[0474] X9 represents the amino acid residue methionine (M) or tyrosine (Y);

[0475] X 10 represents the amino acid residue leucine (L) or alanine (A);

[0476] X 11 represents the amino acid residue tryptophan (W) or serine (S);

[0477] X 12 represents the amino acid residue tryptophan (W) or glycine (G);

[0478] X 13 represents the amino acid residue serine (S) or arginine (R);

[0479] X 14 represents the amino acid residue glycine (G) or serine (S);

[0480] X 15 represents the amino acid residue arginine (R) or threonine (T);

[0481] X 16 Indicates the amino acid residue valine (V) or isoleucine (I)

[0482] X 17 Indicates the amino acid residues tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0483] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48.

[0484] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52.

[0485] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55, and 56. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55, and 56.

[0486] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35 and 36. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 35 and 36.

[0487] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40.

[0488] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, and 45. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, and 45.

[0489] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48, except for one, two, three, or four amino acid substitutions.

[0490] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52, except for one, two, three, or four amino acid substitutions.

[0491] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55, and 56, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55, and 56, except for one, two, three, or four amino acid substitutions.

[0492] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35 and 36, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 35 and 36, except for one, two, three, or four amino acid substitutions.

[0493] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40, except for one, two, three, or four amino acid substitutions.

[0494] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, and 45, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, and 45, except for one, two, three, or four amino acid substitutions.

[0495] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48; a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52; and a VL-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55, and 56. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48; a VL-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52; and a VL-CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55, and 56.

[0496] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35 and 36; a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39 and 40; and a VH-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44 and 45. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 35 and 36; a VH-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39 and 40; and a VH-CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44 and 45.

[0497] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 46, 47 and 48, except for one, two, three or four amino acid substitutions; a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51 and 52, except for one, two, three or four amino acid substitutions; and a VL-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55 and 56, except for one, two, three or four amino acid substitutions.

[0498] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48, except at one, two, three, or four amino acid substitutions; a VL-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52, except at one, two, three, or four amino acid substitutions; and a VL-CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55, and 56, except at one, two, three, or four amino acid substitutions.

[0499] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35 and 36, except one, two, three or four amino acid substitutions; a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39 and 40, except one, two, three or four amino acid substitutions; and a VH-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44 and 45, except one, two, three or four amino acid substitutions.

[0500] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 35 and 36, except at one, two, three or four amino acid substitutions; a VH-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39 and 40, except at one, two, three or four amino acid substitutions; a VH-CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44 and 45, except at one, two, three or four amino acid substitutions.

[0501] In certain aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises modifications to CDR1 and / or CDR2 and / or CDR3 of the heavy chain and / or light chain, and further comprises modifications to FW1 and / or FW2 and / or FW3 and / or FW4 of the heavy chain and / or light chain.

[0502] In some aspects, FW1 comprises SEQ ID NO: 25 or 26, FW2 comprises SEQ ID NO: 27 or 28, FW3 comprises SEQ ID NO: 29, FW4 comprises SEQ ID NO: 30, FW5 comprises SEQ ID NO: 31, FW6 comprises SEQ ID NO: 32, FW7 comprises SEQ ID NO: 33, and FW8 comprises SEQ ID NO: 34.

[0503] In some aspects, FW1 comprises SEQ ID NO: 25 or 26, except for one, two, three or four amino acid substitutions; FW2 comprises SEQ ID NO: 27 or 28, except for one, two, three or four amino acid substitutions; FW3 comprises SEQ ID NO: 29, except for one, two, three or four amino acid substitutions; FW4 comprises SEQ ID NO: 30, except for one, two, three or four amino acid substitutions; FW5 comprises SEQ ID NO: 31, except for one, two, three or four amino acid substitutions; FW6 comprises SEQ ID NO: 32, except for one, two, three or four amino acid substitutions; FW7 comprises SEQ ID NO: 33, except for one, two, three or four amino acid substitutions; and FW8 comprises SEQ ID NO: 34.

[0504] In certain aspects, the anti-CD733 antibody or antigen-binding fragment thereof comprises a VL and a VH comprising VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequences that are identical or identical except for one, two, three or four amino acid substitutions in one or more CDRs, wherein such VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, respectively, are:

[0505] SEQ ID NOs: 46, 49, 53, 35, 37, and 41; or

[0506] SEQ ID NOs: 47, 49, 53, 35, 37, and 41; or

[0507] SEQ ID NOs: 47, 49, 54, 36, 37, and 42; or

[0508] SEQ ID NOs: 46, 50, 54, 36, 38, and 43; or

[0509] SEQ ID NOs: 46, 51, 55, 36, 39, and 44; or

[0510] SEQ ID NOs: 48, 52, 54, 36, 40, and 44; or

[0511] SEQ ID NOs: 46, 49, 56, 35, 37, and 41; or

[0512] SEQ ID NOs: 46, 49, 53, 35, 37, and 45; or

[0513] SEQ ID NOs: 47, 49, 56, 36, 37, and 45; or

[0514] SEQ ID NOs: 46, 50, 56, 36, 38, and 45; or

[0515] SEQ ID NOs: 46, 51, 56, 36, 39, and 45; or

[0516] SEQ ID NOs: 48, 52, 56, 36, 40, and 45; or

[0517] SEQ ID NOs: 46, 49, 56, 35, 37 and 45.

[0518] In certain aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises an antibody VL and an antibody VH, wherein the VL comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70.

[0519] In other aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises an antibody VL and an antibody VH, wherein the VH comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.

[0520] In other aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL comprising a sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70. NO:70, and further comprising a VH comprising a sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, and SEQ ID NO:84.

[0521] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL comprising the sequence of SEQ ID NO: 68 and a VH comprising the sequence of SEQ ID NO: 82. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL consisting of the sequence of SEQ ID NO: 68 and a VH consisting of the sequence of SEQ ID NO: 82.

[0522] "Clone 10.3 antibody" (also named "73combo3" or "MEDI9447") is an IgG1 comprising two CD730010-derived light chains (VL) of SEQ ID NO: 68 (including three CDRs, CDR1, CDR2 and CDR3, having the sequences of SEQ ID NOs: 46, 51 and 56, respectively) and two CD730010-derived heavy chains (VH) of SEQ ID NO: 82 (including three CDRs, CDR1, CDR2 and CDR3, having the sequences of SEQ ID NOs: 36, 39 and 45, respectively).

[0523] In certain aspects, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein bind CD73 with substantially the same or better affinity than the 10.3 antibody comprising the 10.3 heavy chain VH of SEQ ID NO: 82 and the 10.3 light chain VL of SEQ ID NO: 68.

[0524] (ii) CD730002-derived anti-CD73 antibodies

[0525] In certain aspects, the anti-CD73 antibodies of the present disclosure include modifications to the CDR1 and / or CDR2 and / or CDR3 of the light chain of the CD730002 antibody, including but not limited to:

[0526] 1) a light chain CDR1 comprising the sequence SGDKVGDKYAS; and / or

[0527] 2) Includes consensus sequence EDX 18 KX 19 X 20 S light chain CDR2, where X 18 represents the amino acid residue serine (S) or threonine (T), X 19 represents the amino acid residue arginine (R) or tyrosine (Y), and X 20 represents the amino acid residue histidine (H), proline (P) or leucine (L); and / or

[0528] 3) A light chain CDR3 comprising the sequence QAWDTSFWV.

[0529] In certain aspects, the anti-CD73 antibodies of the present disclosure include modifications to the CDR1 and / or CDR2 and / or CDR3 of the heavy chain of CD730002, including but not limited to:

[0530] 1) Include sequence SX 21 AX 22 S heavy chain CDR1, where X 21 represents the amino acid residue tyrosine (Y) or valine (V), and X 22 represents the amino acid residue methionine (M) or arginine (R); and / or

[0531] 2) Include the sequence AISGSGGSX 23 YY X 24 DSVKX 25 The heavy chain CDR2 of 23 represents the amino acid residue threonine (T) or proline (P); X 24 represents the amino acid residue alanine (A) or G (glycine); and X 25 represents the amino acid residue glycine (G) or arginine (R); and / or

[0532] 3) A heavy chain CDR3 comprising the sequence DKGYYWYM.

[0533] In one aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL region comprising the consensus amino acid sequence:

[0534] [FW9]SGDKVGDKYAS[FW 10 ]EDX 18 KX 19 X 20 S[FW 11 ]QAWDTSFWV[FW 12 ]

[0535] Among them, [FW9], [FW 10 ]、[FW 11 ] and [FW 12 ] represent amino acid residues of VL framework region 1 (SEQ ID NO: 90 or 91), VL framework region 2 (SEQ ID NO: 92), VL framework region 3 (SEQ ID NO: 93, 94 or 122), and VL framework region 4 (SEQ ID NO: 30), respectively; and wherein X 18 represents the amino acid residue proline (P) or leucine (L); X 19 represents the amino acid residue arginine (R) or tyrosine (Y); and X 20 Indicates the amino acid residues histidine (H), proline (P), or leucine (L).

[0536] In one aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH region comprising the consensus amino acid sequence:

[0537] [FW 13 ]SX 21 AX 22 S[FW 14 ]AISGSGGSX 23 YYX 24 DSVKX 25 [FW 15 ]DKGYYWYM[FW 16 ]

[0538] Among them [FW 13 ]、[FW 14 ]、[FW 15 ] and [FW 16] represent amino acid residues of VH framework region 1 (SEQ ID NO: 31), VH framework region 2 (SEQ ID NO: 32), VH framework region 3 (SEQ ID NO: 33), and VH framework region 4 (SEQ ID NO: 89), respectively; and wherein X 21 represents the amino acid residue tyrosine (Y) or valine (V); X 22 represents the amino acid residue methionine (M) or arginine (R); X 23 represents the amino acid residue threonine (T) or proline (P); X 24 represents the amino acid residue alanine (A) or G (glycine); and X 25 represents the amino acid residue glycine (G) or arginine (R).

[0539] In one aspect, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL region comprising the consensus amino acid sequence:

[0540] [FW9]SGDKVGDKYAS[FW 10 ]EDX 18 KX 19 X 20 S[FW 11 ]QAWDTSFWV[FW 12 ]

[0541] Among them, [FW9], [FW 10 ]、[FW 11 ] and [FW 12 ] represent amino acid residues of VL framework region 1 (SEQ ID NO: 90 or 91), VL framework region 2 (SEQ ID NO: 92), VL framework region 3 (SEQ ID NO: 93, 94 or 122), and VL framework region 4 (SEQ ID NO: 30), respectively; and wherein X 18 represents the amino acid residue proline (P) or leucine (L); X 19 represents the amino acid residue arginine (R) or tyrosine (Y); and X20 represents the amino acid residue histidine (H), proline (P) or leucine (L),

[0542] and wherein the anti-CD73 antibody or antigen-binding fragment thereof further comprises a VH region comprising the consensus amino acid sequence:

[0543] [FW 13 ]SX 21 AX 22 S[FW 14 ]AISGSGGSX 23 YYX 24 DSVKX25 [FW 15 ]DKGYYWYM[FW 16 ]

[0544] Among them [FW 13 ]、[FW 14 ]、[FW 15 ] and [FW 16 ] represent amino acid residues of VH framework region 1 (SEQ ID NO: 31), VH framework region 2 (SEQ ID NO: 32), VH framework region 3 (SEQ ID NO: 33), and VH framework region 4 (SEQ ID NO: 89), respectively; and wherein X 21 represents the amino acid residue tyrosine (Y) or valine (V); X 22 represents the amino acid residue methionine (M) or arginine (R); X 23 represents the amino acid residue threonine (T) or proline (P); X 24 represents the amino acid residue alanine (A) or G (glycine); and X 25 represents the amino acid residue glycine (G) or arginine (R).

[0545] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence consisting of SEQ ID NO: 97. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising a sequence consisting of SEQ ID NO: 97.

[0546] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129.

[0547] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 consisting of a sequence consisting of SEQ ID NO: 100. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 comprising a sequence consisting of SEQ ID NO: 100.

[0548] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124.

[0549] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126.

[0550] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 consisting of a sequence consisting of SEQ ID NO: 96. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 comprising a sequence consisting of SEQ ID NO: 96.

[0551] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence consisting of SEQ ID NO: 97, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence consisting of SEQ ID NO: 97, except for one, two, three, or four amino acid substitutions.

[0552] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129, except for one, two, three, or four amino acid substitutions.

[0553] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 consisting of a sequence consisting of SEQ ID NO: 100, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 consisting of a sequence consisting of SEQ ID NO: 100, except for one, two, three, or four amino acid substitutions.

[0554] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124, except for one, two, three, or four amino acid substitutions.

[0555] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126, except for one, two, three, or four amino acid substitutions.

[0556] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 consisting of a sequence consisting of SEQ ID NO: 96, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 consisting of a sequence consisting of SEQ ID NO: 96, except for one, two, three, or four amino acid substitutions.

[0557] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence consisting of SEQ ID NO: 97; a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129; and a VL-CDR3 consisting of a sequence consisting of SEQ ID NO: 100. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising a sequence consisting of SEQ ID NO: 97; a VL-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129; and a VL-CDR3 consisting of a sequence consisting of SEQ ID NO: 100.

[0558] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124; a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126; and a VH-CDR3 consisting of a sequence consisting of SEQ ID NO: 96. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124; a VH-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126; and a VH-CDR3 consisting of a sequence consisting of SEQ ID NO: 96.

[0559] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of a sequence consisting of SEQ ID NO: 97, except for one, two, three or four amino acid substitutions; a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128 and 129, except for one, two, three or four amino acid substitutions; and a VL-CDR3 consisting of a sequence consisting of SEQ ID NO: 100, except for one, two, three or four amino acid substitutions.

[0560] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising the sequence consisting of SEQ ID NO: 97, except for one, two, three or four amino acid substitutions; a VL-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128 and 129, except for one, two, three or four amino acid substitutions; and a VL-CDR3 comprising the sequence consisting of SEQ ID NO: 100, except for one, two, three or four amino acid substitutions.

[0561] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124, except for one, two, three, or four amino acid substitutions; a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126, except for one, two, three, or four amino acid substitutions; and a VH-CDR3 consisting of a sequence consisting of SEQ ID NO: 96, except for one, two, three, or four amino acid substitutions.

[0562] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124, except at one, two, three, or four amino acid substitutions; a VH-CDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126, except at one, two, three, or four amino acid substitutions; and a VH-CDR3 comprising a sequence consisting of SEQ ID NO: 96, except at one, two, three, or four amino acid substitutions.

[0563] In certain aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises modifications to CDR1 and / or CDR2 and / or CDR3 of the heavy chain and / or light chain, and further comprises modifications to FW1 and / or FW2 and / or FW3 and / or FW4 of the heavy chain and / or light chain.

[0564] In some aspects, FW9 comprises SEQ ID NO: 90 or 91, FW 10 Including SEQ ID NO: 92, FW 11 including SEQ ID NO: 93, 94, or 122, FW 12 including SEQ ID NO: 30, FW13 Including SEQ ID NO: 31, FW 14 including SEQ ID NO: 32, FW 15 including SEQ ID NO: 33, and FW 16 Including SEQ ID NO:89.

[0565] In some aspects, FW9 comprises SEQ ID NO: 90 or 91 except for one, two, three, or four amino acid substitutions, FW 10 comprising SEQ ID NO: 92, FW except for one, two, three or four amino acid substitutions 11 comprising SEQ ID NO: 93, 94, or 122 except for one, two, three, or four amino acid substitutions, FW 12 comprising SEQ ID NO: 30, FW except for one, two, three or four amino acid substitutions 13 SEQ ID NO: 31, FW except for one, two, three or four amino acid substitutions 14 comprising SEQ ID NO: 32, FW except for one, two, three or four amino acid substitutions 15 comprising SEQ ID NO: 33 except for one, two, three or four amino acid substitutions, and FW 16 Including SEQ ID NO:89.

[0566] In certain aspects, the anti-CD733 antibody or antigen-binding fragment thereof comprises a VL and a VH comprising VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequences that are identical or identical except for one, two, three or four amino acid substitutions in one or more CDRs, wherein such VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, respectively, are:

[0567] SEQ ID NOs: 97, 98, 100, 35, 37, and 96; or

[0568] SEQ ID NOs: 97, 99, 100, 35, 95, and 96; or

[0569] SEQ ID NOs: 97, 98, 100, 35, 37, and 96; or

[0570] SEQ ID NOs: 97, 99, 100, 123, 37, and 96; or

[0571] SEQ ID NOs: 97, 99, 100, 124, 37, and 96; or

[0572] SEQ ID NOs: 97, 99, 100, 35, 125, and 96; or

[0573] SEQ ID NOs: 97, 99, 100, 35, 126, and 96; or

[0574] SEQ ID NOs: 97, 99, 100, 35, 95, and 96; or

[0575] SEQ ID NOs: 97, 127, 100, 35, 95, and 96; or

[0576] SEQ ID NOs: 97, 128, 100, 35, 95, and 96; or

[0577] SEQ ID NOs: 97, 129, 100, 35, 95, and 96; or

[0578] SEQ ID NOs: 97, 99, 100, 35, 95 and 96.

[0579] In certain aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises an antibody VL and an antibody VH, wherein the VL comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 88, 112, 118, 119, 120, and 121.

[0580] In other aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises an antibody VL and an antibody VH, wherein the VH comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 87, 111, 113, 114, 115, 116, and 117.

[0581] In other aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL comprising a sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 88, 112, 118, 119, 120, and 121; and further comprises a VH comprising a sequence at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 87, 111, 113, 114, 115, 116, and 117.

[0582] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL comprising the sequence of SEQ ID NO: 88 and a VH comprising the sequence of SEQ ID NO: 87. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL consisting of the sequence of SEQ ID NO: 87 and a VH consisting of the sequence of SEQ ID NO: 87.

[0583] The "clone 2C5 antibody" is an IgG1 comprising two CD730002-derived light chains (VL) of SEQ ID NO: 88 (comprising three CDRs, CDR1, CDR2 and CDR3, having the sequences of SEQ ID NOs: 97, 99 and 100, respectively) and a CD7300002-derived heavy chain (VH) of SEQ ID NO: 87 (comprising three CDRs, CDR1, CDR2 and CDR3, having the sequences of SEQ ID NOs: 35, 95 and 96, respectively).

[0584] In certain aspects, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein bind CD73 with substantially the same or better affinity than the 2C5 antibody comprising the 2C5 heavy chain VH of SEQ ID NO: 87 and the 2C5 light chain VL of SEQ ID NO: 88.

[0585] (iii) Anti-CD73 antibodies having a parent antibody other than CD730002 or CD730010

[0586] In other aspects, the parent antibody of the anti-CD73 antibodies or antigen-binding fragments disclosed herein is CD730004 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 104 and a VH of SEQ ID NO: 103), CD730008 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 106 and a VH of SEQ ID NO: 107), CD7300011 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 5 and a VH of SEQ ID NO: 6), CD730021 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 7 and a VH of SEQ ID NO: 8), CD730042 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 9 and a VH of SEQ ID NO: 10), CD730046 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 11 and a VH of SEQ ID NO: 12), No. 100047 (i.e., an anti-CD73 antibody comprising the VL of SEQ ID NO: 13 and the VH of SEQ ID NO: 14), CD730068 (i.e., an anti-CD73 antibody comprising the VL of SEQ ID NO: 108 and the VH of SEQ ID NO: 107), or CD730069 (i.e., an anti-CD73 antibody comprising the VL of SEQ ID NO: 110 and the VH of SEQ ID NO: 109). Modifications to these parent antibodies, such as CD730004, can include mutations in the CDR regions and / or FW regions compared to the parent antibody.

[0587] In certain aspects, the anti-CD73 antibody comprises modifications to the CDR1 and / or CDR2 and / or CDR3 of the light chain of CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0588] In certain aspects, the anti-CD73 antibody comprises modifications to the CDR1 and / or CDR2 and / or CDR3 of the heavy chain of CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0589] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0590] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0591] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions.

[0592] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions. In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions.

[0593] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069; 021, CD730042, CD730046, CD730047, CD730068, or CD730069; and a VL-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0594] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069; 021, CD730042, CD730046, CD730047, CD730068, or CD730069; and a VH-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0595] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions; 2. VL-CDR2 from CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions; and VL-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions.

[0596] In some aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions; 2. VH-CDR2 from CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions; and VH-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions.

[0597] In certain aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises modifications to CDR1 and / or CDR2 and / or CDR3 of the heavy and / or light chain from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, and further comprises modifications to FW1 and / or FW2 and / or FW3 and / or FW4 of the heavy and / or light chain from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0598] In certain aspects, the anti-CD733 antibody or antigen-binding fragment thereof comprises a VL and a VH comprising VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences that are identical or identical except for one, two, three, or four amino acid substitutions in one or more CDRs, wherein such VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 are from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0599] In certain aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises an antibody VL and an antibody VH, wherein the VL comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from a VL sequence from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0600] In other aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises an antibody VL and an antibody VH, wherein the VH comprises an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from a VH sequence from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0601] In other aspects, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL that comprises at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from a Vl sequence from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. 00% identical sequence and further comprises a VH comprising a sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from a VH sequence from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0602] In certain aspects, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein bind to CD73 with substantially the same or better affinity than the CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069 antibodies.

[0603] (iv) Mixed and matched anti-CD73 antibodies

[0604] The VH and VL sequences from the anti-CD73 binding molecules disclosed herein (e.g., CD730002, CD730004, CD730008, CD730010, CD730011, CD73021, CD730042, CD730046, CD730047, CD730068, or CD730069) or variants of such sequences (e.g., clone 10GL9, clone 10P32E, clone 10C1, clone 10C2, clone 10D3, clone 10G10, clone 10HPT, clone 10GRVE, clone 10combo1, clone 10combO2, clone 10combo3, clone 10combo5, or clone combo6) can be “mixed and matched” to create additional anti-CD73 binding molecules.

[0605] In certain aspects, the VH sequences of the 10.3 antibody and the 2C5 antibody are mixed and matched. In another aspect, the VL sequences of the 10.03 antibody and the 2C5 antibody are mixed and matched. Additionally or alternatively, the VL and / or VH sequences of the Clone 10 (CD730010) variants disclosed herein can be mixed and matched. Additionally or alternatively, the VL and / or VH sequences of the Clone 2 (CD730002) variants disclosed herein can be mixed and matched. Additionally or alternatively, the VL and / or VH sequences of the Clone 10 (CD730010) and Clone 2 (CD730002) variants disclosed herein can be mixed and matched.

[0606] In some aspects, VL and / or VH mixing and matching can occur between sequences derived from antibodies grouped in the same epitope bin (see Example 2). As used herein, the term "epitope bin" refers to a grouping of antibodies or antigen-binding fragments thereof that bind to the same epitope or overlapping epitopes, or compete with each other for binding to the same epitope or overlapping epitopes. For example, sequences from CD730003, CD730010, CD730021, CD730042, CD730046, and CD730047, all of which belong to "epitope bin B," can be mixed and matched. In other aspects, VL and / or VH mixing and matching can occur between sequences derived from anti-CD73 antibodies grouped in different epitope bins. Thus, sequences from antibodies belonging to "Epitope Box B" can be mixed and matched with sequences from anti-CD73 antibodies in "Epitope Box A" (CD730002, CD730004, CD730008, and CD730011) or "Epitope Box C" (CD730068 and CD730069).

[0607] (v) Mutant anti-CD73 antibodies

[0608] In certain aspects, the anti-CD73 antibodies disclosed herein (e.g., MEDI9447, clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments thereof include mutations that improve binding to human FcRn and improve the half-life of the anti-CD73 antibodies or antigen-binding fragments thereof. In some aspects, such mutations are methionine (M) to tyrosine (Y) mutations at position 252, serine (S) to threonine (T) mutations at position 254, and threonine (T) to glutamate (E) mutations at position 256 introduced into the constant domain of IgG1, which are numbered according to the EU index in Kabat (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, D.C. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. Mutant IgGs of this type (referred to as "YTE mutants") have been shown to exhibit approximately a four-fold increase in half-life compared to the wild-type form of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006). In some aspects, an anti-CD73 antibody or antigen-binding fragment thereof comprising an IgG constant domain comprises one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat, wherein such mutations increase the serum half-life of the anti-CD73 antibody or antigen-binding fragment thereof.

[0609] In some aspects, the YTE mutant further includes the replacement (numbered according to the EU index in Kabat) of the position 434 of the amino acid pair IgG constant domain selected from the group consisting of tryptophan (W), methionine (M), tyrosine (Y) and serine (S). In other aspects, the YTE mutant further includes the replacement (numbered according to the EU index in Kabat) of the position 434 of the amino acid pair IgG constant domain selected from the group consisting of tryptophan (W), methionine (M), tyrosine (Y) and serine (S); and the replacement (numbered according to the EU index in Kabat) of the amino acid pair IgG constant domain selected from the group consisting of threonine (T), leucine (L), phenylalanine (F) and serine (S).

[0610] Still in other aspects, the YTE mutant further comprises a substitution of tyrosine (Y) at position 434 of the IgG constant domain (numbered according to the EU index in Kabat), and a substitution of leucine (L) at position 257 of the IgG constant domain (numbered according to the EU index in Kabat). In some aspects, the YTE mutant further comprises a substitution of serine (S) at position 434 of the IgG constant domain (numbered according to the EU index in Kabat), and a substitution of leucine (L) at position 428 of the IgG constant domain (numbered according to the EU index in Kabat).

[0611] In certain aspects, an anti-CD73 antibody (e.g., MEDI9447, a clone 10.3 antibody, or a clone 2C5 antibody) or antigen-binding fragment thereof disclosed herein comprises an IgG1 constant domain comprising a methionine (M) to tyrosine (Y) mutation at position 252, a serine (S) to threonine (T) mutation at position 254, and a threonine (T) to glutamic acid (E) mutation at position 256 of the IgG1 constant domain (numbering according to the EU index as in Kabat).

[0612] In certain aspects, an anti-CD73 antibody (e.g., MEDI9447, a clone 10.3 antibody, or a clone 2C5 antibody) or antigen-binding fragment thereof disclosed herein comprises at least one IgG constant domain amino acid substitution selected from the group consisting of:

[0613] (a) the amino acid at position 252 is substituted with tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T);

[0614] (b) the amino acid at position 254 is substituted with threonine (T);

[0615] (c) the amino acid at position 256 is substituted with serine (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), or threonine (T);

[0616] (d) the amino acid at position 257 is substituted with leucine (L);

[0617] (e) the amino acid at position 309 is substituted with proline (P);

[0618] (f) the amino acid at position 311 is substituted with serine (S);

[0619] (g) the amino acid at position 428 is substituted with threonine (T), leucine (L), phenylalanine (F), or serine (S);

[0620] (h) the amino acid at position 433 is substituted with arginine (R), serine (S), isoleucine (I), proline (P), or glutamine (Q);

[0621] (i) the amino acid at position 434 is substituted with tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F), or tyrosine; and

[0622] (j) a combination of two or more of the above substitutions,

[0623] wherein positions are numbered according to the EU index as in Kabat, and wherein the modified IgG has an increased serum half-life compared to the serum half-life of an IgG having a wild-type IgG constant domain.

[0624] In some aspects, the VH and / or VL amino acid sequences of the anti-CD73 antibodies disclosed herein (e.g., MEDI9447, a clone 10.3 antibody or a clone 2C5 antibody) or antigen-binding fragments thereof may be 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to the VH and VL sequences listed above and include 1, 2, 3, 4, 5 or more conservative substitutions. CD73 antibodies having VH and VL regions having high (i.e., 80% or greater) sequence similarity or sequence identity to the VH region of SEQ ID NO: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 111, 113, 114, 115, 116, or 117, and / or the VL region of SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 86, 88, 112, 118, 119, 120, or 121, respectively, can be obtained by encoding a VH and VL region of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121 and then testing the encoded altered antibody for retention of function using the functional assays described herein.

[0625] In some aspects, the Fc domain of an anti-CD73 antibody disclosed herein or the Fc domain of a fusion protein comprising a CD73 binding fragment of an antibody disclosed herein has reduced binding to an Fc receptor to reduce cytotoxicity, for example, by ADCC. In some aspects, the Fc domain of the antibody or Fc fusion protein has increased binding to an Fc receptor to increase cytotoxicity, for example, via ADCC. In some aspects, the Fc domain of the antibody or Fc fusion protein comprises a non-naturally occurring ADCC-lowering amino acid residue at one or more positions selected from the group consisting of: 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 301, 302, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315 , 263, 264, 265, 266, 267, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440, and 443. Many specific mutations that can reduce the ADCC activity of an antibody are known in the art and include, for example, 234F, 235E, 235F, 235Q (or 235Y), 239A, 332Q, 331S, and combinations thereof. For example, see the mutations described in U.S. Patent Nos. 5,624,821, 5,648,260, 7,597,889, 8,961,967, 7,371,826, 7,785,791, 7,790,858; U.S. Patent Publication Nos. 20140378663, 20130071390, 20110212087, 20150118227, 20060194290, 20060194291, 20080274105, 20080274506, US 20130089541, and US 20130108623, which are incorporated herein by reference in their entireties. Antibodies with reduced ADCC effector function also include those with one or more substitutions in the Fc region residues 238, 265, 269, 270, 297, 327 and 329 (see, e.g., U.S. Patent No. 6,737,056). Such Fc mutants also include Fc mutants with substitutions at two or more positions in amino acid positions 265, 269, 270, 297 and 327, including Fc mutants with substitutions of residues 265 and 297 to alanine (see, e.g., U.S. Patent No. 7,332,581). Optionally, mutations that reduce both ADCC and CDC can be incorporated.In some aspects, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein that include mutations that reduce or eliminate ADCC and / or CDC can be used to generate antibody drug conjugates (ADCs).

[0626] In one aspect, the disclosure provides an anti-CD73 antibody, wherein the antibody is an IgG1, IgG2 or IgG3 and comprises at least one modification at one or more positions selected from the group consisting of 234, 235 and 331, as numbered by the EU index as set forth in Kabat. In yet another specific aspect, the Fc region is an IgG1, IgG2 or IgG3 Fc region and the non-naturally occurring amino acids are selected from the group consisting of 234F, 235E, 235F, 235Q (or 235Y), 239A, 332Q, 331S, 332Q, as numbered by the EU index as set forth in Kabat.

[0627] In another aspect, the disclosure provides an anti-CD73 antibody, wherein the antibody is an IgG4 and comprises at least one modification at one or more positions selected from the group consisting of 228 and 235, as numbered by the EU index as set forth in Kabat. In yet another specific aspect, the Fc region is an IgG4 Fc region and the non-naturally occurring amino acids are selected from the group consisting of 228P, 235E, and 235Y, as numbered by the EU index as set forth in Kabat. In a specific aspect, the disclosure provides an anti-CD73 antibody, wherein the antibody is an IgG1, IgG2, or IgG3 and comprises modifications at the following positions: (i) 234F, 235E, and 331S; (ii) 234F, 235F, and 331S; (iii) 234F, 235Q, and 322Q. In another specific aspect, the present disclosure provides an anti-CD73 antibody, wherein the antibody is IgG4 and comprises modifications 228P and 235E.

[0628] III. Epitope-competitive CD73-binding molecules

[0629] In another aspect, the disclosure provides CD73 binding molecules that bind to the same epitope as a different anti-CD73 antibody described herein, e.g., molecules that bind to the same epitope as MEDI9447, the clone 10.3 antibody, or the same epitope as the clone 2C5 antibody.

[0630] Such antibodies can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with the anti-CD73 antibodies disclosed herein, such as the CD730010 antibody, CD730002 antibody, CD730004 antibody, and antigen-binding fragments thereof, in a standard CD73 binding assay (e.g., a flow cytometry assay, surface plasmon resonance, or a solution assay).

[0631] Thus, in one aspect, the present disclosure provides anti-CD73 antibodies and antigen-binding fragments thereof, e.g., human monoclonal antibodies, that compete with another anti-CD73 antibody or antigen-binding fragment thereof, such as CD730010 antibody, CD730002 antibody, CD730004 antibody, variants thereof (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody), or antigen-binding fragment thereof for binding to CD73. The ability of a test antibody to inhibit binding of, e.g., CD730010 antibody (or clone 10.3 antibody, or antigen-binding fragment thereof) or CD730002 antibody (or clone 2C5 antibody, or antigen-binding fragment thereof) demonstrates that the test antibody can compete with that antibody for binding to CD73; according to non-limiting theory, such an antibody may bind to an epitope on CD73 that is the same as or related (e.g., structurally similar or spatially proximal) to the epitope bound by the anti-CD73 antibody or antigen-binding fragment thereof with which it competes. In one aspect, the anti-CD73 antibody or antigen-binding fragment thereof that binds to the same epitope on CD73 as, e.g., the CD730010 antibody (or clone 10.3 antibody or antigen-binding fragment thereof) or the CD730002 antibody (or clone 2C5 antibody or antigen-binding fragment thereof) binds is a human monoclonal antibody.

[0632] As described here, the epitope of MEDI9447, a monoclonal antibody that directly inhibits the enzymatic activity of CD73, was identified to elucidate its mechanism of action. This epitope is located within the apical surface of the N-terminal domain of CD73, a region distal to substrate binding and active site residues. Structural and mechanistic studies reveal that MEDI9447 antagonizes CD73 through a dual mechanism that prevents CD73 from adopting a catalytically active conformation. These results provide the first report of a finely mapped epitope that can be targeted for selective, potent, and noncompetitive inhibition of CD73 as a means of modulating adenosine signaling in the tumor microenvironment.

[0633] Using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) and mutagenesis strategies, we defined the epitope of MEDI9447 and examined the potential impact of antibody binding on the overall CD73 structure. The antibody binds to a site in the N-terminal domain of CD73 that enables non-competitive inhibition of AMP hydrolysis. In various aspects, the epitope includes one or more CD73 amino acid residues corresponding to V144, K180, and N185. In various aspects, the epitope further includes one or more CD73 amino acid residues corresponding to Y135, K136, and N187 of CD73. Remarkably, the epitope is positioned so that antibody binding interferes with the conversion of CD73 from an open conformer to a catalytically active closed conformer. Furthermore, our studies show that MEDI9447 can inhibit both anchored and soluble CD73 through a dual mechanism of inhibition mediated by the potency of the antibody's interaction with CD73.

[0634] IV. Functional Characterization of Anti-CD73 Antibodies

[0635] The affinity or avidity of an antibody for an antigen can be determined using any suitable method known in the art (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., BIACORE TM The affinity of an antibody-antigen interaction can be determined experimentally using direct binding assays as well as competitive binding assay formats. (See, e.g., Berzofsky et al., "Antibody-Antigen Interactions," in Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, Immunology, WH Freeman and Company: New York, NY (1992); and the methods described herein.) The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other antigen binding parameters (e.g., K) may vary. D or Kd, K on , K off ) are measured with standardized solutions of antibody and antigen and a standardized buffer (as known in the art and such as the buffer described herein).

[0636] It is also known in the art to use surface plasmon resonance analysis (e.g., BIACORE TM) can vary depending on which reactant is bound to the chip. In this regard, affinity can be measured using a format in which a targeting antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) is immobilized to the chip (referred to as an "IgG down" format) or using a format in which a target protein (e.g., CD73) is immobilized to the chip (referred to as, for example, a "CD73 down" format).

[0637] In one aspect of the present disclosure, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment thereof is expressed in less than 10 -6 M, or less than 10 -7 M, or less than 10 -8 M, or less than 10 -9 M, or less than 10 -10 M, or less than 10 -11 M, or less than 10 -12 M, or less than 10 -13 The dissociation constant or k of M d (k off / k on ) specifically binds to CD73 and / or its antigenic fragment.

[0638] In another aspect, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof is expressed in an amount of less than 1×10 -3 s -1 , or less than 2×10 -3 s -1 K off In other aspects, the anti-CD73 antibody or antigen-binding fragment thereof binds to CD73 and / or an antigenic fragment thereof at a concentration of less than 10 -3 s -1 , less than 5×10 -3 s -1 , less than 10 -4 s -1 , less than 5×10 -4 s -1 , less than 10 -5 s -1 , less than 5×10 -5 s -1 , less than 10 -6 s -1 , less than 5×10 -6 s -1 Less than less than 5×10 -7 s -1 , less than 10 -8 s -1 , less than 5×10 -8 s-1 , less than 10 -9 s -1 , less than 5×10 -9 s -1 , or less than 10 -10 s -1 K off Binds to CD73 and its antigenic fragments.

[0639] In another aspect, the anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) or antigen-binding fragment thereof is expressed in an amount of at least 10 5 M -1 s -1 , at least 5×10 5 M -1 s -1 , at least 10 6 M -1 s -1 , at least 5×10 6 M -1 s -1 , at least 10 7 M -1 s -1 , at least 5×10 7 M -1 s -1 , or at least 10 8 M -1 s -1 , or at least 10 9 M -1 s -1 The binding rate constant or k on The binding rate to CD73 and / or its antigenic fragments.

[0640] In some aspects, the anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) or antigen-binding fragment thereof has a K of at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 110 pM, at least about 120 pM, at least about 130 pM, at least about 140 pM, at least about 150 pM, at least about 160 pM, or at least about 170 pM (as measured by flow cytometry). D Binds to CD73 on the surface of MB-MDA-231 cells. In a specific aspect, the anti-CD73 antibody is a clone 10.3 antibody, and it binds to CD73 on the surface of MB-MDA-231 cells with a K of about 150 pM (as measured by flow cytometry). D Binds to CD73 on the surface of MB-MDA-231 cells. In another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody, and it binds to CD73 on the surface of MB-MDA-231 cells with a K of about 80 pM (as measured by flow cytometry). DBinds to CD73 on the surface of MB-MDA-231 cells.

[0641] In some aspects, the anti-CD73 antibody (e.g., MEDI9447, a clone 10.3 antibody, or a clone 2C5 antibody) or antigen-binding fragment thereof has a K of at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 120 pM, or at least about 130 pM (as measured by flow cytometry). D Binds to CD73 on the surface of murine 3T1 cells. In a specific aspect, the anti-CD73 antibody is a clone 10.3 antibody, and it binds to CD73 on the surface of murine 3T1 cells with a K of about 110 pM (as measured by flow cytometry). D In another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody and binds to CD73 on the surface of murine 3T1 cells with a K of about 55 pM (as measured by flow cytometry). D Binds to CD73 on the surface of murine 3T1 cells.

[0642] In some aspects, the anti-CD73 antibody (e.g., MEDI9447, a clone 10.3 antibody, or a clone 2C5 antibody) or antigen-binding fragment thereof has a K of at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, or at least about 100 pM (as measured by flow cytometry). D Binds to CD73 on the surface of cynomolgus monkey MK-1 cells. In a specific aspect, the anti-CD73 antibody is a clone 10.3 antibody, and it binds to CD73 on the surface of cynomolgus monkey MK-1 cells with a K of about 80 pM (as measured by flow cytometry). D In another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody and binds to CD73 on the surface of cynomolgus monkey MK-1 cells with a K of about 60 pM (as measured by flow cytometry). D Binds to CD73 on the surface of cynomolgus macaque MK-1 cells.

[0643] In some aspects, the anti-CD73 antibody (e.g., MEDI9447, a clone 10.3 antibody, or a clone 2C5 antibody) or antigen-binding fragment thereof is expressed at a concentration of at least about 3 pM, at least about 4 pM, at least about 5 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, or at least about 10 pM (e.g., by surface plasmon resonance). K D In one specific aspect, the anti-CD73 antibody is a clone 10.3 antibody and binds to human CD73 at about 4 pM (as determined by surface plasmon resonance). K DIn another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody and binds to human CD73 at about 9 pM (as determined by surface plasmon resonance). K D Binds to human CD73.

[0644] In some aspects, an anti-CD73 antibody disclosed herein (e.g., MEDI9447, a clone 10.3 antibody, or a clone 2C5 antibody) or an antigen-binding fragment thereof is expressed at a concentration of at least about 1 pM, at least about 2 pM, at least about 3 pM, at least about 4 pM, at least about 5 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, at least about 10 pM, at least about 11 pM, at least about 12 pM, at least about 13 pM, at least about 14 pM, at least about 15 pM, at least about 16 pM, at least about 17 pM, at least about 18 pM, at least about 19 pM, at least about 20 pM, at least about 21 pM, at least about 22 pM, at least about 23 pM, at least about 24 pM, or at least about 25 pM (e.g., by surface plasmon resonance spectroscopy). K D Binds to murine CD73. In a specific aspect, the anti-CD73 antibody is a clone 10.3 antibody, and it binds to murine CD73 at about 1 pM (as measured by surface plasmon resonance K D In another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody and binds to murine CD73 at about 22 pM (as measured by surface plasmon resonance). K D Binds to murine CD73.

[0645] In some aspects, an anti-CD73 antibody (e.g., MEDI9447, a clone 10.3 antibody, or a clone 2C5 antibody) or an antigen-binding fragment thereof is expressed at a concentration of at least about 3 pM, at least about 4 pM, at least about 5 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, or at least about 10 pM (e.g., by surface plasmon resonance). K D Binds to cynomolgus monkey CD73. In a specific aspect, the anti-CD73 antibody is a clone 10.3 antibody, and it binds to cynomolgus monkey CD73 with a K of about 7 pM (as measured by SPR (Proteon)). D In another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody and binds to cynomolgus monkey CD73 at about 9 pM (as measured by surface plasmon resonance). K D Binds to cynomolgus monkey CD73.

[0646] In some aspects, the anti-CD73 antibody (e.g., MEDI9447, a clone 10.3 antibody, or a clone 2C5 antibody) or antigen-binding fragment thereof has a K of at least about 40 pM, at least about 50 pM, at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, or at least about 110 pM (as measured by solution binding). D Binds to human CD73. In a specific aspect, the anti-CD73 antibody is a clone 10.3 antibody, and it binds to human CD73 with a K of about 80 pM (as measured by solution binding). D In another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody and binds to human CD73 with a K of about 80 pM (as measured by solution binding). D Binds to human CD73.

[0647] In some aspects, the anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) or antigen-binding fragment thereof has a K of at least about 100 pM, at least about 200 pM, at least about 300 pM, at least about 400 pM, at least about 500 pM, at least about 600 pM, at least about 700 pM, at least about 800 pM, at least about 900 pM, at least about 1000 pM, at least about 1100 pM, at least about 1200 pM, at least about 1300 pM, at least about 1400 pM, at least about 1500 pM, at least about 1600 pM, at least about, or at least about 1700 pM (as measured by solution binding). D Binds to murine CD73. In a specific aspect, the anti-CD73 antibody is a clone 10.3 antibody, and it binds to murine CD73 with a K of about 130 pM (as measured by solution binding). D In another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody and binds to murine CD73 with a K of about 1500 pM (as measured by solution binding). D Binds to murine CD73.

[0648] In some aspects, the anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) or antigen-binding fragment thereof has a K of at least about 60 pM, at least about 70 pM, at least about 80 pM, at least about 90 pM, at least about 100 pM, at least about 110 pM, or at least about 120 pM (as measured by solution binding). D In one specific aspect, the anti-CD73 antibody is a clone 10.3 antibody and binds to cynomolgus monkey CD73 with a K of about 90 pM (as measured by solution binding). D In another specific aspect, the anti-CD73 antibody is a clone 2C5 antibody and binds to cynomolgus monkey CD73 with a K of about 100 pM (as measured by solution binding).D Binds to cynomolgus monkey CD73. In a specific aspect, MEDI9447 binds to cynomolgus monkey CD73 at a concentration of about 1x10 -12 , 5x10 -12 , 10x10 -12 , 100x10 -12 , or 150x10 -12 K D Binds to CD73.

[0649] In some aspects, the CD73 binding molecules disclosed herein, such as anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibodies, or clone 2C5 antibodies), or antigen-binding fragments thereof, can relieve AMP-mediated inhibition of T cell division. In other aspects, the CD73 binding molecules disclosed herein, such as anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibodies, or clone 2C5 antibodies), or antigen-binding fragments thereof, can rescue ATP-induced inhibition of T cell division. 调节 T 效应 inhibition.

[0650] In some aspects, the CD73 binding molecules disclosed herein, such as anti-CD73 antibodies or antigen-binding fragments thereof (e.g., clone 10.3 antibodies or clone 2C5 antibodies), can significantly inhibit syngeneic tumor growth. In one aspect, the tumor is a non-small cell lung tumor, an ovarian tumor, a breast tumor, a head and neck tumor, a pancreatic tumor, a colorectal cancer tumor, a melanoma tumor, or a lymphoma tumor. In one aspect, the tumor is a CT26 mouse syngeneic CRC tumor, a B16F10 melanoma tumor, an EG7-OVA lymphoma tumor, or a LL2 (Lewis lung) tumor. In some aspects, the CD73 binding molecules disclosed herein, such as anti-CD73 antibodies or antigen-binding fragments thereof (e.g., clone 10.3 antibodies or clone 2C5 antibodies), can significantly inhibit tumor growth, wherein the tumor is unresponsive to therapy with anti-PD-1 and / or anti-PD-L1 and / or anti-PD-L2 and / or anti-CTLA-4 antibodies. In some aspects, the CD73-binding molecules disclosed herein, such as anti-CD73 antibodies or antigen-binding fragments thereof (e.g., a clone 10.3 antibody or a clone 2C5 antibody), can significantly inhibit tumor growth when administered at a concentration of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg.PD1.

[0651] In some aspects, the CD73 binding molecules disclosed herein, such as anti-CD73 antibodies or antigen-binding fragments thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), can be internalized after binding to a cell. In some aspects, the CD73 binding molecule is an antibody drug conjugate (ADC).

[0652] V. Preparation of Anti-CD73 Antibodies and Antigen-Binding Fragments

[0653] Monoclonal anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody), and antigen-binding fragments thereof, can be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma method, mice, hamsters, or other suitable host animals are immunized as described above to elicit lymphocytes that produce antibodies that will specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specific for the selected antigen (by immunoprecipitation, immunoblotting, or by in vitro binding assays (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA))) can then be grown in vitro using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in animals. Monoclonal antibodies can then be purified from the culture medium or ascites as described above for polyclonal antibodies.

[0654] Alternatively, anti-CD73 monoclonal antibodies (e.g., MEDI9447, clone 10.3 antibody or clone 2C5 antibody) and antigen-binding fragments thereof can also be prepared using recombinant DNA methods (as described, for example, in U.S. Patent No. 4,816,567). Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, such as by RT-PCR, using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody, and their sequences are determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into appropriate expression vectors, which, when transfected into host cells that do not produce immunoglobulins, such as Escherichia coli cells, anthropoid COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, produce monoclonal antibodies by these host cells. Also, recombinant anti-CD73 monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing the CDRs of the desired species as described (McCafferty et al., 1990, Nature 348:552-554; Clarkson et al., 1991, Nature 352:624-628; and Marks et al., 1991, J. Mol. Biol. 222:581-597).

[0655] One or more polynucleotides encoding anti-CD73 antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies) or antigen-binding fragments thereof can be further modified in a variety of different ways using recombinant DNA technology to generate antibodies for selection. In some aspects, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be replaced by (1) those of a human antibody, for example, to generate a chimeric antibody or by (2) a non-immunoglobulin polypeptide, for example, to generate a fusion antibody. In some aspects, these constant regions are truncated or removed to generate the desired antibody fragment of the monoclonal antibody. Site-directed mutagenesis or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0656] In certain aspects, the anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment is a human antibody or its antigen-binding fragment. Different techniques known in the art can be used to directly prepare human antibodies. Immortalized human B lymphocytes isolated from immune individuals that produce antibodies to the target antigen or immunized in vitro can be produced (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol. 147 (1): 86-95; and U.S. Pat. No. 5,750,373).

[0657] Additionally, the anti-CD73 human antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) or antigen-binding fragment thereof can be selected from a phage library, wherein the phage library expresses human antibodies, such as those described in, e.g., Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Natl. Acad. Sci. USA 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381, and Marks et al., 1991, J. Mol. Biol. 222:581). Techniques for generating and using antibody phage libraries are also described in U.S. Patent Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2007, J. Mol. Biol., doi: 10.1016 / j.jmb.2007.12.018 (each of which is incorporated by reference in its entirety).

[0658] Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio / Technology 10:779-783, incorporated by reference in its entirety) are known in the art and can be used to generate high-affinity human antibodies or antigen-binding fragments thereof.

[0659] In some aspects, the anti-CD73 monoclonal antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) can be a humanized antibody. Methods for engineering, humanizing, or resurfacing non-human or human antibodies can also be used and are well known in the art. Humanized, resurfaced, or similarly engineered antibodies can have one or more amino acid residues from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are replaced by residues often referred to as "import" residues, which are typically taken from "import" variable domains, constant domains, or other domains of known human sequences. Such import sequences can be used to reduce immunogenicity or reduce, enhance, or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristics as known in the art. In general, CDR residues are directly and most substantially involved in influencing CD73 binding. Thus, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions can be replaced by human or other amino acids.

[0660] The antibodies may also be optionally humanized, resurfaced, engineered, or human antibody engineered, retaining high affinity for the CD73 antigen and other favorable biological properties. To achieve this goal, humanized (or human) or engineered anti-CD73 antibodies and resurfaced antibodies may optionally be prepared by analyzing the parental sequences and various conceptual humanized and engineered products using three-dimensional models of the parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those of ordinary skill in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen, such as CD73. In this way, framework (FW) residues can be selected and combined from the consistent and input sequences so that the desired antibody characteristics, such as increased affinity for one or more target antigens, are achieved.

[0661] Humanization, resurfacing, or engineering of an anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) or an antigen-binding fragment thereof can be performed using any known method, such as, but not limited to, those described in Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988), Sims et al., J. Immunol 151:2296 (1993), Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. USA 237:1547 (1989), and others. USA) 89: 4285 (1992); Presta et al., J. Immunol 151: 2623 (1993), U.S. Patent Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714, 352; 5,9,55,358; 6,204,023; 6,180,370; 6,331,431; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567; 5,969,108; 7,635,666; 7,723,270; 7,557,189; 7,538,195; and 7,342,110; International Application No. PCT / US 98 / 16280; PCT / US 91 / 05939; PCT / US 94 / 01234; PCT / GB 92 / 01755; International Patent Application Publication Nos. WO 90 / 14443; WO 90 / 14424; WO 90 / 14430; and European Patent Publication No. EP 229246; each of which is incorporated herein by reference in its entirety, including the references cited therein.

[0662] Anti-CD73 humanized antibodies and antigen-binding fragments thereof can also be made in transgenic mice containing human immunoglobulin loci that are capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0663] In certain aspects, anti-CD73 antibody fragments (e.g., fragments from clone 10.3 antibodies or from clone 2C5 antibodies) are provided. Various techniques for producing antibody fragments are known. Traditionally, these fragments are derived by proteolysis of intact antibodies (e.g., Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24: 107-117; Brennan et al., 1985, Science 229: 81). In certain aspects, anti-CD73 antibody fragments are recombinantly produced. All Fab, Fv, and scFv antibody fragments can be expressed in E. coli or other host cells and secreted therefrom, thereby allowing the production of large quantities of these fragments. Such anti-CD73 antibody fragments can also be isolated from the antibody phage libraries discussed above. Anti-CD73 antibody fragments can also be linear antibodies as described in U.S. Patent No. 5,641,870. Other techniques for producing antibody fragments (e.g., chemical synthesis) will be clear to skilled practitioners.

[0664] According to the present disclosure, the technology can be adapted to produce single-chain antibodies specific for CD73 (see, e.g., U.S. Patent No. 4,946,778). In addition, the method can be adapted to construct a Fab expression library (see, e.g., Huse et al., Science 246: 1275-1281 (1989)) to allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity for CD73 or its derivatives, fragments, analogs or homologs. Antibody fragments can be produced by techniques in the art, including but not limited to: (a) F(ab')2 fragments produced by pepsin digestion of antibody molecules; (b) Fab fragments produced by reducing disulfide bonds of F(ab')2 fragments, (c) Fab fragments produced by treating antibody molecules with papain and a reducing agent, and (d) Fv fragments.

[0665] The anti-CD733 antibodies disclosed herein (e.g., clone 10.3 antibodies or clone 2C5 antibodies) or antigen-binding fragments thereof can be modified to increase their serum half-life. This can be accomplished by, for example, incorporating a salvage receptor binding epitope into the antibody or antibody fragment by mutating appropriate regions in the antibody or antibody fragment, or by incorporating the epitope into a peptide tag that is subsequently fused to the end or middle of the antibody or antibody fragment (e.g., by DNA or peptide synthesis) or by YTE mutation. Other methods of increasing the serum half-life of antibodies or antigen-binding fragments thereof are known in the art, for example, by conjugation to a heterologous molecule such as PEG.

[0666] Heteroconjugate anti-CD73 antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies) and antigen-binding fragments thereof are also within the scope of this disclosure. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies have, for example, been suggested for targeting immune cells to non-desirable cells (see, for example, U.S. Patent No. 4,676,980). It is expected that heteroconjugate anti-CD73 antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies) and antigen-binding fragments thereof can be prepared in vitro using methods known in synthetic protein chemistry (including those involving cross-linking agents). For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include imine thiols and methyl-4-mercaptobutyrimidate.

[0667] In certain aspects, the CD73 binding molecules disclosed herein, such as antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies), or antigen-binding fragments thereof, can be combined with other therapeutic agents (e.g., in combination therapy) or they can be fused (e.g., genetically fused to form a fusion protein) or conjugated (e.g., chemically conjugated or enzymatically conjugated) to at least one heterologous moiety. Thus, the CD73 binding molecules disclosed herein can be fused or conjugated with other therapeutic agents or toxins to form immunoconjugates and / or fusion proteins. The present disclosure also provides antibody-drug conjugates (ADCs) comprising at least one of the CD73 binding molecules disclosed herein, at least one of which is derivatized or linked (e.g., chemically or recombinantly) to another molecule (e.g., a peptide, small drug molecule, detectable molecule, etc.). Typically, the anti-CD73 antibodies or portions thereof are derivatized so that their binding to CD73 is not adversely affected by derivatization or labeling. Thus, the anti-CD73 antibodies and antibody portions disclosed herein are intended to include both intact and modified forms of the anti-CD73 binding molecules described herein. For example, the anti-CD73 binding molecules disclosed herein, or CD73 binding portions thereof, can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities such as a cytotoxic agent, a pharmaceutical agent, a detection agent, and / or a protein or peptide that can mediate binding of the anti-CD73 binding molecule to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0668] One type of derivatized molecule can be produced by cross-linking two or more molecular entities, such as the anti-CD73 binding molecules disclosed herein and therapeutic agents (e.g., cytotoxins such as tubulysin or MEDI 1508). Suitable cross-linking agents include those that are heterobifunctional, i.e., having two differently reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester); or homobifunctional (e.g., disuccinimide suberate). Such cross-linking agents are available, for example, from Pierce Chemicals, Rockford, IL. Additional bifunctional coupling agents include N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), activated esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0669] Another type of derivatization molecule can be produced by combining detectable labels. Useful detection agents include fluorescent compounds (for example, fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc.), enzymes useful for detection (for example, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc.), epitopes (for example, leucine zipper pairs, binding sites of secondary antibodies, metal binding domains, epitope tags, etc.) of secondary reporter recognition. In some aspects, detectable labels can be attached by at least one spacer arm. Spacer arms can have different lengths to reduce potential steric hindrance.

[0670] The anti-CD73 binding molecules disclosed herein can also be labeled with radiolabeled amino acids. Radiolabels can be used for both diagnostic and therapeutic purposes. For example, radiolabels can be used to detect CD73 expressing cells by X-ray or other diagnostic techniques such as positron emission tomography (PET).

[0671] In addition, radiolabels can be used therapeutically as toxins to CD73-expressing cells, such as those that elicit an unwanted immune response. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides: 3 H. 14 C. 15N. 35 S. 90 y、 99 Tc, 111 In, 125 I and 131 I. In some aspects, the anti-CD73 binding molecules can be labeled with paramagnetic, radioactive, or fluorescent ions that are detectable during imaging. In some aspects, the paramagnetic ion is chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), or erbium (III). In other aspects, the radioactive ion is iodine-123, technetium-99, indium-111, rhenium-188, rhenium-186, copper-67, iodine-131, yttrium-90, iodine-125, astatine-211, and gallium-67. In other aspects, the anti-CD73 binding molecules are labeled with X-ray imaging agents such as lanthanum (III), gold (III), lead (II), and bismuth (III). The anti-CD73 binding molecules disclosed herein can also be derivatized with chemical groups, for example, polymers such as polyethylene glycol (PEG), methyl groups, ethyl groups, or carbohydrate groups. These groups can be used to improve the biological characteristics of the antibody, for example, to increase serum half-life or increase tissue binding.

[0672] The term "cytotoxic agent" as used herein is broadly defined and refers to a substance that inhibits or prevents the function of a cell and / or causes cell destruction (cell death), and / or exerts an anti-tumor / anti-proliferative effect. For example, a cytotoxic agent can directly or indirectly prevent the development, maturation, or spread of neoplastic tumor cells. The term also includes such agents that cause only a cytostatic effect and not merely a cytotoxic effect. The term includes the chemotherapeutic agents noted below, as well as other CD73 antagonists, anti-angiogenic agents, tyrosine kinase inhibitors, protein kinase A inhibitors, members of the cytokine family, radioactive isotopes, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin.

[0673] The term "chemotherapeutic agent" is a subset of the term "cytotoxic agent" that includes natural or synthetic chemical compounds. Examples of chemotherapeutic agents include alkylating agents such as nitrogen mustards, ethyleneimine compounds, alkyl sulfonates, and other compounds with alkylating effects, such as nitrosoureas, cisplatin, and dacarbazine; antimetabolites such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors such as vinca alkaloids, and podophyllotoxin derivatives; cytotoxic antibiotics and camptothecin derivatives. Other chemotherapeutic agents are amifostine. Cisplatin, dacarbazine (DTIC), dactinomycin, dichloromethyl diethylamine (nitrogen mustard), streptomycin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin Doxorubicin lipo Gemcitabine Daunorubicin, daunorubicin lipo Procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel Docetaxel Aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38), gefitinib Dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, irinotecan, mitoxantrone, topotecan, leuprorelin, megestrol acetate, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testosterone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil aromatase inhibitors, and combinations thereof.

[0674] For the purposes of this disclosure, it will be understood that the modified anti-CD73 antibodies or antigen-binding fragments thereof may include any type of variable region that provides for the binding of the antibody or polypeptide to CD73. In this regard, the variable region may comprise or be derived from any type of mammal that can induce an increased humoral response and generate immunoglobulins against the desired tumor-associated antigen. For this reason, the variable region of the modified anti-CD73 antibodies or antigen-binding fragments thereof may be, for example, human, murine, non-human primate (e.g., cynomolgus monkey, macaque, etc.) or lupin origin. In some aspects, the variable and constant regions of the modified anti-CD73 antibodies or antigen-binding fragments thereof are human. In other aspects, the variable regions of compatible antibodies (typically derived from non-human sources) may be engineered or specifically tailored to improve binding properties or reduce the immunogenicity of the molecule. In this regard, the variable region may be humanized or altered by incorporating an imported amino acid sequence.

[0675] In certain aspects, the variable domains in the heavy and light chains of an anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) or an antigen-binding fragment thereof are altered by at least partially replacing one or more CDRs and, if necessary, by replacing a portion of the framework region and sequence changes. Although the CDRs may be derived from antibodies of the same class or even subclass as the antibody from which the framework regions are derived, it is envisioned that the CDRs will be derived from antibodies of different classes and, in certain aspects, from different species. It is not necessary to replace all CDRs with complete CDRs from the donor variable region in order to transfer the antigen binding ability of one variable domain to another. Rather, only those residues necessary to maintain the activity of the antigen binding site need to be transferred. In view of the explanations set forth in U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762, it is fully within the ability of one skilled in the art to obtain functional antibodies with reduced immunogenicity by performing routine experiments or by trial and error testing.

[0676] Despite the changes made to the variable regions, one of ordinary skill in the art will appreciate that the modified anti-CD73 antibodies (e.g., modified clone 10.3 antibodies or modified clone 2C5 antibodies) or antigen-binding fragments thereof will include antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a portion of one or more constant region domains has been deleted or otherwise altered to provide desired biochemical characteristics, such as increased tumor localization or decreased serum half-life when compared to an antibody comprising a native or unaltered constant region and having approximately the same immunogenicity. In some aspects, the constant region of the modified antibody comprises a human constant region. Compatible modifications to the constant region of the anti-CD73 molecules disclosed herein include additions, deletions, or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or to the light chain constant domain (CL). In some aspects, modified constant regions are contemplated in which one or more domains are partially or completely deleted. In some aspects, the modified antibodies will comprise domain-deleted constructs or variants in which the entire CH2 domain is removed (ΔCH2 constructs). In some aspects, the omitted constant region domains will be replaced by a short amino acid spacer (e.g., 10 residues) that provides some molecular flexibility normally conferred by the absent constant region.

[0677] In addition to their configuration, it is known in the art that constant regions mediate several effector functions. For example, the binding of the C1 component of complement to antibodies activates the complement system. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and can also be involved in autoimmune hypersensitivity reactions. In addition, antibodies bind to cells via the Fc region, where the Fc receptor site on the antibody Fc region binds to Fc receptors (FcR) on the cell. There are many Fc receptors that are specific for different classes of antibodies, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (μ receptors). The binding of antibodies to Fc receptors on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, killer cell lysis of antibody-coated target cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transport, and control of immunoglobulin production.

[0678] In certain aspects, the anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) or an antigen-binding fragment thereof provides altered effector function, thereby affecting the biological characteristics of the administered antibody or antigen-binding fragment thereof. For example, deletion or inactivation of the constant region (via point mutation or other means) can reduce Fc receptor binding of circulating modified antibodies, thereby increasing tumor localization. In other cases, consistent with the present disclosure, constant region modifications can mitigate complement binding and thereby reduce the serum half-life and non-specific association of conjugated cytotoxins. Yet other modifications of the constant region can be used to eliminate disulfide bonds or oligosaccharide moieties, thereby allowing enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region according to the present disclosure can be easily performed using well-known biochemical or molecular engineering techniques within the scope of the skilled artisan's cognition.

[0679] In certain aspects, the CD73 binding molecules disclosed herein that are antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies) or antigen-binding fragments thereof do not have one or more effector functions. For example, in some aspects, the antibody or antigen-binding fragment thereof has no antibody-dependent cellular cytotoxicity (ADCC) activity and / or no complement-dependent cytotoxicity (CDC) activity. In certain aspects, the anti-CD73 antibody or antigen-binding fragment thereof does not bind to Fc receptors and / or complement factors. In certain aspects, the antibody or antigen-binding fragment thereof has no effector function.

[0680] It should be noted that in certain aspects, the anti-CD73 modified antibodies or antigen-binding fragments thereof can be engineered to fuse the CH3 domain directly to the hinge region of the corresponding modified antibody or fragment thereof. In other constructs, it may be desirable to provide a peptide spacer between the hinge region and the modified CH2 and / or CH3 domains. For example, a compatibility construct can be expressed in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is bound to a hinge region having a 5-20 amino acid spacer. This spacer may be added, for example, to ensure that regulatory elements of the constant domain remain free and accessible, or that the hinge region remains flexible. However, it should be noted that in some cases, the amino acid spacer may prove to be immunogenic and elicit an undesirable immune response against the construct. Therefore, in certain aspects, any spacer added to the construct will be relatively non-immunogenic, or even omitted entirely, to maintain the desired biochemical properties of the modified antibody.

[0681] In addition to the deletion of the entire constant region domain, it will be understood that the anti-CD73 antibodies and antigen-binding fragments thereof disclosed herein can be provided by partial deletion or substitution of a few or even a single amino acid. For example, mutation of a single amino acid in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby increase tumor localization. Similarly, it may be desirable to delete only a portion of one or more constant region domains that control the effector function to be modulated (e.g., complement C1Q binding). Such partial deletions of the constant region can improve selected properties of the antibody or antigen-binding fragment thereof (e.g., serum half-life) while leaving other desired functions associated with the subject constant region domain intact. In addition, as mentioned above, the constant region of the disclosed anti-CD73 antibodies and antigen-binding fragments thereof can be modified by mutation or substitution of one or more amino acids, which enhances the characteristics of the final construct. In this regard, the activity provided by the conserved binding site (e.g., Fc binding) can be interfered with while substantially maintaining the configuration and immunogenic characteristics of the modified antibody or antigen-binding fragment thereof. Certain aspects may include adding one or more amino acids to the constant region to enhance desired properties, such as reducing or increasing effector function, or providing for more cytotoxin or carbohydrate attachment. In these aspects, it may be desirable to insert or replicate a specific sequence derived from a selected constant region domain.

[0682] The present disclosure also provides variants and equivalents that are substantially homologous to the chimeric, humanized, and human anti-CD73 antibodies or antigen-binding fragments thereof listed herein. These may comprise, for example, conservative substitution mutations, i.e., replacement of one or more amino acids by similar amino acids. For example, a conservative substitution refers to the replacement of one amino acid with another within the same general class, such as, for example, replacing an acidic amino acid with another acidic amino acid, replacing a basic amino acid with another basic amino acid, or replacing a neutral amino acid with another neutral amino acid. Conservative amino acid substitutions are well known in the art.

[0683] The anti-CD73 antibodies or antigen-binding fragments thereof can be further modified to include additional chemical moieties that are not normally part of the protein. Those derivatized moieties can improve the solubility, biological half-life, or absorption of the protein. These moieties can also reduce or eliminate any desired side effects of the protein, etc. A review of those moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0684] VI. Polynucleotides Encoding CD73 Binding Molecules

[0685] In certain aspects, the present disclosure encompasses polynucleotides comprising nucleic acid sequences encoding polypeptides that specifically bind to CD73 or an antigen-binding fragment thereof. For example, the present disclosure provides polynucleotides comprising nucleic acid sequences encoding anti-CD73 antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies) or encoding an antigen-binding fragment of such an antibody. The polynucleotides of the present disclosure may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand.

[0686] In some aspects, the polynucleotide is isolated. In some aspects, the polynucleotide is substantially pure. In some aspects, the polynucleotide comprises a coding sequence for a mature polypeptide fused in the same reading frame to a polynucleotide that facilitates, for example, polypeptide expression and polypeptide secretion from a host cell (e.g., a leader sequence that acts as a secretory sequence for controlling polypeptide transport from a cell). A polypeptide with a leader sequence is a preprotein and can cause the leader sequence to be cleaved by the host cell to form a mature form of the polypeptide. These polynucleotides can also encode a CD73 binding proprotein, which is a mature protein plus additional 5' amino acid residues.

[0687] In certain aspects, the polynucleotides include the coding sequence of a mature CD73 binding polypeptide, such as an anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody), or an antigen-binding fragment thereof, fused in frame to a marker sequence that allows, for example, purification of the encoded polypeptide. For example, in the case of a bacterial host, the marker sequence can be a hexahistidine tag supplied by the pQE-9 vector to provide for purification of the mature polypeptide fused to the marker, or when a mammalian host (e.g., COS-7 cells) is used, the marker sequence can be a hemagglutinin (HA) tag derived from the influenza virus hemagglutinin protein.

[0688] The present disclosure also provides variants of the polynucleotides encoding, for example, CD73-binding fragments, analogs, and derivatives of the CD73-binding molecules disclosed herein (e.g., a clone 10.3 antibody or a clone 2C5 antibody).

[0689] In some aspects, these polynucleotide variants contain changes in coding region, noncoding region or both. In some aspects, these polynucleotide variants contain silent replacements, additions or deletions that do not change the characteristic or active change of the polypeptide encoded. In some aspects, nucleotide variants are produced by the silent replacement due to the degeneracy of the genetic code. Polynucleotide variants can be produced for various reasons, for example, in order to optimize the codon expression of a specific host (the codons in the human mRNA are changed into those preferred codons of the bacterial host such as Escherichia coli). Carriers and cells comprising the polynucleotide described herein are also provided.

[0690] In some aspects, a DNA sequence encoding a CD73 binding molecule, such as an anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody), or an antigen-binding fragment thereof, can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are preferred in the host cell (where the recombinant polypeptide of interest will be produced). Standard methods can be used to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. For example, the complete amino acid sequence can be used to construct a reverse-translation gene. In addition, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding desired polypeptide portions can be synthesized and then connected. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0691] In case of assembly (by synthesis, site-directed mutagenesis or another method), the polynucleotide sequence encoding the specific isolated polypeptide of interest will be inserted into the expression vector and operably connected to an expression control sequence suitable for expressing the protein in the desired host. Suitable assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping and expression of the bioactive polypeptide suitable for the host. As well known in the art, in order to obtain a high expression level of the transfected gene in the host, the gene must be operably connected to transcription and translation expression control sequences that work in the selection expression host.

[0692] In certain aspects, recombinant expression vectors are used to amplify and express DNA encoding anti-CD73 antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies) or antigen-binding fragments thereof. A recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding an anti-CD73 antibody polypeptide chain or antigen-binding fragment thereof, operatively linked to suitable transcriptional and translational regulatory elements derived from mammalian, microbial, viral, or insect genes.

[0693] A transcription unit generally comprises the following components: (1) one or more genetic elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural sequence or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulatory elements may include an operator sequence for controlling transcription. An origin of replication, which generally confers the ability to replicate in the host, and a selection gene that aids in identifying transformants may also be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation. Structural elements intended for use in yeast expression systems include a leader sequence that enables the host cell to secrete the translated protein extracellularly. Alternatively, in the case where the recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. Subsequently, this residue can optionally be cleaved from the expressed recombinant protein to provide the final product.

[0694] The selection of expression control sequences and expression vectors will depend on the selection of the host. A variety of expression host / vector combinations can be adopted. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9, and derivatives thereof, and more widely host range plasmids, such as M13 and filamentous single-stranded DNA phage.

[0695] Suitable host cells for expressing CD73 binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody), or antigen-binding fragments thereof, under the control of an appropriate promoter include prokaryotes, yeast, insects, or higher eukaryotic cells. Prokaryotic cells include gram-negative or gram-positive organisms, such as Escherichia coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems can also be employed. Appropriate cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosure of which is incorporated herein by reference. Additional information regarding methods of protein production, including antibody production, can be found, for example, in U.S. Patent Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746, 6,660,501, and 7,932,087, each of which is incorporated herein by reference in its entirety.

[0696] Various mammalian or insect cell culture systems can also be advantageously used to express recombinant CD73 binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments thereof. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified, and fully functional.

[0697] Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 line of monkey kidney cells described by Gluzman (Cell 23: 175, 1981), and other cell lines including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), NSO, HeLa, and BHK cell lines. Mammalian expression vectors may contain non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, as well as 5' or 3' non-translated sequences (such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites), and transcription termination sequences. The baculovirus system for producing heterologous proteins in insect cells was reviewed by Luckow and Summers, BioTechnology 6: 47 (1988).

[0698] CD73 binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies), or antigen-binding fragments thereof, produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and size fractionation column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow the protein to be more easily purified after passing through a suitable affinity column. Such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallization can also be used to physically characterize the isolated protein.

[0699] For example, one can first use a commercially available protein concentration filter such as AMICON or Millipore PELLICON The ultrafiltration unit concentrates the supernatant from the system that secretes the recombinant protein into the culture medium. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be used, such as a matrix or substrate with a diethylaminoethyl (DEAE) group on the side. The matrix can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium (e.g., silica gel with methyl or other aliphatic groups on the side) can be used to further purify the CD73 binding molecule (e.g., clone 10.3 antibody or clone 2C5 antibody). Some or all of the above purification steps in different combinations can also be used to provide a homogeneous recombinant protein.

[0700] Recombinant CD73 binding proteins, such as anti-CD73 antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies), or antigen-binding fragments thereof, produced in bacterial culture can be isolated, for example, by initial extraction from a cell pellet, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. Final purification steps can be performed using high performance liquid chromatography (HPLC). Microbial cells used for recombinant protein expression can be disrupted by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

[0701] Art-known methods for purifying antibodies and other proteins also include, for example, those described in US Patent Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is incorporated herein by reference in its entirety.

[0702] In certain aspects, the CD73 binding molecule is a polypeptide that is not an antibody. Various methods for identifying and producing non-antibody polypeptides that bind to protein targets with high affinity are known in the art. See, for example, Skerra, Curr. Opin. Biotechnol. 18:295-304 (2007), Hosse et al., Protein Science 15:14-27 (2006), Gill et al., Curr. Opin. Biotechnol. 17:653-658 (2006), Nygren, FEBS J. 275:2668-76 (2008), and Skerra, FEBS J. 275:2677-83 (2008), each of which is incorporated herein by reference in its entirety. In certain aspects, phage display technology can be used to identify / produce CD73 binding polypeptides. In certain aspects, the polypeptide comprises a protein scaffold selected from the group consisting of protein A, lipocalin, a fibronectin domain (e.g., a fibronectin domain such as tenascin-3 Fn III domain), an ankyrin consensus repeat domain, and thioredoxin.

[0703] VI. Therapeutic Methods Using Therapeutic Anti-CD73 Antibodies

[0704] The present disclosure provides methods for using anti-CD73 binding molecules, such as antibodies (including antigen-binding fragments, variants, and derivatives thereof) (e.g., clone 10.3 antibody or clone 2C5 antibody) to treat patients suffering from diseases associated with CD73 expression or CD73-expressing cells (e.g., cancer). In some specific aspects, such cancers are lung cancer, breast cancer, ovarian cancer, colorectal cancer, bladder cancer, pancreatic cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, colon cancer, and lymphoma.

[0705] "CD73-expressing cells" means cells that express CD73. CD73 can be membrane-bound through a glycosyl-phosphatidylinositol anchor and is also present as a soluble protein. Methods for detecting CD73 expression in cells and other suitable samples are well known in the art and include, but are not limited to, immunohistochemistry, flow cytometry, Western blotting, ELISA, and the like.

[0706] Although the following discussion refers to diagnostic methods and treatments for various diseases and disorders using the CD73 binding molecules disclosed herein (e.g., clone 10.3 antibodies or clone 2C5 antibodies), the methods described herein are also applicable to any other anti-CD73 antibodies that retain the desirable properties of the anti-CD73 antibodies disclosed herein, such as the ability to specifically bind to CD73 and neutralize its 5'-nucleotidase activity, as well as antigen-binding fragments, variants, and derivatives (e.g., fusion proteins or conjugates) of these anti-CD73 antibodies. In some aspects, the CD73 binding molecule is a human or humanized antibody that does not mediate human ADCC, or an anti-CD73 antibody that is engineered so that it does not mediate ADCC.

[0707] In some aspects, the CD73 binding molecule is a CD730010 antibody or an antigen-binding fragment thereof, a clone 10.3 antibody or an antigen-binding fragment thereof, a CD730002 antibody or an antigen-binding fragment thereof, a clone 2C5 antibody or an antigen-binding fragment thereof, or a CD73004 antibody or an antigen-binding fragment thereof. In other aspects, the CD73 binding molecule is a clone 10.3 mutant antibody. In some aspects, the CD73 binding molecule is a clone 10.3 monoclonal antibody. In some aspects, the CD73 binding molecule is a clone 10.3 monoclonal antibody engineered to extend serum half-life. In other aspects, the CD73 binding molecule is a clone 10.3 YTE mutant antibody. In other aspects, the CD73 binding molecule is a clone 2C5 mutant antibody. In some aspects, the CD73 binding molecule is a clone 2C5 monoclonal antibody. In some aspects, the CD73 binding molecule is a clone 2C5 monoclonal antibody engineered to extend serum half-life. In other aspects, the CD73 binding molecule is a clone 2C5 YTE mutant antibody.

[0708] In one aspect, treatment comprises administering or administering an anti-CD73 binding molecule of the disclosure, e.g., an antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody), or an antigen-binding fragment, variant, or derivative thereof, to a subject or patient, or administering or administering the anti-CD73 binding molecule to a tissue or cell line isolated from a subject or patient, wherein the subject or patient has a disease, a symptom of a disease, or a predisposition to a disease. In another aspect, treatment is also intended to comprise administering or administering a pharmaceutical composition comprising an anti-CD73 binding molecule of the disclosure, e.g., an antibody, or an antigen-binding fragment, variant, or derivative thereof, to a subject or patient, or administering or administering the pharmaceutical composition comprising the anti-CD73 binding molecule to a tissue or cell line isolated from a subject or patient, wherein the subject or patient has a disease, a symptom of a disease, or a predisposition to a disease.

[0709] The anti-CD73 binding molecules disclosed herein, such as antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies) or antigen-binding fragments, variants, or derivatives thereof can be used to treat different cancers. In one aspect, the present disclosure provides anti-CD73 binding molecules, such as antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies) or antigen-binding fragments, variants, or derivatives thereof, for use as medicaments, specifically the medicaments for treating or preventing cancer (e.g., colon cancer, melanoma, breast cancer, lymphoma or non-small cell lung cancer Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Burkitt's lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer). In some aspects, the cancer exhibits a pro-metastatic phenotype. In some aspects, the cancer exhibiting a pro-metastatic phenotype is melanoma or breast cancer. In some aspects, the cancer is a metastatic cancer. In some aspects, the anti-CD73 binding molecules disclosed herein can trigger adaptive anti-tumor activity and / or inhibit metastasis. In some specific aspects, the anti-CD73 binding molecules disclosed herein can inhibit metastasis in breast cancer.

[0710] According to the methods of the present disclosure, at least one anti-CD73 binding molecule, e.g., an antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody), or an antigen-binding fragment, variant, or derivative thereof, as defined elsewhere herein, is used to promote a positive therapeutic response to cancer. The term "positive therapeutic response" for the treatment of cancer refers to an improvement in the disease and / or an improvement in symptoms associated with the disease associated with the activity of these anti-CD73 binding molecules, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof. Thus, for example, an improvement in the disease can be characterized as a complete response. By the term "complete response" is intended the absence of clinically detectable disease, as normalized by any prior test results. Alternatively, an improvement in the disease can be classified as a partial response. A "positive therapeutic response" encompasses a reduction or inhibition of the progression and / or duration of a cancer, a reduction or improvement in the severity of a cancer, and / or an improvement in one or more symptoms thereof, resulting from the administration of an anti-CD73 binding molecule disclosed herein.

[0711] In specific aspects, such terms refer to one, two, or three or more of the following outcomes following administration of an anti-CD73 binding molecule disclosed herein: (1) stabilization, reduction, or elimination of cancer cell populations; (2) stabilization or reduction of cancer growth; (3) impairment in cancer development; (4) eradication, removal, or control of primary, regional, and / or metastatic cancer; (5) reduction in mortality; (6) increase in disease-free, relapse-free, progression-free, and / or overall survival, duration, or rate; (7) increase in response rate, durability of response, or number of patients who respond or are in remission; (8) reduction in hospitalization rate, (9) reduction in length of hospitalization, (10) maintenance of cancer size and no increase or increase by less than 10%, preferably less than 5%, preferably less than 4%, preferably less than 2%, and (12) increase in the number of patients in remission.

[0712] Clinical responses can be assessed using screening techniques such as magnetic resonance imaging (MRI) scans, x-ray imaging, computed tomography (CT) scans, flow cytometry or fluorescence activated cell sorter (FACS) analysis, histology, gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, RIA, chromatography, and the like. In addition to these positive therapeutic responses, subjects undergoing treatment with an anti-CD73 binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof, can experience beneficial effects in terms of improvement in symptoms associated with the disease.

[0713] The anti-CD73 binding molecules, such as the antibodies disclosed herein (e.g., a clone 10.3 antibody or a clone 2C5 antibody), or antigen-binding fragments, variants, or derivatives thereof, can be used in combination with any therapy known for cancer, including any agent or combination of agents known to be useful for treating, or used or currently used to treat cancer (e.g., colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Burkitt's lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer). The second agent or combination of agents of the pharmaceutical combination formulation or dosing regimen preferably has complementary activities to the antibodies or polypeptides disclosed herein such that they do not adversely affect each other.

[0714] Anticancer agents include drugs used to treat malignant tumors, such as cancerous growths. Drug therapy can be used alone or in combination with other treatments, such as surgery or radiotherapy. Depending on the nature of the organ involved, several types of drugs can be used to treat cancer. For example, breast cancer is usually stimulated by estrogen and can be treated with drugs that inactivate hormones. Similarly, prostate cancer can be treated with drugs that inactivate androgens, male sex hormones. Anti-cancer agents used in certain methods of the present disclosure include, among others, antibodies (e.g., an antibody that binds to IGF-1R, an antibody that binds to EGFR, an antibody that binds to Her2, or an antibody that binds to cMET), small molecules targeting IGF1R, small molecules targeting EGFR, small molecules targeting Her2, antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule targeting agents, kinase inhibitors, protein synthesis inhibitors, immunotherapeutics, hormone therapeutics, glucocorticoids, aromatase inhibitors, mTOR inhibitors, chemotherapeutics, protein kinase B inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, cyclin-dependent kinase (CDK) inhibitors, RLr9, CD289, enzyme inhibitors, anti-TRAIL, MEK inhibitors, and the like.

[0715] In certain aspects, the CD73 binding molecules disclosed herein, such as antibodies (e.g., clone 10.3 antibodies or clone 2C5 antibodies), or antigen-binding fragments thereof, can be administered in combination with antibodies or antibody fragments that target, for example, PD-1 (programmed death 1 protein), its two ligands PD-L1 (programmed death ligand 1) and / or PD-L2, or CTLA-4 (cytotoxic T lymphocyte antigen 4 protein). See, for example, Stagg et al. PNAS 107: 1547-1552 (2010); Jin et al. Cancer Res. 70(6): (2010); Allard et al. Clin. Cancer Res. 19: 5626 (2013), which are incorporated herein by reference in their entireties. In some aspects, the anti-CTLA-4 antibody is ipilimumab or an antigen-binding fragment thereof. In other aspects, the anti-CTLA-4 antibody is tremelimumab (tesimumab, CP-675,206) or an antigen-binding fragment thereof. In some aspects, the anti-PD-1 antibody is pembrolizumab ( In some aspects, the anti-PD-1 antibody is nivolumab (BMS-936558, MDX-1106, ONO-4538, ) or an antigen-binding fragment thereof. In some aspects, the anti-PD-L1 antibody is BMS-936559 or an antigen-binding fragment thereof. In other aspects, the anti-PD-L1 antibody is MPDL3280A. In other aspects, the anti-PD-1 antibody is AMP-224 (anti-PD-1 Fc fusion protein) or an antigen-binding fragment thereof. In different aspects, the anti-PD-L1 antibody is MEDI4736 or an antigen-binding fragment thereof.

[0716] In some aspects, the CD73 binding molecules disclosed herein (e.g., clone 10.3 antibodies or clone 2C5 antibodies) can be administered in combination with anti-PD-1 or anti-PD-1 antibodies. In various embodiments, the anti-CD73 antibody is administered at a concentration of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg. In some aspects, the CD73-binding molecules disclosed herein (e.g., a clone 10.3 antibody or a clone 2C5 antibody) can be administered in combination with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, wherein the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody is administered at a concentration of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, or about 20 mg / kg. In some aspects, the anti-CD73 antibody and the anti-PD-1 antibody, anti-PD-L1, or anti-CTLA4 are administered at a ratio of about 1:1, 1:2, 1:3, or 1:4. In some aspects, the anti-CD73 antibody and the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody are administered at a ratio of about 1:2. In a specific aspect, the concentration of the anti-CD73 antibody (e.g., a clone 10.3 antibody or a clone 2C5 antibody) is about 10 mg / kg, and the concentration of the anti-PD-1 antibody is about 20 mg / kg. In some aspects, the CD73 binding molecules disclosed herein (e.g., a clone 10.3 antibody or a clone 2C5 antibody) can be administered in combination with an anti-PD-1 antibody. In some aspects, administration of a combination therapy comprising a CD73-binding molecule disclosed herein (e.g., MEDI9447, a clone 10.3 antibody or a clone 2C5 antibody) in combination with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody can increase survival by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, compared to untreated subjects or subjects treated with monotherapy (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody without anti-CD73 antibody).In some aspects, administration of a combination therapy comprising a CD73-binding molecule disclosed herein (e.g., a clone 10.3 antibody or a clone 2C5 antibody) in combination with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody can increase survival by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold compared to untreated subjects or subjects treated with monotherapy (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody without anti-CD73 antibody).

[0717] Where the combination therapy comprises administration of an anti-CD73 binding molecule in combination with administration of another therapeutic agent (e.g., an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody), the methods disclosed herein encompass co-administration using separate formulations or a single pharmaceutical formulation, as well as sequential administration in either order. In some aspects, an anti-CD73 antibody described herein (e.g., a clone 10.3 antibody or a clone 2C5 antibody) is administered in combination with other drugs, wherein the antibody or antigen-binding fragment, variant, or derivative thereof and the one or more therapeutic agents can be administered sequentially in any order or simultaneously (i.e., at the same time or within the same time frame).

[0718] The combination therapy may provide and be demonstrated to be "synergistic," i.e., the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds when used separately. Synergy may be obtained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined unit dosage formulation; (2) delivered as separate formulations by alternation or parallel delivery; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., continuously, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[0719] In other aspects, the CD73 binding molecules disclosed herein (e.g., clone 10.3 antibodies or clone 2C5 antibodies) can be administered in combination with tyrosine kinase inhibitors. In some other specific aspects, the CD73 binding molecules disclosed herein can be administered in combination with inhibitors of tyrosine kinase activity associated with EGFR and / or HER2 / neu, such as lapatinib. In some aspects, the CD73 binding molecules disclosed herein can be administered in combination with anti-mitotic agents. In some specific aspects, the CD73 binding molecules disclosed herein can be administered in combination with agents that stabilize mitotic spindle microtubule assembly, such as paclitaxel or docetaxel. Another aspect is the use of anti-CD73 binding molecules, such as antibodies or antigen-binding fragments, variants, or derivatives thereof (e.g., clone 10.3 antibodies or clone 2C5 antibodies), for diagnosing protein levels in monitoring tissues (as part of a clinical testing program), such as to determine the effectiveness of a given treatment regimen. For example, detection can be facilitated by coupling the antibody to a detectable substance.

[0720] Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0721] VIII. Anti-CD73 Antibody Therapeutic Combinations and Co-therapy

[0722] The present disclosure provides methods for treating patients with cancer, including colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer, using therapeutic combinations including anti-CD73 binding molecules, such as antibodies, including antigen-binding fragments, variants, and derivatives thereof (e.g., a clone 10.3 antibody or a clone 2C5 antibody).

[0723] Although the following discussion refers to therapeutic combinations featuring the CD73 binding molecules of the disclosure (e.g., a clone 10.3 antibody or a clone 2C5 antibody), the methods described herein are also applicable to any other anti-CD73 antibodies that retain the desirable properties of the anti-CD73 antibodies disclosed herein, such as the ability to specifically bind to CD73 and neutralize its 5'-nucleotidase activity, as well as antigen-binding fragments, variants, and derivatives (e.g., fusion proteins or conjugates) of these anti-CD73 antibodies. In some aspects, the CD73 binding molecule is a human or humanized antibody that does not mediate human ADCC, or an anti-CD73 antibody that is engineered so that it does not mediate ADCC.

[0724] Treatment of patients with solid tumors using combinations of the present invention, such as an anti-CD73 antibody or antigen-binding fragment thereof in combination with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or antigen-binding fragment thereof, can result in additive or synergistic effects. The term "synergistic," as used herein, refers to a therapeutic combination (e.g., a combination of an anti-CD73 antibody (e.g., MEDI9447) and an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody) that is more effective than the additive effects of the monotherapies.

[0725] A synergistic effect of a therapeutic combination (e.g., a combination of an anti-CD73 antibody (e.g., MEDI9447) and an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody) allows for the use of lower doses of one or more of these therapeutic agents to patients with solid tumors and / or less frequent administration of the therapeutic agents to patients with solid tumors. The ability to utilize lower doses of therapeutic agents and / or administer the therapy less frequently reduces toxicities associated with administering the therapy to a subject without reducing the efficacy of the therapy in the treatment of solid tumors. In addition, a synergistic effect can result in increased efficacy of the therapeutic agents in the management, treatment, or improvement of solid tumors. A synergistic effect of a combination of therapeutic agents can avoid or reduce adverse or unwanted side effects associated with the use of either monotherapy.

[0726] In co-therapy, the combination of an anti-CD73 antibody (e.g., MEDI9447) or an antigen binding fragment thereof and an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or its antigen binding fragment can optionally be included in the same pharmaceutical composition, or can be included in a separate pharmaceutical composition. In the latter case, the pharmaceutical composition comprising an anti-CD73 antibody (e.g., MEDI9447) or an antigen binding fragment thereof is suitable for administration before, simultaneously with, or after administration of a pharmaceutical composition comprising an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or its antigen binding fragment. In some cases, the anti-CD73 antibody (e.g., MEDI9447) or an antigen binding fragment thereof and an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody are administered in a separate composition at overlapping times.

[0727] Anti-CD73 antibodies (e.g., MEDI9447) or antigen-binding fragments thereof and anti-PD-1, anti-PD-L1, or anti-CTLA4 antibodies or antigen-binding fragments thereof can be administered only once or occasionally while still providing benefit to the patient. In another aspect, the patient is administered additional subsequent doses. Subsequent doses may be administered at varying time intervals depending on the patient's age, weight, clinical evaluation, tumor burden, and / or other factors, including the discretion of the attending physician.

[0728] These methods provided herein can weaken or delay tumor growth. In some aspects, the reduction or delay can be statistically significant. The reduction of tumor growth can be measured by comparing the growth of the patient's tumor at baseline, for expected tumor growth, for expected tumor growth based on a large patient population, or for the tumor growth of a control population. In other embodiments, the method of the present invention improves survival rate.

[0729] IX. Anti-PD-L1 Antibodies

[0730] Antibodies that specifically bind to and inhibit PD-L1 activity (e.g., binding to PD-1 and / or CD80) are useful for treating tumors. B7-H1, also known as PD-L1, is a type I transmembrane protein with a size of approximately 53 kDa. In humans, B7-H1 is expressed on many immune cell types (including activated and anergic / exhausted T cells), naive and activated B cells, as well as myeloid dendritic cells (DCs), monocytes, and mast cells. It is also expressed on non-immune cells, including pancreatic islets, liver Kupffer cells, vascular endothelium, and selected epithelial cells (e.g., airway epithelial cells and renal tubular epithelial cells), where its expression is enhanced during inflammatory episodes. Increased levels of B7-H1 expression are also found on many tumors, including but not limited to breast, colon, colorectal, lung, renal cell tumors, including renal cell carcinoma, gastric cancer, bladder cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), and pancreatic cancer, as well as melanoma.

[0731] B7-H1 is known to bind two alternative ligands. The first of these, PD-1, is a 50-55 kDa type I transmembrane receptor originally identified in T cell lines undergoing activation-induced apoptosis. PD-1 is expressed on activated T cells, B cells, and monocytes, as well as other cells of the immune system, and binds both B7-H1 (PD-L1) and the related B7-DC (PD-L2). The second is the B7 family member B7-1, which is expressed on activated T cells, B cells, monocytes, and antigen-presenting cells.

[0732] Signaling through the PD-1 / B7-H1 axis is believed to play an important non-redundant function within the immune system by negatively regulating T cell responses. B7-H1 expression on tumor cells is thought to help tumors evade detection and elimination by the immune system. B7-H1 acts in this regard through several alternative mechanisms, including driving exhaustion and anergy of tumor-infiltrating T lymphocytes, stimulating the secretion of immunosuppressive cytokines into the tumor microenvironment, stimulating suppressive regulatory T cell function, and protecting B7-H1-expressing tumor cells from lysis by tumor cell-specific cytotoxic T cells.

[0733] MEDI4736 is an exemplary anti-PD-L1 antibody that is selective for B7-H1 and blocks the binding of B7-H1 to PD-1 and CD80 receptors. MEDI4736 can relieve B7-H1-mediated inhibition of human T-cell activation in vitro and inhibit tumor growth in xenograft models through T-cell-dependent mechanisms. Other agents that can be used include agents (AB or other) that inhibit PD-L1 and / or PD-1.

[0734] Information regarding MEDI4736 (or fragments thereof) for use in the methods provided herein can be found in US20130034559 / US8779108 and US20140356353, the disclosures of which are incorporated herein by reference in their entireties. The fragment crystallizable (Fc) domain of MEDI4736 contains a triple mutation in the constant domain of the IgG1 heavy chain that reduces binding to complement component C1q and Fcγ receptors responsible for mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0735] MEDI4736 and its antigen-binding fragments used in the methods provided herein comprise a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. In a specific aspect, MEDI4736 or its antigen-binding fragments used in the methods provided herein comprise a light chain variable region and a heavy chain variable region, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 130, and the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 131. In a specific aspect, MEDI4736 or its antigen-binding fragments used in the methods provided herein comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 132-134, and wherein the light chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 135-137. Those skilled in the art will readily be able to identify Chothia's definition, Abm's definition, or other CDR definitions known to those skilled in the art. In a specific aspect, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H90T antibody as disclosed in US 20130034559 / US 8779108 and US 20140356353, each of which disclosures are incorporated herein by reference in their entirety.

[0736] X. Anti-CTLA4 Antibodies

[0737] Thus, in one embodiment, the therapeutic combination of the invention includes a CTLA4 blocking antibody (e.g., tremelimumab) and / or multiple antibodies that reduce the PD1 / PD-L1 interaction. Two T cell regulatory pathways that have received significant attention to date signal through cytotoxic T lymphocyte antigen-4 (CTLA4, CD152) and programmed death ligand 1 (PD-L1, also known as B7H-1 or CD274).

[0738] CTLA4 is expressed on activated T cells and acts as a co-inhibitor to suppress T cell responses following CD28-mediated T cell activation. CTLA4 is thought to regulate the magnitude of early activation of naive and memory T cells following TCR engagement and is part of a central inhibitory pathway that influences both anti-tumor immunity and autoimmunity. CTLA4 is primarily expressed on T cells, and expression of its ligands, CD80 (B71) and CD86 (B7.2), is primarily restricted to antigen-presenting cells, T cells, and other immune-mediating cells. Antagonistic anti-CTLA4 antibodies that block the CTLA4 signaling pathway have been reported to enhance T cell activation. One such antibody, ipilimumab, was approved by the FDA in 2011 for the treatment of metastatic melanoma. Another anti-CTLA4 antibody, tremelimumab, was tested in a Phase III clinical trial for the treatment of advanced melanoma but did not significantly increase overall survival compared to the then-standard treatment (temozolomide or dacarbazine).

[0739] Information about tremelimumab (or an antigen-binding fragment thereof) for use in the methods provided herein can be found in US 6,682,736 (where it is referred to as 11.2.1), the disclosure of which is incorporated herein by reference in its entirety. Tremelimumab (also known as CP-675,206, CP-675, CP-675206, and tesitumumab) is a human IgG2 monoclonal antibody that is highly selective for CTLA4 and blocks the binding of CTLA4 to CD80 (B7.1) and CD86 (B7.2). It has been shown to cause immune activation in vitro, and some patients treated with tremelimumab have shown tumor regression.

[0740] Tremelimumab used in these methods provided herein comprises a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. In a specific aspect, tremelimumab or its antigen-binding fragment used in the methods provided herein comprises a light chain variable region and a heavy chain variable region. In a specific aspect, tremelimumab or its antigen-binding fragment used in the methods provided herein comprises a heavy chain variable region and a light chain variable region identified herein. Those skilled in the art will be able to easily identify the Chothia definition, Abm definition or other CDR definitions known to those skilled in the art. In a specific aspect, tremelimumab or its antigen-binding fragment used in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 11.2.1 antibody as disclosed in US 6,682,736, which is incorporated herein by reference in its entirety.

[0741] XI.VII. Pharmaceutical Compositions and Methods of Administration

[0742] Methods of preparing and...

Claims

1. Use of an antibody or an antigen-binding fragment thereof in the manufacture of a medicament for treating colorectal cancer or non-small cell lung cancer, wherein the antibody specifically binds to CD73 and comprises V L and V H , the V L and V H comprising VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2 and VH-CDR3, wherein VL-CDR1 is shown as SEQ ID NO:46, VL-CDR2 is shown as SEQ ID NO:51, VL-CDR3 is shown as SEQ ID NO:56, VH-CDR1 is shown as SEQ ID NO:36, VH-CDR2 is shown as SEQ ID NO:39, and VH-CDR3 is shown as SEQ ID NO:

45.

2. The use according to claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to CD73 comprises V L and V H , where V L Comprising the amino acid sequence of SEQ ID NO:68; and wherein said V H Comprising the amino acid sequence of SEQ ID NO:82.

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