Binding molecules specific for CD73 and uses thereof

Abstract

To provide improved compositions and methods for reducing tumor-mediated immunosuppression.SOLUTION: The present disclosure provides anti-CD73 binding molecules, e.g., antibodies and antigen binding fragments thereof. Also provided are pharmaceutical formulations comprising the disclosed compositions, and methods for the diagnosis and treatment of diseases associated with CD73-expression, e.g., cancer. Such diseases can be treated, e.g., by direct therapy with the anti-CD73 binding molecules disclosed herein (e.g., naked antibodies or antibody-drug conjugates that bind 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 therapies where the anti-CD73 molecules are administered before, after, or concurrently with chemotherapy.SELECTED DRAWING: None

Description

[Background technology]

[0001] CD73, or ecto-5'-nucleotidase (5'-NT), is ubiquitously expressed in several tissues. This protein is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage, has extracellular enzymatic activity, and plays a role in signal transduction. The primary function of CD73 is to convert extracellular nucleotides (e.g., 5'-AMP), which are generally cell-impermeable, into their corresponding nucleosides (e.g., adenosine), which can readily enter many cells. Adenosine production by CD73 dephosphorylation of AMP has been shown to regulate adenosine receptor association in many tissues, suggesting that adenosine functions in cytoprotection, cell growth, angiogenesis, and immunosuppression, as well as a role in tumorigenesis.

[0002] 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 has also been associated with tumor invasiveness, metastasis, and reduced patient survival. CD73 creates an immunosuppressive environment characterized by increased adenosine levels, which promotes cancer development and progression. In particular, CD73 expression has been associated with a prometastatic phenotype in melanoma and breast cancer. Summary of the Invention [Problem to be solved by the invention]

[0003] Immune checkpoint inhibitors hold great promise as cancer therapies. Despite this, clinical benefit from immune checkpoint inhibition has been limited. One possible explanation is that tumors use non-overlapping immunosuppressive mechanisms to promote immune escape. Therefore, improved compositions and methods for reducing tumor-mediated immunosuppression are urgently needed. [Means for solving the problem]

[0004] The present invention provides isolated binding molecules or antigen-binding fragments thereof that specifically bind to CD73. In some aspects, such CD73 binding molecules are, for example, antibodies or antigen-binding fragments thereof. In particular embodiments, anti-CD73 antibodies of the invention (e.g., MEDI9447) are useful for reducing tumor-mediated immunosuppression. Accordingly, the present invention also provides therapeutic combinations featuring anti-CD73 antibodies (e.g., MEDI9447) and other agents that target different 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 are useful for reducing tumor-mediated immunosuppression.

[0005] In one aspect, the invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to a CD73 epitope, wherein the binding molecule comprises a heavy chain variable region (V) of an antibody selected from CD730002, CD730003, CD730004, CD730008, CD730010, CD730011, CD730021, CD730042, CD730046, CD730047, or CD730058. H ) and the light chain variable region (V L ) specifically binds to the same CD73 epitope as an antibody or antigen-binding fragment thereof having

[0006] In another aspect, the present invention provides a method for the detection of antibodies that specifically bind to CD73 and are V of CD730002, CD730003, CD730004, CD730008, CD730010, CD730011, CD730021, CD730042, CD730046, CD730047, or CD730058. H and V L The present invention provides an isolated binding molecule or antigen-binding fragment thereof that competitively inhibits CD73 binding by an antibody or antigen-binding fragment thereof comprising:

[0007] In another embodiment, the present invention provides antibody V L and V Lis the amino acid sequence: [FW1]SGSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4] (Wherein, [FW1], [FW2], [FW3] and [FW4] are V L framework regions, wherein X1 represents the amino acid residue proline (P), glutamic acid (E), or aspartic acid (D), and 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); X5 represents the amino acid residue glycine (G) or serine (S); 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 and X8 represents the amino acid residue leucine (L) or threonine (T). In various embodiments of any aspect described herein, the isolated binding molecule or antigen-binding fragment thereof of claim 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.

[0008] In another aspect, the present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, comprising an antibody VH, H is the amino acid sequence: [FW5]SYAX9S[FW6]X 10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8] (wherein [FW5], [FW6], [FW7] and [FW8] represent VH framework regions, X9 represents the amino acid residue methionine (M) or tyrosine (Y); 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), X 13 represents the amino acid residue serine (S) or arginine (R), X 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 represents the amino acid residue tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0009] In another embodiment, the present invention provides antibody V L and antibody V H and V L is the amino acid sequence: [FW1]SGSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4] (Wherein, [FW1], [FW2], [FW3] and [FW4] are V L represents a framework region, where: 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); X3 represents the amino acid residue glutamine (Q) or leucine (L); X4 represents the amino acid residue leucine (L) or proline (P); X5 represents the amino acid residue glycine (G) or serine (S); 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), and V H is the amino acid sequence: [FW5]SYAX9S[FW6]X 10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8] (wherein [FW5], [FW6], [FW7] and [FW8] represent VH framework regions, X9 represents the amino acid residue methionine (M) or tyrosine (Y); 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), X 13 represents the amino acid residue serine (S) or arginine (R), X 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 represents the amino acid residue tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0010] In another embodiment, the present invention provides antibody V L and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: L is identical to SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 52, or is identical thereto except for 4, 3, 2 or 1 amino acid substitutions. L It has a complementarity determining region 2 (VL-CDR2) amino acid sequence.

[0011] In another embodiment, the present invention provides antibody V L and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: L has a complementarity determining region 3 (VL-CDR3) amino acid sequence that is identical to, or identical to, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, or SEQ ID NO:56 except for 4, 3, 2, or 1 amino acid substitutions.

[0012] In another embodiment, the present invention provides antibody V H and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: H has a complementarity determining region 1 (VH-CDR1) amino acid sequence that is identical to SEQ ID NO: 35 or SEQ ID NO: 36, or that is identical thereto except for 4, 3, 2, or 1 amino acid substitutions.

[0013] In another embodiment, the present invention provides antibody V H and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: H has 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 4, 3, 2, or 1 amino acid substitutions.

[0014] In another embodiment, the present invention provides antibody V H and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: Hhas 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 4, 3, 2, or 1 amino acid substitutions.

[0015] In another embodiment, the present invention provides antibody V L and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: L have 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 except for 4, 3, 2, or 1 amino acid substitutions in one or more of the VL-CDRs.

[0016] In another embodiment, the present invention provides antibody V H and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: H have 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 except for 4, 3, 2, or 1 amino acid substitution in one or more of the VH-CDRs.

[0017] In another aspect, the present invention provides 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: 46, 49, 53, 35, 37, and 45; SEQ ID NOs: 47, 49, 56, 3 SEQ ID NOs: 46, 50, 56, 36, 38, and 45; SEQ ID NOs: 46, 51, 56, 36, 39, and 45; SEQ ID NOs: 48, 52, 56, 36, 40, and 45; or VH-CDR1, VH-CDR2, VH-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences that are identical to SEQ ID NOs: 46, 49, 56, 35, 37, and 45, or identical thereto except for 4, 3, 2, or 1 amino acid substitution in one or more CDRs. L and V H The present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD73, having the following structure:

[0018] In another embodiment, the present invention provides antibody V L and antibody V H and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: L has an amino acid sequence that is at least about 90% to about 100% identical to a reference amino acid sequence selected from 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.

[0019] In another embodiment, the present invention provides antibody V L and antibody V H and an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, the isolated binding molecule or antigen-binding fragment thereof having: Hhas an amino acid 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.

[0020] In another aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD73, wherein the antibody or antigen-binding fragment has a sequence that is at least about 90% to about 100% identical to a reference amino acid sequence selected from 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. L and the antibody or antigen-binding fragment has a V 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. H It has.

[0021] In another embodiment, the present invention provides a V L and V consisting essentially of SEQ ID NO: 71 H and

[0022] In another embodiment, the present invention provides a V L and V consisting essentially of SEQ ID NO: 82 H and

[0023] In another aspect, the present invention provides a V L and V consisting of SEQ ID NO: 71 H and

[0024] In another aspect, the present invention provides a V L and V consisting of SEQ ID NO: 82 H and

[0025] In another aspect, the present invention provides a composition comprising the isolated antibody or antigen-binding fragment thereof of the present invention and a carrier.

[0026] In another aspect, the present invention provides a nucleic acid having a sequence encoding the isolated antibody or antigen-binding fragment thereof of the present invention.

[0027] In another aspect, the present invention provides a composition comprising a nucleic acid according to the present invention.

[0028] In another aspect, the present invention provides a vector containing the nucleic acid of the present invention.

[0029] In another aspect, the present invention provides a host cell comprising a nucleic acid sequence, composition or vector according to the present invention.

[0030] In another aspect, the present invention provides a method of making an antibody or antigen-binding fragment thereof according to the invention, the method comprising culturing a cell containing a nucleic acid sequence, composition, or vector according to the invention, and isolating the antibody or antigen-binding fragment thereof.

[0031] In another aspect, the present invention provides a diagnostic reagent comprising a labeled isolated antibody or antigen-binding fragment of the present invention.

[0032] In another aspect, the present invention provides a kit containing the isolated antibody or antigen-binding fragment thereof, composition, or diagnostic reagent of the present invention.

[0033] In another aspect, the invention provides a method of inhibiting the growth of a cell expressing CD73, comprising contacting the cell with an antibody or antigen-binding fragment thereof of the invention.

[0034] In another aspect, the invention provides a method of treating cancer in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the invention.

[0035] In another aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a first agent that is an antibody or antigen-binding fragment of the invention, in combination with a therapeutically effective amount of a second agent that is an anti-cancer agent other than the first agent.

[0036] In another aspect, the invention provides a method of treatment comprising administering an anti-CD73 antibody, or antigen-binding fragment thereof, to a subject identified as having a tumor with increased expression of CD73 relative to a reference.

[0037] In another aspect, the invention provides a method of treatment comprising administering to a subject identified as having a tumor with increased expression of CD73 compared to a reference an anti-CD73 antibody, or antigen-binding fragment thereof, and anti-PD-1, anti-PD-L1, or anti-CTLA4, or antigen-binding fragment thereof.

[0038] In another aspect, the present invention provides a method of treatment comprising administering MEDI9447 or Phen0203 hIgG1, or an antigen-binding fragment thereof, and pembrolizumab (Keytruda®) or nivolumab (Opdiva®), or an antigen-binding fragment thereof, to a subject identified as having a tumor with increased expression of CD73 compared to a reference.

[0039] In another aspect, the invention provides a method of treatment comprising 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 expression of CD73 compared to a reference.

[0040] In another aspect, the invention provides a method of treatment comprising 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 expression of CD73 compared to a reference.

[0041] In another aspect, the invention provides a method for identifying a subject having a cancer that is responsive to anti-CD73 therapy, the method comprising 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 as responsive to anti-CD73 therapy.

[0042] In another aspect, the invention provides a method for identifying a subject having a cancer that will be responsive to anti-CD73 therapy in combination with one or more of anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy, the method comprising detecting an increase in CD73 expression or activity levels in tumor cells or blood cells of the subject relative to a reference, thereby identifying the cancer as being responsive to anti-CD73 therapy in combination with one or more of anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

[0043] In another aspect, the invention provides a method for identifying a subject having a cancer that will respond to anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy, the method comprising detecting a decrease in CD73 expression or activity levels in the subject's tumor cells or blood cells relative to a reference, thereby identifying the cancer as responsive to anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

[0044] In another aspect, the present invention provides a method of inhibiting tumor growth in a subject, the method comprising administering to a subject in need thereof an anti-CD73 antibody, or antigen-binding fragment thereof, and one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or antigen-binding fragments thereof.

[0045] In another aspect, the present invention provides a method of increasing an anti-tumor immune response in a subject, the method comprising administering to a subject in need thereof an anti-CD73 antibody, or antigen-binding fragment thereof, and one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or antigen-binding fragments thereof.

[0046] In another aspect, the present invention provides a method of treating a tumor in a subject, the method comprising administering to a subject in need thereof an anti-CD73 antibody, or antigen-binding fragment thereof, and one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or antigen-binding fragments thereof.

[0047] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of an anti-CD73 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof.

[0048] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of MEDI9447 or an antigen-binding fragment thereof and pembrolizumab (Keytruda®) or an antigen-binding fragment thereof.

[0049] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of MEDI9447 or an antigen-binding fragment thereof and nivolumab (Opdiva®) or an antigen-binding fragment thereof.

[0050] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of Phen0203 hIgG1 or an antigen-binding fragment thereof and pembrolizumab (Keytruda®) or an antigen-binding fragment thereof.

[0051] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of Phen0203 hIgG1 or an antigen-binding fragment thereof and nivolumab (Opdiva®) or an antigen-binding fragment thereof.

[0052] In another aspect, the invention provides a pharmaceutical formulation containing an effective amount of an anti-CD73 antibody, or antigen-binding fragment thereof, and an anti-PD-L1 antibody, or antigen-binding fragment thereof.

[0053] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of MEDI9447, or an antigen-binding fragment thereof, and MEDI4736, or an antigen-binding fragment thereof.

[0054] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of Phen0203 hIgG1 or an antigen-binding fragment thereof and MEDI4736 or an antigen-binding fragment thereof.

[0055] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of an anti-CD73 antibody or antigen-binding fragment thereof and an anti-CTLA4 antibody or antigen-binding fragment thereof.

[0056] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of MEDI9447 or an antigen-binding fragment thereof and tremelimumab or an antigen-binding fragment thereof.

[0057] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of MEDI9447 or an antigen-binding fragment thereof and ipilimumab or an antigen-binding fragment thereof.

[0058] 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.

[0059] 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.

[0060] In another aspect, the present invention provides a kit for increasing anti-tumor activity, comprising an anti-CD73 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof.

[0061] In another aspect, the invention provides a kit for increasing anti-tumor activity, the kit comprising an anti-CD73 antibody, or antigen-binding fragment thereof, and an anti-PD-L1 antibody, or antigen-binding fragment thereof.

[0062] In another aspect, the present invention provides a kit for increasing anti-tumor activity, comprising an anti-CD73 antibody or antigen-binding fragment thereof and an anti-CTLA4 antibody or antigen-binding fragment thereof.

[0063] In various embodiments of any aspect described herein, the VL and VH of CD730002 are or include SEQ ID NOs: 1 and 2, respectively, and the VL and VH of CD730010 are or include SEQ ID NOs: 3 and 4, respectively.

[0064] In various embodiments of any aspect described herein, the isolated binding molecule or antigen-binding fragment thereof comprises an antibody or antigen-binding fragment thereof.

[0065] In various embodiments of any aspect described herein, the binding molecule is affinity matured.

[0066] In various embodiments of any aspect described herein, FW5 is or comprises SEQ ID NO: 31, FW6 is or comprises SEQ ID NO: 32, FW7 is or comprises SEQ ID NO: 33, and FW8 is or comprises SEQ ID NO: 34.

[0067] In various embodiments of any aspect described herein, FW1 is or comprises SEQ ID NO: 25 or 26, FW2 is or comprises SEQ ID NO: 27 or 28, FW3 is or comprises SEQ ID NO: 29, FW4 is or comprises SEQ ID NO: 30, FW5 is or comprises SEQ ID NO: 31, FW6 is or comprises SEQ ID NO: 32, FW7 is or comprises SEQ ID NO: 33, and FW8 is or comprises SEQ ID NO: 34.

[0068] In various embodiments of any aspect described herein, the VL comprises a VL complementarity determining region 1 (VL-CDR1) amino acid sequence identical to SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48, or identical thereto except for 4, 3, 2, or 1 amino acid substitutions.

[0069] In various embodiments of any aspect described 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.

[0070] In various embodiments of any aspect described 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.

[0071] In various embodiments of any aspect described herein, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain constant region or fragment thereof.

[0072] 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.

[0073] In various embodiments of any aspect described 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.

[0074] In various embodiments of any aspect described herein, the IgG constant region comprises one or more amino acid substitutions relative to a wild-type IgG constant region, and the modified IgG has an increased half-life relative to the half-life of an IgG comprising the wild-type IgG constant region.

[0075] In various embodiments of any aspect described herein, the IgG constant region has one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436 (numbering according to the EU index as defined in Kabat).

[0076] In various embodiments of any aspect delineated herein, the at least one IgG constant region amino acid substitution is (a) substitution of the amino acid at position 252 with tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T); (b) substitution of the amino acid at position 254 with threonine (T); (c) substitution of the amino acid at position 256 with serine (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), or threonine (T); (d) substitution of the amino acid at position 257 with leucine (L); (e) substitution of the amino acid at position 309 with proline (P); (f) substitution of the amino acid at position 311 with serine (S); (g) substitution of the amino acid at position 428 with threonine (T), leucine (L), phenylalanine (F), or serine (S); (h) substitution of the amino acid at position 433 with arginine (R), serine (S), isoleucine (I), proline (P), or glutamine (Q); (i) substitution of the amino acid at position 434 with tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F), or tyrosine; and (j) a combination of two or more of the above substitutions (numbering is according to the EU index as defined in Kabat).

[0077] 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: (a) the amino acid at position 252 is replaced by tyrosine (Y); (b) the amino acid at position 254 is replaced by threonine (T), and (c) The amino acid at position 256 is replaced by glutamic acid (E). (Numbering follows the EU index as defined in Kabat).

[0078] In various embodiments of any aspect described herein, the amino acid at position 434 is substituted with an amino acid selected from tryptophan (W), methionine (M), tyrosine (Y), and serine (S), where numbering is according to the EU index as defined in Kabat.

[0079] In various embodiments of any aspect described herein, the amino acid at position 428 is substituted with an amino acid selected from threonine (T), leucine (L), phenylalanine (F), and serine (S), where numbering is according to the EU index as defined in Kabat.

[0080] In various embodiments of any aspect described herein, the amino acid at position 257 is substituted with a leucine (L) and the amino acid at Kabat position 434 is substituted with a tyrosine (Y), where numbering is according to the EU index as defined in Kabat.

[0081] In various embodiments of any aspect described 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).

[0082] 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 (numbering according to the EU index as defined in Kabat), wherein: (a) the amino acid at position 252 is replaced by tyrosine (Y); (b) the amino acid at position 254 is replaced by threonine (T), and (c) The amino acid at position 256 is replaced by glutamic acid (E).

[0083] In various embodiments of any aspect described 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.

[0084] In various embodiments of any aspect described herein, the antigen-binding fragment is an Fv, Fab, F(ab')2, Fab', dsFv, scFv, or sc(Fv)2.

[0085] In various embodiments of any aspect described herein, the isolated antibody or antigen-binding fragment thereof is conjugated to at least one heterologous agent, including, for example, an anti-cancer agent.

[0086] In various embodiments of any aspect described herein, the compositions of the invention further comprise an anti-cancer agent.

[0087] In various embodiments of any aspect described herein, the isolated antibody or antigen-binding fragment thereof does not induce antibody-dependent cell-mediated cytotoxicity (ADCC).

[0088] In various embodiments of any aspect described herein, the isolated antibody or antigen-binding fragment thereof is an antagonist of CD73.

[0089] In various embodiments, the isolated antibody or antigen-binding fragment thereof is an antagonist of CD73 on a cell selected from MB-MDA-231, 4T1, MK1, or a combination of two or more of the listed cells.

[0090] In various embodiments of any aspect delineated herein, the CD73 is human CD73.

[0091] In various embodiments of any aspect described herein, the binding of the antibody or antigen-binding fragment to CD73 may reduce cell proliferation.

[0092] In various embodiments of any aspect described herein, the antibody or antigen-binding fragment against CD73 can bind to human CD73, cynomolgus monkey CD73, and mouse CD73.

[0093] In various embodiments of any aspect described 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.

[0094] In various embodiments of any aspect described herein, the cancer has a pre-metastatic phenotype, including melanoma or breast cancer.

[0095] In various embodiments of any aspect described herein, the subject is a human.

[0096] In various embodiments of any aspect described herein, the combination of the first agent and the second agent has superior anti-tumor activity, which may be additive or synergistic.

[0097] In various embodiments of any aspect described herein, the second agent is an antibody or antigen-binding fragment thereof.

[0098] 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).

[0099] In various embodiments of any aspect described herein, the second agent is an anti-CTLA-4 antibody or an antigen-binding fragment thereof, including, for example, ipilimumab, tremelimumab (ticilimumab, CP-675,206), or an antigen-binding fragment thereof.

[0100] In various embodiments of any aspect described herein, the second agent is an anti-PD-1 antibody or an antigen-binding fragment thereof, including, for example, pembrolizumab (Keytruda®, lambrolizumab, MK-3475), nivolumab (Opdiva®, BMS-936558, MDX-1106, ONO-4538), AMP-224, or an antigen-binding fragment thereof.

[0101] In various embodiments of any aspect described herein, the second agent is an anti-PD-L1 antibody or antigen-binding fragment thereof, including, for example, MEDI4736, BMS-936559, MPDL3280A, or an antigen-binding fragment thereof.

[0102] In various embodiments of any aspect delineated herein, the anti-CD73 antibody is MEDI9447, Phen0203 hIgG1, or an antigen-binding fragment thereof.

[0103] In various embodiments of any aspect described herein, the subject is receiving, has received, or will receive anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

[0104] In various embodiments of any aspect described herein, the anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy comprises administering an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, respectively, or an antigen-binding fragment thereof.

[0105] In various embodiments, the anti-PD-1 antibody is pembrolizumab (Keytruda®, lambrolizumab, MK-3475), nivolumab (Opdiva®, BMS-936558, MDX-1106, ONO-4538), AMP-224, or an antigen-binding fragment thereof.

[0106] In various embodiments, the anti-PD-L1 antibody is MEDI4736, BMS-936559, MPDL3280A, or an antigen-binding fragment thereof.

[0107] In various embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab (ticilimumab, CP-675,206), or an antigen-binding fragment thereof.

[0108] In various embodiments of any aspect described 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.

[0109] In various embodiments of any aspect described herein, CD73 expression or activity is detected in a tumor sample, a blood sample, or a lymph sample.

[0110] In various embodiments of any aspect described herein, CD73 expression is detected on tumor cells or peripheral blood cells, including, for example, lymphoid or myeloid cell subsets (i.e., one or more of B lymphocytes, CD4+, FoxP3+ lymphocytes, or myeloid-derived suppressor cells (MDSCs)).

[0111] In various embodiments of any aspect delineated herein, CD73 expression is detected by flow cytometry, immunohistochemistry (IHC), or CD73 enzyme activity or soluble CD73 levels in the sample.

[0112] In various embodiments of any aspect described 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.

[0113] In various embodiments of any aspect described herein, the method induces or increases a tumor-specific immune response.

[0114] In various embodiments of any aspect delineated herein, the method reduces the immunosuppressive effect of the AMP / CD73 / adenosine pathway.

[0115] In various embodiments of any aspect described herein, the tumor is a CD73-overexpressing tumor.

[0116] In another aspect, the invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds CD73, comprising an antibody VL and an antibody VH, which specifically binds to an epitope of the CD73 protein having one or more amino acids corresponding to Val144, Lys180, and Asn185.

[0117] In various embodiments of any aspect described herein, the isolated binding molecule or antigen binding further contains one or more amino acids corresponding to Tyr135, Lys136, and Asn187.

[0118] In various embodiments of any aspect described herein, the isolated binding molecule or antigen-binding fragment thereof contains amino acids corresponding to Tyr135, Lys136, and Asn187.

[0119] In various embodiments of any aspect described herein, the isolated binding molecule or antigen-binding fragment thereof contains Tyr135, Lys136, Asn187, and amino acids corresponding to Tyr135, Lys136, and Asn187.

[0120] In various embodiments of any aspect described herein, the isolated binding molecule or antigen-binding fragment thereof binds to an epitope in one or more of the following regions of a CD73 protein: Tyr132 to Val144 and / or Lys180 to Asn187.

[0121] In various embodiments of any aspect described herein, the isolated binding molecule or antigen-binding fragment thereof contains or consists of the amino acid sequence from Tyr132 to Val144 and / or Lys180 to Asn187.

[0122] In another aspect, the present invention provides a conformational epitope on the surface of CD73 protein having one or more amino acids corresponding to Val144, Lys180, and Asn185, which can be specifically bound by monoclonal antibody MEDI9447 or an antigen-binding fragment, variant, analog, or derivative thereof.

[0123] In various embodiments of any aspect described herein, the conformational epitope further contains one or more amino acids corresponding to Tyr135, Lys136, and Asn18.

[0124] In various embodiments of any aspect described herein, the conformational epitope contains amino acids corresponding to Tyr135, Lys136, and Asn187.

[0125] In various embodiments of any aspect described herein, the conformational epitope comprises amino acids corresponding to Tyr135, Lys136, Asn187, Tyr135, Lys136, and Asn187.

[0126] In various embodiments of any aspect described herein, the conformational epitope is in one or more of the following regions of the CD73 protein: Tyr132 to Val144 and / or Lys180 to Asn187.

[0127] In various embodiments of any aspect described herein, the conformational epitope contains or is located within the amino acid sequence Tyr132 to Val144 and / or Lys180 to Asn187.

[0128] In various embodiments of any aspect described herein, MEDI9447 binds to CD73 protein in an inactive or catalytically active state, or in an open or closed state.

[0129] In various embodiments of any aspect delineated herein, the CD73 protein is human CD73.

[0130] In various embodiments of any aspect described herein, the isolated binding molecule or antigen-binding fragment thereof, the VL and VH are the VL and VH of MED19447.

[0131] The compositions and articles defined by the present invention have been isolated or otherwise prepared in connection with the examples provided below. Other features and advantages of the present invention will be apparent from the detailed description, and from the claims. [Brief explanation of the drawings]

[0132] [Figure 1A] The nucleotide sequence and amino acid translation of the MEDI9447 VH domain are shown, with the CDRs indicated according to the Kabat numbering convention. [Figure 1B] The nucleotide sequence and amino acid translation of the MEDI9447 VL domain are shown, with the CDRs indicated according to the Kabat numbering convention. [Figure 1C] Alignment of MEDI9447 VH with the closest human VH and JH germline sequences is shown. CDRs are highlighted based on Kabat numbering, and residues that differ from the germline sequences are boxed. [Figure 1D] Alignment of MEDI9447 VL with the closest human VL and JL germline sequences is shown. CDRs are highlighted based on Kabat numbering, and residues that differ from the germline sequences are boxed. [Figure 2] Two graphs are provided showing antibody-mediated internalization of cytotoxic FabZAP reagents into MDA-MB-231 and 4T1 cells, where the antibody is MEDI9447 and the control antibody R347. [Figure 3] Figure 3A is a graph showing the inhibition of 5' ectonucleotidase by the anti-CD73 antibody MEDI9447, and Figure 3B is a graph showing the inhibition of AMP hydrolysis by the anti-CD73 antibody CD370010. [Figure 4] Figure 1 is a graph showing that MEDI9447 inhibited tumor growth in a CT26 syngeneic tumor model. Murine CT26 tumor cells were implanted subcutaneously into the right flank of female Balb / C mice. Tumors were allowed to grow for 3 days and then treated with MEDI9447 or isotype control twice weekly for 2 weeks. Tumors were harvested on day 16 and subjected to flow cytometry analysis. [Figure 5]

[0023] Figure 10 is a graph showing that MEDI9447 inhibited tumor-infiltrating myeloid-derived suppressor cells (MDSCs). On study day 16, MEDI9447-treated CT26 tumor-bearing mice were sacrificed and tumors were harvested. Tumors were dissociated into single cells, stained for CD45 and MDSC markers, and analyzed by flow cytometry. [Figure 6] Six spider plots are included to show the effect of MEDI9447 mIgG1, anti-PD-1, or the combination on tumor volume. Control antibodies included rIgG2a (a rat IgG2a control monoclonal rat antibody specific for Escherichia coli β-galactosidase (β-Gal)) and an isotype control mouse IgG1. Tumor volumes for each group of animals through day 40 of the study were plotted for individual animals. No mice in the control group were tumor-free by the end of the 40-day study period. Anti-CD73 treatment alone resulted in 10% of animals being tumor-free at the end of the study. Anti-PD1 treatment alone also resulted in 10% of animals being tumor-free at the end of the study. Notably, the combination of anti-CD73 and anti-PD-1 treatment resulted in 60% of mice being tumor-free. No mice in the control group were tumor-free by the end of the study. [Figure 7] Graph showing the effect of MEDI9447 mIgG1, anti-PD1 or combination on survival. [Figure 8] Figure 1 shows that the combination of MEDI9447 and anti-PD-1 significantly enhanced (<0.05) tumor growth inhibition of colorectal cancer tumors compared to either agent alone. Mice were injected subcutaneously with syngeneic MC38-OVA colorectal cancer cells and treated twice weekly with 10 mg / kg MEDI9447, 10 mg / kg anti-PD-1 antibody alone, or both antibodies in combination. Tumor volumes were measured twice weekly. [Figure 9] Figure 1 shows that anti-PD-1 induced 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 of anti-PD-1 or an irrelevant isotype control antibody. The day after the first treatment, tumors were excised, and cells were dissociated and analyzed for surface phenotype by flow cytometry. [Figure 10] Figure 1 shows that anti-PD-1 induced 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 of anti-PD-1 or an irrelevant isotype control antibody. The day after the first treatment, tumors were excised, tumor cells were isolated, and peripheral whole blood cells were collected and analyzed for surface CD73 expression by flow cytometry. [Figure 11] Figure 1 shows that anti-PD-1 induced a CD73-rich tumor microenvironment, as measured by CD73 expression on CD4+, FoxP3+ lymphocytes isolated from tumor-bearing mice. Mice (n=4) were injected subcutaneously with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg of anti-PD-1 or an irrelevant isotype control antibody. Three days after the initial treatment, tumors were excised, and peripheral whole blood cells were collected and analyzed for surface CD73 expression by flow cytometry. [Figure 12]Figure 1 shows that the combination of MEDI9447 and anti-PD-L1 significantly enhanced (<0.05) tumor growth inhibition of melanoma tumors compared to either agent alone. 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 alone, or both antibodies in combination. Tumor volumes were measured twice weekly. [Figure 13] Figure 1 shows that the combination of MEDI9447 and anti-PD-L1 significantly enhanced tumor growth inhibition (p<0.01) of lymphoma tumors compared to either agent alone. 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 alone, or both antibodies in combination. Tumor volumes were measured twice weekly. [Figure 14] Figure 1 shows that anti-PD-L1 induced 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 of anti-PD-L1 or an irrelevant isotype control antibody. The day after the first treatment, cells were isolated from the draining lymph nodes and analyzed for surface phenotype by flow cytometry. [Figure 15] Figure 1 shows that anti-PD-L1 induced a CD73-rich tumor microenvironment, as measured by surface expression of CD73 on tumor-infiltrating CD4+,FoxP3+ lymphocytes. Mice (n=4) were injected subcutaneously with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg of anti-PD-L1 or an irrelevant isotype control antibody. Three days after the first treatment, tumors were excised, and cells were dissociated and analyzed for surface phenotype by flow cytometry. [Figure 16]Figure 1 shows that MEDI9447 alone or in combination with anti-PD-L1 reduced CD73 expression on tumor-infiltrating lymphoid cells. Mice bearing colorectal CT26 syngeneic tumors were treated twice weekly (days 12 and 16) with either 30 mg / kg MEDI9447 or 30 mg / kg anti-PD-L1 alone, or a combination of both MEDI9447 and anti-PD-L1. On day 17, tumors were harvested and analyzed for surface CD73 expression by flow cytometry. CD73 expression on tumors is correlated with (A) CD4+ FoxP3+ Tregs and (B) CD8+ T cells. [Figure 17] Figure 1 shows that MEDI9447 alone or in combination with anti-PD-L1 reduced CD73 activity in (A) tumor cells and (B) peripheral whole blood cells. Mice bearing colorectal CT26 syngeneic tumors were treated twice weekly (days 12 and 16) with either 30 mg / kg MEDI9447 or 30 mg / kg anti-PD-L1 alone, or a combination of both MEDI9447 and anti-PD-L1. On day 17, tumor and peripheral whole blood cells were harvested and analyzed for surface CD73 expression and enzyme activity using Cell-Titre Glo. [Figure 18] This set of graphs shows the cytokine profiles of peripheral blood mononuclear cells (PBMCs) treated with MEDI9447 and antibodies or fusion proteins specific for CTLA4, OX40, PD-1, and PD-L1. Primary human PBMCs were incubated in a mixed leukocyte reaction (MLE) with MEDI9447 and / or antibodies or fusion proteins specific for the indicated targets for 72 hours. Cytokines (IFN-γ, IL-1β, TNF-α) in duplicate supernatants were quantified by ELISA. Data shown represent optimal dose combinations of anti-CD73 antibodies with four different partner agents. The combination of anti-PD-1 and anti-CD73 demonstrated significant (p<0.05) synergy as determined by the Bliss surface reaction assay (Zhao et al.). The cytokine profiles indicate that both myeloid and lymphoid lineages were affected. More than 50 donor pairs were tested. [Figure 19A]Figure 19A shows the results of hydrogen-deuterium exchange MS (HDX-MS) analysis of CD73 complexed with MEDI9447. Figure 19A shows a hydrogen-deuterium exchange heat map depicting regions of CD73 (N- to C-terminus) that undergo a decrease in deuterium uptake upon binding to MEDI9447. The relative exchange between antibody-bound and unbound CD73 as a function of exposure time is shown in red for decreased exchange, blue for increased exchange, and white for unchanged exchange. The N-terminal regions at positions 132-143 and 182-187 exhibited the highest degree of differential exchange. Figure 19B shows the crystal structure of the CD73 monomer, depicting the location of the binding interface (cyan) identified by HDX within the N-terminal domain (yellow). The CD73 linker region and C-terminal domain are shown in orange and blue, respectively. [Figure 19B] This is a continuation of Figure 19A. [Figure 20A]Figure 20 shows the results of hydrogen-deuterium exchange MS (HDX-MS) analysis demonstrating regions of differential hydrogen exchange between free and bound CD73 and MEDI9447. Figure 20A shows a plot of relative deuterium uptake (mass change in Daltons) as a function of deuterium exposure time within peptides encompassing the 132-143 region. Figure 20B shows a plot of relative deuterium uptake (mass change in Daltons) as a function of deuterium exposure time within peptides encompassing the 182-187 region. In Figures 20A and 20B, uptake of CD73 alone is indicated by a square, and uptake of CD73 bound to MEDI9447 Fab is indicated by a red color. Peptide sequences, positions, and masses are shown within the plot boxes. Relative mass changes of overlapping peptides were compared to narrow down the regions containing sequences exhibiting altered hydrogen exchange and that may be expected to form epitopes. For example, peptides spanning positions 173-186 showed differential exchange, but peptides spanning positions 173-181 did not. Therefore, it was inferred that residues upstream of 182 were not differentially labeled. Figure 20C shows a DynamX difference chart for the MEDI9447 Fab heavy chain. Figure 20D shows a DynamX difference chart for the MEDI9447 Fab light chain. For Figures 20C and 20D, each data point represents 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). Vertical bars represent the sum of the uptake differences across exposure time points. CDRs that show a decrease in relative uptake when the Fab is bound to CD73 are indicated. Figure 20E shows a DynamX difference chart for CD73 alone (negative values ​​on the y-axis) and CD73 bound to the 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). Overlaid on this chart is a dotted line indicating 1.6 daltons, the 98% confidence interval cutoff for statistically significant changes. [Figure 20B] This is a continuation of Figure 20A. [Figure 20C-E] This is a continuation of Figure 20A. [Figure 21A-D]Figure 21A shows sensor chip data indicating that the MEDI9447 epitope is located within the N-terminal domain of CD73. Figure 21A is a graph showing 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 is a graph showing sensor chip data for CD73 proteins with swapped N-terminal domains. CD73 proteins with swapped N-terminal domains 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 the N-terminal domain was swapped. Figure 21C is a graph showing sensor chip data for CD73 proteins with swapped N- and C-terminal domains. CD73 proteins with swapped N- and C-terminal domains were immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. When both the N- and C-terminal domains were swapped, MEDI9447 did not bind to CD73. Figure 21D shows sensor chip data for CD73 proteins with swapped linker domains. CD73 proteins with swapped linker domains were immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. Swapping only the linker domain did not affect binding. Figure 21E shows sensor chip data for CD73 proteins with swapped C-terminal domains. CD73 proteins with swapped C-terminal domains were immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM to 0.3 nM) was measured by SPR. Swapping only the C-terminal domain did not affect binding. Figure 21F shows the sensor chip data for CD73 proteins with swapped interface E1 (aa 132-143). CD73 proteins with swapped interface E1 (aa 132-143) were immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM-0.3 nM) was measured by SPR.Figure 21G is a graph showing sensor chip data for CD73 protein with swapped interface E2 (aa182-187). CD73 protein with swapped interface E2 (aa182-187) was immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM-0.3 nM) was measured by SPR. Figure 21H is a graph showing sensor chip data for CD73 protein with swapped interface E1 (aa132-143) and interface E2 (aa182-187). CD73 protein with swapped interface E1 (aa132-143) and interface E2 (aa182-187) was immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5 nM-0.3 nM) was measured by SPR. For Figures 21F-H, swapping HDX interface E1 (aa 132-143) (Figure 21F) had little effect on binding, in contrast to swapping HDX interface E2 (aa 182-187) alone (Figure 21G) or in combination with E1 (Figure 21H). For Figures 21A-H, sensorgrams and overlay fits are shown in corresponding colors. Kinetic measurements for each binding analysis are provided in Table 16. [Figure 21E-H] Continuation of Figures 21A-D. [Figure 22-1] Figure 1 shows an alignment of human and chicken CD73 protein sequences. Only the mature protein sequence is shown. Non-conserved residues are highlighted in the chicken sequence. Regions swapped between chicken and human to create knockout mutants are annotated (e.g., DS1a, DS1b, etc.). [Figure 22-2] This is a continuation of Figure 22-1. [Figure 22-3] This is a continuation of Figure 22-1. [Figure 22-4] This is a continuation of Figure 22-1. [Figure 22-5] This is a continuation of Figure 22-1. [Figure 23]Figure 23 shows the binding of MEDI9447 to CD73 mutants. Figure 23 is a table of data showing the binding of MEDI9447 to CD73 mutants. The KD of mutants highlighted in blue is greater than 2-fold changed from the WT or KO parental construct. *Kinetic measurements obtained from a 2:1 heterologous ligand fit. **Numbering corresponds to chicken sequence (129=133, 140=144, and 181=185 in human). [Figure 24A-B] The MEDI9447 epitope is located at the apical end of the N-terminal domain. Figure 13A shows that evaluation of MEDI9447 binding to a panel of CD73 mutants (see Figures 22 and 23) revealed six positions that constitute the interaction site. Two of the three most influential residues (highlighted in boxes) are located outside the HDX interface region (highlighted in blue). The three less influential residues (pink boxes) are located within the HDX interface. Figure 24B is a table showing that knocking in N185 and V144 (K180 is conserved) into a CD73 construct containing chicken N- and C-terminal domain sequences restored binding to less than 20-fold the KD of wild-type CD73 (MEDI9447 dilutions 5 nM to 0.3 nM; compare with Figure 10B). Figure 24C shows a detailed view of the epitope residues located within the N-terminal domain of CD73. The residues most important for binding are highlighted, while less influential positions (Y135, K136, and N187) are colored pink. The HDX interface is overlaid in blue. Figure 24D shows a surface representation demonstrating that the epitope forms a nearly continuous binding surface. Figure 24E shows the crystal structure of the open conformation of CD73, showing the location of the epitope on the apical outer surface of the N-terminal domain. Figure 24F shows the location of the epitope distant from the substrate binding site (adenosine represented as a sphere) and the zinc ion (gray sphere) coordination site (cyan side chain). In both crystal structures, the CD73 N-terminal domain, linker region, and C-terminal domain are colored yellow, orange, and blue, respectively. [Figure 24C-D] This is a continuation of Figures 24A-B. [Figure 24E-F] This is a continuation of Figures 24A-B. [Figure 25] Figure 25A shows that MEDI9447 is a noncompetitive inhibitor of AMP hydrolysis by CD73. Figure 25A is a graph showing the reaction rate of AMP phosphate hydrolysis by CD73 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 by inhibiting hydrolysis equally regardless of substrate concentration. In contrast, APCP, a known competitive inhibitor of CD73, increases Km but not Vmax. Figure 25C is a graph showing the dose response of MEDI9447 IgG, Fab, or control IgG for inhibition of AMP hydrolysis by CD73. MEDI9447 IgG reached maximal inhibition at a 1:1 molar stoichiometry with CD73 dimer (arrow). At higher concentrations where MEDI9447 IgG was in excess (>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 luminescence units). [Figure 26-1]Figure 26A shows that binding of anti-CD73 mAb (clone 0069) depends on residues in the CD73 N- and C-terminal domains. Figure 26A is a graph showing sensorchip 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 CD7 (KO_1-291, green sensorgram), and C-terminal domain-swapped knockout CD7 (KO_311-523, cyan sensorgram) demonstrates that mAb binding is influenced by residues in both the N- and C-terminal domains. Figure 26B shows the crystal structures of open and closed CD73 (N-terminal domain in yellow, linker in orange, and C-terminal domain in blue) showing the location of highlighted mAb A binding hotspots (aa 114-134 and 153-170) located near the N-terminal and C-terminal domain interface. Mapping was based on the binding data in Figures 26A and 26C. Figure 26C shows binding sensorgrams of mAb A to various domain-swap knockout mutants of CD73. Swapping subregions 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. [Figure 26-2] This is a continuation of Figure 26-1. [Figure 26-3] This is a continuation of Figure 26-1. [Figure 27A-B]This shows that MEDI9447 inhibited the transition of CD73 to a conformationally active structure. Figure 27A is a graph showing wild-type CD73 biosensor data. 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 Octet QK384. When CD73 was preincubated with Zn2+ and APCP, MEDI9447 retained binding (black sensorgram), but mAb A binding was abolished (orange sensorgram). Figure 27B is a graph showing that preincubation of CD73 with Zn2+ and APCP resulted in a loss of mAb A binding (orange sensorgram), but preincubation with MEDI9447 before addition of Zn2+ and APCP restored binding (purple sensorgram). Binding of mAb A to CD73 alone and to CD73 preincubated with MEDI9447 (but not Zn2+ or APCP) is shown in blue and brown sensorgrams, respectively. Figure 27C shows a proposed model of how MEDI9447 prevents CD73 from adopting the fully closed, active conformation induced by Zn2+ and APCP. MEDI9447 may restrict the transition to an intermediate state with low affinity for mAb A. [Figure 27C] This is a continuation of Figures 27A-B. [Figure 28]Figure 28A shows the binding of MEDI9447 or mAb A to CD73 under various conditions measured by BLI as described herein unless otherwise noted below. Figure 28A 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 incubated with Zn2+, APCP, and / or EDTA for 900 seconds, and then the biosensor was incubated with 30 nM mAb A for 600 seconds and binding was measured. mAb A bound to CD73 (blue sensorgram) but not to CD73 preincubated with Zn2+ and APCP (purple sensorgram). mAb A maintained binding to CD73 preincubated with APCP and EDTA (green sensorgram) or Zn2+, APCP, and EDTA (gold sensorgram). The chelating effect of EDTA indicates that divalent cations were required for the loss of mAb A binding when CD73 was incubated with Zn2+ and APCP. Figure 28B is a graph showing that MEDI9447 Fab or control IgG did not rescue mAb A binding to CD73 incubated with Zn2+ and APCP. The assay was performed as in Figure 27B. MEDI9447 Fab or isotype-matched control IgG was preincubated with CD73 before the addition of Zn2+ and APCP. mAb A immobilized on the biosensor bound to CD73 preincubated with either CD73 alone (blue sensorgram), MEDI9447 Fab (light blue sensorgram), or control IgG (black sensorgram), but not to CD73 incubated with Zn2+ and APCP (brown sensorgram), or to either Fab (gold sensorgram) or control IgG (purple sensorgram) preincubated with CD73 before the addition of Zn2+ and APCP. [Figure 29-1]Figure 29A shows that anti-CD73 mAb B binding depends on residues in the subregion DS2b (aa 92-134) or DS2c (aa 114-134). Figure 29A is a table showing the SEC-MALS data corresponding to Figures 30A-30C. For each mixture of CD73 with either MEDI9447 or mAb B, the corresponding SEC retention time, Mw, and polydispersity of the complex formed are shown. Figure 29B shows the determination of the binding hotspot of mAb B for CD73. Binding of mAb B to CD73 mutants immobilized on a HIS2 biosensor was measured by BLI as described for mAb A (clone 0069) in Figures 26A-26C according to the methods herein. The binding sensorgrams showed that swapping either the subregion DS2b (aa 92-134) or DS2c (aa 114-134) knocked out binding by mAb B. [Figure 29-2] This is a continuation of Figure 29-1. [Figure 29-3] This is a continuation of Figure 29-1. [Figure 29-4] This is a continuation of Figure 29-1. [Figure 30-1]Figure 30A shows that MEDI9447 forms interdimer bridges between soluble CD73 molecules. CD73 was incubated with various amounts of MEDI9447 or anti-CD73 mAb B and analyzed by SEC-MALS. SEC UV chromatograms are shown with protein retention time on the x-axis and molar mass determined by MALS on the y-axis. Figure 30B shows a chromatogram showing that MEDI9447 formed complexes with CD73 of approximately 1.7 (^) and approximately 6.6 (+) megadaltons at a 1:1 molar ratio (green trace). Complexes of comparable size were formed at lower ratios of MEDI9447:CD73 (0.5:1 blue, 0.1:1 magenta). MEDI9447 and CD73 alone are represented by the black and red UV traces, respectively. Figure 30B is a top-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 in an open conformation at a site close to the central groove between dimers. Figure 30C is a chromatogram showing that when CD73 bound to mAb B, a single predominant complex of approximately 270-290 kD (peak approximately 7.2 min) was formed. 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 in magenta and black, respectively. [Figure 30-2] This is a continuation of Figure 30-1. [Figure 31A-B]Figure 31A shows that surface-bound CD73 was inhibited by MEDI9447 in IgG and Fab formats. Figure 31A is a graph depicting the inhibition of AMP hydrolysis by immobilized CD73 by MEDI9447 IgG, Fab, or control antibody. CD73 was immobilized to a nickel-coated microtiter plate via a C-terminal histidine tag, and inhibition of AMP hydrolysis by MEDI9447 IgG, Fab, or control antibody was measured using the malachite green assay as described herein. MEDI9447 IgG inhibited AMP hydrolysis by CD73 in a dose-dependent manner, whereas control IgG did not. MEDI9447 Fab also inhibited CD73 activity, but to a much lesser extent. Figure 31B shows a complex containing MEDI9447 Fab (green) bound to an anti-Fd antibody (xFd, red). When MEDI9447 Fab (green) was bound to one arm of an anti-Fd antibody (xFd, red) and the other arm to a nonspecific polyclonal Fab (pFab, orange), inhibition increased to the same extent as MEDI9447 IgG (Fab + xFd + pFab vs. MEDI9447 IgG and MEDI9947 IgG + xFd + pFab) (see Figure 31A). Figure 31C is a graph showing 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 measured by 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 complexing it with anti-Fd antibody enhanced inhibition. Figure 31D is a graph showing the 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, binding of MEDI9947 Fab with both xFd arms (MEDI9447 Fab+xFd) conferred bivalency and resulted in CD73 inhibition. [Fig. 31C-D] This is a continuation of Figures 31A-B. [Figure 32] Figure 1 is a graph showing that MEDI94447 IgG and Fab inhibited AMP hydrolysis by CD73. 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, i.e., loss of inhibition, was observed. MEDI9447 Fab also inhibited CD73 function, but the maximal level of inhibition was lower. This experiment was performed twice with comparable results. Only data from one experiment is shown. [Figure 33] Figure 1 shows a model demonstrating that inhibition of CD73 hydrolytic activity by MEDI9447 occurs via a dual mechanism. MEDI9447 IgG (green) inhibits soluble CD73 by forming interdimer crosslinks and preventing the conformational transition to the closed state. Monovalently bound IgG or Fab do not inhibit soluble CD73. When CD73 is surface-bound, inhibition can occur by crosslinking of adjacent CD73 dimers or by steric blocking from monovalently bound IgG or Fab / xFd (red) complexes. DETAILED DESCRIPTION OF THE INVENTION

[0133] The present invention provides isolated binding molecules or antigen-binding fragments thereof that specifically bind to CD73. In some embodiments, such molecules are antibodies and antigen-binding fragments thereof that specifically bind to CD73. Related polynucleotides, vectors, and pharmaceutical compositions comprising anti-CD73 antibodies or antigen-binding fragments thereof are also provided. Also provided are methods of making and using the anti-CD73 antibodies and antigen-binding fragments disclosed herein, e.g., diagnostic methods and methods of treating cancer in a subject (as direct therapy, adjuvant therapy, or in combination therapy). The present invention also provides antibody-drug conjugates derived from the CD73 binding molecules disclosed herein. Furthermore, the present invention provides therapeutic combinations featuring an anti-CD73 antibody (e.g., MEDI9447) and one or more agents that target different aspects of the cancer immune cycle, such as anti-PD-1 antibodies, anti-PD-L1 antibodies (e.g., MEDI4736), or anti-CTLA4 antibodies; and methods of using such combinations to reduce tumor-mediated immunosuppression.

[0134] In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0135] I. Definition Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or method steps, which may vary. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and "one or more" and "at least one" can be used interchangeably herein.

[0136] Furthermore, "and / or," when used herein, should be construed as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or," when 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). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: 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).

[0137] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. 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, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0138] Units, prefixes, and symbols are shown in their International System of Units (SI) accepted format. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects that may be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0139] Whenever embodiments are described herein using the term "comprising," it is understood that otherwise similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0140] Amino acids may be 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. Nucleotides may also be referred to by their commonly accepted single-letter codes.

[0141] The term "CD73 polypeptide," as used herein, 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, e.g., Misumi et al. Eur. J. Biochem. 191(3):563-9 (1990). The sequences of human and mouse 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, not including the signal sequence residues. Thus, for example, an antibody that binds to amino acids Val144, Lys180, and Asn185 refers to the amino acid positions after cleavage of the signal sequence, i.e., the mature protein.

[0142] Exemplary CD73 polypeptides are provided below. >sp|P21589|5NTD_Human 5'-nucleotidase OS=Human (Homo sapiens) GN=NT5E PE=1 SV=1 [ka]

[0143] 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 contains 574 amino acids. The mature CD73 protein is produced after removal of the signal sequence (positions 1-26) and the C-terminal region of the propeptide (positions 550-574). In addition, CD73 isoform 2 has amino acids 404-453 removed after alternative splicing. Naturally occurring variants, such as the C358Y variant, the T376A variant, and the M379T variant, are also known. 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. 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 entireties).

[0144] Typical diseases that lead to changes in CD73 levels in tissue fluids, particularly serum, of patients include 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 trauma or reperfusion injury described above, or by chronic conditions, including allergic conditions, autoimmune diseases, and inflammatory diseases. Examples of such chronic conditions include allergic conditions such as arthritis and asthma, inflammatory conditions such as inflammatory bowel disease or inflammatory skin conditions, psoriasis, Parkinson's disease, Alzheimer's disease, autoimmune diseases, type I or II diabetes, atherosclerosis, multiple sclerosis, Crohn's disease, or rejection due to organ transplantation. In particular, inflammatory diseases such as systemic inflammatory response syndrome (SIRS), acute lung injury (ALI), multiple organ failure (MOF), ischemia-reperfusion injury (IRI), and adverse drug reactions (ADRS) lead to changes in tissue fluid CD73 protein levels. 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 CD73 levels in patient-derived samples (e.g., tissue fluids) using the CD73 binding molecules disclosed herein can be used to monitor the development of the aforementioned diseases, assess the effectiveness of therapy, select patients for treatment with a particular therapy, or make medical decisions, such as initiating, terminating, discontinuing, or modifying a particular treatment.

[0145] The terms "inhibit," "block," "suppress," and grammatical variations thereof are used interchangeably herein and refer to any statistically significant decrease in biological activity, including complete blocking of activity. For example, "inhibition" can refer to about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in biological activity. Thus, for example, when the terms "inhibition" or "suppression" are used to describe the 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 decrease the 5'-nucleotidase activity of CD73 (catabolizing the hydrolysis of adenosine monophosphate, AMP, to adenosine) compared to the CD73-mediated 5'-nucleotidase activity of untreated (control) cells. The CD73-expressing cells 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 embodiments, 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 embodiment, the anti-CD73 binding molecule, e.g., the 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 methods known in the art.

[0146] The term "inhibits CD73 activity," as used herein, refers to the ability of an anti-CD73 binding molecule, e.g., an antibody or antigen-binding fragment thereof, to statistically significantly reduce CD73-dependent 5'-nucleotidase activity in cells expressing or containing CD73. In some embodiments, 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 of the disclosure, e.g., an antibody or antigen-binding fragment thereof, compared to CD73 activity measured in the absence of the anti-CD73 binding molecule, e.g., an antibody or antigen-binding fragment thereof (control conditions).

[0147] The terms "antibody" or "immunoglobulin," as used interchangeably herein, include whole antibodies and any antigen-binding fragment or single chain thereof.

[0148] A typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as VH or V H Each light chain comprises a light chain variable region (herein abbreviated as VL or VL) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. LThe heavy and light chain variable regions contain a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (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, ADCC-deficient optimized antibodies, conjugated antibodies (e.g., ADCs), and other optimized antibodies (e.g., serum half-life optimized antibodies, e.g., YTE mutations, see Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006) and U.S. Pat. No. 7,083,784, which are hereby incorporated by reference in their entireties).

[0149] The term "germlining" refers to backmutating amino acids at specific positions in an antibody to those in the germline.

[0150] The term "antibody" refers to 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 foregoing, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigenic determinant of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibody exhibits the desired biological activity.

[0151] Antibodies can be of any of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated α, δ, ε, γ, and μ, respectively. Different immunoglobulin classes have different 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.

[0152] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds, such as CD73. In certain embodiments, a blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen. Desirably, 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%.

[0153] The terms "CD73 antibody," "antibody that binds to CD73," or "anti-CD73" refer to an antibody or antigen-binding fragment thereof that has the ability to bind to CD73 with sufficient affinity so that the molecule is useful as a therapeutic or diagnostic reagent in targeting CD73. The extent of binding of an anti-CD73 antibody to an unrelated, non-CD73 protein is less than about 10% of the binding of the antibody to CD73 as measured, for example, by radioimmunoassay (RIA), BIACORE™ (using recombinant CD73 as the analyte and the antibody as the ligand, or vice versa), or other binding assays known in the art. In certain embodiments, an antibody that binds to CD73 has a dissociation constant (K D ) is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦10 pM, ≦1 pM, or ≦0.1 pM. The term "anti-CD73" also broadly encompasses molecules comprising the CDRs of an antibody disclosed herein, for example, incorporated into a scaffold. Thus, the phrase "an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73" can refer not only to antibodies and antigen-binding fragments thereof, but also to molecules comprising, for example, one or more scaffolds (such as a fibronectin type III domain from fibronectin or tenascin-3) incorporating the CDRs of an antibody disclosed herein. See, e.g., U.S. Patent Application Publication No. 20150098955 (incorporated herein by reference in its entirety).

[0154] In one embodiment, the anti-CD73 antibody refers to an antibody in an IgG1-TM format in which the IgG1 Fc domain contains the mutations L234, L235E, and P331, and binds to soluble CD73 and cell surface-displayed CD73 and inhibits CD73 enzymatic activity. Figures 1A-1D provide the nucleotide and amino acid sequences of the MEDI9447 VH and VL domains.

[0155] 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, that has T cell inhibitory activity. The sequence of AAL07473.1 is provided below. CTLA4 polypeptide sequence [Human (Homo sapiens)] gi|15778586|gb|AAL07473.1|AF414120_1 [ka]

[0156] By "CTLA4 nucleic acid molecule" is meant a polynucleotide that encodes a CTLA4 polypeptide. An exemplary CTLA4 nucleic acid molecule sequence is provided in GenBank Accession No. AAL07473.

[0157] "Anti-CTLA4 antibody" refers to 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 is provided in the sequence listing herein below (SEQ ID NOs: 130-137).

[0158] By "PD-1 polypeptide" is meant a polypeptide or fragment thereof that has at least about 85% amino acid identity to NCBI Accession No. NP_005009 and that has PD-L1 and / or PD-L2 binding activity. The sequence of NP_005009 is provided below. PD-1 polypeptide sequence NCBI accession number NP_005009 [ka]

[0159] By "PD-1 nucleic acid molecule" is meant a polynucleotide that encodes a PD-1 polypeptide. An exemplary PD-1 nucleic acid molecule sequence is provided in NCBI Accession No. NM_005018.

[0160] "Anti-PD-1 antibody" refers to an antibody or antigen-binding fragment thereof that selectively binds to a PD-1 polypeptide. Exemplary anti-PD-1 antibodies include, for example, pembrolizumab (KEYTRUDA®, lambrolizumab, MK-3475), nivolumab (OPDIVA®, BMS-936558, MDX-1106, ONO-4538), or AMP-224.

[0161] By "PD-L1 polypeptide" is meant a polypeptide or fragment thereof that has at least about 85%, 95%, or 100% amino acid identity to NCBI Accession No. NP_001254635 and that has PD-1 and CD80 binding activity. The sequence of NP_001254635 is provided below. PD-L1 polypeptide sequence NCBI accession number NP_001254635 [ka]

[0162] By "PD-L1 nucleic acid molecule" is meant a polynucleotide that encodes a PD-L1 polypeptide. An exemplary PD-L1 nucleic acid molecule sequence is provided in NCBI Accession No. NM_001267706.

[0163] The term "anti-PD-L1 antibody" refers to 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 U.S. Patent Application Publication No. 20130034559 / U.S. Patent No. 8,779,108 and U.S. Patent Application Publication No. 20140356353, which are incorporated by reference herein. MEDI4736 is an exemplary PD-L1 antibody. The sequence of MEDI4736 is provided in the Sequence Listing herein below (SEQ ID NOS: 138-145).

[0164] The term "antigen-binding fragment" refers to a molecule comprising a portion of an intact antibody, particularly a molecule comprising 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 fragments 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.

[0165] A "monoclonal antibody" refers to a homogeneous antibody population that is responsible for the 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.

[0166] The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain variable fragments (scFv), fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies produced by any method, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals (e.g., expression of human antibodies in transgenic mice).

[0167] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that has been modified to contain minimal non-human (e.g., mouse) sequences. Typically, humanized antibodies are human immunoglobulins in which CDR residues have been replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and capacity (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, framework amino acid residues of the human immunoglobulin are replaced by the corresponding residues of an antibody from a non-human species having the desired specificity, affinity, and / or capacity.

[0168] Humanized antibodies can be further modified by substitution of additional residues within the Fv framework regions and / or the replaced non-human residues to refine and optimize the antibody's specificity, affinity, and / or capacity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to make humanized antibodies are described in U.S. Patent Nos. 5,225,539 or 5,639,641.

[0169] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four FW regions connected by three CDR regions. The CDRs of each chain are held together in close proximity by the FW regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) a method based on interspecies sequence diversity (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)). In addition, a combination of these two methods is sometimes used in the art to determine CDRs.

[0170] The Kabat numbering system is generally used when referring to variable domain residues (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0171] The phrases "amino acid position numbering as in Kabat," "Kabat position," and grammatical variations thereof refer to the numbering system used for heavy or light chain variable domains of antibody sequences in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FW or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.).

[0172] [Table 1]

[0173] The Kabat numbering of residues can be determined for a given antibody by aligning the antibody's sequence with the "standard" Kabat-numbered sequence at the homologous region. Chothia instead refers to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, 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 region represents a compromise between the Kabat CDRs and the Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.

[0174] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, e.g., 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 of over 5,000 sequences, structural data, and characterization of hypervariable loops, allowing for easy comparison of variable and CDR regions across species. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 is located at positions 51-57, VH-CDR3 is located at positions 93-102, VL-CDR1 is located at positions 27-32, VL-CDR2 is located at positions 50-52, and VL-CDR3 is located at positions 89-97.

[0175] The EU index or EU numbering system is based on the sequential numbering of the first human IgG (EU antibody) to be sequenced. The most common reference for this convention is the Kabat sequence manual (Kabat et al., 1991), and therefore the EU index is sometimes incorrectly used synonymously with the Kabat index. The EU index does not account for insertions and deletions, and therefore comparison of IgG positions between IgG subclasses and species can sometimes be uncertain, particularly in the hinge region. Nevertheless, this convention has been sufficient to allow direct comparisons between Fc regions in many Fc structure-function studies. Thus, the numbering scheme used herein for substitutions and insertions in the Fc region is the EU index as in Kabat. In contrast, the numbering scheme used herein for the variable regions (VH and VL) is the standard Kabat numbering.

[0176] As used throughout this specification, the VH CDR sequences described correspond to 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 classical Kabat numbering positions, i.e., positions 24-34, 50-56, and 89-97.

[0177] As used herein, the Fc region includes the polypeptides 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, as well as the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes immunoglobulin domains C gamma 2 and C gamma 3 (C gamma 2 and C gamma 3) and the hinge between C gamma 1 (C gamma 1) and C gamma 2 (C gamma 2).

[0178] Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus (where numbering is according to the EU index as defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991))). Fc may refer to this region independently 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 when numbered by the EU index; therefore, slight differences between the presented sequence and prior art sequences may exist.

[0179] 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, made using any technique known in the art (e.g., recombinant expression in cells in culture or expression in a transgenic animal). Thus, the term human antibody also encompasses antibodies that are originally produced by humans (or modified variants or derivatives thereof), but that have an amino acid sequence corresponding to an antibody expressed in a non-human system (e.g., produced by chemical synthesis; recombinantly expressed in microbial, mammalian, or insect cells; or expressed in an animal subject). Thus, an antibody obtained from a human subject or human cells (e.g., a hybridoma or cell line expressing a recombinant antibody or fragment thereof) and subsequently expressed in an animal, such as a mouse, is considered a human antibody. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide, such as an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0180] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from more than one animal species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and / or capacity, while the constant regions are homologous to the sequences of antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0181] The term "epitope," as used herein, refers to an antigenic protein determinant capable of binding to a CD73 antibody or CD73-binding molecule disclosed herein. Epitopes typically consist 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 an antibody or binding molecule that recognizes an epitope is called a paratope. Epitopes of protein antigens are classified into two types: conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational epitopes are composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the antigen's three-dimensional surface features and shape or tertiary structure. In contrast, linear epitopes interact with the paratope based on its primary structure. Linear epitopes are formed by the continuous amino acid sequence of the antigen.

[0182] The term "antibody combining site" refers to a region within an antigen (e.g., CD73) that comprises a continuous or discontinuous site (i.e., epitope) to which a complementary antibody specifically binds. Thus, an antibody combining site can include additional regions within the antigen beyond the epitope that can determine properties such as binding affinity and / or stability, or that can 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 separate intermolecular contacts with amino acids outside the epitope, such antibodies are considered to bind separate antibody combining sites.

[0183] "Binding affinity" generally refers to the strength of the sum total 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, which 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 is generally determined by the dissociation constant (K D) Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens quickly 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 this disclosure.

[0184] "Potency" is typically expressed as IC in nM unless otherwise specified. 50 It is expressed as a value. 50 is the median inhibitory concentration of an antigen-binding molecule. In functional assays, IC 50 is the concentration that reduces a biological response by 50% of its maximum. In ligand binding studies, the IC 50 is the concentration that reduces receptor binding by 50% of the maximal specific binding level. 50 can be calculated by any means known in the art. The improvement in potency can be determined, for example, by measurement relative to a parent antibody (e.g., the parent antibody before germlining or before affinity optimization).

[0185] The fold improvement in potency of an antibody or polypeptide of the disclosure compared to a 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.

[0186] "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 antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Specific high-affinity IgG antibodies directed to the surface of the target cells "arm" the cytotoxic cells and are essential for such killing. Lysis of the target cells is extracellular, requires direct cell-to-cell contact, and does not involve complement. In addition to antibodies, other proteins containing an Fc region capable of specifically binding to antigen-bearing target cells, particularly Fc fusion proteins, are believed to be capable of achieving cell-mediated cytotoxicity. For simplicity, cell-mediated cytotoxicity resulting from the activity of Fc fusion proteins is also referred to herein as ADCC activity.

[0187] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition 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 the form in which they are found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0188] The term "subject" refers to any animal (e.g., mammal) that will 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 to refer to a human subject.

[0189] The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of an active ingredient (e.g., an anti-CD73 binding molecule disclosed herein) to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the composition is to be administered. Such compositions may be sterile.

[0190] An "effective amount" of an anti-CD73 binding molecule as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be routinely determined empirically for the stated purpose.

[0191] The term "therapeutically effective amount" refers to an amount of an anti-CD73 binding molecule or other drug disclosed herein effective to "treat" a disease or disorder in a subject or mammal.

[0192] The phrase "label," as used herein, refers to a detectable compound or composition directly or indirectly fused (e.g., genetically fused) or conjugated (e.g., chemically conjugated) to an anti-CD73 binding molecule disclosed herein, thereby resulting in a "labeled" anti-CD73 binding molecule. The label may itself be detectable (e.g., a radioisotope label or fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0193] The terms "derivatizable group" and "derivatizable functional group," etc., are used interchangeably and refer to a functional group capable of reacting to enable the formation of a covalent bond between an anti-CD73 binding molecule (e.g., a CD73 antibody) disclosed herein and another substance. In some embodiments, such a substance is a therapeutic agent (e.g., a cytotoxin), a detectable label, a polymer (e.g., PEG), etc. Exemplary derivatizable groups include thiol, hydroxyl, amino, carboxy, and amide, as well as modified forms thereof, such as activated or protected forms.

[0194] The terms "treat" or "treatment" or "treating" or "alleviate" or "alleviating," and the like, refer to both (1) therapeutic measures that cure, slow, relieve the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent and / or delay the onset of the targeted pathological condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to developing the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject has been successfully "treated" for cancer by the methods of the present disclosure if the patient experiences, for example, a complete remission, partial remission, or temporary remission of certain cancers.

[0195] The terms "cancer," "tumor," "cancerous," and "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas such as adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatocarcinoma and hepatocellular carcinoma, 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 head and neck cancers, and cancers of mucinous origin, e.g., mucinous ovarian cancer, cholangiocarcinoma (liver), and papillary renal carcinoma. In some embodiments, the term cancer as used herein specifically refers to cancers that express CD73. In some specific embodiments, the term cancer refers to cancers that express low levels of CD73. In some embodiments, the term cancer as used herein specifically refers to cancers that express CD73 (e.g., colon cancer, breast cancer, lymphoma, non-small cell carcinoma).

[0196] "Polynucleotide," or "nucleic acid," as used interchangeably herein, refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0197] The term "vector" refers to a construct capable of delivering, and in some embodiments expressing, one or more genes or sequences of interest in a host cell. 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 producer cells.

[0198] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. These terms also include amino acid polymers that are modified naturally or by intervention, such as 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, unnatural amino acids, etc.), as well as other modifications known in the art. Because the polypeptides of the present disclosure are based on antibodies, it is understood that in certain embodiments, the polypeptides can occur as single chains or associated chains.

[0199] A "recombinant" polypeptide or protein refers to a polypeptide or protein produced by recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in modified host cells are considered to be isolated, just like naturally occurring or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique. The polypeptides disclosed herein may be recombinantly produced using methods known in the art. Alternatively, the proteins and peptides disclosed herein may be chemically synthesized.

[0200] The term "amino acid substitution" refers to the replacement of an amino acid residue present in a parent sequence with another amino acid residue. Amino acids can be substituted in a parent sequence using, for example, chemical peptide synthesis or recombinant methods known in the art. Thus, a "substitution at position X" or a "substitution at position X" refers to the replacement of the amino acid present at position X with an alternative amino acid residue. In some embodiments, the substitution pattern can be described according to the schema AXY, where A is the single-letter code corresponding to the amino acid naturally occurring at position X, and Y is the substituting amino acid residue. In other embodiments, the substitution pattern can be described according to the schema XY, where Y is the single-letter code corresponding to the amino acid residue substituting for the amino acid naturally occurring at position X.

[0201] 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 having 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, when 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 embodiment, stretches of amino acids can be conservatively replaced with structurally similar stretches that differ in the order and / or composition of side chain family members.

[0202] Non-conservative substitutions include (i) a residue having an electropositive side chain (e.g., Arg, His, or Lys) for or substituted by an electronegative residue (e.g., Glu or Asp); (ii) a hydrophilic residue (e.g., Ser or Thr) for or substituted by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) a cysteine ​​or proline for or substituted by any other residue; or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) for or substituted by one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

[0203] Those skilled in the art can easily identify other substitutions. For example, for the amino acid alanine, the substitution can be selected from any one of D-alanine, glycine, β-alanine, L-cysteine, and D-cysteine. For lysine, the substitution can be any one of D-lysine, arginine, D-arginine, homoarginine, methionine, D-methionine, ornithine, or D-ornithine. In general, substitutions in functionally important regions that may be expected to cause changes in the properties of an isolated polypeptide include (i) polar residues, such as serine or threonine, substituted for (or by) hydrophobic residues, such as leucine, isoleucine, phenylalanine, or alanine; (ii) cysteine ​​residues substituted for (or by) any other residue; (iii) residues with electropositive side chains, such as lysine, arginine, or histidine, substituted for (or by) residues with electronegative side chains, such as glutamic acid or aspartic acid; or (iv) residues with bulky side chains, such as phenylalanine, substituted for (or by) residues without such side chains, such as glycine. The likelihood that one of the aforementioned non-conservative substitutions may alter the functional properties of a protein also correlates with the location of the substitution relative to the functionally important regions of the protein. Thus, some non-conservative substitutions have little or no effect on biological properties.

[0204] The term "amino acid insertion" refers to the introduction of a new amino acid residue between two amino acid residues present in a parent sequence. Amino acids can be inserted into a parent sequence using, for example, chemical peptide synthesis or by recombinant methods known in the art. Thus, as used herein, the phrase "insertion between positions X and Y" or "insertion between Kabat positions X and Y" (where X and Y correspond to amino acid positions) (e.g., a cysteine ​​amino acid insertion between positions 239 and 240) refers to the insertion of an amino acid between positions X and Y, and also refers to the insertion of a codon encoding an amino acid between the codons encoding the amino acids at positions X and Y in a nucleic acid sequence. Insertion patterns can be described according to the schema AXins, where A is the single-letter code corresponding to the amino acid to be inserted and X is the position before the insertion.

[0205] The term "percent sequence identity" between two polypeptide or polynucleotide sequences refers to the number of identical matched positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is provided in both the target sequence and the reference sequence. Gaps provided in the target sequence are not counted, as gaps are not nucleotides or amino acids. Similarly, gaps provided in the reference sequence are not counted, as it is the nucleotides or amino acids of the target sequence that are counted, not the nucleotides or amino acids of the reference sequence.

[0206] The percentage of sequence identity is calculated by determining the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage sequence identity. Sequence comparison and determination of percent sequence identity between two sequences can be accomplished using readily available software for both online use and download. Suitable software programs are available from a variety of sources for aligning both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). bl2seq uses either the BLASTN or BLASTP algorithm to perform comparisons between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics program suite and are also available from the European Bioinformatics Institute (EBI), www.ebi.ac.uk / Tools / psa.

[0207] Different regions within a single polynucleotide or polypeptide target sequence that are aligned with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Note that percent sequence identity values ​​are rounded to two decimal places. 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. Note also that length values ​​are always integers.

[0208] In certain embodiments, the percentage identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y / Z), where Y is the number of amino acid residues scored as identical matches in an alignment of the first and second sequences (when aligned by visual inspection or by a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of 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.

[0209] Those skilled in the art will understand that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence comparisons driven only by primary sequence data. Sequence alignments can be obtained 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 / . Alternatively, ClustalW2 and MUSCLE are available, for example, from EBI.

[0210] It will also be appreciated that sequence alignments may be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available at www.tcoffee.org or available, for example, from EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity may be curated automatically or manually.

[0211] The term "consensus sequence," as used herein with respect to light chain (VL) and heavy chain (VH) variable regions, refers to a composite or generalized VL or VH sequence defined based on information regarding which amino acid residues within the VL or VH chain are suitable for modification without impairing antigen binding. Thus, in a "consensus sequence" for a VL or VH chain, a particular amino acid position is occupied by one of multiple possible amino acid residues at that position. For example, if arginine (R) or serine (S) is present at a particular position, that particular position in the consensus sequence can be either arginine or serine (R or S). Consensus sequences for the VH and VL chains can be defined, for example, by in vitro affinity maturation (e.g., randomizing every amino acid position within a particular CDR using degenerate coding primers), scanning mutagenesis of amino acid residues within the antibody CDRs (e.g., alanine scanning mutagenesis), or any other method known in the art, followed by assessing binding of the mutants to antigen to determine whether the mutated amino acid positions affect antigen binding. In some embodiments, mutations are introduced in the CDR regions. In other embodiments, mutations are introduced in the framework regions. In some other embodiments, mutations are introduced in the CDR regions and in the framework regions.

[0212] II.CD73 binding molecule The present disclosure provides CD73-binding molecules, e.g., 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 embodiments, the anti-CD73 binding molecule is a human antibody (e.g., clone 10.3 antibody, clone 2C5 antibody, MEDI9447). In certain embodiments, the CD73-binding molecule is an antibody or an antigen-binding fragment thereof.

[0213] In some embodiments, the CD73 binding molecule, e.g., an antibody or antigen-binding fragment thereof, is a Fab, Fab', F(ab'), Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgG CH2, minibody, F(ab'), tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv)2, or scFv-Fc. In some embodiments, the antibody is an IgG type antibody, such as an IgG1 type antibody.

[0214] In some embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof comprises a heavy chain constant region or a fragment thereof. In some specific embodiments, the heavy chain constant region is an IgG constant region. The IgG constant region can comprise a light chain constant region selected from the group consisting of a kappa constant region and a lambda constant region.

[0215] In certain embodiments, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein are modified compared to a parent antibody, e.g., a CD730010 antibody or a CD730002 antibody. In some embodiments, the parent antibody is CD730010. In other embodiments, the parent antibody is CD730002. In other embodiments, the parent antibody is CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. The modifications may include mutations in the CDR and / or FW regions compared to the parent antibody, e.g., CD730010 or CD730002.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] (i)CD730010-derived anti-CD73 antibody In particular embodiments, the anti-CD73 antibodies of the present disclosure are directed to, but not limited to: 1) a light chain CDR1 comprising the consensus sequence SGSLSNIGRNX1VN, where X1 represents the amino acid residue proline (P), glutamic acid (E), or aspartic acid (D), and / or 2) a light chain CDR2 comprising the consensus sequence LX2NX3RX4X5, where 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 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)); The present invention also includes modifications to CDR1 and / or CDR2 and / or CDR3 of the light chain of the CD730010 antibody, including:

[0228] In particular embodiments, the anti-CD73 antibodies of the present disclosure are directed to, but not limited to: 1) a heavy chain CDR1 comprising the consensus sequence SYAX9S, where X9 represents the amino acid residue methionine (M) or tyrosine (Y), and / or 2) Consensus sequence X 10 IX 11 GSX 12 GX 13 TYYADSVKG (where X 10 represents the amino acid residue leucine (L) or alanine (A), and X 11 represents the amino acid residue tryptophan (W) or serine (S), and X 12 represents the amino acid residue tryptophan (W) or glycine (G), and X 13 represents the amino acid residue serine (S) or arginine (R), and / or 3) Consensus sequence LGYX 14 X 15 X 16 DX 17 (In the formula, X 14 represents the amino acid residue glycine (G) or serine (S), and X 15 represents the amino acid residue arginine (R) or threonine (T), and X 16represents the amino acid residue valine (V) or isoleucine (I), and X 17 represents the amino acid residue tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E) The present invention also includes modifications to CDR1 and / or CDR2 and / or CDR3 of the heavy chain of the CD730010 antibody, including:

[0229] In one embodiment, the anti-CD73 antibody, or antigen-binding fragment thereof, has the consensus amino acid sequence: [FW1]SGSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4] (In the formula, [FW1], [FW2], [FW3], and [FW4] represent 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, 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); X3 represents the amino acid residue glutamine (Q) or leucine (L); X4 represents the amino acid residue leucine (L) or proline (P); X5 represents the amino acid residue glycine (G) or serine (S); 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 comprises a VL region containing the amino acid residues leucine (L) or threonine (T).

[0230] In one embodiment, the anti-CD73 antibody or antigen-binding fragment thereof has the consensus amino acid sequence: [FW5]SYAX9S[FW6]X 10 IX 11GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8] (In the formula, [FW5], [FW6], [FW7], and [FW8] 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: 34), respectively, X9 represents the amino acid residue methionine (M) or tyrosine (Y); 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), X 13 represents the amino acid residue serine (S) or arginine (R), X 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). X 17 represents the amino acid residues tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0231] In one embodiment, the anti-CD73 antibody or antigen-binding fragment thereof has the consensus amino acid sequence: [FW1]SGSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4] (In the formula, [FW1], [FW2], [FW3], and [FW4] represent 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, 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); X3 represents the amino acid residue glutamine (Q) or leucine (L); X4 represents the amino acid residue leucine (L) or proline (P); X5 represents the amino acid residue glycine (G) or serine (S); 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 and X8 represents the amino acid residue leucine (L) or threonine (T), and the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL region comprising the consensus amino acid sequence: [FW5]SYAX9S[FW6]X 10 IX 11 GSX 12 GX 13 TYYADSVKG[FW7]LGYX 14 X 15 X 16 DX 17 [FW8] (In the formula, [FW5], [FW6], [FW7], and [FW8] 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: 34), respectively, X9 represents the amino acid residue methionine (M) or tyrosine (Y); X 10 represents the amino acid residue leucine (L) or alanine (A), X 11represents the amino acid residue tryptophan (W) or serine (S), X 12 represents the amino acid residue tryptophan (W) or glycine (G), X 13 represents the amino acid residue serine (S) or arginine (R), X 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). X 17 represents the amino acid residue tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

[0232] In some embodiments, 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 embodiments, 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.

[0233] In some embodiments, 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 embodiments, 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.

[0234] In some embodiments, 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 embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VL-CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 53, 54, 55, and 56.

[0235] In some embodiments, 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 embodiments, 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.

[0236] In some embodiments, 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 embodiments, 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.

[0237] In some embodiments, 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 embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VH-CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, and 45.

[0238] In some embodiments, 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 embodiments, 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.

[0239] In some embodiments, 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 embodiments, 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.

[0240] In some embodiments, 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 embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 comprising 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.

[0241] In some embodiments, 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 embodiments, 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.

[0242] In some embodiments, 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 embodiments, 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.

[0243] In some embodiments, 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 embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 comprising 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.

[0244] In some embodiments, 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 embodiments, 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.

[0245] In some embodiments, 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 embodiments, 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.

[0246] In some embodiments, 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.

[0247] In some embodiments, 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; 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; and a VL-CDR3 comprising 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.

[0248] In some embodiments, 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; 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; and 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.

[0249] In some embodiments, 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; 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; and a VH-CDR3 comprising 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.

[0250] In certain embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a modification to CDR1, and / or CDR2, and / or CDR3 of the heavy and / or light chain, and further comprises a modification to FW1, and / or FW2, and / or FW3, and / or FW4 of the heavy and / or light chain.

[0251] In some embodiments, 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.

[0252] In some embodiments, FW1 comprises SEQ ID NO: 25 or 26 except for 1, 2, 3, or 4 amino acid substitutions; FW2 comprises SEQ ID NO: 27 or 28 except for 1, 2, 3, or 4 amino acid substitutions; FW3 comprises SEQ ID NO: 29 except for 1, 2, 3, or 4 amino acid substitutions; FW4 comprises SEQ ID NO: 30 except for 1, 2, 3, or 4 amino acid substitutions; FW5 comprises SEQ ID NO: 31 except for 1, 2, 3, or 4 amino acid substitutions; FW6 comprises SEQ ID NO: 32 except for 1, 2, 3, or 4 amino acid substitutions; FW7 comprises SEQ ID NO: 33 except for 1, 2, 3, or 4 amino acid substitutions; and FW8 comprises SEQ ID NO: 34.

[0253] In certain embodiments, 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 that are 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 SEQ ID NOs: 46, 49, 53, 35, 37, and 41, or SEQ ID NOs: 47, 49, 53, 35, 37 and 41, or SEQ ID NOs: 47, 49, 54, 36, 37 and 42, or SEQ ID NOs: 46, 50, 54, 36, 38 and 43, or SEQ ID NOs: 46, 51, 55, 36, 39 and 44, or SEQ ID NOs: 48, 52, 54, 36, 40 and 44, or SEQ ID NOs: 46, 49, 56, 35, 37 and 41, or SEQ ID NOs: 46, 49, 53, 35, 37 and 45, or SEQ ID NOs: 47, 49, 56, 36, 37 and 45, or SEQ ID NOs: 46, 50, 56, 36, 38 and 45, or SEQ ID NOs: 46, 51, 56, 36, 39 and 45, or SEQ ID NOs: 48, 52, 56, 36, 40 and 45, or SEQ ID NOs: 46, 49, 56, 35, 37 and 45.

[0254] In certain embodiments, 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.

[0255] In other embodiments, 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.

[0256] In other embodiments, the anti-CD73 antibody or antigen-binding fragment thereof 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 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 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.

[0257] In some embodiments, 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 embodiments, 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.

[0258] The "clone 10.3 antibody" (also referred to as "73combo3" or "MEDI9447") is an IgG1 comprising two CD730010-derived light chains (VL) of SEQ ID NO: 68 (comprising 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 (comprising three CDRs, CDR1, CDR2, and CDR3, having the sequences of SEQ ID NOs: 36, 39, and 45, respectively).

[0259] In certain embodiments, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein bind to 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.

[0260] (ii) CD730002-derived anti-CD73 antibody In particular embodiments, the anti-CD73 antibodies of the present disclosure are directed to, but not limited to: 1) a light chain CDR1 comprising the sequence SGDKVGDKYAS, and / or 2) Consensus sequence EDX 18 KX 19 X 20 S (where X 18 represents the amino acid residue serine (S) or threonine (T), and 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 3) a light chain CDR3 comprising the sequence QAWDTSFWV The present invention includes modifications to CDR1 and / or CDR2 and / or CDR3 of the light chain of the CD730002 antibody, including:

[0261] In particular embodiments, the anti-CD73 antibodies of the present disclosure are directed to, but not limited to: 1) Array SX 21 AX 22 S (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 2) Array AISGSGGSX 23 YYX 24 DSVKX 25 (In the formula, 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), and / or 3) a heavy chain CDR3 comprising the sequence DKGYYWYM The present invention includes modifications to CDR1 and / or CDR2 and / or CDR3 of the heavy chain of CD730002, including:

[0262] In one embodiment, the anti-CD73 antibody or antigen-binding fragment thereof has the consensus amino acid sequence: [FW9]SGDKVGDKYAS[FW 10 ]EDX 18 KX 19 X 20 S [FW 11 ]QAWDTSFWV[FW 12 ] (In the formula, [FW9], [FW 10 ], [FW 11 ] and [FW 12 ] represent amino acid residues in 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; 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 represents the amino acid residues histidine (H), proline (P), or leucine (L).

[0263] In one embodiment, the anti-CD73 antibody or antigen-binding fragment thereof has the consensus amino acid sequence: [FW 13 ]SX 21 AX 22 S [FW 14 ]AISGSGGSX 23 YYX 24 DSVKX 25 [FW 15 ]DKGYYWYM[FW 16 ] (In the formula, [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;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).

[0264] In one embodiment, the anti-CD73 antibody or antigen-binding fragment thereof has the consensus amino acid sequence: [FW9]SGDKVGDKYAS[FW 10 ]EDX 18 KX 19 X 20 S [FW 11 ]QAWDTSFWV[FW 12 ] (In the formula, [FW9], [FW 10 ], [FW 11 ] and [FW 12 ] represent amino acid residues in 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; 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 represents the amino acid residue histidine (H), proline (P), or leucine (L), and the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL region comprising the consensus amino acid sequence: [FW 13 ]SX 21 AX 22 S [FW 14 ]AISGSGGSX 23 YYX 24 DSVKX 25 [FW 15 ]DKGYYWYM[FW 16 ] (In the formula, [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; 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).

[0265] In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VL-CDR1 consisting of the sequence consisting of SEQ ID NO: 97. In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VL-CDR1 comprising the sequence consisting of SEQ ID NO: 97.

[0266] In some embodiments, 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 embodiments, 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.

[0267] In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VL-CDR3 consisting of the sequence consisting of SEQ ID NO: 100. In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VL-CDR3 comprising the sequence consisting of SEQ ID NO: 100.

[0268] In some embodiments, 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 embodiments, 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.

[0269] In some embodiments, 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 embodiments, 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.

[0270] In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VH-CDR3 consisting of the sequence consisting of SEQ ID NO: 96. In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VH-CDR3 comprising the sequence consisting of SEQ ID NO: 96.

[0271] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of the sequence of SEQ ID NO: 97 except for one, two, three, or four amino acid substitutions. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising the sequence of SEQ ID NO: 97 except for one, two, three, or four amino acid substitutions.

[0272] In some embodiments, 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 embodiments, 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 except for one, two, three, or four amino acid substitutions.

[0273] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 consisting of the sequence of SEQ ID NO: 100 except for one, two, three, or four amino acid substitutions. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 comprising the sequence of SEQ ID NO: 100 except for one, two, three, or four amino acid substitutions.

[0274] In some embodiments, 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 embodiments, 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 for one, two, three, or four amino acid substitutions.

[0275] In some embodiments, 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 embodiments, 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 except for one, two, three, or four amino acid substitutions.

[0276] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 consisting of the sequence of SEQ ID NO: 96 except for one, two, three, or four amino acid substitutions. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 comprising the sequence of SEQ ID NO: 96 except for one, two, three, or four amino acid substitutions.

[0277] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 consisting of the 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 the sequence consisting of SEQ ID NO: 100. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 comprising the 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 comprising the sequence consisting of SEQ ID NO: 100.

[0278] In some embodiments, 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 the sequence consisting of SEQ ID NO: 96. In some embodiments, 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 comprising the sequence consisting of SEQ ID NO: 96.

[0279] In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VL-CDR1 consisting of the sequence 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 the sequence of SEQ ID NO: 100 except for one, two, three, or four amino acid substitutions.

[0280] In some embodiments, the anti-CD73 antibody, or antigen-binding fragment thereof, comprises a VL-CDR1 comprising the sequence 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 of SEQ ID NO: 100 except for one, two, three, or four amino acid substitutions.

[0281] In some embodiments, 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 the sequence of SEQ ID NO: 96, except for one, two, three, or four amino acid substitutions.

[0282] In some embodiments, 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 for 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 for one, two, three, or four amino acid substitutions; and a VH-CDR3 comprising a sequence consisting of SEQ ID NO: 96, except for one, two, three, or four amino acid substitutions.

[0283] In certain embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a modification to CDR1, and / or CDR2, and / or CDR3 of the heavy and / or light chain, and further comprises a modification to FW1, and / or FW2, and / or FW3, and / or FW4 of the heavy and / or light chain.

[0284] In some embodiments, FW9 comprises SEQ ID NO: 90 or 91, and FW 10 comprises SEQ ID NO: 92, FW 11 comprises SEQ ID NO: 93, 94, or 122, FW 12 comprises SEQ ID NO: 30, FW13 comprises SEQ ID NO: 31, FW 14 comprises SEQ ID NO: 32, FW 15 comprises SEQ ID NO: 33, and FW 16 comprises SEQ ID NO:89.

[0285] In some embodiments, FW9 comprises SEQ ID NO: 90 or 91 except for 1, 2, 3, or 4 amino acid substitutions; 10 comprises SEQ ID NO: 92 except for 1, 2, 3, or 4 amino acid substitutions, and FW 11 comprises SEQ ID NO: 93, 94, or 122 except for 1, 2, 3, or 4 amino acid substitutions; FW 12 comprises SEQ ID NO: 30 except for 1, 2, 3, or 4 amino acid substitutions, and FW 13 comprises SEQ ID NO: 31 except for 1, 2, 3, or 4 amino acid substitutions, and FW 14 comprises SEQ ID NO: 32 except for 1, 2, 3, or 4 amino acid substitutions, and FW 15 comprises SEQ ID NO: 33 except for 1, 2, 3 or 4 amino acid substitutions, and FW 16 comprises SEQ ID NO:89.

[0286] In certain embodiments, 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 that are 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 SEQ ID NOs: 97, 98, 100, 35, 37, and 96, or SEQ ID NOs: 97, 99, 100, 35, 95 and 96, or SEQ ID NOs: 97, 98, 100, 35, 37, and 96, or SEQ ID NOs: 97, 99, 100, 123, 37, and 96, or SEQ ID NOs: 97, 99, 100, 124, 37, and 96, or SEQ ID NOs: 97, 99, 100, 35, 125, and 96, or SEQ ID NOs: 97, 99, 100, 35, 126, and 96, or SEQ ID NOs: 97, 99, 100, 35, 95, and 96, or SEQ ID NOs: 97, 127, 100, 35, 95, and 96, or SEQ ID NOs: 97, 128, 100, 35, 95, and 96, or SEQ ID NOs: 97, 129, 100, 35, 95, and 96, or SEQ ID NOs: 97, 99, 100, 35, 95, and 96.

[0287] In certain embodiments, 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 NOs: 86, 88, 112, 118, 119, 120, and 121.

[0288] In other embodiments, 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.

[0289] In other embodiments, 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.

[0290] In some embodiments, 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 embodiments, 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.

[0291] 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).

[0292] In certain embodiments, the anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein bind to 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.

[0293] (iii) Anti-CD73 Antibodies with Parent Antibodies Other Than CD730002 or CD730010 In other embodiments, the parent antibodies of the anti-CD73 antibodies or antigen-binding fragments disclosed herein include 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), CD7300012 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 7 and a VH of SEQ ID NO: 8), CD7300013 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 5 and a VH of SEQ ID NO: 6), CD7300014 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 5 and a VH of SEQ ID NO: 6), CD7300015 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 5 and a VH of SEQ ID NO: 6), CD7300016 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 7 and a VH of SEQ ID NO: 8), CD7300017 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 7 and 042 (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), CD730047 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 13 and a VH of SEQ ID NO: 14), CD730068 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 108 and a VH of SEQ ID NO: 107), or CD730069 (i.e., an anti-CD73 antibody comprising a VL of SEQ ID NO: 110 and a VH of SEQ ID NO: 109). Modifications to the parent antibody may include mutations in the CDR and / or FW regions when compared to the parent antibody, e.g., CD730004.

[0294] In certain embodiments, the anti-CD73 antibody comprises modifications to CDR1 and / or CDR2 and / or CDR3 of the light chain of CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0295] In certain embodiments, the anti-CD73 antibody comprises modifications to CDR1 and / or CDR2 and / or CDR3 of the heavy chain of CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0296] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0297] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0298] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR2 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions. In some embodiments, 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.

[0299] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions. In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR2 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions. In some embodiments, 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.

[0300] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VL-CDR1 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069; 21, CD730042, CD730046, CD730047, CD730068, or CD730069; and VL-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0301] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a VH-CDR1 and a VH-CDR2 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069; 21, CD730042, CD730046, CD730047, CD730068, or CD730069; and VH-CDR3 from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0302] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof has a VL-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions; and a VL-CDR3 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions.

[0303] In some embodiments, the anti-CD73 antibody or antigen-binding fragment thereof has a VH-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions; and a VH-CDR3 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for one, two, three, or four amino acid substitutions.

[0304] In certain embodiments, the anti-CD73 antibody or antigen-binding fragment thereof comprises a modification 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 a modification 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.

[0305] In certain embodiments, 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 that are 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 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0306] In certain embodiments, 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 a VL sequence from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0307] In other embodiments, 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 a VH sequence from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0308] In other embodiments, the anti-CD73 antibody or antigen-binding fragment thereof 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. 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 a VH sequence from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0309] 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.

[0310] (iv) mixed and matched anti-CD73 antibody The VH and VL sequences of 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 10 GL9, clone 10 P32E, clone 10 C1, clone 10 C2, clone 10 D3, clone 10 G10, clone 10 HPT, clone 10 GRVE, clone 10 combo1, clone 10 combo2, clone 10 combo3, clone 10 combo5, or clone combo6) can be "mixed and matched" to create other anti-CD73 binding molecules.

[0311] In a specific embodiment, the VH sequences of the 10.3 antibody and the 2C5 antibody are mixed and matched. In another embodiment, the VL sequences of the 10.03 antibody and the 2C5 antibody can be mixed and matched. Additionally or alternatively, the VL and / or VH sequences of clone 10 (CD730010) variants disclosed herein can be mixed and matched. Additionally or alternatively, the VL and / or VH sequences of clone 2 (CD730002) variants disclosed herein can be mixed and matched. Additionally or alternatively, the VL and / or VH sequences of clone 10 (CD730010) and clone 2 (CD730002) variants disclosed herein can be mixed and matched.

[0312] In some embodiments, VL and / or VH mix-matching can be performed between sequences derived from antibodies that fall into 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 or overlapping epitopes, or that compete with each other for binding to the same or overlapping epitopes. For example, sequences derived from CD730003, CD730010, CD730021, CD730042, CD730046, and CD730047 (all of these antibodies belong to "epitope bin B") can be mixed and matched. In other embodiments, VL and / or VH mix-matching can be performed between sequences derived from anti-CD73 antibodies that fall into different epitope bins. Thus, antibody sequences belonging to "epitope bin B" can be mixed and matched with sequences of anti-CD73 antibodies in "epitope bin A" (CD730002, CD730004, CD730008, and CD730011) or "epitope bin C" (CD730068 and CD730069).

[0313] (v) mutant anti-CD73 antibody In certain embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof disclosed herein comprises mutations that improve binding to human FcRn and improve the half-life of the anti-CD73 antibody or antigen-binding fragment thereof. In some embodiments, such mutations are introduced into the IgG1 constant domain: 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 (numbered according to the EU index as in Kabat (Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, DC)). See U.S. Patent No. 7,658,921, which is incorporated herein by reference. Mutant IgGs of this type, termed "YTE mutants," have been shown to exhibit approximately a four-fold increase in half-life when compared to the wild-type version of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006)). In some embodiments, 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), where such mutations increase the serum half-life of the anti-CD73 antibody or antigen-binding fragment thereof.

[0314] In some embodiments, the YTE mutant further comprises a substitution at position 434 (numbered according to the EU index as in Kabat) of the IgG constant domain with an amino acid selected from the group consisting of tryptophan (W), methionine (M), tyrosine (Y), and serine (S). In other embodiments, the YTE mutant further comprises a substitution at position 434 (numbered according to the EU index as in Kabat) of the IgG constant domain with an amino acid selected from the group consisting of tryptophan (W), methionine (M), tyrosine (Y), and serine (S), and a substitution at position 428 (numbered according to the EU index as in Kabat) of the IgG constant domain with an amino acid selected from the group consisting of threonine (T), leucine (L), phenylalanine (F), and serine (S).

[0315] In yet other embodiments, the YTE mutant further comprises a substitution at position 434 of the IgG constant domain (numbered according to the EU index as in Kabat) with a tyrosine (Y) and a substitution at position 257 of the IgG constant domain (numbered according to the EU index as in Kabat) with a leucine (L). In some embodiments, the YTE mutant further comprises a substitution at position 434 of the IgG constant domain (numbered according to the EU index as in Kabat) with a serine (S) and a substitution at position 428 of the IgG constant domain (numbered according to the EU index as in Kabat) with a leucine (L).

[0316] In a specific embodiment, an anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or 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 (numbering according to the EU index as in Kabat).

[0317] In certain embodiments, the anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragments thereof disclosed herein are (a) substitution of the amino acid at position 252 with tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T); (b) substitution of the amino acid at position 254 with threonine (T); (c) substitution of the amino acid at position 256 with serine (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), or threonine (T); (d) substitution of the amino acid at position 257 with leucine (L); (e) substitution of the amino acid at position 309 with proline (P); (f) substitution of the amino acid at position 311 with serine (S); (g) substitution of the amino acid at position 428 with threonine (T), leucine (L), phenylalanine (F), or serine (S); (h) substitution of the amino acid at position 433 with arginine (R), serine (S), isoleucine (I), proline (P), or glutamine (Q); (i) substitution of the amino acid at position 434 with tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F), or tyrosine; and (j) a combination of two or more of the above substitutions (positions are numbered according to the EU index as in Kabat), 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.

[0318] In some embodiments, the VH and / or VL amino acid sequences of an anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof disclosed herein are 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to the VH and VL sequences set forth above and contain 1, 2, 3, 4, 5, or more conservative substitutions. CD73 antibodies having VH and VL regions with high sequence similarity or sequence identity (i.e., 80% or greater) to the VH region of SEQ ID NOs: 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 NOs: 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 86, 88, 112, 118, 119, 120, or 121, respectively. , 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, by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis), followed by testing the encoded modified antibody for retained function using the functional assays described herein.

[0319] In some embodiments, 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, e.g., to reduce cytotoxicity by ADCC. In some embodiments, the Fc domain of an antibody or Fc fusion protein has increased binding to an Fc receptor, e.g., to increase ... and a non-naturally occurring ADCC-reducing amino acid residue at one or more positions selected from the group consisting of 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. A number of specific mutations that can reduce the ADCC activity of an antibody are known in the art, including, for example, 234F, 235E, 235F, 235Q (or 235Y), 239A, 332Q, 331S, and combinations thereof.For example, U.S. Patent Nos. 5,624,821, 5,648,260, 7,597,889, 8,961,967, 7,371,826, 7,785,791, and 7,790,858, U.S. Patent Application Publication Nos. 20140378663, 20130071390, and 20110212087, See the mutations described in U.S. Patent Application Publication Nos. 20150118227, 20060194290, 20060194291, 20080274105, 20080274506, U.S. Patent Application Publication Nos. 20130089541, and 20130108623, which are incorporated by reference in their entireties. Antibodies with reduced ADCC effector function also include those with substitution of one or more of 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 of amino acid positions 265, 269, 270, 297, and 327, including Fc mutants with substitutions at residues 265 and 297 to alanine (see, e.g., U.S. Pat. No. 7,332,581). Optionally, mutations that reduce both ADCC and CDC may be incorporated. In some aspects, anti-CD73 antibodies or antigen-binding fragments thereof disclosed herein that contain mutations that reduce or abolish ADCC and / or CDC can be used to generate antibody drug conjugates (ADCs).

[0320] 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 defined in Kabat. In yet another specific aspect, the Fc region is an IgG1, IgG2, or IgG3 Fc region, and the non-naturally occurring amino acid is selected from the group consisting of 234F, 235E, 235F, 235Q (or 235Y), 239A, 332Q, 331S, 332Q, as numbered by the EU index as defined in Kabat.

[0321] In another embodiment, the disclosure provides an anti-CD73 antibody, which 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 defined in Kabat. In yet another specific embodiment, 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 defined in Kabat. In specific embodiments, the disclosure provides an anti-CD73 antibody, which is an IgG1, IgG2, or IgG3, and comprises modifications at positions (i) 234F, 235E, and 331S; (ii) 234F, 235F, and 331S; (iii) 234F, 235Q, and 322Q. In another specific embodiment, the disclosure provides an anti-CD73 antibody, which is an IgG4 and comprises the modifications 228P and 235E.

[0322] III. Epitope-competing CD73-binding molecules In another aspect, the disclosure provides CD73 binding molecules that bind to the same epitope as the various anti-CD73 antibodies described herein, e.g., molecules that bind to the same epitope as MEDI9447, the clone 10.3 antibody, or the clone 2C5 antibody.

[0323] Such antibodies can be identified based on their ability to cross-compete with (e.g., competitively inhibit in a statistically significant manner the binding of) an anti-CD73 antibody disclosed herein, e.g., CD730010 antibody, CD730002 antibody, CD730004 antibody, and antigen-binding fragments thereof, in a standard CD73 binding assay (e.g., flow cytometry assay, surface plasmon resonance, or solution assay).

[0324] 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, e.g., 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; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., structurally similar or spatially proximate) epitope on CD73 as the anti-CD73 antibody or antigen-binding fragment thereof with which it competes. In one embodiment, the anti-CD73 antibody or antigen-binding fragment thereof that binds to the same epitope on CD73 as, for example, 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) is a human monoclonal antibody.

[0325] As described herein, to elucidate the mechanism of action of MEDI9447, a monoclonal antibody that directly inhibits the enzymatic activity of CD73, we identified the epitope of MEDI9447. This epitope resides within the apical surface of the N-terminal domain of CD73, a region distant from the substrate-binding and active site residues. Structural and mechanistic studies revealed 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.

[0326] The inventors used hydrogen-deuterium exchange (HDX) mass spectrometry (MS) and mutagenesis strategies to define the epitope of MEDI9447 and to investigate the potential effect of antibody binding on global CD73 structure. The antibody binds to a site in the N-terminal domain of CD73 that allows noncompetitive inhibition of AMP hydrolysis. In various embodiments, the epitope includes one or more CD73 amino acid residues corresponding to V144, K180, and N185. In various embodiments, the epitope further includes one or more CD73 amino acid residues corresponding to Y135, K136, and N187 of CD73. Notably, the epitope is positioned such that antibody binding prevents conversion of CD73 from the open conformer to the catalytically active closed conformer. Furthermore, our studies demonstrate that MEDI9447 can inhibit both anchored and soluble CD73 using a dual inhibitory mechanism mediated by the valency of antibody interaction with CD73.

[0327] IV. Functional Properties of Anti-CD73 Antibodies The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., BIACORE™ analysis). Direct binding assays as well as competitive binding assay formats are readily available. (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 methods described therein.) Affinity measurements for a particular antibody-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Thus, affinity and other antigen-binding parameters (e.g., K D or Kd, K on , K. off ) measurements are made in standardized solutions of antibody and antigen, and in standardized buffers, such as those known in the art and described herein.

[0328] It is also known in the art that affinity measured using surface plasmon resonance analysis (e.g., BIACORE™) can vary depending on which one of the reactants is bound to the chip. In this regard, affinity can be measured using a format in which the target antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) is immobilized on the chip (referred to as an "IgG-down" format), or a format in which the target protein (e.g., CD73) is immobilized on the chip (referred to as a "CD73-down" format).

[0329] In one embodiment of the present disclosure, an anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof is-6 Less than M or 10 -7 Less than M or 10 -8 Less than M or 10 -9 Less than M or 10 -10 Less than M or 10 -11 Less than M or 10 -12 Less than M or 10 -13 A dissociation constant or k less than M d (k off / k on ) and specifically binds to CD73 and / or its antigenic fragments.

[0330] In another embodiment, an anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof is administered at a concentration of 1 x 10 -3 s -1 Less than or 2 x 10 -3 s -1 Less than K off In other embodiments, the anti-CD73 antibody or antigen-binding fragment thereof binds to CD73 and / or an antigen-binding fragment thereof at 10 -3 s -1 Less than 5 x 10 -3 s -1 Less than 10 -4 s -1 Less than 5 x 10 -4 s -1 Less than 10 -5 s -1 Less than 5 x 10 -5 s -1 Less than 10 -6 s -1 Less than 5 x 10 -6 s -1 Less than 5 x 10 -7 s -1 Less than 10 -8 s -1 Less than 5 x 10 -8 s -1 Less than 10 -9 s -1 Less than 5 x 10 -9 s -1 Less than or 10 -10 s -1 Less than K off It binds to CD73 and its antigenic fragments.

[0331] In another embodiment, the anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment thereof is 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 association rate constant or k on It binds to CD73 and / or its antigenic fragments with high velocity.

[0332] In some embodiments, the anti-CD73 antibody (e.g., clone 10.3 antibody or 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 In one specific embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which has a K of about 150 pM as measured by flow cytometry. D In another specific embodiment, the anti-CD73 antibody is the clone 2C5 antibody, which has a K of about 80 pM as measured by flow cytometry. DBinds to CD73 on the surface of MB-MDA-231 cells.

[0333] In some embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or 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 In one specific embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which has a K of about 110 pM as measured by flow cytometry. D In another specific embodiment, the anti-CD73 antibody is the clone 2C5 antibody, which binds to CD73 on the surface of mouse 3T1 cells at a K of about 55 pM as measured by flow cytometry. D It binds to CD73 on the surface of mouse 3T1 cells.

[0334] In some embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or 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 In one specific embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which has a K of about 80 pM as measured by flow cytometry. D In another specific embodiment, the anti-CD73 antibody is clone 2C5 antibody, which binds to CD73 on the surface of cynomolgus monkey MK-1 cells at a K of about 60 pM as measured by flow cytometry. D It binds to CD73 on the surface of cynomolgus monkey MK-1 cells.

[0335] In some embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof has a K 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 as measured by surface plasmon resonance (PROTEON®). D In one specific embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which has a K of about 4 pM as measured by surface plasmon resonance (PROTEON®). D In another specific embodiment, the anti-CD73 antibody is the clone 2C5 antibody, which binds to human CD73 with a K of about 9 pM as measured by surface plasmon resonance (PROTEON®). D It binds to human CD73.

[0336] In some embodiments, an anti-CD73 antibody disclosed herein (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof has a K 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 as measured by surface plasmon resonance (PROTEON®). D In one specific embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which has a K of about 1 pM as measured by surface plasmon resonance (PROTEON®). DIn another specific embodiment, the anti-CD73 antibody is the clone 2C5 antibody, which binds to mouse CD73 with a K of about 22 pM as measured by surface plasmon resonance (PROTEON®). D It binds to mouse CD73.

[0337] In some embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragment thereof has a K 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 as measured by surface plasmon resonance (PROTEON®). D In one specific embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which has a K of about 7 pM as measured by SPR (Proteon). D In another specific embodiment, the anti-CD73 antibody is the clone 2C5 antibody, which binds to cynomolgus monkey CD73 with a K of about 9 pM as measured by surface plasmon resonance (PROTEON®). D It binds to cynomolgus monkey CD73.

[0338] In some embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or 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 In one specific embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which binds to human CD73 with a K of about 80 pM as measured by solution binding. D In another specific embodiment, the anti-CD73 antibody is the clone 2C5 antibody, which binds to human CD73 with a K of about 80 pM as measured by solution binding. D It binds to human CD73.

[0339] In some embodiments, the anti-CD73 antibody (e.g., clone 10.3 antibody or 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 In one specific embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which binds to mouse CD73 with a K of about 130 pM as measured by solution binding. D In another specific embodiment, the anti-CD73 antibody is the clone 2C5 antibody, which binds to mouse CD73 with a K of about 1500 pM as measured by solution binding. D It binds to mouse CD73.

[0340] In some embodiments, the anti-CD73 antibody (e.g., clone 10.3 antibody or 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 embodiment, the anti-CD73 antibody is the clone 10.3 antibody, which binds to cynomolgus monkey CD73 with a K of about 90 pM as measured by solution binding. D In another specific embodiment, the anti-CD73 antibody is the clone 2C5 antibody, which binds to cynomolgus monkey CD73 with a K of about 100 pM as measured by solution binding. D In a particular embodiment, MEDI9447 binds to cynomolgus monkey CD73 at about 1 x 10 -12 , 5×10 -12 , 10×10 -12 , 100×10 -12 , or 150 x 10 -12 KD It binds to CD73.

[0341] In some embodiments, the CD73 binding molecules disclosed herein, e.g., anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragments thereof, can alleviate AMP-mediated inhibition of T cell division. In other embodiments, the CD73 binding molecules disclosed herein, e.g., anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or antigen-binding fragments thereof, can alleviate AMP-mediated inhibition of T cell division. reg ATP-induced T eff Suppression can be rescued.

[0342] In some embodiments, a CD73 binding molecule disclosed herein, e.g., an anti-CD73 antibody or antigen-binding fragment thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), can significantly inhibit syngeneic tumor growth. In one embodiment, the tumor is a non-small cell lung cancer tumor, an ovarian cancer tumor, a breast cancer tumor, a head and neck cancer tumor, a pancreatic cancer tumor, a colorectal cancer tumor, a melanoma tumor, or a lymphoma tumor. In one embodiment, the tumor is a CT26 murine syngeneic CRC tumor, a B16F10 melanoma tumor, an EG7-OVA lymphoma tumor, or an LL2 (Lewis lung) tumor. In some embodiments, a CD73 binding molecule disclosed herein, e.g., an anti-CD73 antibody or antigen-binding fragment thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), can significantly inhibit tumor growth, wherein the tumor is refractory to therapy with anti-PD-1 and / or anti-PD-L1 and / or anti-PD-L2 and / or anti-CTLA-4 antibodies. In some embodiments, a CD73 binding molecule disclosed herein, e.g., an anti-CD73 antibody or antigen-binding fragment thereof (e.g., clone 10.3 antibody or 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 of PD1.

[0343] In some embodiments, a CD73 binding molecule disclosed herein, e.g., an anti-CD73 antibody or antigen-binding fragment thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), can be internalized after binding to a cell. In some embodiments, the CD73 binding molecule is an antibody drug conjugate (ADC).

[0344] V. Preparation of Anti-CD73 Antibodies and Antigen-Binding Fragments 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, a mouse, hamster, or other suitable host animal is immunized as described above to induce the production by lymphocytes of antibodies capable of specifically binding to the immunizing antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are isolated and fused, for example, using polyethylene glycol, with a suitable myeloma cell line to form hybridoma cells, from which unfused lymphocytes and myeloma cells can then be selected away. Hybridomas that produce monoclonal antibodies specifically directed against the antigen of choice, as determined by immunoprecipitation, immunoblotting, or by in vitro binding assays (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)), can then be grown in vitro in culture 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 fluid as described above for polyclonal antibodies.

[0345] Alternatively, anti-CD73 monoclonal antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) and antigen-binding fragments thereof can also be produced using recombinant DNA methods, for example, as described in U.S. Pat. No. 4,816,567. Polynucleotides encoding the monoclonal antibody are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the antibody heavy and light chains, and sequenced using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, and the expression vector is transfected into host cells, such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not naturally produce immunoglobulin proteins, and the host cells produce the monoclonal antibody. Recombinant anti-CD73 monoclonal antibodies or antigen-binding fragments thereof of a desired species can also 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).

[0346] Polynucleotides encoding anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments thereof can be further modified in any of a variety of ways using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a murine monoclonal antibody can be (1) substituted with those regions of, for example, a human antibody to generate a chimeric antibody, or (2) substituted with a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable regions can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0347] In certain embodiments, the anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be prepared directly using various techniques known in the art. Immobilized human B lymphocytes isolated from in vitro immunized individuals producing antibodies against the target antigen can be generated (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).

[0348] Anti-CD73 human antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments thereof can also be selected from phage libraries, where the phage library expresses human antibodies, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314; Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; and Marks et al., 1991, J. Mol. Biol., 222:581. Techniques for producing and using antibody phage libraries are also described in U.S. Pat. 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. Bio., doi:10.1016 / j.jmb.2007.12.018 (each of which is incorporated by reference in its entirety).

[0349] Affinity maturation 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.

[0350] In some embodiments, the anti-CD73 monoclonal antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) may be a humanized antibody. Methods for modifying, humanizing, or resurfacing non-human or human antibodies can also be used and are well known in the art. Humanized, resurfacing, or similarly modified antibodies can have one or more amino acid residues derived from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammalian source. These non-human amino acid residues are often replaced with residues referred to as "import" residues, which are typically taken from the "import" variable, constant, or other domains of known human sequences. Such imported sequences can be used to reduce immunogenicity or to reduce, enhance, or alter binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable property as known in the art. Generally, CDR residues are directly and most substantially involved in influencing CD73 binding. Thus, some or all of the non-human or human CDR sequences can be maintained while the non-human sequences of the variable and constant regions can be replaced with human or other amino acids.

[0351] Antibodies may also be humanized, resurfacing, engineered, or human antibodies that have been modified, optionally while 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 resurfacing antibodies can be prepared by a process of analysis of 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 are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen, such as CD73. In this manner, framework (FW) residues from the consensus and import sequences can be selected and combined to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0352] Humanization, resurfacing, or modification of an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or an antigen-binding fragment thereof can be performed using techniques described, but not limited to, 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 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; 5,71 Specification No. 4,352; Specification No. 5,9,55,358; Specification No. 6,204,023; Specification No. 6,180,370; Specification No. 6,331,431; Specification No. 5,693,7 Specification No. 62; Specification No. 5,530,101; Specification No. 5,585,089; Specification No. 5,225,539; Specification No. 4,816,567; Specification No. 5,969,108 This can be carried out using any known method, such as those described in PCT / US Patent Application Nos. 7,635,666; 7,723,270; 7,557,189; 7,538,195; and 7,342,110; PCT / US Patent Application Nos. 98 / 16280; PCT / US Patent Application Nos. 91 / 05939; PCT / US Patent Application No. 94 / 01234; PCT / UK Patent Application No. 92 / 01755; WO 90 / 14443; WO 90 / 14424; WO 90 / 14430; and EP 229246 (each of which is incorporated herein by reference in its entirety, including the references cited therein).

[0353] Humanized anti-CD73 antibodies and antigen-binding fragments thereof can also be generated in transgenic mice containing human immunoglobulin loci that are capable, upon immunization, of producing a full repertoire of human antibodies 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.

[0354] In certain embodiments, anti-CD73 antibody fragments (e.g., fragments derived from clone 10.3 or clone 2C5 antibodies) are provided. Various techniques are known for producing antibody fragments. Traditionally, these fragments have been obtained by proteolytic digestion 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 embodiments, anti-CD73 antibody fragments are recombinantly produced. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, allowing for the production of large amounts 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. Pat. No. 5,641,870. Other techniques for producing antibody fragments will be apparent to the skilled practitioner, such as chemical synthesis.

[0355] According to the present disclosure, techniques for producing single-chain antibodies specific for CD73 can be adapted (see, e.g., U.S. Pat. No. 4,946,778). In addition, methods for constructing Fab expression libraries can be adapted to allow rapid and efficient identification of monoclonal Fab fragments, or derivatives, fragments, analogs, or homologs thereof, having the desired specificity for CD73 (see, e.g., Huse et al., Science 246:1275-1281 (1989)). 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 generated by reduction of disulfide bridges of F(ab')2 fragments; (c) Fab fragments generated by treating antibody molecules with papain and a reducing agent; and (d) Fv fragments.

[0356] The anti-CD733 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments thereof disclosed herein can be modified to increase their serum half-life. This can be achieved, for example, by incorporating a salvage receptor binding epitope into the antibody or antibody fragment by mutation of the appropriate region of the antibody or antibody fragment, or by incorporating the epitope into a peptide tag that is subsequently fused to the antibody or antibody fragment at either end or in the middle (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, such as conjugation with heterologous molecules such as PEG, are known in the art.

[0357] Heteroconjugate anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) and antigen-binding fragments thereof are also within the scope of the present disclosure. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune cells to unwanted cells (see, e.g., U.S. Pat. No. 4,676,980). It is contemplated that heteroconjugate anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) and antigen-binding fragments thereof can be prepared in vitro using known methods in synthetic protein chemistry, including methods involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0358] In certain embodiments, the CD73 binding molecules disclosed herein, e.g., antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments thereof, can be combined (e.g., in combination therapy) with other therapeutic agents or fused (e.g., genetically fused, thereby forming a fusion protein) or conjugated (e.g., chemically or enzymatically conjugated) with 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 CD73 binding molecule disclosed herein that is derivatized or linked (e.g., chemically or recombinantly) to another molecule (e.g., a peptide, a small drug molecule, a detectable molecule, etc.). Generally, the anti-CD73 antibodies or portions thereof are derivatized such that their CD73 binding properties are not adversely affected by the derivatization or labeling. Thus, the anti-CD73 antibodies and antibody portions of the present disclosure 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 operably linked (by chemical coupling, genetic fusion, non-covalent association, etc.) to one or more other molecular entities, e.g., a cytotoxic agent, a pharmaceutical agent, a detection agent, and / or a protein or peptide that can mediate association of the anti-CD73 binding molecule with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0359] Certain derivatized molecules can be generated by crosslinking two or more molecular entities, for example, an anti-CD73 binding molecule disclosed herein and a therapeutic agent (e.g., a cytotoxin such as tubulysin or MEDI 1508). Suitable crosslinkers include heterobifunctional ones, i.e., those with two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester); or homobifunctional ones (e.g., disuccinimidyl suberate). Such crosslinkers are available, for example, from Pierce Chemical Company, Rockford, II. Further bifunctional coupling agents include N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolylene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0360] Other types of derivatized molecules can be created by introducing detectable labels. Useful detection agents include fluorescent compounds (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide fluorophores, etc.), enzymes useful for detection (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc.), and epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags, etc.). In some embodiments, at least one spacer arm can be added to the detectable label. The spacer arm can be of various lengths to reduce potential steric hindrance.

[0361] The anti-CD73 binding molecules disclosed herein can also be labeled with a radiolabeled amino acid. Radiolabeling can be used for both diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect CD73-expressing cells by X-ray or other diagnostic techniques, such as positron emission tomography (PET).

[0362] Additionally, radioactive labels can be used therapeutically as toxins to CD73-expressing cells, such as those that provoke an unwanted immune response. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionuclides: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I and 131In some embodiments, the anti-CD73 binding molecule can be labeled with a paramagnetic, radioactive, or fluorogenic ion that is detectable during imaging. In some embodiments, 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 embodiments, 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 embodiments, the anti-CD73 binding molecules are labeled with X-ray contrast 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, such as polymers such as polyethylene glycol (PEG), methyl groups, ethyl groups, or carbohydrate groups. These groups are useful for improving the biological properties of the antibody, such as increasing serum half-life or increasing tissue binding.

[0363] The term "cytotoxic agent," as used herein, is broadly defined and refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells (cell death), and / or exerts an anti-neoplastic / anti-proliferative effect. For example, a cytotoxic agent can directly or indirectly interfere with the development, maturation, or spread of neoplastic tumor cells. The term also includes agents that produce solely a cytostatic effect, rather than merely a cytotoxic effect. The term includes chemotherapeutic agents, as identified below, as well as other CD73 antagonists, anti-angiogenic agents, tyrosine kinase inhibitors, protein kinase A inhibitors, cytokine family members, radioisotopes, and toxins, such as enzymatically active toxins of bacterial, fungal, plant, or animal origin.

[0364] The term "chemotherapeutic agent" is a subset of the term "cytotoxic agent," which includes natural or synthetic chemical compounds. Examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustards, ethylenimine compounds, alkyl sulfonates, and other compounds with alkylating activity, 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 include amifostine (ETHYOL®), cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin®), doxorubicin lipoprotein (DOXIL®), gemcitabine (GEMZAR®), daunorubicin, daunorubicin lipoprotein (DAUNOXOME®), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (Taxol®), docetaxel (Taxotere®), aldesleukin, asparagus, fluticasone, fluoxetine ... ginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38), gefitinib (IRESSA®), dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, aromatase inhibitors, and combinations thereof.

[0365] For purposes of this disclosure, it should be understood that a modified anti-CD73 antibody or antigen-binding fragment thereof can comprise any type of variable region that provides for the association of the antibody or polypeptide with CD73. In this regard, the variable region can comprise or be derived from any type of mammal that can be induced to mount a humoral response and produce immunoglobulins against a desired tumor-associated antigen. Thus, the variable region of a modified anti-CD73 antibody or antigen-binding fragment thereof can be of, for example, human, murine, non-human primate (e.g., cynomolgus monkey, macaque, etc.), or lupine origin. In some embodiments, both the variable and constant regions of a modified anti-CD73 antibody or antigen-binding fragment thereof are human. In other embodiments, the variable region of a matched antibody (usually derived from a non-human source) is modified or specifically tailored to improve the binding characteristics or reduce the immunogenicity of the molecule. In this regard, the variable region may be humanized or otherwise altered by including imported amino acid sequences.

[0366] In certain embodiments, the variable domains of both the heavy and light chains of an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment thereof are altered by at least partial replacement of one or more CDRs, and, if necessary, partial framework region replacement and sequence changes. The CDRs can be derived from antibodies of the same class or even subclass as the antibody from which the framework regions are derived, although it is contemplated that the CDRs will be derived from antibodies of a different class, or in certain embodiments, a different species. To transfer the antigen-binding capacity of one variable domain to another, it is not necessary to replace all CDRs with the complete CDRs of the donor variable region. Rather, it is sufficient to transfer only those residues necessary to maintain the activity of the antigen-binding site. Given the descriptions in U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, obtaining functional antibodies with reduced immunogenicity is well within the capabilities of one of ordinary skill in the art through routine experimentation or trial and error.

[0367] Despite the alterations in the variable regions, those skilled in the art will understand that modified anti-CD73 antibodies (e.g., modified clone 10.3 antibodies or modified clone 2C5 antibodies) or antigen-binding fragments thereof can also include antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a portion of one or more of the constant region domains has been deleted or otherwise altered to provide desired biochemical properties, such as increased tumor localization or decreased serum half-life, when compared to an antibody of substantially the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the modified antibody can comprise a human constant region. Constant region modifications compatible with the anti-CD73 molecules disclosed herein include the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein can include alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or the light chain constant domain (CL). In some embodiments, modified constant regions in which one or more domains are partially or completely deleted are contemplated. In some embodiments, modified antibodies may comprise domain-deleted constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the deleted constant region domain may be replaced with a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility typically conferred by the missing constant region.

[0368] In addition to their structure, the art knows that constant regions mediate several effector functions. For example, binding of the C1 component of complement to an antibody activates the complement system. Complement activation is important for opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, antibodies bind to cells via their Fc region, where Fc receptor sites on the antibody Fc region bind to Fc receptors (FcRs) on the cell. There are several Fc receptors specific for different antibody classes, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of an antibody to an Fc receptor on the cell surface initiates several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental passage, and regulation of immunoglobulin production.

[0369] In certain embodiments, an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment thereof provides altered effector functions, thereby affecting the biological profile of the administered antibody or antigen-binding fragment thereof. For example, deleting or inactivating the constant region domain (by point mutation or other means) can reduce Fc receptor binding of the modified antibody in circulation, thereby increasing tumor localization. In other cases, constant region modifications, consistent with the present disclosure, can modulate complement binding and thus reduce the serum half-life and nonspecific association of conjugated cytotoxins. Still other modifications of the constant region can be used to remove disulfide bonds or oligosaccharide moieties, allowing for enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region in the present disclosure can be readily made using well-known biochemical or molecular engineering techniques well within the skill of one in the art.

[0370] In certain embodiments, a CD73 binding molecule disclosed herein that is an antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or an antigen-binding fragment thereof does not have one or more effector functions. For example, in some embodiments, the antibody or antigen-binding fragment thereof does not have antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the anti-CD73 antibody or antigen-binding fragment thereof does not bind to Fc receptors and / or complement factors. In certain embodiments, the antibody or antigen-binding fragment thereof does not have effector functions.

[0371] It will be noted that in certain embodiments, anti-CD73 modified antibodies or antigen-binding fragments thereof can be engineered so that the CH3 domain of each modified antibody or fragment thereof is fused directly to the hinge region. In other constructs, it may be desirable to provide a peptide spacer between the hinge region and the modified CH2 and / or CH3 domain. For example, a compatible construct can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is attached to the hinge region by a spacer of 5 to 20 amino acids. Adding such a spacer can ensure, for example, that regulatory elements of the constant domain remain free and available or that the hinge region remains flexible. However, it should be noted that in some cases, amino acid spacers may prove to be immunogenic and induce undesirable immune responses against the construct. Thus, in certain embodiments, any spacer added to the construct may be relatively non-immunogenic or even omitted entirely to maintain the desired biochemical qualities of the modified antibody.

[0372] It will be understood that, in addition to deletion of entire constant region domains, the anti-CD73 antibodies and antigen-binding fragments thereof of the present disclosure can be provided by partial deletion or substitution of a few, or even a single, amino acids. 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 simply delete a portion of one or more constant region domains that control the effector function to be modulated (e.g., complement C1Q binding). Such partial deletion 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 that constant region domain intact. Furthermore, as alluded to above, the constant regions of the disclosed anti-CD73 antibodies and antigen-binding fragments thereof can be modified by mutation or substitution of one or more amino acids that enhance the profile of the resulting construct. In this regard, it is possible to disrupt an activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the conformation and immunogenic profile of the modified antibody or antigen-binding fragment thereof. Certain embodiments may include the addition of one or more amino acids to the constant region to enhance a desired property, such as reduced or increased effector function, or to provide more cytotoxicity or carbohydrate binding. In such embodiments, it may be desirable to insert or duplicate specific sequences from a selected constant region domain.

[0373] 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, set forth herein. These can include, for example, conservative substitution mutations, i.e., the replacement of one or more amino acids with similar amino acids. For example, a conservative substitution refers to the substitution of an amino acid for another amino acid within the same general class, e.g., the substitution of one acidic amino acid for another acidic amino acid, one basic amino acid for another basic amino acid, or one neutral amino acid for another neutral amino acid. What is meant by conservative amino acid substitution is well known in the art.

[0374] The anti-CD73 antibodies or antigen-binding fragments thereof can be further modified to contain additional chemical moieties not normally part of the protein. These derivatized moieties can improve the solubility, biological half-life, or absorption of the protein. These moieties can also reduce or eliminate any undesirable side effects of the protein. For a review of such moieties, see Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0375] VI. Polynucleotides Encoding CD73-Binding Molecules In certain embodiments, the present disclosure encompasses polynucleotides comprising a nucleic acid sequence encoding a polypeptide or antigen-binding fragment thereof that specifically binds to CD73. For example, the present disclosure provides polynucleotides comprising a nucleic acid sequence encoding an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or encoding an antigen-binding fragment of such an antibody. The polynucleotides of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and, if single-stranded, may be the coding strand or non-coding strand (antisense strand).

[0376] In certain embodiments, the polynucleotide is isolated. In certain embodiments, the polynucleotide is substantially pure. In certain embodiments, the polynucleotide comprises a coding sequence for a mature polypeptide fused in the same reading frame to a polynucleotide that aids in the expression and secretion of the polypeptide, e.g., from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide from the cell). Polypeptides with leader sequences are preproteins, and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotide can also encode a CD73-binding proprotein, which is the mature protein plus additional 5' amino acid residues.

[0377] In certain embodiments, the polynucleotide comprises the coding sequence for a mature CD73-binding polypeptide, e.g., an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment thereof, fused in the same reading frame to a marker sequence that, e.g., allows for purification of the encoded polypeptide. For example, the marker sequence can be a hexahistidine tag provided by the pQE-9 vector in the case of a bacterial host, which provides for purification of the mature polypeptide fused to the marker, or the marker sequence can be a hemagglutinin (HA) tag, derived from the influenza hemagglutinin protein, when a mammalian host (e.g., COS-7 cells) is used.

[0378] The present disclosure also provides variants of the described polynucleotides that encode, for example, CD73-binding fragments, analogs, and derivatives of the CD73 binding molecules disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody).

[0379] Polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, nucleotide variants are made by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be made for a variety of reasons, such as to optimize codon expression for a particular host (e.g., to change the codons of human mRNA to those preferred by a bacterial host, such as E. coli). Vectors and cells containing the polynucleotides described herein are also provided.

[0380] In some embodiments, DNA sequences encoding CD73-binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments 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 the codon selection that is favorable in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. For example, a complete amino acid sequence can be used to construct a reverse-translated gene. Additionally, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0381] Once assembled (by synthesis, site-directed mutagenesis, or otherwise), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into an expression vector and operably linked to expression control sequences appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to achieve high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0382] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment thereof. Recombinant expression vectors are replicable DNA constructs containing synthetic or cDNA-derived DNA fragments encoding the polypeptide chains of an anti-CD73 antibody and / or antigen-binding fragment thereof operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes.

[0383] A transcription unit generally comprises an assembly of (1) one or more genetic elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural 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 more detail below. Such regulatory elements may include an operator sequence to control transcription. The ability to replicate in a host, generally conferred by an origin of replication, and a selection gene to facilitate recognition of 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 that polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue, which can optionally be later cleaved from the expressed recombinant protein to provide the final product.

[0384] The choice of expression control sequence and expression vector will depend on the host chosen. A wide variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences derived from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids such as E. coli-derived plasmids, including pCR1, pBR322, pMB9, and their derivatives, as well as broader host range plasmids such as M13 and filamentous single-stranded DNA phages.

[0385] 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, include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli (E. coli) or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin, as described below. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use in 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 hereby incorporated by reference. For further information regarding methods of protein production, including antibody production, see, e.g., U.S. Patent Application Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746, 6,660,501, and 7,932,087, each of which is hereby incorporated by reference in its entirety.

[0386] 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 is feasible because such proteins are generally correctly folded, appropriately modified, and fully functional.

[0387] 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 such as L cells, C127, 3T3, Chinese hamster ovary (CHO), NSO, HeLa, and BHK cell lines. Mammalian expression vectors may contain nontranscribed 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 nontranscribed sequences, and 5'- or 3'-untranslated sequences, such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for heterologous protein production in insect cells are reviewed by Luckow and Summers, BioTechnology 6:47 (1988).

[0388] CD73-binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments thereof, produced by transformed hosts can be purified by any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Addition of affinity tags, such as hexahistidine, maltose-binding domain, influenza coat sequence, and glutathione-S-transferase, to the protein can allow for easy purification by passage through an appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0389] For example, supernatants from systems secreting recombinant proteins into culture media can be initially concentrated using commercially available protein concentration filters, such as Amicon® or Millipore Pellicon® ultrafiltration units. Following this concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate bearing pendant diethylaminoethyl (DEAE) groups, can be used. 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, CD73-binding molecules (e.g., clone 10.3 or clone 2C5 antibodies) can be further purified using one or more reverse-phase high-performance liquid chromatography (RP-HPLC) steps using hydrophobic RP-HPLC media, such as silica gel bearing pendant methyl or other aliphatic groups. Various combinations of some or all of the above purification steps can also be used to provide homogeneous recombinant proteins.

[0390] Recombinant CD73 binding proteins, such as anti-CD73 antibodies (e.g., clone 10.3 or clone 2C5 antibodies) or antigen-binding fragments thereof, produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size-exclusion chromatography steps. High-performance liquid chromatography (HPLC) can be used for final purification steps. Microbial cells used to express recombinant proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

[0391] Methods known in the art for purifying antibodies and other proteins also include those described, for example, in U.S. Patent Application Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is hereby incorporated by reference in its entirety.

[0392] In certain embodiments, the CD73-binding molecule is a polypeptide that is not an antibody. Various methods for identifying and generating non-antibody polypeptides that bind to protein targets with high affinity are known in the art. See, e.g., 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 embodiments, phage display technology can be used to identify / generate CD73-binding polypeptides. In certain aspects, the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, lipocalin, a fibronectin domain (e.g., a fibronectin domain such as a tenascin-3 Fn III domain), an ankyrin consensus repeat domain, and thioredoxin.

[0393] VI. Treatment Methods Using Therapeutic Anti-CD73 Antibodies The present disclosure provides methods related to treating patients having diseases associated with CD73 expression or CD73-expressing cells, e.g., cancer, by using anti-CD73 binding molecules, e.g., antibodies, e.g., antigen-binding fragments, variants, and derivatives thereof (e.g., clone 10.3 antibody or clone 2C5 antibody). In some specific embodiments, such cancers are lung cancer, breast cancer, ovarian cancer, colorectal cancer, bladder cancer, pancreatic cancer, renal cancer, gastric cancer, prostate cancer, breast cancer, lung and colon cancer, and lymphoma.

[0394] "CD73-expressing cells" refers to cells that express CD73. CD73 may be membrane-bound via glycosylphosphatidylinositol anchoring or may exist 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 blot, ELISA, etc.

[0395] Although the following discussion refers to methods of diagnosing and treating various diseases and disorders with the CD73 binding molecules of the present disclosure (e.g., clone 10.3 antibody or clone 2C5 antibody), the methods described herein are also applicable to any other anti-CD73 antibodies and antigen-binding fragments, variants, and derivatives (e.g., fusion proteins or conjugates) of these anti-CD73 antibodies that retain the desired properties of the anti-CD73 antibodies disclosed herein, e.g., the ability to specifically bind to CD73 and neutralize its 5'-nucleotidase activity. In some embodiments, the CD73 binding molecule is a human or humanized antibody that does not mediate human ADCC, or an anti-CD73 antibody that has been modified so that it does not mediate ADCC.

[0396] In some embodiments, the CD73 binding molecule is a CD730010 antibody or antigen-binding fragment thereof, a clone 10.3 antibody or antigen-binding fragment thereof, a CD730002 antibody or antigen-binding fragment thereof, a clone 2C5 antibody or antigen-binding fragment thereof, or a CD73004 antibody or antigen-binding fragment thereof. In other embodiments, the CD73 binding molecule is a clone 10.3 mutant antibody. In some embodiments, the CD73 binding molecule is a clone 10.3 monoclonal antibody. In some embodiments, the CD73 binding molecule is a clone 10.3 monoclonal antibody modified to have an extended serum half-life. In other embodiments, the CD73 binding molecule is a clone 10.3 YTE mutant antibody. In other embodiments, the CD73 binding molecule is a clone 2C5 mutant antibody. In some embodiments, the CD73 binding molecule is a clone 2C5 monoclonal antibody. In some embodiments, the CD73 binding molecule is a clone 2C5 monoclonal antibody modified to have an extended serum half-life. In other embodiments, the CD73 binding molecule is the clone 2C5 YTE mutant antibody.

[0397] In one embodiment, treatment comprises application or administration of an anti-CD73 binding molecule of the present disclosure, e.g., an antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment, variant, or derivative thereof, to a subject or patient, or application or administration of an anti-CD73 binding molecule to an isolated tissue or cell line derived 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 embodiment, treatment is also intended to comprise application or administration of a pharmaceutical composition comprising an anti-CD73 binding molecule of the present disclosure, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, to a subject or patient having a disease, a symptom of a disease, or a predisposition to a disease, or application or administration of a pharmaceutical composition comprising an anti-CD73 binding molecule to an isolated tissue or cell line derived from a subject or patient.

[0398] The anti-CD73 binding molecules of the present disclosure, e.g., antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments, variants, or derivatives thereof, are useful in the treatment of various cancers. In one aspect, the present disclosure provides anti-CD73 binding molecules, e.g., antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments, variants, or derivatives thereof, for use as a medicament, particularly for use in the treatment or prevention of 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 embodiments, the cancer exhibits a pre-metastatic phenotype. In some embodiments, the cancer exhibiting a pre-metastatic phenotype is melanoma or breast cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the anti-CD73 binding molecules disclosed herein can elicit adaptive anti-tumor activity and / or inhibit metastasis. In some detailed embodiments, the anti-CD73 binding molecules disclosed herein can inhibit metastasis in breast cancer.

[0399] The methods of the present disclosure use at least one anti-CD73 binding molecule, e.g., an antibody (e.g., clone 10.3 antibody or clone 2C5 antibody), or an antigen-binding fragment, variant, or derivative thereof, as defined elsewhere herein, to promote a favorable therapeutic response to cancer. The term "favorable therapeutic response" to cancer treatment refers to an improvement in the disease and / or an improvement in symptoms associated with the activity of these anti-CD73 binding molecules, e.g., an antibody, or an antigen-binding fragment, variant, or derivative thereof. Thus, for example, an improvement in the disease can be characterized as a complete response. "Complete response" is intended to mean the absence of clinically detectable disease, standardized against any previous test results. Alternatively, an improvement in the disease can be classified as a partial response. A "favorable therapeutic response" encompasses a reduction or inhibition of the progression and / or duration of cancer, a reduction or improvement in the severity of cancer, and / or an improvement in one or more symptoms thereof, achieved by administration of an anti-CD73 binding molecule disclosed herein.

[0400] In specific embodiments, such terms refer to one, two, or three or more results following administration of an anti-CD73 binding molecule disclosed herein: (1) stabilization, reduction, or elimination of cancer cell population; (2) stabilization or reduction in cancer growth; (3) impairment of cancer formation; (4) eradication, removal, or control of primary, regional, and / or metastatic cancer; (5) reduction in mortality; (6) increase in disease-free, recurrence-free, progression-free, and / or overall survival time or rate; (7) response rate, durability of response, or increase in the number of patients responding or in remission; (8) reduction in hospitalization rate; (9) reduction in length of hospitalization; (10) cancer size that remains constant, does not increase, or increases 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.

[0401] 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 changes detectable by blood chemistry, including, but not limited to, ELISA, RIA, chromatography, etc. In addition to these favorable therapeutic responses, subjects undergoing therapy with an anti-CD73 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, can experience beneficial effects in ameliorating symptoms associated with the disease.

[0402] The anti-CD73 binding molecules disclosed herein, e.g., antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragments, variants, or derivatives thereof, can be used in combination with any known cancer therapy, e.g., any agent or combination of agents known to be useful in or that has been used or is currently being 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 have complementary activities relative to the antibodies or polypeptides of the disclosure so that they do not adversely affect each other.

[0403] Anticancer drugs include drugs used to treat malignant lesions such as cancerous growths. Drug therapy can be used alone or in combination with other treatments such as surgery or radiation therapy. Several drug classes can be used in cancer treatment depending on the nature of the affected organ. For example, breast cancer is generally stimulated by estrogen and can be treated with drugs that inactivate sex hormones. Similarly, prostate cancer can be treated with drugs that inactivate the male hormone androgen. Anti-cancer agents for use in certain methods of the disclosure include, among others, antibodies (e.g., antibodies that bind IGF-1R, antibodies that bind EGFR, antibodies that bind Her2, or antibodies that bind cMET), small molecules that target IGF1R, small molecules that target EGFR, small molecules that target Her2, antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule-targeting agents, kinase inhibitors, protein synthesis inhibitors, immunotherapeutics, hormone therapy, 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.

[0404] In specific embodiments, the CD73 binding molecules disclosed herein, e.g., antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) 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, e.g., 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 embodiments, the anti-CTLA-4 antibody is ipilimumab or an antigen-binding fragment thereof. In other embodiments, the anti-CTLA-4 antibody is tremelimumab (ticilimumab, CP-675,206) or an antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody is pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475) or an antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody is nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or an antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody is BMS-936559 or an antigen-binding fragment thereof. In other embodiments, the anti-PD-L1 antibody is MPDL3280A. In other embodiments, the anti-PD-1 antibody is AMP-224 (anti-PD-1 Fc fusion protein) or an antigen-binding fragment thereof. In various embodiments, the anti-PD-L1 antibody is MEDI4736 or an antigen-binding fragment thereof.

[0405] In some aspects, a CD73 binding molecule disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody) can be administered in combination with an anti-PD-1 or anti-PD-1 antibody. 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 embodiments, a CD73 binding molecule disclosed herein (e.g., clone 10.3 antibody or 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 embodiments, the anti-CD73 antibody and the anti-PD-1 antibody, anti-PD-L1, or anti-CTLA4 antibody are administered in a ratio of about 1:1, 1:2, 1:3, or 1:4. In some embodiments, the anti-CD73 antibody and the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody are administered in a ratio of about 1:2. In a specific embodiment, the concentration of the anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) is about 10 mg / kg, and the concentration of the anti-PD-1 antibody is about 20 mg / kg. In some embodiments, a CD73 binding molecule disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody) can be administered in combination with an anti-PD-1 antibody.In some embodiments, administration of a combination therapy comprising a CD73 binding molecule disclosed herein (e.g., MEDI9447, Clone 10.3 antibody, or 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 an untreated subject or a subject treated with a monotherapy (e.g., an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody without an anti-CD73 antibody). In some embodiments, administration of a combination therapy comprising a CD73 binding molecule disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody) in combination with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody increases 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 an untreated subject or a subject treated with a monotherapy (e.g., an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody without an anti-CD73 antibody).

[0406] Where the combination therapy involves 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, and sequential administration in any order. In some embodiments, an anti-CD73 antibody described herein (e.g., clone 10.3 antibody or clone 2C5 antibody) is administered in combination with another agent, wherein the antibody or antigen-binding fragment, variant, or derivative thereof and the therapeutic agent can be administered sequentially, in any order, or simultaneously (i.e., at the same time or within the same time frame).

[0407] Combination therapy can provide "synergistic effects," i.e., the effect achieved when active ingredients are used together can be found to be greater than the sum of the effects achieved when the compounds are used individually. Synergistic effects can be achieved when the active ingredients are (1) co-formulated and administered or delivered simultaneously in a combined unit dosage formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, in alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., sequentially, while in combination therapy, effective dosages of two or more active ingredients are administered together.

[0408] In other embodiments, the CD73 binding molecules disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody) can be administered in combination with a tyrosine kinase inhibitor. In some other specific embodiments, the CD73 binding molecules disclosed herein can be administered in combination with an inhibitor of tyrosine kinase activity associated with EGFR and / or HER2 / neu, e.g., lapatinib. In some embodiments, the CD73 binding molecules disclosed herein can be administered in combination with an antimitotic agent. In some specific embodiments, the CD73 binding molecules disclosed herein can be administered in combination with an agent that stabilizes spindle microtubule assembly, e.g., paclitaxel or docetaxel. A further embodiment is the use of an anti-CD73 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), for diagnostic monitoring of protein levels in tissues as part of a clinical trial procedure, thereby, for example, determining the efficacy of a given therapeutic regimen. Detection can be facilitated, for example, by coupling the antibody to a detectable substance.

[0409] 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; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H is one example.

[0410] VIII. Anti-CD73 Antibody Therapeutic Combinations and Co-Therapies The present disclosure provides methods related to 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) with therapeutic combinations comprising anti-CD73 binding molecules, e.g., antibodies, e.g., antigen-binding fragments, variants, and derivatives thereof (e.g., clone 10.3 antibody or clone 2C5 antibody).

[0411] While the following discussion refers to therapeutic combinations featuring a CD73 binding molecule of the present disclosure (e.g., clone 10.3 antibody or clone 2C5 antibody), the methods described herein are also applicable to any other anti-CD73 antibody, and antigen-binding fragments, variants, and derivatives (e.g., fusion proteins or conjugates) of these anti-CD73 antibodies that retain the desired properties of the anti-CD73 antibodies disclosed herein, e.g., the ability to specifically bind to CD73 and neutralize its 5'-nucleotidase activity. In some embodiments, the CD73 binding molecule is a human or humanized antibody that does not mediate human ADCC, or an anti-CD73 antibody that has been modified so that it does not mediate ADCC.

[0412] Treatment of patients with solid tumors with the combinations of the invention, such as an anti-CD73 antibody, or an antigen-binding fragment thereof, in combination with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, or an antigen-binding fragment thereof, may result in additive or synergistic effects. As used herein, the term "synergistic" refers to a combination of therapies that is more effective than the additive effects of the monotherapies, e.g., the combination of an anti-CD73 antibody (e.g., MEDI9447) with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody.

[0413] A synergistic effect of a combination of therapies, e.g., a combination of an anti-CD73 antibody (e.g., MEDI9447) with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, may allow for the use of lower dosages of one or more of the therapeutic agents and / or less frequent administration of the therapeutic agents in patients with solid tumors. The ability to utilize lower dosages of the therapeutic agents and / or administer the therapies less frequently reduces the toxicity associated with administration of the therapies to a subject without reducing the effectiveness of the therapies in treating the solid tumor. Additionally, a synergistic effect may increase the effectiveness of the therapeutic agents in managing, treating, or ameliorating a solid tumor. A synergistic effect of a combination of therapeutic agents may avoid or reduce adverse or unwanted side effects associated with the use of either monotherapy.

[0414] In cotherapy, the combination of an anti-CD73 antibody (e.g., MEDI9447) or antigen-binding fragment thereof and an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, or antigen-binding fragment thereof, can optionally be included in the same pharmaceutical composition or in separate pharmaceutical compositions. In this latter example, the pharmaceutical composition comprising the anti-CD73 antibody (e.g., MEDI9447) or antigen-binding fragment thereof is suitable for administration before, simultaneously with, or after the administration of the pharmaceutical composition comprising the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, or antigen-binding fragment thereof. Optionally, the anti-CD73 antibody (e.g., MEDI9447) or antigen-binding fragment thereof and the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody are administered at overlapping times in separate compositions.

[0415] The anti-CD73 antibody (e.g., MEDI9447) or antigen-binding fragment thereof and the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody, or antigen-binding fragment thereof, can be administered only once or less frequently while still providing benefit to the patient. In a further embodiment, the patient is administered additional follow-on doses. The follow-on doses may be administered at various time intervals depending on the patient's age, weight, clinical evaluation, tumor burden, and / or other factors, including the judgment of the attending physician.

[0416] The methods provided herein can reduce or delay tumor growth. In some aspects, this reduction or delay can be statistically significant. Reduction in tumor growth can be measured by comparison to the patient's tumor growth at baseline, comparison to expected tumor growth, comparison to expected tumor growth based on a large patient population, or comparison to tumor growth in a control population. In other embodiments, the methods of the present invention increase survival.

[0417] IX. Anti-PD-L1 antibody Antibodies that specifically bind to PD-L1 and inhibit its activity (e.g., binding to PD-1 and / or CD80) are useful for tumor treatment. B7-H1, also known as PD-L1, is a type I transmembrane protein approximately 53 kDa in size. In humans, B7-H1 is expressed on several 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. B7-H1 is also expressed on non-immune cells, including pancreatic islets, hepatic Kupffer cells, vascular endothelium, and certain epithelia, such as airway epithelium and renal tubular epithelium, and its expression is enhanced during inflammatory episodes. B7-H1 expression is also found at elevated levels in several tumors, including, but not limited to, breast cancer, colon cancer, colorectal cancer, lung cancer, renal cancer including renal cell carcinoma, gastric cancer, bladder cancer, non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), and pancreatic cancer, and melanoma.

[0418] B7-H1 is known to bind to two alternative ligands. The first, PD-1, is a 50-55 kDa type I transmembrane receptor originally identified in T cell lines that undergo activation-induced apoptosis. PD-1 is expressed on activated T cells, B cells, and monocytes, as well as other immune system cells, and binds both B7-H1 (PD-L1) and the related B7-DC (PD-L2). The second, a B7 family member called B7-1, is expressed on activated T cells, B cells, monocytes, and antigen-presenting cells.

[0419] Signaling through the PD-1 / B7-H1 axis is thought to play an important, essential role in 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. In this regard, B7-H1 functions through several alternative mechanisms, including depletion and anergy of tumor-infiltrating T lymphocytes, stimulation of the secretion of immunosuppressive cytokines into the tumor microenvironment, stimulation of suppressive regulatory T cell function, and protection of B7-H1-expressing tumor cells from lysis by tumor-cell-specific cytotoxic T cells.

[0420] MEDI4736 is an exemplary anti-PD-L1 antibody that is selective for B7-H1 and blocks B7-H1 binding to the PD-1 and CD80 receptors. MEDI4736 can attenuate B7-H1-mediated suppression of human T cell activation in vitro and inhibits tumor growth through a T cell-dependent mechanism in xenograft models. Other agents that may be used include agents (such as AB) that inhibit PD-L1 and / or PD-1.

[0421] For information regarding MEDI4736 (or fragments thereof) for use in the methods provided herein, see U.S. Patent Application Publication No. 20130034559 / U.S. Patent No. 8,779,108 and U.S. Patent Application Publication No. 20140356353, the disclosures of each 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, which are involved in mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0422] MEDI4736 and antigen-binding fragments thereof 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 embodiment, MEDI4736 or an antigen-binding fragment thereof used in the methods provided herein comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 130 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 131. In a specific embodiment, MEDI4736 or an antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences according to the Kabat definition of SEQ ID NOs: 132-134, and the light chain variable region comprises CDR1, CDR2, and CDR3 sequences according to the Kabat definition of SEQ ID NOs: 135-137. One of skill in the art would be able to readily identify CDR definitions using the Chothia definition, the Abm definition, or other CDR definitions known to one of skill in the art. In certain specific embodiments, MEDI4736 or an antigen-binding fragment thereof used in the methods provided herein comprises the variable heavy and variable light chain CDR sequences of the 2.14H9OPT antibody as disclosed in U.S. Patent Application Publication No. 20130034559 / U.S. Patent No. 8,779,108 and U.S. Patent Application Publication No. 20140356353 (the disclosures of each of which are incorporated herein by reference in their entirety).

[0423] X. Anti-CTLA4 antibody Thus, in one embodiment, the therapeutic combination of the invention comprises a CTLA4 blocking antibody (e.g., tremelimumab) and / or an antibody that reduces the PD1 / PD-L1 interaction. Two T cell regulatory pathways that have received the most attention to date signal through cytotoxic T lymphocyte-associated antigen 4 (CTLA4, CD152) and programmed death-ligand 1 (PD-L1, also known as B7H-1 or CD274).

[0424] CTLA4 is expressed on activated T cells and acts as a co-inhibitor that suppresses T cell responses after CD28-mediated T cell activation. CTLA4 regulates the amplitude of initial activation of naive and memory T cells after TCR engagement and is thought to be part of a central inhibitory pathway that influences both antitumor immunity and autoimmunity. CTLA4 is primarily expressed on T cells, while expression of its ligands, CD80 (B71) and CD86 (B7.2), is generally restricted to antigen-presenting cells, T cells, and other immune mediators. Antagonist 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 investigated in a phase III trial for the treatment of advanced melanoma, but did not significantly increase overall patient survival compared with standard treatments (temozolomide or dacarbazine) at the time.

[0425] For information regarding tremelimumab (or its antigen-binding fragment) used in the methods provided herein, see U.S. Patent No. 6,682,736 (referred to therein 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 ticilimumab) 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). Tremelimumab has been shown to result in immune activation in vitro, and some patients treated with tremelimumab have shown tumor regression.

[0426] The tremelimumab used in the 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 embodiment, the tremelimumab or antigen-binding fragment thereof used in the methods provided herein comprises a light chain variable region and a heavy chain variable region. In a specific embodiment, the tremelimumab or antigen-binding fragment thereof used in the methods provided herein comprises a heavy chain variable region and a light chain variable region as defined herein. Those skilled in the art will be able to readily identify the Chothia definition, the Abm definition, or other CDR definitions known to those of skill in the art. In a specific embodiment, the tremelimumab or antigen-binding fragment thereof 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 U.S. Patent No. 6,682,736 (incorporated herein by reference in its entirety).

[0427] XI.VII. Pharmaceutical Compositions and Methods of Administration Methods for preparing and administering anti-CD73 binding molecules, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), to a subject in need thereof are well known to, or can be readily determined by, those of skill in the art. The route of administration of anti-CD73 binding molecules, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be, for example, oral, parenteral, by inhalation, or topical. As used herein, the term parenteral includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration. However, in other methods consistent with the teachings herein, the anti-CD73 binding molecules of the present disclosure, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be delivered directly to the site of a harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0428] As discussed herein, the anti-CD73 binding molecules of the present disclosure, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), can be administered in a pharmaceutically effective amount for the in vivo treatment of diseases mediated by CD73-expressing cells, such as certain cancers.

[0429] Methods for preparing and administering to a subject in need thereof therapeutic combinations comprising an anti-CD73 binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), in combination with an anti-PD-1, anti-PD-L1, and / or anti-CTLA4 antibody, or antigen-binding fragment thereof, are well known to, or readily determined by, those of skill in the art. Routes of administration of these combinations can be, for example, oral, parenteral, by inhalation, or topical. As used herein, the term parenteral includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration. However, in other methods consistent with the teachings herein, the combinations of the present disclosure can be delivered directly to the site of the harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent. As discussed herein, an anti-CD73 antibody (e.g., MEDI9447) in combination with an anti-PD-1, anti-PD-L1, and / or anti-CTLA4 antibody can be administered in a pharmaceutically effective amount for the in vivo treatment of diseases mediated by CD73-expressing cells, such as certain cancers.

[0430] Pharmaceutical compositions used in the present disclosure may contain pharmaceutically acceptable carriers, including, for example, water, ion exchangers, proteins, buffer substances, and salts. Preservatives and other additives may also be present. The carrier may be a solvent or dispersion medium. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.), 16th ed. (1980).

[0431] In either case, a sterile injectable solution can be prepared by combining the therapeutic combinations of the invention in the required amount of active compound (e.g., an anti-CD73 antibody, or antigen-binding fragment, variant, or derivative thereof, e.g., clone 10.3 antibody or clone 2C5 antibody, alone or in combination with other active agents) in an appropriate solvent, followed by sterile filtration. Moreover, such preparations can be packaged and sold in kit form. Such articles can have a label or package insert indicating that the associated composition is useful for treating a subject suffering from or susceptible to a disease or disorder.

[0432] Parenteral formulations may be a single bolus dose, an infusion, or a loading bolus dose followed by a maintenance dose. These compositions may be administered at specific fixed or variable intervals, for example, once daily, or "as needed."

[0433] The compositions may be administered as a single dose, multiple doses, or over a defined period of time in an infusion. Dosage regimens may also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0434] Therapeutically effective doses of the compositions of the present disclosure for treating diseases mediated by CD73-expressing cells, such as certain cancers, including 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, will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or an animal, other medications being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, although non-human mammals, including transgenic mammals, may also be treated. Treatment dosages can be titrated to optimize safety and efficacy using routine methods known to those of skill in the art.

[0435] The dosage of at least one anti-CD73 binding molecule of the present invention, e.g., an antibody, or a binding fragment, variant, or derivative thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), or a therapeutic combination, can be readily determined by one of ordinary skill in the art without undue experimentation given the disclosure herein. Factors that influence the method of administration and the respective amounts of at least one anti-CD73 binding molecule of the present invention, e.g., an antibody, antigen-binding fragment, variant, or derivative thereof, or a therapeutic combination, include, but are not limited to, the severity of the disease, medical history, and the age, height, weight, health, and physical condition of the individual receiving treatment. Similarly, the dosage of an anti-CD73 binding molecule of the present invention, e.g., an antibody, or a fragment, variant, or derivative thereof, or a therapeutic combination, will depend on the method of administration and whether the subject receives a single or multiple doses of the agent.

[0436] The present disclosure also provides the use of an anti-CD73 binding molecule of the invention, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), or therapeutic combination, in the manufacture of a medicament for the treatment of certain cancers, including, for example, 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.

[0437] The present disclosure also provides for the use of an anti-CD73 binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), in the manufacture of a medicament for treating a subject for the treatment of certain cancers. In certain embodiments, the medicament is used in a subject who has been pretreated with at least one other therapy.

[0438] By "pretreated" or "pretreatment" is intended that the subject has undergone one or more other therapies (e.g., been treated with at least one other anti-cancer therapy) before being administered a medicament comprising an anti-CD73 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof (e.g., clone 10.3 antibody or clone 2C5 antibody). It is not necessary that the subject was a responder to the prior treatment with the prior therapy or therapies. Thus, a subject administered a medicament comprising an anti-CD73 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, may or may not have responded to the prior therapy, or to one or more of the prior therapies if the prior treatment included multiple therapies.

[0439] The present disclosure also provides for the co-administration of an anti-CD73 binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), with at least one other therapy. The anti-CD73 antibody and at least one other therapy can be co-administered together in a single composition, or can be co-administered together in separate compositions at the same time or at overlapping times. In some embodiments, the anti-CD73 antibody can be co-administered with, e.g., an antibody that targets PD-1 (programmed death 1 protein). The present disclosure also provides for the use of an anti-CD73 binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), in the manufacture of a medicament for treating a subject for treating cancer, wherein the anti-CD73 binding molecule is administered before the subject is treated with at least one other therapy.

[0440] VIII. Diagnosis The present disclosure further provides diagnostic methods useful in diagnosing diseases mediated by CD73-expressing cells, such as certain types of cancer, comprising measuring the expression level of CD73 protein in tissue or other cells or bodily fluids from an individual and comparing the measured expression level to a standard CD73 expression level in a normal tissue or bodily fluid, wherein an increased expression level compared to the standard is indicative of a disorder.

[0441] The anti-CD73 antibodies and antigen-binding fragments, variants, and derivatives thereof (e.g., clone 10.3 antibody or clone 2C5 antibody) disclosed herein can be used to assay CD73 protein levels in biological samples using classical immunohistological methods known to those skilled in the art (see, e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting CD73 protein expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable assays are described in more detail elsewhere herein.

[0442] By "assaying the expression level of a CD73 polypeptide" is intended to mean qualitatively or quantitatively measuring or estimating the level of a CD73 polypeptide in a first biological sample directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing to a disease-associated polypeptide level in a second biological sample). The CD73 polypeptide expression level in a first biological sample can be measured or estimated and compared to a standard CD73 polypeptide level, which can be taken from a second biological sample obtained from an individual without the disorder or determined by averaging the levels of a population of individuals without the disorder. As will be understood in the art, once a "standard" CD73 polypeptide level is known, it can be used repeatedly as a standard for comparison.

[0443] By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other cell source that potentially expresses CD73. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0444] IX. Kits containing CD73-binding molecules The present disclosure also provides kits comprising at least one of the CD73 binding molecules described herein, e.g., an anti-CD73 antibody or antigen-binding fragment thereof, a variant, or derivative of a molecule disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody), which can be used to practice the methods described herein. In certain embodiments, the kit comprises at least one purified anti-CD73 antibody or antigen-binding fragment thereof in one or more containers. In some embodiments, the kit contains all components necessary and / or sufficient to conduct a detection assay, including all controls, instructions for conducting the assay, and any software necessary for analyzing and publishing the results. One of skill in the art will readily recognize that the disclosed CD73 binding molecules, e.g., an anti-CD73 antibody or antigen-binding fragment thereof of the present disclosure (e.g., clone 10.3 antibody or clone 2C5 antibody), can be readily incorporated into one of the established kit formats well known in the art.

[0445] X. Immunoassay Anti-CD73 binding molecules disclosed herein, e.g., anti-CD73 antibodies or antigen-binding fragments thereof, variants, or derivatives of the molecules disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody), can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay, to name a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds. (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, incorporated herein by reference in its entirety).

[0446] CD73-binding molecules, e.g., anti-CD73 antibodies or antigen-binding fragments thereof, and variants or derivatives thereof (e.g., clone 10.3 antibody or clone 2C5 antibody), can be used histologically for in situ detection of CD73 or its conserved variants or peptide fragments, such as by immunofluorescence, immunoelectron microscopy, or non-immunological assays. In situ detection can be achieved by removing a tissue specimen from a patient and applying a labeled CD73-binding molecule, e.g., an anti-CD73 antibody or its antigen-binding fragment, variant, or derivative, to the specimen, preferably by overlaying the labeled CD73-binding molecule (e.g., antibody or fragment) on the biological sample. Using such procedures, it is possible to determine not only the presence of CD73 or its conserved variants or peptide fragments, but also its distribution in the tissue under test. Using the present disclosure, one of skill in the art will readily understand that any of a wide variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0447] The binding activity of a given lot of a CD73 binding molecule, e.g., an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or an antigen-binding fragment, variant, or derivative thereof, can be determined by well-known methods. One skilled in the art will be able to determine effective and optimal assay conditions for each determination using routine experimentation.

[0448] Methods and reagents suitable for determining the binding properties of an isolated CD73-binding molecule, e.g., an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or antigen-binding fragment thereof, variant, or modified / mutated derivative thereof, are known in the art and / or commercially available. Instruments and software designed for such kinetic analyses are commercially available (e.g., BIAcore, BIAevaluation software, GE Healthcare; KinExa software, Sapidyne Instruments).

[0449] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art and are explained fully in the literature. For example, Sambrook et al...

Claims

1. Antibody V Ｌ 1. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, comprising: Ｌ But the amino acid sequence: []. １ \$ AlnSn\\G want １ [#] ２ ️ ２ ︚ ３ ︹ ４ ︸ ５ []. ３ ________ ６ ︸ ７ ︃︸ ８ []. ４ ] (In the formula, [FW １ ], [FW ２ ], [FW ３ ] and [FW ４ ]is V Ｌ represents a framework region, １ represents the amino acid residue proline (P), glutamic acid (E) or aspartic acid (D); ２ represents the amino acid residue asparagine (N) or aspartic acid (D), X ３ represents the amino acid residue glutamine (Q) or leucine (L), X ４ represents the amino acid residue leucine (L) or proline (P), X ５ represents the amino acid residue glycine (G) or serine (S), X ６ represents the amino acid residue leucine (L) or histidine (H), X ７ represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N), and X ８ represents the amino acid residue leucine (L) or threonine (T), or

2. Forward １ comprises SEQ ID NO: 25 or 26, FW ２ comprises SEQ ID NO: 27 or 28, FW ３ comprises SEQ ID NO: 29, and FW ４ The isolated binding molecule or antigen-binding fragment thereof of claim 1, wherein said binding molecule or antigen-binding fragment comprises SEQ ID NO:

30.

3. 1. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, comprising an antibody VH, Ｈ But the amino acid sequence: [FW ５ ]SYAX ９ S[FW ６ ]X １０ IX １１ GSX １２ GX １３ TYYADSVKG[FW ７ ]LGYX １４ X １５ X １６ DX １７ [FW ８ ] (In the formula, [FW ５ ], [FW ６ ], [FW ７ ] and [FW ８ ] represents the VH framework region, X ９ represents the amino acid residue methionine (M) or tyrosine (Y), 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), X １３ represents the amino acid residue serine (S) or arginine (R), X 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 represents the amino acid residue tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

4. Forward ５ comprises SEQ ID NO: 31, FW ６ comprises SEQ ID NO: 32, FW ７ comprises SEQ ID NO: 33, and FW ８ The isolated binding molecule or antigen-binding fragment thereof of claim 3, wherein said binding molecule or antigen-binding fragment comprises SEQ ID NO:

34.

5. Antibody V Ｌ and antibody V Ｈ 1. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, comprising: Ｌ But the amino acid sequence: []. １ \$ AlnSn\\G want １ [#] ２ ️ ２ ︚ ３ ︹ ４ ︸ ５ []. ３ ________ ６ ︸ ７ ︃︸ ８ []. ４ ] (In the formula, [FW １ ], [FW ２ ], [FW ３ ] and [FW ４ ]is V Ｌ represents a framework region, where: X １ represents the amino acid residue proline (P), glutamic acid (E) or aspartic acid (D), X ２ represents the amino acid residue asparagine (N) or aspartic acid (D), X ３ represents the amino acid residue glutamine (Q) or leucine (L), X ４ represents the amino acid residue leucine (L) or proline (P), X ５ represents the amino acid residue glycine (G) or serine (S), X ６ represents the amino acid residue leucine (L) or histidine (H), X ７ represents the amino acid residue lysine (K), proline (P), isoleucine (I) or asparagine (N), and X ８ represents the amino acid residue leucine (L) or threonine (T), Ｈ But the amino acid sequence: [FW ５ ]SYAX ９ S[FW ６ ]X １０ IX １１ GSX １２ GX １３ TYYADSVKG[FW ７ ]LGYX １４ X １５ X １６ DX １７ [FW ８ ] (In the formula, [FW ５ ], [FW ６ ], [FW ７ ] and [FW ８ ] represents the VH framework region, X ９ represents the amino acid residue methionine (M) or tyrosine (Y), 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), X １３ represents the amino acid residue serine (S) or arginine (R), X 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 represents the amino acid residue tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

6. Forward １ comprises SEQ ID NO: 25 or 26, FW ２ comprises SEQ ID NO: 27 or 28, FW ３ comprises SEQ ID NO: 29, FW ４ comprises SEQ ID NO: 30, FW ５ comprises SEQ ID NO: 31, FW ６ comprises SEQ ID NO: 32, FW ７ comprises SEQ ID NO: 33, and FW ８ The isolated binding molecule or antigen-binding fragment thereof of claim 5, wherein said binding molecule or antigen-binding fragment comprises SEQ ID NO:

34.

7. 7. The isolated binding molecule or antigen-binding fragment thereof of any one of claims 1 to 6, comprising an antibody or antigen-binding fragment thereof.

8. Antibody V Ｌ 1. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, comprising: Ｌ or SEQ ID NOs:48, 52, and 54; SEQ ID NOs:46, 52, and 56; SEQ ID NOs:47, 49, and 56; 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, or identical thereto except for 4, 3, 2, or 1 amino acid substitutions in one or more of the VL-CDRs,

9. Antibody V Ｈ 1. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, comprising: Ｈ or SEQ ID NOs:36, 40, and 45; or SEQ ID NOs:36, 40, and 45, respectively, which are identical to, or identical to, except for 4, 3, 2, or 1 amino acid substitutions in one or more of the VH-CDRs.

10. 10. The isolated binding molecule or antigen-binding fragment thereof of claim 8 or 9, comprising an antibody or antigen-binding fragment thereof.

11. 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: 46, 49, 53, 35, 37, and 45; SEQ ID NOs: 47, 49, 56, 36, 37, and and 45; SEQ ID NOs: 46, 50, 56, 36, 38, and 45; SEQ ID NOs: 46, 51, 56, 36, 39, and 45; SEQ ID NOs: 48, 52, 56, 36, 40, and 45; or VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences that are identical to SEQ ID NOs: 46, 49, 56, 35, 37, and 45, or identical thereto except for 4, 3, 2, or 1 amino acid substitutions in one or more CDRs. Ｌ and V Ｈ 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to CD73, comprising:

12. 12. The isolated antibody or antigen-binding fragment thereof of claim 11, comprising an antibody or antigen-binding fragment thereof.

13. V comprising SEQ ID NO:68 Ｌ and V comprising SEQ ID NO:82 Ｈ and an isolated antibody or antigen-binding fragment thereof.

14. V consisting essentially of SEQ ID NO:68 Ｌ and V consisting essentially of SEQ ID NO:82 Ｈ and an isolated antibody or antigen-binding fragment thereof.

15. V consisting of SEQ ID NO:68 Ｌ and V consisting of SEQ ID NO:82 Ｈ and an isolated antibody or antigen-binding fragment thereof.

16. 8. The isolated binding molecule or antigen-binding fragment thereof of claim 7, comprising a heavy chain constant region or fragment thereof.

17. 11. The isolated binding molecule or antigen-binding fragment thereof of claim 10, comprising a heavy chain constant region or fragment thereof.

18. 16. The isolated antibody or antigen-binding fragment thereof of any one of claims 11 to 15, comprising a heavy chain constant region or fragment thereof.

19. A nucleic acid comprising a sequence encoding the isolated antibody or antigen-binding fragment thereof of any one of claims 7 or 10 to 18.

20. A host cell comprising the nucleic acid sequence of claim 19.

21. 20. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 7 or 10 to 18, comprising the steps of: (a) culturing a cell according to claim 20; and (b) isolating the antibody or antigen-binding fragment thereof.

22. 8. The isolated binding molecule or antigen-binding fragment thereof of claim 7, which is an antagonist of CD73.

23. 11. The isolated binding molecule or antigen-binding fragment thereof of claim 10, which is an antagonist of CD73.

24. 16. The isolated antibody or antigen-binding fragment thereof of any one of claims 11 to 15, which is an antagonist of CD73.

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

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

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

28. 28. Use of the antibody or antigen-binding fragment thereof according to any one of claims 7 to 27 in the manufacture of a medicament for the treatment of cancer.

29. 29. The use of claim 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.

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 expression of CD73 compared to a reference.

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

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.

33. 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 antigen-binding fragment thereof.

34. 34. The use of claim 33, wherein the anti-PD-1 antibody is pembrolizumab (KEYTRUDA®, lambrolizumab, MK-3475), nivolumab (OPDIVA®, BMS-936558, MDX-1106, ONO-4538), AMP-224, or an antigen-binding fragment thereof.

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

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

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.

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

39. 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.

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

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

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

43. Antibody V Ｌ and antibody V Ｈ 1. An isolated binding molecule, or antigen-binding fragment thereof, that specifically binds to CD73, having the formula:

44. 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.

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

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

47. 45. The isolated binding molecule or antigen-binding fragment thereof of claim 43 or 44, which binds to an epitope in one or more of the following regions of CD73 protein: Tyr132 to Val144 and / or Lys180 to Asn187.

48. 45. The isolated binding molecule or antigen-binding fragment thereof of claim 43 or 44, comprising the amino acid sequence at Tyr132 to Val144 and / or Lys180 to Asn187.

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