CD73-specific binding molecule and its use

Abstract

The present invention provides anti-CD73 binding molecules, such as antibodies and their antigen-binding fragments. [Solution] Provided are isolated binding molecules or antigen-binding fragments thereof that contain an antibody VL and specifically bind to CD73 having a specific sequence. Furthermore, pharmaceutical formulations comprising the disclosed composition, as well as methods for diagnosing and treating diseases associated with CD73 expression, such as cancer, are also provided. Such diseases can be treated, for example, by direct therapy with the disclosed anti-CD73 binding molecule (e.g., a naked antibody or antibody-drug conjugate that binds to CD73), by adjuvant therapy with other antigen-binding anticancer agents such as immune checkpoint inhibitors (e.g., anti-CTLA-4 and anti-PD-1 monoclonal antibodies), and / or by combination therapy in which the anti-CD73 molecule is administered before, after, or concurrently with chemotherapy.

Description

[Background technology]

[0001] CD73, or ecto-5'-nucleotidase (5'-NT), is ubiquitously expressed in many tissues. This protein anchors to the cell membrane via glycosylphosphatidylinositol (GPI) ligation, possesses extracellular enzymatic activity, and plays a role in signal transduction. The main function of CD73 is to convert extracellular nucleotides (e.g., 5'-AMP), which are generally impermeable to cells, into their corresponding nucleosides (e.g., adenosine), which can readily enter many cells. Adenosine production by AMP dephosphorylation of CD73 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 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. Elevated CD73 expression has also been associated with reduced tumor invasiveness, metastasis, and 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 premetastatic phenotypes in melanoma and breast cancer. [Overview of the project] [Problems that the invention aims to solve]

[0003] Immune checkpoint inhibitors hold immense potential as a cancer therapy. Despite this, the clinical benefits of immune checkpoint inhibition have not been fully realized. One possible explanation is that tumors promote immune escape using uninterrupted immunosuppressive mechanisms. Therefore, improved compositions and methods to reduce 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 embodiments, such CD73 binding molecules are, for example, antibodies or antigen-binding fragments thereof. In detailed embodiments, the anti-CD73 antibody of the present invention (e.g., MEDI9447) is useful for reducing tumor-mediated immunosuppression. Accordingly, the present invention also provides therapeutic combinations characterized by an anti-CD73 antibody (e.g., MEDI9447) and other agents that target other aspects of the cancer immune cycle (i.e., anti-PD-1 or anti-PD-L1 antibodies; anti-CTLA4 antibodies, A2aR antagonists, STAT-3 inhibitors), the use of which such combinations is useful for reducing tumor-mediated immunosuppression.

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

[0006] In another embodiment, the present invention specifically binds to CD73 and to the 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 its antigen-binding fragment that competitively inhibits CD73 binding by an antibody or its antigen-binding fragment.

[0007] In another aspect, the present invention relates to antibody V L This provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and includes V Lis the amino acid sequence: [FW1]SGSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4] (where [FW1], [FW2], [FW3] and [FW4] are V L represent framework regions, 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)). In various embodiments of any aspect described herein, it is the isolated binding molecule or its antigen-binding fragment according to 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 its antigen-binding fragment that specifically binds to CD73 and comprises an antibody VH, where 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] (where [FW5], [FW6], [FW7] and [FW8] represent VH framework regions, where 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 aspect, the present invention relates to antibody V L and antibody V H This provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and includes V L The amino acid sequence is: [FW1]SGSLSNIGRNX1VN[FW2]LX2NX3RX4X5[FW3]ATWDDSX6X7GWX8[FW4] (In the formula, [FW1], [FW2], [FW3] and [FW4] are V L This represents the framework domain, and here, 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 has an amino acid residue (representing leucine (L) or threonine (T)), V H The amino acid sequence is: [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 the VH framework region, where, 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 aspect, the present invention relates to antibody V L The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V L This is identical to SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52, or identical 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 aspect, the present invention relates to antibody V L The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V L It has a complementarity-determining region 3 (VL-CDR3) amino acid sequence that is identical to, or identical to, sequence number 53, sequence number 54, sequence number 55, or sequence number 56, except for 4, 3, 2, or 1 amino acid substitution.

[0012] In another aspect, the present invention relates to antibody V H The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V H It has a complementarity-determining region 1 (VH-CDR1) amino acid sequence that is identical to or identical to sequence number 35 or 36, except for 4, 3, 2, or 1 amino acid substitutions.

[0013] In another aspect, the present invention relates to antibody V H The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V H It has a complementarity-determining region 2 (VH-CDR2) amino acid sequence that is identical to, or identical to, sequence number 37, sequence number 38, sequence number 39, or sequence number 40, except for 4, 3, 2, or 1 amino acid substitution.

[0014] In another aspect, the present invention relates to antibody V H The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V HIt has a complementarity-determining region 3 (VH-CDR3) amino acid sequence that is identical to, or identical to, sequence number 41, sequence number 42, sequence number 43, sequence number 44, or sequence number 45, except for 4, 3, 2, or 1 amino acid substitution.

[0015] In another aspect, the present invention relates to antibody V L The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V L These VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences are identical to or identical to SEQ ID NOs. 46, 49, and 53; SEQ ID NOs. 47, 49, and 53; SEQ ID NOs. 47, 49, and 54; SEQ ID NOs. 46, 50, and 54; SEQ ID NOs. 46, 51, and 55; SEQ ID NOs. 48, 52, and 54; SEQ ID NOs. 46, 49, and 56; SEQ ID NOs. 47, 49, and 56; SEQ ID NOs. 46, 50, and 56; SEQ ID NOs. 46, 51, and 56; or SEQ ID NOs. 48, 52, and 56, respectively.

[0016] In another aspect, the present invention relates to antibody V H The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V H These each have the same VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences as 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, or are identical to them except for 4, 3, 2, or 1 amino acid substitution in one or more VH-CDRs.

[0017] In another aspect, the present invention relates to Sequence IDs 46, 49, 53, 35, 37, and 41; Sequence IDs 47, 49, 53, 35, 37, and 41; Sequence IDs 47, 49, 54, 36, 37, and 42; Sequence IDs 46, 50, 54, 36, 38, and 43; Sequence IDs 46, 51, 55, 36, 39, and 44; Sequence IDs 48, 52, 54, 36, 40, and 44; Sequence IDs 46, 49, 56, 35, 37, and 41; Sequence IDs 46, 49, 53, 35, 37, and 45; Sequence IDs 47, 49, 56, 3 VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences that are identical to or identical to sequence numbers 46, 49, 56, 35, 37, and 45, 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 its antigen-binding fragment that specifically binds to CD73.

[0018] In another aspect, the present invention relates to antibody V L and antibody V H The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V L It has an amino acid sequence that is at least approximately 90% to approximately 100% identical to a reference amino acid sequence selected from SEQ ID NOs. 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70.

[0019] In another aspect, the present invention relates to antibody V L and antibody V H The present invention provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and V HIt has an amino acid sequence that is at least approximately 90% to approximately 100% identical to a reference amino acid sequence selected from SEQ ID NOs. 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84.

[0020] In another embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to CD73, wherein the antibody or antigen-binding fragment has a sequence that is at least about 90% to about 100% identical to a reference amino acid sequence selected from SEQ ID NOs. 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70. L 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 NOs. 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84. H It holds.

[0021] In another embodiment, the present invention essentially comprises V from Sequence ID No. 57. L And V essentially derived from sequence number 71. H The present invention provides an isolated antibody or its antigen-binding fragment having the above characteristics.

[0022] In another embodiment, the present invention essentially consists of V from Sequence ID No. 68. L And V essentially becomes from sequence number 82. H The present invention provides an isolated antibody or its antigen-binding fragment having the above characteristics.

[0023] In another embodiment, the present invention relates to V, which is set to sequence number 57. L V consisting of sequence number 71 H The present invention provides an isolated antibody or its antigen-binding fragment having the above characteristics.

[0024] In another embodiment, the present invention relates to V, which is set to sequence number 68. L V consisting of sequence number 82 H The present invention provides an isolated antibody or its antigen-binding fragment having the above characteristics.

[0025] In another embodiment, the present invention provides a composition containing an isolated antibody or an antigen-binding fragment thereof and a carrier according to the present invention.

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

[0027] In another embodiment, the present invention provides compositions comprising nucleic acids according to the present invention.

[0028] In another embodiment, the present invention provides a vector containing nucleic acid according to the present invention.

[0029] In another embodiment, 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 for producing an antibody or an antigen-binding fragment thereof according to the present invention, comprising the steps of culturing cells containing a nucleic acid sequence, composition, or vector according to the present invention, and isolating the antibody or an antigen-binding fragment thereof.

[0031] In another embodiment, the present invention provides a diagnostic reagent containing a labeled isolated antibody or antigen-binding fragment according to the present invention.

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

[0033] In another embodiment, the present invention provides a method for inhibiting the growth of cells expressing CD73, comprising the step of contacting the cells with an antibody or antigen-binding fragment thereof according to the present invention.

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

[0035] In another embodiment, the present invention provides a method for treating a target cancer, comprising the step of administering to the target a therapeutically effective amount of a first agent, which is an antibody or antigen-binding fragment according to the present invention, in combination with a therapeutically effective amount of a second agent, which is an anticancer agent other than the first agent.

[0036] In another embodiment, the present invention provides a therapeutic method comprising the step of administering an anti-CD73 antibody or its antigen-binding fragment to a subject identified as having a tumor with increased CD73 expression compared to a reference.

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

[0038] In another embodiment, the present invention provides a therapeutic method comprising the step of 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 CD73 expression compared to a reference.

[0039] In another embodiment, the present invention provides a therapeutic method comprising the step of administering MEDI9447 or Phen0203 hIgG1 or its antigen-binding fragment and MEDI4736 or its antigen-binding fragment to a subject identified as having a tumor with increased CD73 expression compared to a reference.

[0040] In another embodiment, the present invention provides a therapeutic method comprising the step of administering MEDI9447 or Phen0203 hIgG1 or its antigen-binding fragment and tremelimumab or its antigen-binding fragment to a subject identified as having a tumor with increased CD73 expression compared to a reference.

[0041] In another embodiment, the present invention provides a method for identifying a subject having cancer responsive to anti-CD73 therapy, the method comprising the step of detecting an increase in CD73 expression or activity levels in the subject's tumor cells or blood cells compared to a reference, thereby identifying the cancer as responsive to anti-CD73 therapy.

[0042] In another embodiment, the present invention provides a method for identifying a subject having cancer responsive to anti-CD73 therapy in combination with one or more anti-PD-1, anti-PD-L1, or anti-CTLA4 therapies, the method comprising the step of detecting an increase in CD73 expression or activity levels in the subject's tumor cells or blood cells compared to a reference, thereby identifying the cancer as responsive to anti-CD73 therapy in combination with one or more anti-PD-1, anti-PD-L1, or anti-CTLA4 therapies.

[0043] In another embodiment, the present invention provides a method for identifying a subject having cancer responsive to anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy, the method comprising the step of identifying the cancer as responsive to anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy by detecting a decrease in CD73 expression or activity levels in the subject's tumor cells or blood cells compared to a reference.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0058] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of Phen0203 hIgG1 or its antigen-binding fragment and tremelimumab or its antigen-binding fragment.

[0059] In another aspect, the present invention provides a pharmaceutical formulation containing an effective amount of Phen0203 hIgG1 or its antigen-binding fragment and ipilimumab or its antigen-binding fragment.

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

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

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

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

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

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

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

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

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

[0069] In various embodiments of any model described herein, the isolated antibody or its antigen-binding fragment comprises VL having SEQ ID NO: 57 and VH having SEQ ID NO: 71.

[0070] In various embodiments of any model described herein, the isolated antibody or its antigen-binding fragment comprises VL having SEQ ID NO: 68 and VH having SEQ ID NO: 82.

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

[0072] In various embodiments, the heavy chain steady region or a fragment thereof is an IgG steady region including, for example, an IgG1 steady region, an IgG2 steady region, an IgG3 steady region, or an IgG4 steady region.

[0073] In various embodiments of any model described herein, an isolated antibody or its antigen-binding fragment comprises a light chain constant region selected from a human κ constant region and a human λ constant region.

[0074] In various embodiments of any aspect described herein, the IgG constant region has one or more amino acid substitutions compared to the wild-type IgG constant region, and this modified IgG has an increased half-life compared to IgG having 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 (numbered according to the EU index as defined in Kabat).

[0076] In various embodiments of any of the features described herein, 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 by 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 by leucine (L), (e) Substitution of the amino acid at position 309 by proline (P), (f) Substitution of the amino acid at position 311 by 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) Two or more combinations of the above substitutions (Numbering follows the EU index as defined in Kabat)

[0077] In various embodiments of any of the features described herein, the human IgG constant region has amino acid substitutions at positions 252, 254, and 256 compared to the wild-type human IgG constant region, where, (a) The amino acid at position 252 is substituted by tyrosine (Y), (b) The amino acid at position 254 is substituted with threonine (T), and (c) The amino acid at position 256 is substituted with glutamic acid (E). (Numbering follows the EU index as defined in Kabat.)

[0078] In various embodiments of any part 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 the numbering follows 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 the numbering follows the EU index as defined in Kabat.

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

[0081] In various embodiments of any aspect described herein, the amino acid at position 428 of Kabat is substituted with leucine (L), and the amino acid at position 434 of Kabat is substituted with serine (S).

[0082] In various embodiments of any model described herein, the human IgG constant region has amino acid substitutions at positions 252, 254, and 256 compared to the wild-type human IgG constant region (numbered according to the EU index as defined in Kabat), where, (a) The amino acid at position 252 is substituted by tyrosine (Y), (b) The amino acid at position 254 is substituted with threonine (T), and (c) The amino acid at position 256 is substituted with 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 Fv, Fab, F(ab')2, Fab', dsFv, scFv, or sc(Fv)2.

[0085] In various embodiments of any model described herein, the isolated antibody or its antigen-binding fragment is conjugated to at least one heterologous drug, for example, an anticancer drug.

[0086] In various embodiments of any model described herein, the composition according to the present invention further comprises an anticancer agent.

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

[0088] In various embodiments of any model described herein, the isolated antibody or its antigen-binding fragment is a CD73 antagonist.

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

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

[0091] In various embodiments of any model described herein, cell proliferation can be reduced by the binding of the antibody or antigen-binding fragment to CD73.

[0092] In various embodiments of any model described herein, antibodies or antigen-binding fragments against CD73 can bind to human CD73, cynomolgus monkey CD73, and mouse CD73.

[0093] In various embodiments of any of the features described herein, 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 model described herein, the cancer, including melanoma or breast cancer, has a premetastatic phenotype.

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

[0096] In various embodiments of any model described herein, the combination of the first agent and the second agent may have excellent antitumor activity, which may be additive or synergistic.

[0097] In various embodiments of any model described herein, the second agent is an antibody or an 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 model described herein, the second agent is an anti-CTLA-4 antibody or its antigen-binding fragment, including, for example, ipilimumab, tremelimumab (tisilimmab, CP-675,206), or its antigen-binding fragment.

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

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

[0102] In various embodiments of any model described herein, the anti-CD73 antibody is MEDI9447, Phen0203 hIgG1, or its antigen-binding fragment.

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

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

[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 its antigen-binding fragment.

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

[0107] In various embodiments, the anti-CTLA-4 antibody is ipilimumab, tremelimumab (tisilimunab, 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 lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma, ovarian cancer, breast cancer, head and neck cancer, or pancreatic cancer.

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

[0110] In various embodiments of any model described herein, CD73 expression is detected in tumor cells or peripheral blood cells, including, for example, lymphoid cells 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 described herein, CD73 expression is detected by flow cytometry, immunohistochemistry (IHC), or by CD73 enzyme activity or soluble CD73 levels in the sample.

[0112] In various embodiments of any model described herein, an anti-CD73 antibody or its antigen-binding fragment is administered simultaneously with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or its antigen-binding fragment.

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

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

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

[0116] In another embodiment, the present invention provides an isolated binding molecule or antigen-binding fragment thereof having antibody VL and 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 model described herein, the isolated binding molecule or antigen binding further comprises one or more amino acids corresponding to Tyr135, Lys136, and Asn187.

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

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

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

[0121] In various embodiments of any model described herein, the isolated binding molecule or its antigen-binding fragment contains or is contained within the amino acid sequence Tyr132-Val144 and / or Lys180-Asn187.

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

[0123] In various embodiments of any model described herein, the stereoepitope further comprises one or more amino acids corresponding to Tyr135, Lys136, and Asn18.

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

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

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

[0127] In various embodiments of any aspect described herein, the stereoepitope contains or is contained within the amino acid sequence Tyr132-Val144 and / or Lys180-Asn187.

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

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

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

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

[0132] [Figure 1A] The nucleotide sequence and amino acid translation of the MEDI9447 VH domain are shown, and the CDR is indicated according to the Kabat numbering rules. [Figure 1B] The nucleotide sequence and amino acid translation of the MEDI9447 VL domain are shown, and the CDR is indicated according to the Kabat numbering rules. [Figure 1C] This shows the alignment of MEDI9447 VH with its closest human VH and JH germline sequences. The CDR (Chronic Derivative Rating) based on Kabat numbering rules is highlighted, and residues differing from the germline sequence are enclosed in boxes. [Figure 1D] This shows the alignment of MEDI9447 VL with its closest relatives, the human VL and JL germline sequences. The CDR based on Kabat numbering rules is highlighted, and residues differing from the germline sequence are enclosed in boxes. [Figure 2] Two graphs are provided showing antibody-mediated internalization of cytotoxic FabZAP reagents into MDA-MB-231 cells and 4T1 cells, where the antibodies are 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. Figure 3B is a graph showing the inhibition of AMP hydrolysis by the anti-CD73 antibody CD370010. [Figure 4] This graph shows that MEDI9447 inhibited tumor growth in a CT26 syngeneic tumor model. Mouse CT26 tumor cells were subcutaneously transplanted into the right flank of female Balb / C mice. The tumors were allowed to grow for 3 days and then treated twice a week for 2 weeks with either MEDI9447 or an isotype control. The tumors were harvested on day 16 and subjected to flow cytometry analysis. [Figure 5] This graph shows that MEDI9447 inhibited tumor-infiltrating myeloid suppressor cells (MDSCs). On day 16 of the study, CT26 tumor-bearing mice treated with MEDI9447 were sacrificed, and the tumors were recovered. The tumors were dissociated into single cells, stained for CD45 and MDSC markers, and analyzed by flow cytometry. [Figure 6] The study includes six spider plots showing the effects of MEDI9447 mIgG1, anti-PD-1, or combination therapy on tumor volume. Control antibodies include rIgG2a (a rat IgG2a-controlled monoclonal rat antibody specific to E. coli β-galactosidase (β-Gal)) and isotype control mouse IgG1. Tumor volume for each animal group up to day 40 of the study was plotted for individual animals. In the control group, no mice were tumor-free by the end of the 40-day study period. With anti-CD73 therapy alone, 10% of animals were tumor-free at the end of the study. Similarly, with anti-PD1 therapy alone, 10% of animals were tumor-free at the end of the study. Notably, with combination therapy of anti-CD73 and anti-PD, 60% of mice were tumor-free. In the control group, no mice were tumor-free by the end of the study. [Figure 7] This graph shows the effects of MEDI9447 mIgG1, anti-PD1, or combination therapy on survival. [Figure 8] This graph shows that the combination of MEDI9447 and anti-PD-1 significantly enhanced tumor growth inhibition in colorectal cancer tumors (<0.05) compared to the use of either drug alone. Mice were subcutaneously injected with syngeneic MC38-OVA colorectal cancer cells and treated twice weekly with 10 mg / kg of MEDI9447, 10 mg / kg of anti-PD-1 antibody alone, or a combination of both antibodies. Tumor volume was measured twice weekly. [Figure 9] This graph 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 subcutaneously injected with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg of anti-PD-1 or an unrelated isotype control antibody. The tumor was excised the day after the first treatment, the cells were dissociated, and their surface phenotype was analyzed by flow cytometry. [Figure 10] This graph 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 subcutaneously injected with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg of anti-PD-1 or an unrelated isotype control antibody. The day after the initial treatment, tumors were excised, tumor cells were isolated, peripheral whole blood cells were collected, and surface CD73 expression was analyzed by flow cytometry. [Figure 11] This graph 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 subcutaneously injected with syngeneic CT26 colorectal cells and treated twice weekly with 10 mg / kg of anti-PD-1 or an unrelated isotype control antibody. Three days after the initial treatment, the tumors were excised, peripheral whole blood cells were collected, and surface CD73 expression was analyzed by flow cytometry. [Figure 12]This is a graph showing that the combination of MEDI9447 and anti-PD-L1 significantly enhanced the inhibition of tumor growth in melanoma tumors (<0.05) when compared to either drug alone. Syngeneic B16F10 melanoma cells were subcutaneously injected into mice and treated twice weekly with 10 mg / kg of MEDI9447 or 10 mg / kg of anti-PD-L1 antibody alone or in combination with both antibodies. Tumor volume was measured twice weekly. [Figure 13] This is a graph showing that the combination of MEDI9447 and anti-PD-L1 significantly enhanced the inhibition of tumor growth (p<0.01) in lymphoma tumors when compared to either drug alone. Syngeneic EG7-OVA lymphoma cells were subcutaneously injected into mice and treated twice weekly with 10 mg / kg of MEDI9447 or 10 mg / kg of anti-PD-L1 antibody alone or in combination with both antibodies. Tumor volume was measured twice weekly. [Figure 14] This is a graph showing that anti-PD-L1 induced a CD73-rich tumor microenvironment as measured by the surface expression of CD73 on draining lymph node B lymphocytes. Syngeneic CT26 colorectal cells were subcutaneously injected into mice (n = 4) and treated twice weekly with 10 mg / kg of anti-PD-L1 or an irrelevant isotype control antibody. Cells were isolated from the draining lymph nodes on the day after the first treatment and analyzed for surface phenotype by flow cytometry. [Figure 15] This is a graph showing that anti-PD-L1 induced a CD73-rich tumor microenvironment as measured by the surface expression of CD73 on tumor infiltrating CD4+, FoxP3+ lymphocytes. Syngeneic CT26 colorectal cells were subcutaneously injected into mice (n = 4) 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, dissociated, and analyzed for surface phenotype by flow cytometry. [Figure 16]Graph showing that the expression of CD73 on tumor infiltrating lymphocytes was reduced by MEDI9447 alone or in combination with anti-PD-L1. Mice bearing colorectal CT26 syngeneic tumors were treated twice weekly (on days 12 and 16) with either 30 mg / kg MEDI9447 or 30 mg / kg anti-PD-L1 alone or in combination with 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 infiltrates into (A) CD4+ FoxP3+ Tregs and (B) CD8+ T cells. [Figure 17] Graph showing that the CD73 activity of (A) tumor cells and (B) peripheral whole blood cells was reduced by MEDI9447 alone or in combination with anti-PD-L1. Mice bearing colorectal CT26 syngeneic tumors were treated twice weekly (on days 12 and 16) with either 30 mg / kg MEDI9447 or 30 mg / kg anti-PD-L1 alone or in combination with both MEDI9447 and anti-PD-L1. On day 17, tumors and peripheral whole blood cells were harvested and the enzyme activity was analyzed for surface CD73 expression by using Cell-Titre Glo. [Figure 18] Set of graphs representing the cytokine profiles of peripheral blood mononuclear cells treated with MEDI9447 and antibodies or fusion proteins specific for CTLA4, OX40, PD-1, and PD-L1. Primary human peripheral blood mononuclear cells were incubated for 72 hours in a mixed leukocyte reaction with MEDI9447 and / or an antibody or fusion protein specific for the indicated target. Cytokines (IFN-γ, IL-1β, TNF-α) in duplicate supernatants were quantified by ELISA. The data shown correspond to the optimal dose combinations of anti-CD73 antibody with four different partner drugs. The combination of anti-PD-1 and anti-CD73 showed significant (p<0.05) synergy when determined by the Bliss surface reaction method (Zhao et al.). The cytokine profiles indicate that both the myeloid and lymphoid lineages were affected. Over 50 donor pairs were tested. [Figure 19A]This shows the results of hydrogen-deuterium exchange MS (HDX-MS) analysis of CD73 compounded with MEDI9447. Figure 19A shows a hydrogen-deuterium exchange heatmap of CD73 (from the N-terminus to the C-terminus) showing the region where deuterium uptake decreases upon binding with MEDI9447. The relative exchange between antibody-bound CD73 and unbound CD73 is shown as a function of exposure time, with decreased exchange shown in red, increased exchange in blue, and no change in white. The N-terminal regions at positions 132-143 and 182-187 showed the highest differential exchange. Figure 8B shows the crystal structure of the CD73 monomer, showing 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 20A shows the results of hydrogen-deuterium exchange MS (HDX-MS) analysis comparing the free and bound states to identify regions in CD73 and MEDI9447 where differential hydrogen exchange occurs. Figure 20B shows a plot representing the relative deuterium uptake (mass change in Dalton units) as a function of deuterium exposure time within a peptide encompassing regions 132-143. Figure 20B shows a plot representing the relative deuterium uptake (mass change in Dalton units) as a function of deuterium exposure time within a peptide encompassing regions 182-187. In Figures 20A and 20B, the uptake of CD73 alone is shown as a square, and the uptake of CD73 bound to MEDI9447 Fab is shown in red. The peptide sequence, position, and mass are shown in the plot boxes. Relative mass changes of duplicate peptides were compared to narrow down the regions that contain sequences showing changes in hydrogen exchange and are expected to form epitopes. For example, peptides between positions 173 and 186 showed differential exchange, but peptides between 173 and 181 showed no difference. Therefore, it was inferred that residues upstream of 182 are not differentially labeled. Figure 20C shows the DynamX difference chart of the MEDI9447 Fab heavy chain. Figure 20D shows the DynamX difference chart of the MEDI9447 Fab light chain. In Figures 20C and 20D, each data point shows the difference in deuterium uptake between the CD73+Fab complex (positive value on the y-axis) and Fab alone (negative value on the y-axis). The vertical bars represent the sum of the uptake differences over the exposure time points. When Fab is bound to CD73, it indicates a CDR, which shows a decrease in relative uptake. Figure 9E shows the DynamX difference chart between CD73 alone (negative value on the y-axis) and CD73 bound to Fab (positive value on the y-axis). This chart indicates regions E1 (aa132~143) and E2 (aa182~187). The horizontal axis corresponds to the analyzed peptides from the N-terminus to the C-terminus (from left to right). A dotted line is overlaid on this chart, indicating a 1.6 Dalton cutoff, or a 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]This graph 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 (5nM to 0.3nM) was measured by surface plasmon resonance (SPR). Figure 21B is a graph showing sensor chip data for CD73 protein with the N-terminal domain swapped. CD73 protein with the N-terminal domain swapped was immobilized on an HTG sensor chip, and binding of MEDI9447 dilutions (5nM to 0.3nM) was measured by SPR. When the N-terminal domain was swapped, MEDI9447 did not bind to CD73. Figure 21C is a graph showing sensor chip data for CD73 protein with both the N-terminal and C-terminal domains swapped. CD73 proteins with swapped N-terminal and C-terminal domains were immobilized on an HTG sensor chip, and binding to MEDI9447 dilutions (5nM~0.3nM) was measured by SPR. When both the N-terminal and C-terminal domains were swapped, MEDI9447 did not bind to CD73. Figure 21D is a graph showing sensor chip data for CD73 proteins with swapped linker regions. CD73 proteins with swapped linker regions were immobilized on an HTG sensor chip, and binding to MEDI9447 dilutions (5nM~0.3nM) was measured by SPR. Swapping only the linker region did not affect binding. Figure 21E is a graph showing 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 to MEDI9447 dilutions (5nM~0.3nM) was measured by SPR. Swapping only the C-terminal domain did not affect binding. Figure 21F is a graph showing sensor chip data for CD73 protein with interface E1 (aa132~143) swapped. CD73 protein with interface E1 (aa132~143) swapped was immobilized on an HTG sensor chip, and binding to MEDI9447 dilutions (5nM~0.3nM) 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 to MEDI9447 dilutions (5nM~0.3nM) was measured by SPR. Figure 21H is a graph showing sensor chip data for CD73 protein with swapped interfaces E1 (aa132~143) and E2 (aa182~187). CD73 protein with swapped interfaces E1 (aa132~143) and E2 (aa182~187) was immobilized on an HTG sensor chip, and binding to MEDI9447 dilutions (5nM~0.3nM) was measured by SPR. Regarding Figures 21F to 21H, the swapping of HDX interface E1 (aa132 to 143) (Figure 21F) had only a slight effect on the coupling, in contrast to the swapping of HDX interface E2 (aa182 to 187) alone (Figure 21G) or in combination with E1 (Figure 21H). For Figures 21A to 21H, the sensorgrams and overlay fits are shown in corresponding colors. The reaction rate measurements for each coupling analysis are provided in Table 16. [Figure 21E-H] This is a continuation of Figure 21A-D. [Figure 22-1] This shows the alignment of human and chicken CD73 protein sequences. Only mature protein sequences are shown. Non-conserved residues are highlighted in the chicken sequence. Annotations are added to regions swapped between chicken and human sequences to create knockout mutants (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. The table of data showing the binding of MEDI9447 to CD73 mutants is shown. Mutants highlighted in blue have a KD of more than 2x from the WT or KO parent construct. *Rituation values ​​obtained from 2:1 heterologous ligand fitting. **Numbering corresponds to chicken sequences (in humans, 129=133, 140=144, and 181=185). [Figure 24A-B] This shows that the MEDI9447 epitope is located at the apex 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 locations that constitute the interaction site. Two of these three most influential residues (highlighted in boxes) are located outside the HDX interface region (highlighted in blue). Three less influential residues (boxed in pink) are located within the HDX interface. Figure 24B is a table showing that knocking in N185 and V144 (K180 is conserved) into the CD73 construct containing the chicken N-terminal and C-terminal domain sequences restored binding to less than 20 times the KD of wild-type CD73 (MEDI9447 dilutions 5nM~0.3nM; compare with Figure 10B). Figure 24C shows a detailed diagram of the epitope residue 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 shown in pink. The HDX interface is overlaid in blue. Figure 24D shows a surface representation illustrating that the epitopes form a nearly continuous binding surface. Figure 24E shows the crystal structure of the open conformation of CD73, indicating the location of the epitope on the apical outer surface of the N-terminal domain. Figure 24F shows that the epitope is located away from the substrate binding site (adenosine represented by a sphere) and the zinc ion (gray sphere) coordination site (cyan side chain). In all crystal structures, the CD73 N-terminal domain, linker region, and C-terminal domain are shown in yellow, orange, and blue, respectively. [Figure 24C-D] This is a continuation of Figure 24A-B. [Figure 24E-F] This is a continuation of Figure 24A-B. [Figure 25] This shows that MEDI9447 is a non-competitive 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-corresponding control mAb. Figure 25B is a graph showing that MEDI9447 acts as a non-competitive inhibitor by equally inhibiting hydrolysis 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 achieved maximum inhibition at a 1:1 molar stoichiometric ratio with the CD73 dimer (arrow). At high concentrations of excess MEDI9447 IgG (>10 nM), loss of inhibition or a "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]This shows that the binding of the anti-CD73 mAb (clone 0069) depends on the N-terminal and C-terminal domain residues of CD73. Figure 26A is a graph showing sensor chip data for histidine-tagged CD73. Histidine-tagged CD73 was immobilized on an HIS2 biosensor, and binding by mAb A was measured by biolayer interferometry (BLI). The binding of mAb A to WT CD7 (blue sensorgram), N-terminal domain swap knockout CD7 (KO_1-291, green sensorgram), and C-terminal domain swap knockout CD7 (KO_311-523, cyan sensorgram) shows that mAb binding is influenced by residues in both the N-terminal 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) highlighting the locations of mAb A binding hotspots (aa114-134 and 153-170) located near the N-terminal and C-terminal domain interfaces. Mapping was based on binding data from Figures 26A and 26C. Figure 26C shows binding sensograms of mAb A for various domain swap knockout mutants of CD73. Swapping of the subregions DS2c (aa114-134) or DS3a (aa153-170) knocked out binding. All binding analyses were performed using 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 an HIS2 biosensor, and the binding of MEDI9447 (blue sensorgram) and anti-CD73 mAb A (brown sensorgram) was measured by BLI using an Octet QK384. When CD73 was pre-incubated with Zn2+ and APCP, MEDI9447 maintained binding (black sensorgram), but mAb A binding disappeared (orange sensorgram). Figure 27B is a graph showing that pre-incubation with Zn2+ and APCP resulted in the disappearance of mAb A binding (orange sensorgram), but binding was restored when pre-incubated with MEDI9447 before adding Zn2+ and APCP (purple sensorgram). The binding of mAb A to CD73 alone and to CD73 pre-incubated with MEDI9447 (but not with Zn2+ and APCP) is shown in blue and brown sensorograms, respectively. Figure 27C shows a proposed model illustrating how MEDI9447 prevents CD73 from adopting a fully closed-activity 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 Figure 27A-B. [Figure 28]Unless otherwise noted below, the binding of MEDI9447 or mAb A to CD73 under various conditions measured by BLI as described herein is shown. Figure 28A is a graph showing the binding of anti-CD73 mAb A to histidine-tagged wild-type CD73 immobilized on an 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 to measure binding. mAb A bound to CD73 (blue sensorgram), but not to CD73 pre-incubated with Zn2+ and APCP (purple sensorgram). mAb A maintained binding to CD73 pre-incubated with APCP and EDTA (green sensorgram) or Zn2+, APCP, and EDTA (gold sensorgram). The chelating effect of EDTA indicates that a divalent cation was required for the disappearance 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 the binding of mAb A to CD73 incubated with Zn2+ and APCP. This assay was performed as shown in Figure 27B. MEDI9447 Fab or isotype-specific control IgG was pre-incubated with CD73 before adding Zn2+ and APCP. mAb A immobilized on the biosensor bound to CD73 pre-incubated with 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), nor to Fab (gold sensorgram) or control IgG (purple sensorgram) pre-incubated with CD73 before adding Zn2+ and APCP. [Figure 29-1]This shows that anti-CD73 mAb B binding depends on residues in the subregions DS2b (aa92-134) or DS2c (aa114-134). Figure 29A is a table showing SEC-MALS data corresponding to Figures 30A-30C. For each mixture of CD73 and either MEDI9447 or mAb B, the corresponding SEC retention time, Mw, and polydispersity of the formed complex are shown. Figure 29B represents the determination of the mAb B binding hotspot to CD73. mAb B binding to CD73 mutants immobilized on HIS2 biosensors was measured by BLI as described in Figures 26A-26C relating to the method herein for mAb A (clone 0069). The binding sensorgrams showed that swapping of either the subregions DS2b (aa92-134) or DS2c (aa114-134) knocked out mAb B binding. [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]This shows that MEDI9447 forms intermeric crosslinks 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 30A is a chromatogram showing that MEDI9447 formed a complex with CD73 at a 1:1 molar ratio (green trace) of approximately 1.7(^) and 6.6(+) megadaltons. 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 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 to a site near the central groove between the dimers. Figure 30C is a chromatogram showing the formation of a single dominant complex with a peak of approximately 270–290 kD (peak approximately 7.2 min) when CD73 binds to mAb B. The UV traces shown represent 1:1 mAb B:CD73 (red), 0.5:1 (blue), and 0.1:1 (green). mAb A and CD73 alone are magenta and black, respectively. [Figure 30-2] This is a continuation of Figure 30-1. [Figure 31A-B]This shows that surface-bound CD73 was inhibited by MEDI9447 in IgG and Fab formats. Figure 31A is a graph showing the inhibition of AMP hydrolysis of immobilized CD73 by MEDI9447 IgG, Fab, or control antibody. CD73 was immobilized on nickel-coated microtiter plates 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 described herein. MEDI9447 IgG dose-dependently inhibited AMP hydrolysis by CD73, while control IgG did not. MEDI9447 Fab also inhibited CD73 activity, but to a much lower degree. Figure 31B shows a complex containing MEDI9447 Fab (green) bound to an anti-Fd antibody (xFd, red). When MEDI9447 Fab (green) bound to one arm of the anti-Fd antibody (xFd, red) and the other arm bound to a nonspecific polyclonal Fab (pFab, orange), the inhibition increased to a level comparable to that of 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 of 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 the CellTiterGlo assay. Similar to immobilized recombinant CD73, MEDI9447 IgG inhibits AMP hydrolysis more strongly than Fab, but the inhibition is enhanced when the effective size of MEDI9447 Fab increases by forming a complex with the anti-Fd antibody. Figure 31D is a graph showing the inhibition of AMP hydrolysis of soluble CD73 (sCD73) by MEDI9447 IgG, Fab, or a control antibody. To test whether xFd+MEDI9447 can inhibit soluble CD73, AMP hydrolysis was measured using a malachite green assay. MEDI9447 Fab did not inhibit soluble CD73 activity, either alone or when bound to a single xFd arm.In contrast, when MEDI9947 Fab binds to both xFd arms (MEDI9447 Fab+xFd), bivalency is conferred and CD73 inhibition results. [Figure 31C-D] Continuation of FIGS. 31A-B. [Figure 32] Graph showing that MEDI94447 IgG and Fab inhibited AMP hydrolysis by CD73. As described herein, CD73 activity was measured using a malachite green assay in the presence of increasing concentrations of antibody. MEDI9447 IgG inhibited CD73 hydrolytic 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 maximum inhibition level was low. This experiment was performed twice with equivalent results. Only the data from one experiment are shown. [Figure 33] Model representing that inhibition of CD73 hydrolytic activity by MEDI9447 occurs via a dual mechanism. MEDI9447IgG (green) inhibits soluble CD73 by forming inter-dimer cross-links and preventing conformational transition to the closed state. Monovalent IgG or Fab does not inhibit soluble CD73. When CD73 is surface-bound, inhibition can occur either by cross-linking of adjacent CD73 dimers or by steric blockade from monovalent IgG or Fab / xFd (red) complexes.

Mode for Carrying Out 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 that specifically bind to CD73 and their antigen-binding fragments. Related polynucleotides, vectors, and pharmaceutical compositions comprising anti-CD73 antibodies or their antigen-binding fragments are also provided. Furthermore, methods for producing and using the anti-CD73 antibodies and antigen-binding fragments disclosed herein are also provided, such as diagnostic methods and methods for treating cancer in a subject (as direct therapy, adjuvant therapy, or in combination therapy). The present invention also provides antibody-drug conjugates obtained from the CD73 binding molecules disclosed herein. Moreover, the present invention provides therapeutic combinations characterized by an anti-CD73 antibody (e.g., MEDI9447) and one or more agents that target other aspects of the cancer immune cycle, such as anti-PD-1 antibodies, anti-PD-L1 antibodies (e.g., MEDI4736), and anti-CTLA4 antibodies; and methods for using such combinations to reduce tumor-mediated immunosuppression.

[0134] To facilitate a better understanding of this disclosure, certain terms are defined at the outset. Additional definitions are provided throughout the detailed description.

[0135] I. Definition Before describing the present invention in detail, it should be understood that this invention is not limited to any particular composition or method step, and that they may vary. When used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless specifically indicated by the context. The terms “a” (or “an”), as well as the terms “one or more” and “at least one,” can be used synonymously herein.

[0136] Furthermore, when used herein, “and / or” should be interpreted as a specific disclosure of each of the two designated features or components, with or without the other. Accordingly, when the term “and / or” is used herein in phrases such as “A and / or B,” it is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, when the term “and / or” is used in phrases such as “A, B, and / or C,” it is intended to include 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 those generally understood by those skilled in the art in the field relating to this disclosure. 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 many common dictionaries of the terms used herein.

[0138] Units, prefixes, and symbols are shown in the form recognized by their International System of Units (SI). Numerical ranges include the number defining the range. Unless otherwise specified, amino acid sequences are written from left to right in the amino-carboxyl direction. The headings provided herein are not limitations on the various aspects that may be obtained by referring to this specification as a whole. Accordingly, the terms defined immediately thereafter are further fully defined by referring to this specification as a whole.

[0139] Whenever an aspect is described using the term "comprising" in this specification, it is understood that other similar aspects are also provided, which are described using the terms "consisting of" and / or "consisting essentially of."

[0140] In this specification, amino acids are referred to by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides are referred to by their commonly accepted single-letter codes.

[0141] The term “CD73 polypeptide,” as used herein, refers to the CD73 (differentiation cluster 73) protein encoded by the NT5E gene, also known in the literature as 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase. See, for example, 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 represents the amino acid residues of the mature CD73 protein, excluding signal sequence residues. Therefore, for example, antibodies that bind to amino acids Val144, Lys180, and Asn185 refer to the amino acid positions of the mature protein after cleavage of the signal sequence.

[0142] An example of a CD73 polypeptide is 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. Mature CD73 protein is produced after the removal of the signal sequence (positions 1-26) and the C-terminal region of the propeptide (positions 550-574). In addition, in isoform 2 of CD73, amino acids 404-453 are removed after alternative splicing. Native mutants are also known, such as mutant C358Y, mutant T376A, and mutant M379T. See Misumi et al., Eur.J.Biochem.191:563-569(1990); Otsuki et al.DNA Res.12:117-126(2005); Mungall et al.Nature 425:805-811(2003); Hansen et al.Gene 167:307-312(1995); Klemens et al.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 these are incorporated herein by reference as a whole).

[0144] Typical diseases that lead to changes in a patient's CD73 levels in tissue fluid, particularly serum, include tissue trauma; reperfusion injury resulting from myocardial infarction or stroke, organ transplantation or other surgical procedures; cancer or cancer metastasis; or inflammatory conditions resulting from the aforementioned trauma or reperfusion injury, or from chronic conditions including allergic conditions, autoimmune diseases, and inflammatory diseases. Examples of such chronic conditions include arthritis, allergic conditions such as asthma, inflammatory conditions such as inflammatory bowel disease or cutaneous inflammation, psoriasis, Parkinson's disease, Alzheimer's disease, autoimmune diseases, type 1 or type 2 diabetes, atherosclerosis, multiple sclerosis, Crohn's disease, or rejection reactions resulting from organ transplantation. In particular, systemic inflammatory response syndrome (SIRS), acute lung injury (ALI), multiple organ failure (MOF), ischemia-reperfusion injury (IRI), and adverse drug reactions (ADRS) in inflammatory diseases lead to changes in the tissue fluid CD73 protein. Accordingly, the CD73-binding molecules disclosed herein can be used, for example, in the treatment or diagnosis of cancer (e.g., colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer). In addition, by measuring CD73 levels in patient-derived samples (e.g., tissue fluid) using the CD73-binding molecules disclosed herein, it is possible to monitor the occurrence of the aforementioned diseases, evaluate the effectiveness of therapies, select patients for treatment with specific therapies, or make medical decisions, such as initiating, terminating, interrupting, or modifying specific treatments.

[0145] The terms “inhibit,” “block,” and “suppress,” and their grammatical variations, are used synonymously herein and refer to any statistically significant reduction in biological activity, including complete blockade. For example, “inhibit” may refer to a reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of biological activity. Thus, when the terms “inhibit” or “suppress” are used, for example, to describe an effect on the enzymatic activity of CD73, the terms refer to the ability of an anti-CD73 antibody or its antigen-binding fragment to statistically significantly reduce 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. Cells expressing CD73 may be naturally occurring cells or cell lines (e.g., cancer cells), or they may be recombinantly produced by introducing nucleic acids encoding CD73 into host cells. In some embodiments, anti-CD73 antibodies or their antigen-binding fragments can statistically significantly reduce the 5'-nucleotidase activity of soluble CD73 in biological fluids. In one embodiment, an anti-CD73 binding molecule, e.g., an antibody or its antigen-binding fragment, 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%, when determined by, for example, the methods described in the following examples and / or methods known in the art.

[0146] The term "inhibit CD73 activity," as used herein, refers to the ability of an anti-CD73 binding molecule, such as an antibody or its antigen-binding fragment, to statistically significantly reduce CD73-dependent 5'-nucleotidase activity in cells expressing CD73 or in a sample containing CD73. In some embodiments, the suppression of CD73 activity may be a reduction of 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 about 100% when cells or samples are brought into contact with the anti-CD73 binding molecule of the Disclosure, e.g., an antibody or its antigen-binding fragment, compared to CD73 activity measured in the absence of the anti-CD73 binding molecule, e.g., an antibody or its antigen-binding fragment (control condition).

[0147] The terms “antibody” or “immunoglobulin” as used synonymously in this specification include the whole antibody 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 has a heavy chain variable region (VH or V in this specification). H It includes a heavy chain constant region (abbreviated as and includes a heavy chain constant region). The heavy chain constant region includes three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region (VL or V in this specification). LThe antibody contains a constant region of the heavy and light chain (abbreviated as ). The constant region of the light chain contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FWs). Each VH and VL contains three CDRs and four FWs, arranged from the amino terminus to the carboxyl terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the classical complement system (C1q). Exemplary antibodies of this disclosure include anti-CD73 antibodies (original and germline-derived), affinity-optimized clones, ADCC-deficient-optimized antibodies, conjugate 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. Patent No. 7,083,784, which is incorporated herein by reference in its entirety).

[0149] The term "germlineization" refers to the reversion of an amino acid at a specific location in an antibody to one that is characteristic of germline cells.

[0150] The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen-recognizing site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes 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 made from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing the antigen-determining portion of an antibody, and any other modified immunoglobulin molecules containing an antigen-recognizing site, insofar as the antibody exhibits the desired biological activity.

[0151] Antibodies may belong to one 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, which are designated α, δ, ε, γ, and μ, respectively. Different immunoglobulin classes have different well-known subunit structures and three-dimensional arrangements. Antibodies may be naked or conjugated to other molecules such as toxins or radioisotopes to form ADCs.

[0152] A “blocking” antibody or “antagonist” antibody inhibits or reduces the biological activity of an antigen to which it binds, such as CD73. In certain embodiments, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. Preferably, 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,” “CD73-binding antibody,” or “anti-CD73” refer to an antibody or its antigen-binding fragment that has the ability to bind to CD73 with sufficient affinity to be useful as a therapeutic or diagnostic agent in targeting CD73. The degree of binding of an anti-CD73 antibody to unrelated non-CD73 proteins is less than about 10% of the binding of this antibody to CD73 when 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 containing the CDR of the antibodies disclosed herein, for example, incorporated into a scaffold. Thus, the phrase “isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73” may refer not only to the antibody and its antigen-binding fragment, but also to molecules containing one or more scaffolds (such as fibronectin or fibronectin type III domains derived from tenascin-3) that incorporate the CDR of the antibodies disclosed herein. See, for example, U.S. Patent Application Publication No. 20150098955 (in whole, incorporated herein by reference).

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

[0155] "CTLA4 polypeptide" means a polypeptide or fragment thereof that has T cell inhibitory activity and has at least 85% amino acid sequence identity with GenBank accession number AAL07473.1. The sequence of AAL07473.1 is provided below. CTLA4 polypeptide sequence [Human (Homo sapiens)] gi|15778586|gb|AAL07473.1|AF414120_1 [ka]

[0156] The term "CTLA4 nucleic acid molecule" refers to a polynucleotide that encodes the CTLA4 polypeptide. An exemplary CTLA4 nucleic acid molecule sequence is provided under GenBank accession number AAL07473.

[0157] An “anti-CTLA4 antibody” means an antibody that selectively binds to the 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 (where tremelimumab is 11.2.1) (these are incorporated herein by reference). Tremelimumab is an exemplary anti-CTLA4 antibody. The tremelimumab sequence is provided in the following sequence listings herein (SEQ ID NOs. 130-137).

[0158] "PD-1 polypeptide" means a polypeptide or fragment thereof that has at least approximately 85% amino acid identity with NCBI accession number NP_005009 and possesses 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] The term "PD-1 nucleic acid molecule" refers to a polynucleotide that encodes the PD-1 polypeptide. An exemplary PD-1 nucleic acid molecule sequence is provided under NCBI accession number NM_005018.

[0160] An "anti-PD-1 antibody" refers to an antibody or its antigen-binding fragment that selectively binds to the PD-1 polypeptide. Examples of anti-PD-1 antibodies include pembrolizumab (KEYTRUDA®, lambrolizumab, MK-3475), nivolumab (OPDIVA®, BMS-936558, MDX-1106, ONO-4538), or AMP-224.

[0161] "PD-L1 polypeptide" means a polypeptide or fragment thereof that has at least approximately 85%, 95%, or 100% amino acid identity with NCBI accession number NP_001254635 and possesses 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] The term "PD-L1 nucleic acid molecule" refers to a polynucleotide that encodes the PD-L1 polypeptide. An exemplary PD-L1 nucleic acid molecule sequence is provided under NCBI accession number NM_001267706.

[0163] "Anti-PD-L1 antibody" means an antibody or its antigen-binding fragment that selectively binds to the PD-L1 polypeptide. Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Application Publication No. 20130034559 / U.S. Patent No. 8779108 and U.S. Patent Application Publication No. 20140356353 (these are incorporated herein by reference). MEDI4736 is an exemplary PD-L1 antibody. The sequence of MEDI4736 is provided in the sequence listing below (SEQ ID NOs: 138-145).

[0164] The term "antigen-binding fragment" refers to a molecule containing a portion of an intact antibody, and more specifically, a molecule containing the antigen-determining variable region of an intact antibody. In the art, it is known that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of antibody fragments, but not limited to, include 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 population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies 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 containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to such antibodies produced by any method, including, but not limited to, 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, a humanized antibody is a human immunoglobulin in which CDR residues are replaced with CDR residues from a non-human species (e.g., mouse, rat, rabbit, or hamster) that has the desired specificity, affinity, and competence (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 (FW) amino acid residues of a human immunoglobulin are replaced with corresponding residues from an antibody derived from a non-human species that has the desired specificity, and / or affinity, and / or competence.

[0168] Humanized antibodies can be further modified by substitution of additional residues in the Fv framework region and / or within replaced non-human residues to refine and optimize their specificity, affinity, and / or capabilities. Generally, humanized antibodies will contain at least one, typically two or three, variable domains containing all or substantially all of the CDR region corresponding to non-human immunoglobulins, while all or substantially all of the FR region is the human immunoglobulin consensus sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region or domain (Fc), typically that of human immunoglobulins. Examples of methods used to prepare humanized antibodies are described in U.S. Patent No. 5,225,539 or No. 5,639,641.

[0169] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either individually or in combination. The variable regions of the heavy and light chains 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 CDR of the other chain, contribute to the formation of the antibody's antigen-binding site. There are at least two techniques for determining the CDR: (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, in this field, a combination of these two methods is sometimes used to determine the CDR.

[0170] The Kabat numbering system is generally used to refer to residues in the variable domain (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 terms “Kabat amino acid position numbering,” “Kabat position,” and their grammatical variations refer to the numbering scheme used for the heavy chain variable domain or light chain variable domain in antibody synthesis, as described 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 scheme, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortenings or insertions into the FW or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and residues inserted after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat).

[0172] [Table 1]

[0173] Kabat numbering of residues can be determined for a given antibody by aligning the antibody sequence with a "standard" Kabat numbered sequence in the homology region. Chothia, on the other hand, refers to the position of the structural loop instead (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)). When numbered using the Kabat numbering rules, the end of the Chothia CDR-H1 loop differs 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 exists, the loop ends at 32; if only 35A exists, the loop ends at 33; if both 35A and 35B exist, the loop ends at 34). The AbM hypervariable region corresponds to a compromise between Kabat CDR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.

[0174] IMGT (ImMunoGeneTics) also provides a numbering scheme for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, MPet al., Dev. Comp. Immunol. 27:55-77 (2003) (which is incorporated herein by reference). The IMGT numbering scheme is based on alignment, structural data, and characterization of over 5,000 sequences, enabling easy comparison of all types of variable regions and CDR regions. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 at positions 51-57, VH-CDR3 at positions 93-102, VL-CDR1 at positions 27-32, VL-CDR2 at positions 50-52, and VL-CDR3 at positions 89-97.

[0175] The EU index, or EU numbering scheme, is based on the sequential numbering of the first sequenced human IgG (EU antibody). Since the most common reference for this rule is the Kabat Sequence Manual (Kabat et al., 1991), the EU index is sometimes mistakenly used as synonymous with the Kabat index. The EU index does not provide insertions and deletions, and therefore, in some cases, comparisons of IgG positions between IgG subclasses and species can be uncertain, particularly in hinge regions. Nevertheless, this rule has been sufficient to enable direct comparisons between Fc regions in numerous Fc structure-function studies. Therefore, the numbering scheme used herein for substitutions and insertions in Fc regions is the EU index as found in Kabat. In contrast, the numbering scheme used herein for 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, namely 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, namely positions 24-34, 50-56, and 89-97.

[0177] As used herein, the Fc region comprises a polypeptide containing 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 N-terminal mobile hinges of these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes the immunoglobulin domains C-gamma 2 and C-gamma 3 (Cγ2 and Cγ3) as well as the hinge between C-gamma 1 (Cγ1) and C-gamma 2 (Cγ2).

[0178] The boundaries of the Fc region can vary, but the human IgG heavy chain Fc region is typically defined as containing residue C226 or P230 at its carboxyl terminus (where numbering follows the EU index as defined in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Fc can refer to this region independently or in relation to an antibody, antibody fragment, or Fc fusion protein. Polymorphism has been observed in several different Fc positions, including, but not limited to, positions 270, 272, 312, 315, 356, and 358 when numbered by the EU index, and therefore there may be slight differences between the presented sequence and the sequence of prior art.

[0179] The term "human antibody" means an antibody produced by a human, or an antibody having an amino acid sequence corresponding to a human-produced antibody, prepared using any technique known in the art (e.g., recombinant expression in cultured cells, or expression in transgenic animals). Therefore, the term "human antibody" also includes antibodies that are originally produced by humans (or modified variants or derivatives thereof) but have an amino acid sequence corresponding to an antibody expressed in a non-human system (e.g., prepared 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 human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide, such as an antibody containing a mouse light chain and a human heavy chain polypeptide.

[0180] The term "chimeric antibody" refers to an antibody whose immunoglobulin molecule's amino acid sequence originates from two or more animal species. Typically, both the light and heavy chain variable regions correspond to the variable regions of an antibody from one mammalian species (e.g., mouse, rat, rabbit, etc.) that possesses the desired specificity and / or affinity and / or capability, while the constant region is homogeneous with the sequence of an antibody from another species (usually human), thus avoiding the induction of an immune response in that species.

[0181] The term “epitope,” as used herein, refers to an antigen-protein determinant having the ability to bind to the 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 usually possess specific three-dimensional structural properties as well as specific charge properties. 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 based on their structure and interaction with paratopes: stereoepitopes and linear epitopes. Stereoepitopes consist of discontinuous sections of the amino acid sequence of the antigen. These epitopes interact with paratopes based on the three-dimensional surface features and shape or tertiary structure of the antigen. In contrast, linear epitopes interact with paratopes based on their primary structure. Linear epitopes are formed by continuous amino acid sequences of the antigen.

[0182] The term "antibody binding site" refers to a region within an antigen (e.g., CD73) that contains a continuous or discontinuous site (i.e., an epitope) to which a complementary antibody specifically binds. Therefore, an antibody binding site may include additional areas within the antigen beyond the epitope that can determine properties such as binding affinity and / or stability, or influence properties such as antigenic enzyme activity or dimerization. Thus, even if two antibodies bind to the same epitope within an antigen, if the antibody molecules establish individual intermolecular contacts with amino acids outside the epitope, such antibodies are considered to bind to individual antibody binding sites.

[0183] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, when used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). DAffinity can be expressed by the following: Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind rapidly to antigens and remain bound for a long time. Various methods for measuring binding affinity are known in the art, and any of them may be used for the purposes of this disclosure.

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

[0185] The improvement factor of the potency of the antibody or polypeptide of this disclosure compared to the parent antibody may be at least about 2, at least about 4, at least about 6, at least about 8, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, or at least about 180 or more.

[0186] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) are bound to secretory immunoglobulins that bind to Fc receptors (FcRs), allowing these cytotoxic effector cells to specifically bind to antigen-carrying target cells, and subsequently kill those target cells with cytotoxicity. Specific high-affinity IgG antibodies directed toward the surface of the target cells "arm" the cytotoxic cells and are essential for such death. Lysis of the target cells occurs extracellularly, requires direct intercellular contact, and does not involve complement. In addition to antibodies, other proteins containing an Fc region capable of specifically binding to antigen-carrying target cells, specifically Fc fusion proteins, are thought 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] "Isolated" polypeptides, antibodies, polynucleotides, vectors, cells, or compositions are polypeptides, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to such an extent that they are no longer in a form found in nature. In some embodiments, isolated antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

[0188] The term "subject" refers to any animal (e.g., mammal) that is a recipient of a particular treatment, including but not limited to humans, non-human primates, and rodents. Typically, the terms "subject" and "patient" are used synonymously in this specification to refer to human subjects.

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

[0190] The “effective amount” of the anti-CD73 binding molecule disclosed herein is an amount sufficient to accomplish the purpose specifically described. The “effective amount” can be determined experimentally by conventional methods with respect to the purpose described.

[0191] The term “therapeutic dose” refers to the amount of an anti-CD73 conjugate molecule or other drug disclosed herein that is effective in “treating” a disease or disorder in a subject or mammal.

[0192] When used herein, the term “labeled” means a detectable compound or composition that is directly or indirectly fused (e.g., by gene fusion) or conjugated (e.g., chemically conjugated) to an anti-CD73 binding molecule disclosed herein, thereby producing a “labeled” anti-CD73 binding molecule. The label may be detectable in itself (e.g., by radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze the chemical modification of a detectable substrate compound or composition.

[0193] The terms “derivativeable group” and “derivativeable functional group” are used synonymously and refer to functional groups that, upon reaction, 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 substances are therapeutic agents (e.g., cytotoxicities), detectable labels, polymers (e.g., PEG), etc. Exemplary derivatable groups include thiols, hydroxyls, aminos, carboxyls, and amides, as well as modified forms thereof, such as activated or protected forms.

[0194] Terms such as “to treat,” “treatment,” “the act of treating,” “to alleviate,” or “the act of alleviating” refer to both (1) therapeutic means that cure, slow down, alleviate the symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) preventive or protective means that prevent and / or delay the onset of a targeted pathological condition or disorder. Accordingly, those who require treatment include those who already have a disorder; those who are prone to developing a disorder; and those who should be prevented from developing a disorder. In certain embodiments, the subject has been successfully “treated” of cancer by the method of this disclosure if the patient exhibits, for example, complete remission, partial remission, or temporary remission of a certain type of cancer.

[0195] The terms “cancer,” “tumor,” “cancerous,” and “malignant” refer to or describe a physiological condition in mammals 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 specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin and non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as 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, such as mucinous ovarian cancer, bile duct cancer (liver), and renal papillary carcinoma. In some embodiments, the term "cancer" as used herein specifically refers to cancer that expresses CD73. In some specific embodiments, the term "cancer" as used herein refers to cancer that expresses low levels of CD73. In some embodiments, the term "cancer" as used herein specifically refers to cancer that expresses CD73 (e.g., colorectal cancer, breast cancer, lymphoma, non-small cell carcinoma).

[0196] As used synonymously herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. A nucleotide may be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or analogues thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogues. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0197] The term "vector" refers to a construct that has the ability to deliver one or more target genes or sequences to a host cell and, in some embodiments, to express them. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells, such as producer cells.

[0198] The terms “polypeptide,” “peptide,” and “protein” are used herein synonymously to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be fragmented by non-amino acids. These terms also encompass amino acid polymers that are naturally modified or modified by intervention (e.g., disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeling component). Furthermore, this definition includes, for example, polypeptides containing one or more analogues of amino acids (e.g., non-natural amino acids), as well as other modifications known in the art. Since the polypeptides of this disclosure are antibody-based, it is understood that, in certain embodiments, polypeptides may exist as single chains or associated chains.

[0199] "Recombinant" polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA technology. Recombinant-produced polypeptides and proteins expressed in modified host cells are considered isolated, similar to natural or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any preferred technology. Polypeptides disclosed herein may be recombinantly produced using methods known in the art. Alternatively, proteins and peptides disclosed herein may be chemically synthesized.

[0200] The term "amino acid substitution" refers to replacing an amino acid residue present in a parent sequence with another amino acid residue. Amino acids can be substituted in the parent sequence, for example, by chemical peptide synthesis or by recombination methods known in the art. Therefore, when we say "substitution at position X" or "substitution at position X," we mean replacing the amino acid present at position X with an alternative amino acid residue. In some embodiments, substitution patterns can be described according to the schema AXY, where A is a single-letter code corresponding to the amino acid naturally present at position X, and Y is the amino acid residue to be substituted. In other embodiments, substitution patterns can be described according to the schema XY, where Y is a single-letter code corresponding to the amino acid residue that replaces the naturally present amino acid at position X.

[0201] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged 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). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, this substitution is considered conservative. In another embodiment, a sequence of amino acids can be conservatively replaced by a structurally similar sequence with different order and / or composition of side-chain family members.

[0202] Non-conservative substitutions include (i) a residue having an electronegative side chain (e.g., Arg, His, or Lys) being replaced by or with an electronegative residue (e.g., Glu or Asp); (ii) a hydrophilic residue (e.g., Ser or Thr) being replaced by or with a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) a cysteine ​​or proline being replaced by or with any other residue; or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) being replaced by one having a smaller side chain (e.g., Ala, Ser) or one without a 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 may be any one of D-lysine, arginine, D-arginine, homoarginine, methionine, D-methionine, ornithine, or D-ornithine. Generally, substitutions in functionally important regions that can be expected to cause changes in the properties of isolated polypeptides are: (i) a polar residue, e.g., serine or threonine, being substituted for (or thereby) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine ​​residue being substituted for (or thereby) any other residue; (iii) a residue with an electronegative side chain, e.g., lysine, arginine, or histidine, being substituted for (or thereby) a residue with an electronegative side chain, e.g., glutamic acid or aspartic acid; or (iv) a residue with a bulky side chain, e.g., phenylalanine, being substituted for (or thereby) a residue without such a side chain, e.g., 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 in a functionally important region of the protein. Therefore, some non-conservative substitutions have little or no effect on the 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 the parent sequence, for example, by chemical peptide synthesis or by recombination methods known in the art. Therefore, as used herein, the phrase "insertion between positions X and Y" or "Kabat insertion between positions X and Y" (where X and Y correspond to amino acid positions) (e.g., 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 a particular amino acid between codons encoding 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 prior to the insertion.

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

[0206] The percentage of sequence identity is calculated by determining the number of matching positions by identifying the number of identical amino acid residues or nucleic acid bases in both sequences, dividing that number by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Sequence comparison and percentage sequence identity determination between two sequences can be achieved using readily available software, both for online use and download. Suitable software programs are available from various suppliers for both protein and nucleotide sequence alignments. One suitable program for determining percentage sequence identity is bl2seq, part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information (USA). bl2seq compares two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used for nucleic acid sequence comparison, while BLASTP is used for amino acid sequence comparison. Other suitable programs include, 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] Various regions within a single polynucleotide or polypeptide target sequence aligned with a polynucleotide or polypeptide reference sequence may each have their own percentage sequence identity. Note that the percentage sequence identity value is 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 that the length value is always an integer.

[0208] In a particular embodiment, the percentage identity "X" of the amino acid sequence of the first sequence to the amino acids of the second sequence is calculated as 100 × (Y / Z) (wherein Y is the number of amino acid residues that score as identical matches in the alignment of the first and second sequences (either by visual inspection or by a specific 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 percentage identity of the first sequence to the second sequence will be higher than the percentage 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 solely 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 understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structure), functional data (e.g., mutation sites), or phylogenetic data. A suitable program for integrating heterogeneous data and generating multiple sequence alignments is T-Coffee, available at www.tcoffee.org or, for example, from EBI. It will also be understood that the final alignment used for calculating percent sequence identity can be curated automatically or manually.

[0211] When the term “consensus sequence” is used herein in relation to the light chain (VL) and heavy chain (VH) variable regions, it refers to a complex 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 the “consensus sequence” of the VL or VH chain, a particular amino acid position is occupied by one of several 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 may be either arginine or serine (R or S). The consensus sequences of the VH and VL chains can be defined, for example, by in vitro affinity maturation (e.g., randomizing any amino acid position within a specific CDR using degenerate coding primers), scanning mutagenesis of amino acid residues within the antibody CDR (e.g., alanine scanning mutagenesis), or any other method known in the art, followed by evaluation of the binding of the mutant to the antigen, to determine whether the mutated amino acid position affects antigen binding. In some embodiments, the mutation is introduced in the CDR region. In other embodiments, the mutation is introduced in the framework region. In some other embodiments, the mutation is introduced in both the CDR and framework regions.

[0212] II.CD73 binding molecule This disclosure provides CD73-binding molecules, such as antibodies that specifically bind to CD73, such as human CD73, and antigen-binding fragments thereof. The full-length amino acid (aa) and nucleotide (nt) sequences of CD73 are known in the art (see, for example, 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., antibody or its antigen-binding fragment, is Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgG CH2, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2 It includes (scFv)2 or scFv-Fc. In some embodiments, the antibody is an IgG type antibody, for example, an IgG1 type antibody.

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

[0215] In certain embodiments, the anti-CD73 antibodies or their antigen-binding fragments disclosed herein are modified compared to a parent antibody, e.g., CD730010 antibody or 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. Modifications may include mutations in the CDR region and / or FW region compared to a parent antibody, e.g., CD730010 or CD730002.

[0216] The term "CD730002 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 1 and two VH domains with the amino acid sequence of SEQ ID NO: 2.

[0217] The term "CD730004 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 104 and two VH domains with the amino acid sequence of SEQ ID NO: 103.

[0218] The term "CD730008 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 106 and two VH domains with the amino acid sequence of SEQ ID NO: 107.

[0219] The term "CD730010 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 3 and two VH domains with the amino acid sequence of SEQ ID NO: 4.

[0220] The term "CD730011 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 5 and two VH domains with the amino acid sequence of SEQ ID NO: 6.

[0221] The term "CD730021 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 7 and two VH domains with the amino acid sequence of SEQ ID NO: 8.

[0222] The term "CD730042 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 9 and two VH domains with the amino acid sequence of SEQ ID NO: 10.

[0223] The term "CD730046 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 11 and two VH domains with the amino acid sequence of SEQ ID NO: 12.

[0224] The term "CD730047 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 13 and two VH domains with the amino acid sequence of SEQ ID NO: 14.

[0225] The term "CD730068 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 108 and two VH domains with the amino acid sequence of SEQ ID NO: 107.

[0226] The term "CD730069 antibody" refers to IgG1 containing two VL domains with the amino acid sequence of SEQ ID NO: 110 and two VH domains with the amino acid sequence of SEQ ID NO: 109.

[0227] (i)CD730010-derived anti-CD73 antibody In certain embodiments, the anti-CD73 antibody of this disclosure may, but is not limited to: 1) Light chain CDR1 containing the consensus sequence SGSLSNIGRNX1VN (wherein X1 represents the amino acid residue proline (P), glutamic acid (E), or aspartic acid (D)), and / or 2) A light chain CDR2 comprising a 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 a consensus sequence ATWDDSX6X7GWX8 (where 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)) Including modifications to CDR1 and / or CDR2 and / or CDR3 of the light chain of the CD730010 antibody.

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

[0229] In one embodiment, the anti-CD73 antibody or its antigen-binding fragment has a 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, 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 contains a VL region which includes the amino acid residue leucine (L) or threonine (T).

[0230] In one embodiment, the anti-CD73 antibody or its antigen-binding fragment has a 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, where, 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 The VH region contains an amino acid residue (representing tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E)).

[0231] In one embodiment, the anti-CD73 antibody or its antigen-binding fragment has a 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, 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 contains a VL region containing the amino acid residue leucine (L) or threonine (T), and the anti-CD73 antibody or its antigen-binding fragment has a 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, where, 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 The VH region further includes an amino acid residue (where represents tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E)).

[0232] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VL-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48.

[0233] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VL-CDR2 consisting of 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 its antigen-binding fragment comprises a VL-CDR3 consisting of 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 its antigen-binding fragment comprises a VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs. 35 and 36.

[0236] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VH-CDR2 consisting of 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 its antigen-binding fragment comprises a VH-CDR3 consisting of 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 its antigen-binding fragment comprises a VL-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs. 46, 47, and 48, excluding one, two, three, or four amino acid substitutions.

[0239] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52, excluding one, two, three, or four amino acid substitutions.

[0240] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VL-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs. 53, 54, 55, and 56, excluding one, two, three, or four amino acid substitutions.

[0241] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs. 35 and 36, excluding one, two, three, or four amino acid substitutions.

[0242] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40, excluding one, two, three, or four amino acid substitutions.

[0243] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VH-CDR3 consisting of a sequence selected from the group consisting of SEQ ID NOs: 41, 42, 43, 44, and 45, excluding one, two, three, or four amino acid substitutions.

[0244] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VL-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48; VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52; and 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 its antigen-binding fragment comprises VL-CDR1 containing a sequence selected from the group consisting of SEQ ID NOs: 46, 47, and 48; VL-CDR2 containing a sequence selected from the group consisting of SEQ ID NOs: 49, 50, 51, and 52; and VL-CDR3 containing 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 its antigen-binding fragment comprises VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35 and 36; VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40; and 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 its antigen-binding fragment comprises VH-CDR1 containing a sequence selected from the group consisting of SEQ ID NOs: 35 and 36; VH-CDR2 containing a sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40; and VH-CDR3 containing 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 its antigen-binding fragment comprises: VL-CDR1, which consists of a sequence selected from the group consisting of SEQ ID NOs. 46, 47, and 48, excluding one, two, three, or four amino acid substitutions; VL-CDR2, which consists of a sequence selected from the group consisting of SEQ ID NOs. 49, 50, 51, and 52, excluding one, two, three, or four amino acid substitutions; and VL-CDR3, which consists of a sequence selected from the group consisting of SEQ ID NOs. 53, 54, 55, and 56, excluding one, two, three, or four amino acid substitutions.

[0247] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises: VL-CDR1 containing a sequence selected from the group consisting of SEQ ID NOs. 46, 47, and 48, excluding one, two, three, or four amino acid substitutions; VL-CDR2 containing a sequence selected from the group consisting of SEQ ID NOs. 49, 50, 51, and 52, excluding one, two, three, or four amino acid substitutions; and VL-CDR3 containing a sequence selected from the group consisting of SEQ ID NOs. 53, 54, 55, and 56, excluding one, two, three, or four amino acid substitutions.

[0248] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises: VH-CDR1, which consists of a sequence selected from the group consisting of SEQ ID NOs. 35 and 36, excluding one, two, three, or four amino acid substitutions; VH-CDR2, which consists of a sequence selected from the group consisting of SEQ ID NOs. 37, 38, 39, and 40, excluding one, two, three, or four amino acid substitutions; and VH-CDR3, which consists of a sequence selected from the group consisting of SEQ ID NOs. 41, 42, 43, 44, and 45, excluding one, two, three, or four amino acid substitutions.

[0249] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR1 containing a sequence selected from the group consisting of SEQ ID NOs. 35 and 36, excluding one, two, three, or four amino acid substitutions; VH-CDR2 containing a sequence selected from the group consisting of SEQ ID NOs. 37, 38, 39, and 40, excluding one, two, three, or four amino acid substitutions; and VH-CDR3 containing a sequence selected from the group consisting of SEQ ID NOs. 41, 42, 43, 44, and 45, excluding one, two, three, or four amino acid substitutions.

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

[0251] In some embodiments, FW1 includes sequence number 25 or 26, FW2 includes sequence number 27 or 28, FW3 includes sequence number 29, FW4 includes sequence number 30, FW5 includes sequence number 31, FW6 includes sequence number 32, FW7 includes sequence number 33, and FW8 includes sequence number 34.

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

[0253] In a particular embodiment, the anti-CD733 antibody or its antigen-binding fragment comprises VL and VH containing the same or identical VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences, wherein such VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 are, respectively, Sequence IDs 46, 49, 53, 35, 37, and 41, or Sequence IDs 47, 49, 53, 35, 37 and 41, or Sequence IDs 47, 49, 54, 36, 37 and 42, or Sequence IDs 46, 50, 54, 36, 38 and 43, or Sequence IDs 46, 51, 55, 36, 39 and 44, or Sequence IDs 48, 52, 54, 36, 40 and 44, or Sequence IDs 46, 49, 56, 35, 37 and 41, or Sequence IDs 46, 49, 53, 35, 37 and 45, or Sequence IDs 47, 49, 56, 36, 37 and 45, or Sequence IDs 46, 50, 56, 36, 38 and 45, or Sequence IDs 46, 51, 56, 36, 39 and 45, or Sequence IDs 48, 52, 56, 36, 40 and 45, or Sequence numbers 46, 49, 56, 35, 37, and 45.

[0254] In a particular embodiment, an anti-CD73 antibody or its antigen-binding fragment comprises antibody VL and antibody VH, wherein VL contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 57, SEQ ID NOs: 58, SEQ ID NOs: 59, SEQ ID NOs: 60, SEQ ID NOs: 61, SEQ ID NOs: 62, SEQ ID NOs: 63, SEQ ID NOs: 64, SEQ ID NOs: 65, SEQ ID NOs: 66, SEQ ID NOs: 67, SEQ ID NOs: 68, SEQ ID NOs: 69, and SEQ ID NOs: 70.

[0255] In another embodiment, the anti-CD73 antibody or its antigen-binding fragment comprises antibody VL and antibody VH, wherein VH contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 71, SEQ ID NOs: 72, SEQ ID NOs: 73, SEQ ID NOs: 74, SEQ ID NOs: 75, SEQ ID NOs: 76, SEQ ID NOs: 77, SEQ ID NOs: 78, SEQ ID NOs: 79, SEQ ID NOs: 80, SEQ ID NOs: 80, SEQ ID NOs: 81, SEQ ID NOs: 82, SEQ ID NOs: 83, and SEQ ID NOs: 84.

[0256] In another embodiment, the anti-CD73 antibody or its antigen-binding fragment is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70. The present invention further comprises a VL containing one sequence, and a VH containing a sequence that is at least approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84.

[0257] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VL containing the sequence of SEQ ID NO: 68 and a VH containing the sequence of SEQ ID NO: 82.

[0258] "Clone 10.3 antibody" (also known as "73combo3" or "MEDI9447") is an IgG1 antibody containing two CD730010-derived light chains (VL) of SEQ ID NO: 68 (containing three CDRs, CDR1, CDR2, and CDR3, each having the sequences of SEQ ID NOs: 46, 51, and 56, respectively) and two CD730010-derived heavy chains (VH) of SEQ ID NO: 82 (containing three CDRs, CDR1, CDR2, and CDR3, each having the sequences of SEQ ID NOs: 36, 39, and 45, respectively).

[0259] In certain embodiments, the anti-CD73 antibodies or antigen-binding fragments disclosed herein bind to CD73 with substantially the same or better affinity than a 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 certain embodiments, the anti-CD73 antibody of this disclosure may, but is not limited to: 1) Light chain CDR1 containing the sequence SGDKVGDKYAS, and / or 2) Consensus Sequence EDX 18 KX 19 X 20 S (where X 18 X 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 A light chain CDR2 containing the amino acid residue histidine (H), proline (P), or leucine (L), and / or 3) Light chain CDR3 containing sequence QAWDTSFWV This includes modifications to the CDR1 and / or CDR2 and / or CDR3 of the light chain of the CD730002 antibody, including the above.

[0261] In certain embodiments, the anti-CD73 antibody of this disclosure may, but is 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 A heavy chain CDR1 containing the amino acid residue methionine (M) or arginine (R), and / or 2) Sequence AISGSGGSX 23 YY X 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 A heavy chain CDR2 containing the amino acid residue glycine (G) or arginine (R), and / or 3) Heavy chain CDR3 containing sequence DKGYYWYM This includes modifications to the heavy chain CDR1 and / or CDR2 and / or CDR3 of CD730002, including those mentioned above.

[0262] In one embodiment, the anti-CD73 antibody or its antigen-binding fragment has a 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 ] represents an amino acid residue in VL framework region 1 (sequence number 90 or 91), VL framework region 2 (sequence number 92), VL framework region 3 (sequence number 93, 94 or 122), and VL framework region 4 (sequence number 30), respectively; where X 18 represents the amino acid residue proline (P) or leucine (L); X 19 represents the amino acid residue arginine (R) or tyrosine (Y); and X 20 The VL region contains an amino acid residue (representing histidine (H), proline (P), or leucine (L)).

[0263] In one embodiment, the anti-CD73 antibody or its antigen-binding fragment has a consensus amino acid sequence: [FW 13 SX 21 AX 22 S[FW 14 ]AISGSGGSX 23 YY X 24 DSVKX 25 [FW 15 ]DKGYYWYM[FW 16 ] (In the formula, [FW 13 ], [FW 14 ], [FW 15 ] and [FW 16 ] represents the amino acid residues of VH framework region 1 (SEQ ID NO: 31), VH framework region 2 (SEQ ID NO: 32), VH framework region 3 (SEQ ID NO: 33), and VH framework region 4 (SEQ ID NO: 89), respectively; where X21 represents the amino acid residue tyrosine (Y) or valine (V); X 22 X 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 The VH region contains an amino acid residue (representing glycine (G) or arginine (R)).

[0264] In one embodiment, the anti-CD73 antibody or its antigen-binding fragment has a 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 ] represents an amino acid residue in VL framework region 1 (sequence number 90 or 91), VL framework region 2 (sequence number 92), VL framework region 3 (sequence number 93, 94 or 122), and VL framework region 4 (sequence number 30), respectively; where X 18 represents the amino acid residue proline (P) or leucine (L); X 19 represents the amino acid residue arginine (R) or tyrosine (Y); and X 20 The VL region contains an amino acid residue (representing histidine (H), proline (P), or leucine (L)), and the anti-CD73 antibody or its antigen-binding fragment has a consensus amino acid sequence: [FW 13 SX 21 AX 22 S[FW 14 ]AISGSGGSX 23 YY X 24 DSVKX 25 [FW 15 ]DKGYYWYM[FW 16 ] (In the formula, [FW 13 ], [FW 14 ], [FW 15 ] and [FW 16 ] represents the amino acid residues of VH framework region 1 (SEQ ID NO: 31), VH framework region 2 (SEQ ID NO: 32), VH framework region 3 (SEQ ID NO: 33), and VH framework region 4 (SEQ ID NO: 89), respectively; where X 21 represents the amino acid residue tyrosine (Y) or valine (V); X 22 X 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 The VH region further includes an amino acid residue (where represents glycine (G) or arginine (R)).

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

[0266] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VL-CDR2 consisting of 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 its antigen-binding fragment comprises a VL-CDR3 consisting of the sequence SEQ ID NO: 100.

[0268] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124.

[0269] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VH-CDR2 consisting of 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 its antigen-binding fragment comprises a VH-CDR3 consisting of the sequence SEQ ID NO: 96.

[0271] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VL-CDR1, which consists of a sequence comprising SEQ ID NO: 97, except for 1, 2, 3, or 4 amino acid substitutions.

[0272] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs. 98, 99, 127, 128, and 129, excluding one, two, three, or four amino acid substitutions.

[0273] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VL-CDR3, which consists of the sequence of SEQ ID NO: 100 except for 1, 2, 3, or 4 amino acid substitutions.

[0274] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs. 35, 123, and 124, excluding one, two, three, or four amino acid substitutions.

[0275] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises a VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126, excluding one, two, three, or four amino acid substitutions.

[0276] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR3, which consists of the sequence of SEQ ID NO: 96, except for 1, 2, 3, or 4 amino acid substitutions.

[0277] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VL-CDR1 consisting of the sequence of SEQ ID NO: 97; VL-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129; and VL-CDR3 consisting of the sequence of SEQ ID NO: 100. In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VL-CDR1 containing the sequence of SEQ ID NO: 97; VL-CDR2 containing a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129; and VL-CDR3 containing the sequence of SEQ ID NO: 100.

[0278] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR1 consisting of a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124; VH-CDR2 consisting of a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126; and VH-CDR3 consisting of a sequence consisting of SEQ ID NO: 96. In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR1 containing a sequence selected from the group consisting of SEQ ID NOs: 35, 123, and 124; VH-CDR2 containing a sequence selected from the group consisting of SEQ ID NOs: 37, 95, 125, and 126; and VH-CDR3 containing a sequence consisting of SEQ ID NO: 96.

[0279] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises: VL-CDR1, which consists of a sequence corresponding to SEQ ID NO: 97 except for 1, 2, 3, or 4 amino acid substitutions; VL-CDR2, which consists of a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129 except for 1, 2, 3, or 4 amino acid substitutions; and VL-CDR3, which consists of a sequence corresponding to SEQ ID NO: 100 except for 1, 2, 3, or 4 amino acid substitutions.

[0280] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises: VL-CDR1 containing the sequence corresponding to SEQ ID NO: 97 except for 1, 2, 3, or 4 amino acid substitutions; VL-CDR2 containing a sequence selected from the group consisting of SEQ ID NOs: 98, 99, 127, 128, and 129 except for 1, 2, 3, or 4 amino acid substitutions; and VL-CDR3 containing the sequence corresponding to SEQ ID NO: 100 except for 1, 2, 3, or 4 amino acid substitutions.

[0281] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises: VH-CDR1, consisting of a sequence selected from the group consisting of SEQ ID NOs. 35, 123, and 124, excluding one, two, three, or four amino acid substitutions; VH-CDR2, consisting of a sequence selected from the group consisting of SEQ ID NOs. 37, 95, 125, and 126, excluding one, two, three, or four amino acid substitutions; and VH-CDR3, consisting of a sequence corresponding to SEQ ID NO. 96, excluding one, two, three, or four amino acid substitutions.

[0282] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR1 containing a sequence selected from the group consisting of SEQ ID NOs. 35, 123, and 124, excluding 1, 2, 3, or 4 amino acid substitutions; VH-CDR2 containing a sequence selected from the group consisting of SEQ ID NOs. 37, 95, 125, and 126, excluding 1, 2, 3, or 4 amino acid substitutions; and VH-CDR3 containing a sequence consisting of SEQ ID NO. 96, excluding 1, 2, 3, or 4 amino acid substitutions.

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

[0284] In some embodiments, FW9 includes sequence number 90 or 91, FW 10 It includes sequence number 92, FW 11 This includes sequence numbers 93, 94, or 122, and FW 12 This includes sequence number 30, FW13 It includes sequence number 31, FW 14 This includes sequence number 32, FW 15 This includes sequence number 33, and FW 16 This includes sequence number 89.

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

[0286] In a particular embodiment, the anti-CD733 antibody or its antigen-binding fragment comprises VL and VH containing the same or identical VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences, respectively, where such VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 are, Sequence IDs 97, 98, 100, 35, 37, and 96, or Sequence IDs 97, 99, 100, 35, 95 and 96, or Sequence IDs 97, 98, 100, 35, 37, and 96, or Sequence numbers 97, 99, 100, 123, 37, and 96, or Sequence numbers 97, 99, 100, 124, 37, and 96, or Sequence IDs 97, 99, 100, 35, 125, and 96, or Sequence IDs 97, 99, 100, 35, 126, and 96, or Sequence IDs 97, 99, 100, 35, 95, and 96, or Sequence IDs 97, 127, 100, 35, 95, and 96, or Sequence IDs 97, 128, 100, 35, 95, and 96, or Sequence IDs 97, 129, 100, 35, 95, and 96, or These are sequence numbers 97, 99, 100, 35, 95, and 96.

[0287] In a particular embodiment, an anti-CD73 antibody or its antigen-binding fragment comprises antibody VL and antibody VH, wherein VL contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 86, 88, 112, 118, 119, 120, and 121.

[0288] In another embodiment, an anti-CD73 antibody or its antigen-binding fragment comprises antibody VL and antibody VH, wherein VH contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 85, 87, 111, 113, 114, 115, 116, and 117.

[0289] In another embodiment, the anti-CD73 antibody or its antigen-binding fragment comprises a VL containing a sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 86, 88, 112, 118, 119, 120, and 121; and further comprises a VH containing a sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from 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 its antigen-binding fragment comprises a VL containing the sequence of SEQ ID NO: 88 and a VH containing the sequence of SEQ ID NO: 87. In some embodiments, the anti-CD73 antibody or its antigen-binding fragment 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 antibody containing two light chains (VL) derived from CD730002 of SEQ ID NO: 88 (containing three CDRs, CDR1, CDR2, and CDR3, each having the sequences of SEQ ID NOs: 97, 99, and 100, respectively) and a heavy chain (VH) derived from CD7300002 of SEQ ID NO: 87 (containing three CDRs, CDR1, CDR2, and CDR3, each having the sequences of SEQ ID NOs: 35, 95, and 96, respectively).

[0292] In certain embodiments, the anti-CD73 antibodies or antigen-binding fragments disclosed herein bind to CD73 with substantially the same or better affinity than a 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 having 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 the VL of SEQ ID NO: 104 and the VH of SEQ ID NO: 103), CD730008 (i.e., an anti-CD73 antibody comprising the VL of SEQ ID NO: 106 and the VH of SEQ ID NO: 107), CD7300011 (i.e., an anti-CD73 antibody comprising the VL of SEQ ID NO: 5 and the VH of SEQ ID NO: 6), CD730021 (i.e., an anti-CD73 antibody comprising the VL of SEQ ID NO: 7 and the VH of SEQ ID NO: 8), CD730 These include CD730042 (i.e., an anti-CD73 antibody containing the VL of SEQ ID NO: 9 and the VH of SEQ ID NO: 10), CD730046 (i.e., an anti-CD73 antibody containing the VL of SEQ ID NO: 11 and the VH of SEQ ID NO: 12), CD730047 (i.e., an anti-CD73 antibody containing the VL of SEQ ID NO: 13 and the VH of SEQ ID NO: 14), CD730068 (i.e., an anti-CD73 antibody containing the VL of SEQ ID NO: 108 and the VH of SEQ ID NO: 107), or CD730069 (i.e., an anti-CD73 antibody containing the VL of SEQ ID NO: 110 and the VH of SEQ ID NO: 109). Modifications to the parent antibody may include mutations in the CDR region and / or FW region compared to the parent antibody, e.g., CD730004.

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

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

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

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

[0298] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment contains VL-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, with the exception of 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the anti-CD73 antibody or its antigen-binding fragment contains VL-CDR2 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, with the exception of 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VL-CDR3 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, with the exception of one, two, three, or four amino acid substitutions.

[0299] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment contains VH-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, with the exception of 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the anti-CD73 antibody or its antigen-binding fragment contains VH-CDR2 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, with the exception of 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the anti-CD73 antibody or its antigen-binding fragment comprises VH-CDR3 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, with the exception of one, two, three, or four amino acid substitutions.

[0300] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment is VL-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069; CD730004, CD730008, CD7300011, CD7300 21, VL-CDR2 derived from CD730042, CD730046, CD730047, CD730068, or CD730069; and VL-CDR3 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0301] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment is VH-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069; CD730004, CD730008, CD7300011, CD7300 21, VH-CDR2 derived from CD730042, CD730046, CD730047, CD730068, or CD730069; and VH-CDR3 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0302] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment is VL-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for 1, 2, 3, or 4 amino acid substitutions; CD730004, CD730008, CD7300 The compound comprises VL-CDR2 derived from 011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069; and VL-CDR3 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, with the exception of 1, 2, 3, or 4 amino acid substitutions.

[0303] In some embodiments, the anti-CD73 antibody or its antigen-binding fragment is VH-CDR1 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, except for 1, 2, 3, or 4 amino acid substitutions; CD730004, CD730008, CD7300 The compound comprises VH-CDR2 derived from 011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069; and VH-CDR3 derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, with the exception of 1, 2, 3, or 4 amino acid substitutions.

[0304] In a particular embodiment, an anti-CD73 antibody or its antigen-binding fragment comprises modifications to CDR1 and / or CDR2 and / or CDR3 of the heavy and / or light chain derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069, and further comprises modifications to FW1 and / or FW2 and / or FW3 and / or FW4 of the heavy and / or light chain derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0305] In a particular embodiment, the anti-CD733 antibody or its antigen-binding fragment comprises VL and VH containing the same VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences, which are identical or identical except for one, two, three, or four amino acid substitutions in one or more CDRs, wherein such VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 are derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0306] In a particular embodiment, an anti-CD73 antibody or its antigen-binding fragment comprises an antibody VL and an antibody VH, wherein the VL contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from a VL sequence derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0307] In another embodiment, an anti-CD73 antibody or its antigen-binding fragment comprises antibody VL and antibody VH, wherein VH contains an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to a reference amino acid sequence selected from a VH sequence derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0308] In another embodiment, the anti-CD73 antibody or its antigen-binding fragment 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 VL sequences derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069. The VL comprises the sequence of and further comprises a VH which contains a sequence that is at least approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical to a reference amino acid sequence selected from a VH sequence derived from CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068, or CD730069.

[0309] In certain embodiments, the anti-CD73 antibodies or their antigen-binding fragments disclosed herein bind to CD73 with substantially the same or better affinity as 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 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 "mix-fitted" to create other anti-CD73 binding molecules.

[0311] In a particular embodiment, the VH sequences of the 10.3 antibody and the 2C5 antibody are co-fitted. In another embodiment, the VL sequences of the 10.03 antibody and the 2C5 antibody can be co-fitted. In addition to or instead, the VL and / or VH sequences of the clone 10 (CD730010) variant disclosed herein can be co-fitted. In addition to or instead, the VL and / or VH sequences of the clone 2 (CD730002) variant disclosed herein can be co-fitted. In addition to or instead, the VL and / or VH sequences of the clone 10 (CD730010) and clone 2 (CD730002) variants disclosed herein can be co-fitted.

[0312] In some embodiments, VL and / or VH mixed matching can be performed between sequences derived from antibodies classified as the same epitope bin (see Example 2). As used herein, the term “epitope bin” refers to a grouping of antibodies or their antigen-binding fragments that bind to the same epitope or duplicate epitope, or compete with each other for binding to the same epitope or duplicate epitope. For example, sequences derived from CD730003, CD730010, CD730021, CD730042, CD730046, and CD730047 (all of these antibodies belong to “epitope bin B”) can be mixed-matched. In other embodiments, VL and / or VH mixed matching can be performed between sequences derived from anti-CD73 antibodies classified as different epitope bins. Therefore, the sequences of antibodies belonging to "epitope bin B" can be mixed and matched with the sequences of anti-CD73 antibodies of "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 antibodies disclosed herein (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or their antigen-binding fragments include mutations that improve binding to human FcRn and improve the half-life of the anti-CD73 antibody or its antigen-binding fragment. In some embodiments, such mutations are mutations introduced into the constant domain of IgG1, from methionine (M) to tyrosine (Y) at position 252, from serine (S) to threonine (T) at position 254, and from threonine (T) to glutamic acid (E) at position 256 (numbered according to the EU index as found in Kabat (Kabat, et al. (1991) Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, DC)). See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, referred to as the "YTE mutant," has been shown to exhibit approximately a four-fold increase in half-life compared to the wild-type version of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006)). In some embodiments, the anti-CD73 antibody or its antigen-binding fragment containing the IgG constant domain contains one or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436 (numbered according to the EU index as found in Kabat), where such mutations increase the serum half-life of the anti-CD73 antibody or its antigen-binding fragment.

[0314] 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 defined in Kabat) 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 of the IgG constant domain (numbered according to the EU index as defined in Kabat) 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 of the IgG constant domain (numbered according to the EU index as defined in Kabat) with an amino acid selected from the group consisting of threonine (T), leucine (L), phenylalanine (F), and serine (S).

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

[0316] In a specific embodiment, an anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or its antigen-binding fragment disclosed herein comprises an IgG1 constant domain including a mutation from methionine (M) to tyrosine (Y) at position 252, a mutation from serine (S) to threonine (T) at position 254, and a mutation from threonine (T) to glutamic acid (E) at position 256 (numbered according to the EU index as found in Kabat).

[0317] In certain embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or its antigen-binding fragment disclosed herein 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 by 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 by leucine (L), (e) Substitution of the amino acid at position 309 by proline (P), (f) Substitution of the amino acid at position 311 by 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) Two or more combinations of the above substitutions The modified IgG comprises at least one IgG constant domain amino acid substitution selected from the group consisting of (positions numbered according to the EU index as found in Kabat), where the modified IgG has an increased serum half-life compared to IgG with the wild-type IgG constant domain.

[0318] In some embodiments, the VH and / or VL amino acid sequences of anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or their antigen-binding fragments disclosed herein are 85%, 90%, 95%, 96%, 97%, 98%, or 99% similar to the above VH and VL sequences and contain one, two, three, four, five, or more conservative substitutions. CD73 antibodies having VH and VL regions that have high (i.e., 80% or more) sequence similarity or sequence identity with 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, are CD73 antibodies having VH and VL regions that have high (i.e., 80% or more) sequence similarity or sequence identity with the VH region of SEQ ID NOs. 57, 58, 59, 60 These can be obtained by mutagenes (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121, and then testing the encoded modified antibody for retention function using a functional assay described herein.

[0319] In some embodiments, the Fc domain of an anti-CD73 antibody disclosed herein or the Fc domain of a fusion protein containing a CD73 binding fragment of an antibody disclosed herein has reduced binding affinity to the Fc receptor in order to reduce, for example, ADCC-mediated cytotoxicity. In some embodiments, the Fc domain of an antibody or Fc fusion protein has increased binding affinity to the Fc receptor in order to increase, for example, ADCC-mediated cytotoxicity. In some embodiments, the Fc domain of an antibody or Fc fusion protein is numbered by the EU index as defined in Kabat as 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 269, 279, 280, The molecule contains one or more ADCC-reducing amino acid residues that do not exist in nature, 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. Numerous specific mutations capable of reducing the ADCC activity of antibodies are known in the art, 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, 7,790,858, U.S. Patent Application Publication Nos. 20140378663, 20130071390, 20110212087, and See the mutations described in U.S. Patent Publication No. 20150118227, No. 20060194290, No. 20060194291, No. 20080274105, No. 20080274506, U.S. Patent Publication No. 20130089541, and U.S. Patent Publication No. 20130108623 (these are incorporated herein by reference as a whole). Antibodies with reduced ADCC effector function also include those having one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (see, for example, U.S. Patent No. 6,737,056). Such Fc mutants also include Fc mutants having substitutions to alanine at residues 265 and 297, as well as Fc mutants having two or more substitutions at amino acid positions 265, 269, 270, 297, and 327 (see, for example, U.S. Patent No. 7,332,581). Optionally, mutations that reduce both ADCC and CDC may be incorporated. In some embodiments, antibody-drug conjugates (ADCs) can be created using anti-CD73 antibodies or their antigen-binding fragments disclosed herein that contain mutations that reduce or eliminate ADCC and / or CDC.

[0320] In one embodiment, the Disclosure provides an anti-CD73 antibody which is IgG1, IgG2, or IgG3 and includes at least one modification at one or more positions selected from the group consisting of 234, 235, and 331 when numbered by the EU index as defined in Kabat. In yet another specific embodiment, the Fc region is an IgG1, IgG2, or IgG3 Fc region, and the amino acids not found in nature are selected from the group consisting of 234F, 235E, 235F, 235Q (or 235Y), 239A, 332Q, 331S, and 332Q when numbered by the EU index as defined in Kabat.

[0321] In another embodiment, the Disclosure provides an anti-CD73 antibody which is IgG4 and includes at least one modification at one or more positions selected from the group consisting of 228 and 235 when numbered by the EU index as defined in Kabat. In yet another specific embodiment, the Fc region is an IgG4 Fc region and the naturally occurring amino acids are selected from the group consisting of 228P, 235E and 235Y when numbered by the EU index as defined in Kabat. In a specific embodiment, the Disclosure provides an anti-CD73 antibody which is IgG1, IgG2, or IgG3 and includes modifications at positions (i) 234F, 235E, and 331S; (ii) 234F, 235F, and 331S; (iii) 234F, 235Q, and 322Q. In yet another specific embodiment, the Disclosure provides an anti-CD73 antibody which is IgG4 and includes modifications 228P and 235E.

[0322] III. Epitope-competitive CD73-binding molecules In another embodiment, the present disclosure provides CD73-binding molecules that bind to the same epitopes as various anti-CD73 antibodies described herein, for example, molecules that bind to the same epitope as MEDI9447, clone 10.3 antibody, or clone 2C5 antibody.

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

[0324] Accordingly, in one embodiment, the Disclosure provides an anti-CD73 antibody and its antigen-binding fragment, such as a human monoclonal antibody, that competes with another anti-CD73 antibody or its antigen-binding fragment, e.g., CD730010 antibody, CD730002 antibody, CD730004 antibody, its variants (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody), or its antigen-binding fragment, for binding to CD73. The ability of the test antibody to inhibit the binding of, for example, CD730010 antibody (or clone 10.3 antibody or its antigen-binding fragment), or CD730002 antibody (or clone 2C5 antibody or its antigen-binding fragment), demonstrates that the test antibody may compete with its antibody for binding to CD73; such antibody may, according to an unrestricted theory, bind to the same or related (e.g., structurally similar or spatially adjacent) epitopes on CD73 as the competing anti-CD73 antibody or its antigen-binding fragment. In one embodiment, for example, an anti-CD73 antibody or its antigen-binding fragment that binds to the same epitope on CD73 as the CD730010 antibody (or clone 10.3 antibody or its antigen-binding fragment), or the CD730002 antibody (or clone 2C5 antibody or its antigen-binding fragment), is a human monoclonal antibody.

[0325] To elucidate the mechanism of action of MEDI9447, a monoclonal antibody that directly inhibits the enzymatic activity of CD73, as described herein, we identified the epitope of MEDI9447. This epitope is located on the apical surface of the N-terminal domain of CD73, in a region distant from the substrate-binding and active site residues. Structural and mechanistic studies revealed that MEDI9447 antagonistizes CD73 via a dual mechanism that prevents CD73 from adopting a catalytically active conformation. These results provide the first report of a precisely mapped epitope that could be targeted for selective, potent, and non-competitive inhibition of CD73 as a means of modulating adenosine signaling in the tumor microenvironment.

[0326] The inventors determined the epitope of MEDI9447 using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) and mutagenesis strategies and investigated the potential effect of antibody binding to the global CD73 structure. The antibody binds to a site in the N-terminal domain of CD73 that enables non-competitive inhibition of AMP hydrolysis. In various embodiments, the epitope comprises one or more CD73 amino acid residues corresponding to V144, K180, and N185. In various embodiments, the epitope further comprises one or more CD73 amino acid residues corresponding to Y135, K136, and N187 of CD73. Notably, the epitope is located such that antibody binding prevents the conversion of CD73 from an open conformational isomer to a catalytically active closed conformational isomer. Furthermore, our tests have shown that MEDI9447 can inhibit both anchored CD73 and soluble CD73 using a dual inhibition mechanism mediated by the binding titer of antibody interactions with CD73.

[0327] IV. Functional properties of anti-CD73 antibodies The affinity or avidity of an antibody to an antigen can be experimentally determined using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or reaction kinetics (e.g., BIACORE® analysis). Direct binding assays and competitive binding assay formats are readily available. (See, for example, Berzofsky et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Immunology, WH Freeman and Company: New York, NY (1992); and the methods described herein.) Affinity measurements of specific antibody-antigen interactions may differ when measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other antigen-binding parameters (e.g., K) may differ. D Or Kd, K on , K off The measurement of ) is performed using standardized solutions of antibodies and antigens, and standardized buffers such as those known in the art and those described herein.

[0328] Furthermore, it is known in the art that affinity measured using surface plasmon resonance analysis (e.g., BIACORE®) may differ depending on which of the reactants binds to the chip. In this regard, affinity can be measured using a format in which a target antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) is immobilized on the chip (referred to as the "IgG down" format), or a format in which a target protein (e.g., CD73) is immobilized on the chip (referred to as the "CD73 down" format).

[0329] In one aspect of this disclosure, an anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or its antigen-binding fragment is 10-6 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 Dissociation constant less than M or k d (k off / k on ) specifically binds to CD73 and / or its antigenic fragment.

[0330] In another embodiment, an anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or its antigen-binding fragment is 1 × 10 -3 s -1 Less than, or 2 × 10 -3 s -1 K less than off It binds to CD73 and / or its antigenic fragment. In another embodiment, the anti-CD73 antibody or its antigenic fragment is 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 K less than off It binds to CD73 and its antigenic fragment.

[0331] In another embodiment, an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment 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 fragment at a rapid rate.

[0332] In some embodiments, the anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment has a K content 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 when measured by flow cytometry. D It binds to CD73 on the surface of MB-MDA-231 cells. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, which has a K content of approximately 150 pM when measured by flow cytometry. D It binds to CD73 on the surface of MB-MDA-231 cells. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody, with a K content of approximately 80 pM when measured by flow cytometry. DIt then binds 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 its antigen-binding fragment has a K content 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 It binds to CD73 on the surface of mouse 3T1 cells. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, and when measured by flow cytometry, it has a K content of approximately 110 pM. D It binds to CD73 on the surface of mouse 3T1 cells. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody, with a K content of approximately 55 pM when measured by flow cytometry. D It then 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 its antigen-binding fragment has a K content 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 when measured by flow cytometry. D It binds to CD73 on the surface of cynomolgus monkey MK-1 cells. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, which has a K content of approximately 80 pM when measured by flow cytometry. D It binds to CD73 on the surface of cynomolgus monkey MK-1 cells. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody, which has a K2 value of approximately 60 pM when 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 its antigen-binding fragment has a K2 level 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 when measured by surface plasmon resonance (PROTEON®). D It binds to human CD73. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, and when measured by surface plasmon resonance (PROTEON®), it has a K2 value of approximately 4 pM. D It binds to human CD73. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody and has a K2 value of approximately 9 pM when measured by surface plasmon resonance (PROTEON®). D It then binds to human CD73.

[0336] In some embodiments, the anti-CD73 antibody disclosed herein (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or its antigen-binding fragment has a K content 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 when measured by surface plasmon resonance (PROTEON®). D It binds to mouse CD73. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, and when measured by surface plasmon resonance (PROTEON®), it has a K2 value of approximately 1 pM. DIt binds to mouse CD73. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody and has a K2 of approximately 22 pM when measured by surface plasmon resonance (PROTEON®). D Then connect to mouse CD73.

[0337] In some embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or its antigen-binding fragment has a K2 level 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 when measured by surface plasmon resonance (PROTEON®). D It binds to cynomolgus monkey CD73. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, and when measured by SPR (Proteon), it has a K content of approximately 7 pM. D It binds to cynomolgus monkey CD73. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody and has a K2 of approximately 9 pM when measured by surface plasmon resonance (PROTEON®). D It binds to the cynomolgus macaque CD73.

[0338] In some embodiments, the anti-CD73 antibody (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or its antigen-binding fragment has a K content 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 when measured by solution binding. D It binds to human CD73. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, and when measured by solution binding, it has a K content of approximately 80 pM. D It binds to human CD73. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody, and when measured by solution binding, it has a K2 value of approximately 80 pM. D It then binds to human CD73.

[0339] In some embodiments, the anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment has a K content 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 when measured by solution binding. D It binds to mouse CD73. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, and when measured by solution binding, it has a K content of approximately 130 pM. D It binds to mouse CD73. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody, and when measured by solution binding, it has a K content of approximately 1500 pM. D Then connect to mouse CD73.

[0340] In some embodiments, the anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment contains 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 of K when measured by solution binding. D It binds to cynomolgus monkey CD73. In one specific embodiment, the anti-CD73 antibody is clone 10.3 antibody, and when measured by solution binding, it has a K content of approximately 90 pM. D It binds to cynomolgus monkey CD73. In another specific embodiment, the anti-CD73 antibody is a clone 2C5 antibody, and when measured by solution binding, it has a K2 of approximately 100 pM. D It binds to the cynomolgus macaque CD73. In a detailed embodiment, MEDI9447 is approximately 1 × 10 -12 , 5×10 -12 , 10×10 -12 , 100×10 -12 , or 150 x 10 -12 KD Then it is combined with CD73.

[0341] In some embodiments, the CD73-binding molecules disclosed herein, for example, anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or their antigen-binding fragments can reduce the AMP-mediated suppression of T cell division. In other embodiments, the CD73-binding molecules disclosed herein, for example, anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) or their antigen-binding fragments can reduce the AMP-mediated suppression of T cell division. reg ATP-induced T eff It can rescue the suppression.

[0342] In some embodiments, CD73-binding molecules disclosed herein, such as anti-CD73 antibodies or their antigen-binding fragments (e.g., clone 10.3 antibody or clone 2C5 antibody), can significantly inhibit syngeneic tumor growth. In one embodiment, the tumors are non-small cell lung cancer tumors, ovarian cancer tumors, breast cancer tumors, head and neck cancer tumors, pancreatic cancer tumors, colorectal cancer tumors, melanoma tumors, and lymphoma tumors. In another embodiment, the tumors are CT26 mouse syngeneic CRC tumors, B16F10 melanoma tumors, EG7-OVA lymphoma tumors, or LL2 (Lewis lung) tumors. In some embodiments, CD73-binding molecules, such as anti-CD73 antibodies or their antigen-binding fragments (e.g., clone 10.3 antibody or clone 2C5 antibody), can significantly inhibit tumor growth, where the tumors are 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, the CD73-binding molecules disclosed herein, such as anti-CD73 antibodies or their antigen-binding fragments (e.g., clone 10.3 antibody or clone 2C5 antibody), can significantly inhibit tumor growth when administered at PD1 concentrations of approximately 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0343] In some embodiments, the CD73-binding molecules disclosed herein, such as anti-CD73 antibodies or their antigen-binding fragments (e.g., clone 10.3 antibodies or clone 2C5 antibodies), can be internalized after binding to cells. In some embodiments, the CD73-binding molecule is an antibody-drug conjugate (ADC).

[0344] V. Preparation of anti-CD73 antibody and antigen-binding fragments Monoclonal anti-CD73 antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) and their antigen-binding fragments can be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. In the hybridoma method, mice, hamsters, or other suitable host animals are immunized as described above to induce lymphocyte production of antibodies that can specifically bind to the immune antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes can be isolated and fused with suitable myeloma cell lines, for example using polyethylene glycol, to form hybridoma cells, from which unfused lymphocytes and myeloma cells can be selectively removed. Next, hybridomas that produce monoclonal antibodies specifically targeting the antigen of choice, as determined by immunoprecipitation, immunoblotting, or in vitro binding assays (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)), can be grown in vitro using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in animals. Then, as described above for polyclonal antibodies, monoclonal antibodies can be purified from culture medium or ascites fluid.

[0345] Alternatively, anti-CD73 monoclonal antibodies (e.g., MEDI9447, clone 10.3 antibody, or clone 2C5 antibody) and their antigen-binding fragments can also be produced using recombinant DNA methods, as described, for example, in U.S. Patent No. 4,816,567. Polynucleotides encoding the monoclonal antibody are isolated from mature B cells or hybridoma cells by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences are determined using conventional procedures. Next, the isolated polynucleotides encoding the heavy and light chains are cloned into a suitable expression vector, and the expression vector is transfected into host cells that do not normally produce immunoglobulin proteins, such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, resulting in the production of monoclonal antibodies by the host cells. Furthermore, as described, recombinant anti-CD73 monoclonal antibodies or their antigen-binding fragments of a desired species can also be isolated from phage display libraries expressing the CDR of the desired species (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] Anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or polynucleotides encoding their antigen-binding fragments can be further modified in various ways using recombinant DNA technology to create alternative antibodies. In some embodiments, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be (1) replaced with those regions of a human antibody to create a chimeric antibody, or (2) replaced with a non-immunoglobulin polypeptide to create a fusion antibody. In some embodiments, the constant region is truncated or removed to create a desired antibody fragment of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable region 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 its antigen-binding fragment is a human antibody or its antigen-binding fragment. Human antibodies can be prepared directly using various techniques known in the art. Immobilized human B lymphocytes can be prepared by immunization in vitro or by isolating from immunized individuals that produce antibodies against the target antigen (see, for example, 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. Patent No. 5,750,373).

[0348] Furthermore, as described, for example, 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, anti-CD73 human antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments can also be selected from a phage library (where the phage library expresses human antibodies). Techniques for creating and using antibody phage libraries are also described in U.S. Patent Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2007, J.Mol.Bio., doi:10.1016 / j.jmb.2007.12.018 (each of these is incorporated by reference as a whole).

[0349] Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio / Technology 10:779-783, as used collectively) are known in the art and can be used to create high-affinity human antibodies or their antigen-binding fragments.

[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 are also available and are well known in the art. Humanized antibodies, resurfacing antibodies, or similarly modified antibodies may 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 mammal. These non-human amino acid residues are often replaced by residues referred to as “import” residues, which are typically taken from “import” variable, constant, or other domains of known human sequences. Using such imported sequences, immunogenicity can be reduced, or binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other desirable properties as known in the art can be reduced, enhanced, or altered. Generally, CDR residues are directly and most substantially involved in the effect on CD73 binding. Therefore, while some or all of the non-human or human CDR sequence can be maintained, the non-human sequences in the variable and constant regions can be replaced with human or other amino acids.

[0351] Antibodies may also be, optionally, humanized antibodies, resurfacing antibodies, modified antibodies, or human antibodies that retain high affinity for the CD73 antigen and other advantageous biological properties. To achieve this goal, optionally, humanized (or human) or modified anti-CD73 antibodies and resurfacing antibodies can be prepared by analytical processes of the parent sequence and various conceptual humanized and modified products using three-dimensional models of the parent sequence, modified sequence, and humanized sequence. Three-dimensional immunoglobulin models are generally available and are well known to those skilled in the art. Computer programs are available that illustrate and display the putative three-dimensional structure of selected candidate immunoglobulin sequences. By examining these displays, it is possible to analyze the expected role of residues in the function of the candidate immunoglobulin sequence, i.e., the residues that affect the candidate immunoglobulin's ability to bind to its antigen, such as CD73. In this way, by selecting and combining framework (FW) residues from consensus and import sequences, desired antibody properties, such as increased affinity for the target antigen, can be achieved.

[0352] Humanization, resurfacing, or modification of anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments is permitted, but is 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 implemented using any known method, such as those described in the following documents: the detailed specifications; Nos. 7,635,666; Nos. 7,723,270; Nos. 7,557,189; Nos. 7,538,195; and Nos. 7,342,110; PCT / US Patent Application No. 98 / 16280; PCT / US Patent Application No. 91 / 05939; PCT / US Patent Application No. 94 / 01234; PCT / UK Patent Application No. 92 / 01755; International Publication Brochure No. 90 / 14443; International Publication Brochure No. 90 / 14424; International Publication Brochure No. 90 / 14430; and European Patent No. 229246 (each of these, including the documents cited therein, is incorporated herein by reference as a whole).

[0353] Anti-CD73 humanized antibodies and their antigen-binding fragments can also be produced in transgenic mice containing a human immunoglobulin locus capable of producing a complete repertoire of human antibodies without producing endogenous immunoglobulins during immunization. This method is described in U.S. Patents No. 5,545,807; No. 5,545,806; No. 5,569,825; No. 5,625,126; No. 5,633,425; and No. 5,661,016.

[0354] In certain embodiments, anti-CD73 antibody fragments (e.g., fragments derived from clone 10.3 antibody or clone 2C5 antibody) are provided. Various techniques for preparing antibody fragments are known. Conventionally, 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 prepared by recombinant means. Fab, Fv, and scFv antibody fragments can all be expressed in Escherichia coli (E. coli) or other host cells and secreted therefrom, thus enabling the preparation of large quantities of these fragments. Such anti-CD73 antibody fragments can also be isolated from the antibody phage libraries discussed above. Anti-CD73 antibody fragments may also be linear antibodies as described in U.S. Patent No. 5,641,870. Other techniques for producing antibody fragments, such as chemical synthesis, will be obvious to those skilled in the art.

[0355] The present disclosure allows for the adaptation of techniques for producing single-chain antibodies specific to CD73 (see, for example, U.S. Patent No. 4,946,778). In addition, it allows for the adaptation of methods for constructing Fab expression libraries that enable the rapid and effective identification of monoclonal Fab fragments, or their derivatives, fragments, analogs, or homologs having desired specificity for CD73 (see, for example, 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 an antibody molecule; (b) Fab fragments produced by reduction of disulfide crosslinks of an F(ab')2 fragment; (c) Fab fragments produced by treating an antibody molecule with papain and a reducing agent; and (d) Fv fragments.

[0356] Anti-CD733 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments 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 in a suitable region of the antibody or antibody fragment, or by incorporating the epitope into a peptide tag that is later fused to the antibody or antibody fragment at either terminal or central (e.g., by DNA or peptide synthesis), or by YTE mutation. Other methods for increasing the serum half-life of an antibody or its antigen-binding fragment, such as conjugation with heterologous molecules like PEG, are known in the art.

[0357] Heteroconjugate anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) and their antigen-binding fragments are also within the scope of this disclosure. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune cells to undesirable cells (see, for example, U.S. Patent No. 4,676,980). Heteroconjugate anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) and their antigen-binding fragments are intended to be prepared in vitro using known methods in synthetic protein chemistry, including methods involving crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutylimidate.

[0358] In certain embodiments, CD73-binding molecules disclosed herein, such as antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments, can be combined with other therapeutic agents (e.g., in combination therapy), or fused with (e.g., by gene fusion, thereby forming a fusion protein) or conjugated (e.g., chemically or enzymatically) with at least one heterologous moiety. Thus, CD73-binding molecules disclosed herein can be fused or conjugated with other therapeutic agents or toxins to form immunoconjugates and / or fusion proteins. The 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, anti-CD73 antibodies or portions thereof are derivatized in such a way that their CD73-binding ability is not adversely affected by derivatization or labeling. Accordingly, the anti-CD73 antibodies and antibody moieties of this 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 or their Cd73 binding moieties disclosed herein can be functionally linked (by chemical coupling, gene fusion, non-covalent association, etc.) to one or more other molecular entities, such as cytotoxic agents, pharmaceuticals, detection agents, and / or proteins or peptides that can mediate the association of the anti-CD73 binding molecule with another molecule (such as a streptavidin core region or polyhistidine tag).

[0359] Certain derivatized molecules can be prepared by crosslinking two or more molecular entities, for example, an anti-CD73 binding molecule disclosed herein with a therapeutic agent (e.g., a cytotoxic agent such as tubulicin or MEDI 1508). Suitable crosslinking agents include heterobifunctional agents, i.e., those having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide); or homobifunctional agents (e.g., disuccinimidyl suberate). Such crosslinking agents are available, for example, from Pierce Chemical Company, Rockford, II. Further examples of bifunctional coupling agents include N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imide esters (such as dimethyl HCl adipimidoate), 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 torylene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0360] Other types of derivatized molecules can be prepared by introducing detectable labels. Useful detection agents include fluorescent compounds (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, 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, the detectable label may have at least one spacer arm attached. The spacer arm may be of varying lengths to reduce potential steric hindrance.

[0361] The anti-CD73 binding molecules disclosed herein can also be labeled with radiolabeled amino acids. 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] Furthermore, radiolabeling can be used therapeutically as a toxin against CD73-expressing cells, such as those that induce undesirable immune responses. Examples of polypeptide labeling 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 131I is one example. In some embodiments, the anti-CD73 binding molecule can be labeled with a paramagnetic, radioactive, or fluorescent ion that can be detected during imaging. In some embodiments, the paramagnetic ions are 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 ions are 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 conjugating molecule is labeled with an X-ray contrast agent such as lanthanum(III), gold(III), lead(II), and bismuth(III). The anti-Cd73 conjugating 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 the serum half-life or tissue binding.

[0363] The term “cytotoxic agent,” as used herein, is broadly defined to refer to substances that inhibit or interfere with the function of cells and / or cause cell destruction (cell death), and / or exert antineoplastic / antiproliferative effects. For example, cytotoxic agents can directly or indirectly interfere with the development, maturation, or propagation of neoplastic tumor cells. The term also includes agents that produce only cell proliferation inhibitory effects, and not merely cytotoxic effects. The term includes chemotherapeutic agents as specified 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 part of the term "cytotoxic agent," which includes natural or synthetic chemical compounds. Examples of chemotherapeutic agents include alkylating agents, e.g., nitrogen mustard, ethyleneimine compounds, alkyl sulfonates, and other compounds with alkylating activity, e.g., nitrosoureas, cisplatin, and dacarbazine; antimetabolites, e.g., folic acid, purine, or pyrimidine antagonists; mitotic inhibitors, e.g., vinca alkaloids and podophyllotoxin derivatives; and cytotoxic antibiotics and camptothecin derivatives. Other chemotherapy agents include amifostine (ETHYOL®), cisplatin, dacarbazine (DTIC), dactinomycin, mechloretamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (Adriamycin®), doxorubicin lipo (DOXIL®), gemcitabine (GEMZAR®), daunorubicin, daunorubicin lipo (DAUNOXOME®), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (Taxol®), docetaxel (Taxotere®), aldezleukin, and asparagus. These include ginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38), gefitinib (IRESSA®), dacarbazine, phloxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon α, interferon β, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegasparagase, pentostatin, pipobromane, plicamycin, streptozocin, tamoxifen, teniposide, testactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, aromatase inhibitors, and combinations thereof.

[0365] For the purposes of this disclosure, it should be understood that a modified anti-CD73 antibody or its antigen-binding fragment may include any type of variable region that provides association between the antibody or polypeptide and CD73. In this regard, the variable region may include or be derived from any type of mammal capable of initiating a humoral response to a desired tumor-associated antigen and inducing the production of immunoglobulins. Accordingly, the variable region of a modified anti-CD73 antibody or its antigen-binding fragment may be of human, mouse, non-human primate (e.g., cynomolgus monkey, macaque, etc.), or lupine origin. In some embodiments, both the variable and constant regions of the modified anti-CD73 antibody or its antigen-binding fragment are human. In other embodiments, the variable region of a compatible antibody (usually derived from a non-human source) is modified or specifically adjusted to improve the molecular binding properties or decrease immunogenicity. In this regard, the variable region may be humanized or modified by including other introduced 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 its antigen-binding fragment are modified by at least partial substitution of one or more CDRs, and, if necessary, by partial framework region substitution and sequence changes. The CDRs may originate from antibodies of the same class or even subclass as the antibody from which the framework region originates, but it is assumed that the CDRs originate from antibodies of different classes, and in certain embodiments, from different species of antibodies. It is not necessary to replace all CDRs with the complete CDR of the donor variable region in order to transfer the antigen-binding ability of one variable domain to another. Rather, it is sufficient to transfer only the residues necessary to maintain the activity of the antigen-binding site. Given the descriptions in U.S. Patents No. 5,585,089, No. 5,693,761, and No. 5,693,762, obtaining functional antibodies with reduced immunogenicity is well within the capabilities of those skilled in the art by performing routine experiments or by trial and error.

[0367] Despite the modification of the variable region, those skilled in the art will understand that a modified anti-CD73 antibody (e.g., a modified clone 10.3 antibody or a modified clone 2C5 antibody) or its antigen-binding fragment may contain an antibody (e.g., a full-length antibody or its immunoresponsive fragment) in which one or more portions of the constant region domain are deleted or otherwise modified, and that this may result in desired biochemical properties, such as increased tumor localization or decreased serum half-life, compared to an antibody of substantially the same immunogenicity containing the natural or unmodified constant region. In some embodiments, the constant region of the modified antibody may include the human constant region. Modifications of the constant region compatible with this anti-CD73 molecule 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 may include modifications or alterations 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, a modified constant region in which one or more domains are partially or completely deleted is intended. In some embodiments, the modified antibody may include a domain deletion construct or variant (ΔCH2 construct) in which the entire CH2 domain is removed. In some embodiments, the deleted constant region domain may be replaced with a short amino acid spacer (e.g., 10 residues) that typically provides some of the molecular mobility conferred by the missing constant region.

[0368] In addition to its structure, the constant region is known in this field to mediate several effector functions. For example, when the C1 component of complement binds to an antibody, the complement system is activated. 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 the Fc region, where the Fc receptor site on the antibody Fc region binds to Fc receptors (FcRs) on the cell. There are several Fc receptors specific to different antibody classes, including IgG (γ receptor), IgE (η receptor), IgA (α receptor), and IgM (μ receptor). When antibodies bind to Fc receptors on the cell surface, several important and diverse biological responses are triggered, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental cross-transfer, and regulation of immunoglobulin production.

[0369] In certain embodiments, an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment provides an altered effector function, thus affecting the biological profile of the administered antibody or its antigen-binding fragment. For example, deletion or inactivation of 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, modification of the constant region can, in accordance with this disclosure, regulate complement binding, thus reducing the serum half-life and nonspecific association of the conjugated cytotoxin. Further modifications of the constant region can be used to remove disulfide bonds or oligosaccharide moieties, which allow for enhanced localization by increasing antigen specificity or antibody mobility. Similarly, modifications to the constant region in this disclosure can be readily fabricated using well-known biochemical or molecular engineering techniques that are well within the scope of the art.

[0370] In certain embodiments, the CD73-binding molecules disclosed herein, which are antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments, do not have one or more effector functions. For example, in some embodiments, the antibody or its antigen-binding fragment does not have antibody-dependent cell-mediated cytotoxicity (ADCC) activity and / or complement-dependent cell-mediated cytotoxicity (CDC) activity. In certain embodiments, the anti-CD73 antibody or its antigen-binding fragment does not bind to Fc receptors and / or complement factors. In certain embodiments, the antibody or its antigen-binding fragment does not have effector functions.

[0371] It should be noted that, in certain embodiments, anti-CD73 modified antibodies or their antigen-binding fragments may be modified so that the CH3 domain of each modified antibody or fragment fuses 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 domains. For example, a compatible construct can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is bound to the hinge region with a 5-20 amino acid spacer. Adding such a spacer can ensure, for example, that the regulatory element of the constant domain remains vacant and available, or that the hinge region remains mobile. However, it should be noted that in some cases the amino acid spacer may be immunogenic and induce an undesirable immune response to the construct. Therefore, in certain embodiments, any spacers added to the construct may be relatively unimmunogenic or even omitted entirely in order to maintain the desired biochemical quality of the modified antibody.

[0372] In addition to deletion of an entire constant region domain, it will be understood that the anti-CD73 antibodies and their antigen-binding fragments of this disclosure may be provided by partial deletions or substitutions of several, or even a single, amino acid. For example, a single amino acid mutation in a select range of the CH2 domain may be sufficient to substantially reduce Fc binding, thereby increasing 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 a constant region can improve selected properties of the antibody or its antigen-binding fragment (e.g., serum half-life) while leaving other desirable functions associated with that constant region domain intact. Furthermore, as suggested above, the constant region of the disclosed anti-CD73 antibodies and their antigen-binding fragments may be modified by mutations or substitutions of one or more amino acids that enhance the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by the conserved binding site (e.g., Fc binding) while substantially maintaining the composition and immunogenicity profile of the modified antibody or its antigen-binding fragment. Certain embodiments may involve the addition of one or more amino acids to a constant region to enhance desirable properties such as a decrease or increase in effector function, or to provide more cytotoxic or carbohydrate binding. In such embodiments, it may be desirable to insert or replicate a specific sequence derived from a selected constant region domain.

[0373] This disclosure also provides variants and equivalents substantially homologous to the chimeric, humanized and human anti-CD73 antibodies or their antigen-binding fragments shown herein. These may include, for example, conservative substitution mutations, i.e., the substitution of one or more amino acids with similar amino acids. For example, a conservative substitution means substituting one amino acid with another amino acid within the same general class, e.g., substituting one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. What is meant by conservative amino acid substitution is well known in the art.

[0374] Anti-CD73 antibodies or their antigen-binding fragments can be further modified to include additional chemical moieties that are not normally part of the protein. These derivatized moieties can improve the protein's solubility, biological half-life, or absorption. These moieties can also reduce or eliminate any desirable side effects of the protein. For an overview of these moieties, see Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0375] VI. Polynucleotide encoding a CD73-binding molecule In certain embodiments, the Disclosure comprises polynucleotides comprising nucleic acid sequences encoding a polypeptide or an antigen-binding fragment thereof that specifically binds to CD73. For example, the Disclosure provides polynucleotides comprising nucleic acid sequences encoding an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or an antigen-binding fragment of such antibody. The polynucleotides of the 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 a coding strand or a 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 of a mature polypeptide fused within the same reading frame to a polynucleotide (e.g., a leader sequence that functions as a secretory sequence to control the transport of polypeptides from the cell) that assists in the expression and secretion of polypeptides from a host cell. The polypeptide having the leader sequence is a preprotein and may have a leader sequence that is cleaved by the host cell to form a mature form of polypeptide. The polynucleotide may also encode a CD73-binding proprotein, which is the mature protein plus an additional 5' amino acid residue.

[0377] In certain embodiments, the polynucleotide includes the coding sequence of a mature CD73-conjugated polypeptide, such as an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody), or its antigen-binding fragment, fused within the same reading frame to a marker sequence that enables the purification of the encoded polypeptide. For example, the marker sequence may be a hexahistidine tag supplied by a pQE-9 vector in the case of a bacterial host, thereby providing the purification of the mature polypeptide fused to the marker, or the marker sequence may be a hemagglutinin (HA) tag derived from influenza hemagglutinin protein when using a mammalian host (e.g., COS-7 cells).

[0378] This disclosure also provides, for example, variants of the described polynucleotides that encode 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 may involve modifications to coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants involve modifications that result in silent substitutions, additions, or deletions, but do not alter the properties or activity of the encoded polypeptide. In some embodiments, nucleotide variants are made by silent substitutions resulting from degeneracy of the gene code. Polynucleotide variants may be made for a variety of reasons, for example, to optimize codon expression for a specific host (to alter the codons of human mRNA to those preferred by a bacterial host such as Escherichia coli). Vectors and cells containing the polynucleotides described herein are also provided.

[0380] In some embodiments, a CD73-binding molecule, such as an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or a DNA sequence encoding its antigen-binding fragment, 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 selection of codons favorable in the host cell producing the recombinant polypeptide of interest. By applying standard methods, an isolated polynucleotide sequence encoding the desired isolated polypeptide can be synthesized. For example, a reverse-translated gene can be constructed using the complete amino acid sequence. Furthermore, DNA oligomers containing nucleotide sequences encoding a specific isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary assembly.

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

[0382] In certain embodiments, a recombinant expression vector is used to amplify and express DNA encoding an anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment. The recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding the polypeptide chain of the anti-CD73 antibody and / or its antigen-binding fragment, operably linked to a suitable transcriptional or translational regulatory element derived from a mammalian, microorganism, virus, or insect gene.

[0383] A transcription unit generally comprises, as described in more detail below, (1) one or more genetic elements that play a regulatory role in gene expression, such as a transcription promoter or enhancer; (2) a structure or coding sequence that is transcribed into mRNA and translated into a protein; and (3) appropriate transcription and translation start and termination sequences. Such regulatory elements may include operator sequences to control transcription. Selective genes that promote host replication ability, generally conferred by the origin of replication, and transformant recognition may be further incorporated. DNA regions are operably linked when they are functionally related to each other. For example, the DNA of a signal peptide (secretion leader) is operably linked to the DNA of that polypeptide if it is expressed as a precursor involved in the secretion of the 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 located in a position that enables translation. Structural elements intended for use in yeast expression systems include leader sequences that enable extracellular secretion of translated proteins by host cells. Alternatively, if a recombinant protein is expressed without a leader or transport sequence, it may contain an N-terminal methionine residue. This residue can be optionally cleaved from a subsequently expressed recombinant protein to provide the final product.

[0384] The selection of expression regulatory sequences and expression vectors will depend on the host selection. A wide variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as E. coli plasmids containing pCR 1, pBR322, pMB9, and their derivatives, as well as plasmids with a broader host range, such as M13 and filamentous single-stranded DNA phages.

[0385] Suitable host cells for the expression of CD73-binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments, include prokaryotes, yeasts, insects, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli (E. coli) or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems can also be used. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985) (this relevant disclosure is incorporated herein by reference). For further information regarding methods for producing proteins, including antibodies, see, for example, U.S. Patent Publication No. 2008 / 0187954, U.S. Patent No. 6,413,746, U.S. Patent No. 6,660,501, and U.S. Patent No. 7,932,087 (each of which is incorporated herein by reference as a whole).

[0386] Various mammalian or insect cell culture systems can also be advantageously used for the expression of recombinant CD73-binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments. Recombinant protein expression may also be carried out in mammalian cells 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 strain 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 include non-transcription elements, such as origins of replication, suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcription sequences, and 5' or 3' untranslated sequences, such as essential ribosome binding sites, polyadenylation sites, splice donor / receptor sites, and transcription termination sequences. Baculovirus systems for heterologous protein production in insect cells have been reviewed by Luckow and Summers, BioTechnology 6:47 (1988).

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

[0389] For example, the supernatant from a system that secretes recombinant protein into culture medium can first be concentrated using a commercially available protein concentration filter, such as an Amicon® or Millipore Pellicon® ultrafiltration unit. After this concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate having pendant diethylaminoethyl (DEAE) groups, may be used. The matrix may be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step may be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, the CD73-binding molecule (e.g., clone 10.3 antibody or clone 2C5 antibody) can be further purified using one or more reversed-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel having pendant methyl groups or other aliphatic groups. Some or all of the above purification steps can be used in various combinations to provide a homogeneous recombinant protein.

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

[0391] Other methods known in the art for purifying antibodies and other proteins include, for example, those described in U.S. Patent Publication No. 2008 / 0312425, No. 2008 / 0177048, and No. 2009 / 0187005 (each of which is incorporated herein by reference as a whole).

[0392] In certain embodiments, the CD73-binding molecule is a non-antibody polypeptide. Various methods for identifying and producing non-antibody polypeptides that bind to protein targets with high affinity are known in the art. See, for example, Skerra, Curr. Opin. Biotechnol. 18:295-304 (2007), Hosse et al., Protein Science, 15:14-27 (2006), Gill et al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J., 275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008) (each of these is incorporated herein by reference as a whole). In certain embodiments, CD73-binding polypeptides can be identified / produced using phage display technology. In certain embodiments, 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. Therapeutic methods using therapeutic anti-CD73 antibodies This disclosure provides methods for treating patients with diseases associated with CD73 expression or CD73-expressing cells, such as cancer, by using anti-CD73 binding molecules, such as antibodies, such as antigen-binding fragments, variants, and derivatives thereof (e.g., clone 10.3 antibody or clone 2C5 antibody). In some specific embodiments, such cancers include lung cancer, breast cancer, ovarian cancer, colorectal cancer, bladder cancer, pancreatic cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, lung-colon cancer, and lymphoma.

[0394] "CD73-expressing cells" refers to cells that express CD73. CD73 may be membrane-bound via glycosylphosphatidylinositol anchoring, or it may exist as a soluble protein. Methods for detecting CD73 expression in cells and other suitable samples are well known in the art and are not limited to, but include immunohistochemistry, flow cytometry, Western blotting, and ELISA.

[0395] The following discussion will refer to diagnostic and therapeutic methods for various diseases and disorders using the CD73-binding molecules of this disclosure (e.g., clone 10.3 antibody or clone 2C5 antibody), but 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, for example, having the ability to specifically bind to CD73 and neutralize its 5'-nucleotidase activity. In some embodiments, the CD73-binding molecule is a non-human ADCC-mediated human or humanized antibody, or an anti-CD73 antibody modified not to mediate ADCC.

[0396] In some embodiments, the CD73-binding molecule is a CD730010 antibody or its antigen-binding fragment, a clone 10.3 antibody or its antigen-binding fragment, a CD730002 antibody or its antigen-binding fragment, a clone 2C5 antibody or its antigen-binding fragment, or a CD73004 antibody or its antigen-binding fragment. 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 another embodiment, the CD73-binding molecule is a clone 2C5 YTE mutant antibody.

[0397] In one embodiment, the treatment includes the application or administration of an anti-CD73 conjugating molecule of the Disclosure, e.g., an antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment, variant, or derivative, to a subject or patient, or the application or administration of an anti-CD73 conjugating molecule to an isolated tissue or cell line derived from a subject or patient, wherein the subject or patient has a disease, symptoms of a disease, or a predisposition to a disease. In another embodiment, the treatment is also intended to include the application or administration of a pharmaceutical composition comprising an anti-CD73 conjugating molecule of the Disclosure, e.g., an antibody or its antigen-binding fragment, variant, or derivative, to a subject or patient having a disease, symptoms of a disease, or a predisposition to a disease, or the application or administration of a pharmaceutical composition comprising an anti-CD73 conjugating molecule to an isolated tissue or cell line derived from a subject or patient.

[0398] The anti-CD73 conjugating molecules of this disclosure, such as antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments, variants, or derivatives, are useful for the treatment of various cancers. In one aspect, this disclosure provides anti-CD73 conjugating molecules, such as antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments, variants, or derivatives, for use as pharmaceuticals, and more specifically, for use in the treatment or prevention of cancer (e.g., colon cancer, melanoma, breast cancer, lymphoma, or non-small cell lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer). In some aspects, the cancer exhibits a premetastatic phenotype. In some aspects, the cancer exhibiting a premetastatic phenotype is melanoma or breast cancer. In some aspects, the cancer is metastatic cancer. In some aspects, the anti-CD73 conjugating molecules disclosed herein can induce adaptive antitumor 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 this disclosure promote a favorable therapeutic response to cancer using at least one anti-CD73 conjugating molecule, e.g., an antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) or its antigen-binding fragment, variant, or derivative, as defined in other parts of this specification. The term “favorable therapeutic response” to cancer treatment refers to improvement of the disease and / or improvement of disease-related symptoms associated with the activity of these anti-CD73 conjugating molecules, e.g., an antibody or its antigen-binding fragment, variant, or derivative. Thus, for example, improvement of the disease may be characterized as complete remission. “Complete remission” is intended to mean that there is no clinically detectable disease when any past test results are standardized. Alternatively, improvement of the disease may be classified as partial remission. “Fair therapeutic response” includes reduction or inhibition of cancer progression and / or duration, reduction or improvement of cancer severity, and / or improvement of one or more of its symptoms, obtained by administration of the anti-CD73 conjugating molecules disclosed herein.

[0400] In specific embodiments, such terms refer to one, two, three or more outcomes following administration of an anti-CD73 binding molecule disclosed herein: (1) stabilization, reduction or disappearance of cancer cell populations; (2) stabilization or reduction of cancer growth; (3) impaired cancer formation; (4) eradication, removal or control of primary, local and / or metastatic cancers; (5) reduced mortality; (6) increased disease-free, recurrence-free, progression-free, and / or overall survival or ratio; (7) increased response rate, duration of response, or number of responsive or remission patients; (8) reduced hospitalization rate; (9) reduced length of hospital stay; (10) preserved and non-increasing tumor size, or increase of less than 10%, preferably less than 5%, preferably less than 4%, preferably less than 2%; and (12) increased number of patients in remission.

[0401] Clinical responses can be evaluated using screening techniques such as magnetic resonance imaging (MRI) scans, X-ray imaging, computed tomography (CT) scans, flow cytometry or fluorescence-activated cell sorting (FACS) analysis, histology, macroscopic pathology, and blood chemistry, including, but not limited to, changes detectable by ELISA, RIA, chromatography, etc. In addition to these favorable therapeutic responses, subjects receiving therapy with anti-CD73 binding molecules, such as antibodies or their antigen-binding fragments, variants, or derivatives, may achieve beneficial effects in improving disease-related symptoms.

[0402] The anti-CD73 binding molecules disclosed herein, such as antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments, variants, or derivatives, can be used in combination with any known agent or combination of agents that has been used or is currently used to treat any known cancer therapies, e.g., cancers (e.g., colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer). The second agent or combination of agents in the drug combination formulation or administration regimen preferably has complementary activity so as not to adversely affect each other with the antibody or polypeptide of the disclosure.

[0403] Anticancer drugs include medications used to treat malignant lesions such as cancerous growth. Drug therapy can be used alone or in combination with other treatments such as surgery or radiation therapy. In cancer treatment, several classes of drugs may be used 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 androgens, which are male hormones. Anticancer agents used in the particular methods of this disclosure include, in particular, antibodies (e.g., antibodies that bind to IGF-1R, antibodies that bind to EGFR, antibodies that bind to Her2, or antibodies that bind to 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, immunotherapies, hormone therapies, glucocorticoids, aromatase inhibitors, mTOR inhibitors, chemotherapeutic agents, 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, such as antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments, can be administered in combination with antibodies or antibody fragments that target, for example, PD-1 (programmed death 1 protein), its two ligands PD-L1 (programmed death ligand 1) and / or PD-L2, or CTLA-4 (cytotoxic T lymphocyte antigen 4 protein). See, for example, Stagg et al. PNAS 107:1547-1552 (2010); Jin et al. Cancer Res. 70(6): (2010); Allard et al. Clin. Cancer Res. 19:5626 (2013) (these are incorporated herein by reference as a whole). In some embodiments, the anti-CTLA-4 antibody is ipilimumab or its antigen-binding fragment. In other embodiments, the anti-CTLA-4 antibody is tremelimumab (tisilimumbab, CP-675, 206) or its antigen-binding fragment. In some embodiments, the anti-PD-1 antibody is pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475) or its antigen-binding fragment. In some embodiments, the anti-PD-1 antibody is nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or its antigen-binding fragment. In some embodiments, the anti-PD-L1 antibody is BMS-936559 or its antigen-binding fragment. 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 its antigen-binding fragment. In various embodiments, the anti-PD-L1 antibody is MEDI4736 or its antigen-binding fragment.

[0405] In some embodiments, the CD73-binding molecules 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 concentrations of approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, or approximately 20 mg / kg. In some embodiments, the CD73-binding molecules 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, where the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody is administered at concentrations of approximately 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg. In some embodiments, the anti-CD73 antibody is administered in a ratio of approximately 1:1, 1:2, 1:3, or 1:4 with the anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA4 antibody. In some embodiments, the anti-CD73 antibody is administered in a ratio of approximately 1:2 with the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody. In specific embodiments, the concentration of the anti-CD73 antibody (e.g., clone 10.3 antibody or clone 2C5 antibody) is approximately 10 mg / kg, and the concentration of the anti-PD-1 antibody is approximately 20 mg / kg. In some embodiments, the CD73-binding molecule disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody) can be administered in combination with the anti-PD-1 antibody.In some embodiments, administration of 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 approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to untreated subjects or subjects treated with monotherapy (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody without an anti-CD73 antibody). In some embodiments, administration of 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 approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to untreated subjects or subjects treated with monotherapy (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody without an anti-CD73 antibody).

[0406] Where combination therapy involves the administration of an anti-CD73 conjugating molecule in combination with the administration of another therapeutic agent (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody), the methods disclosed herein include co-administration using separate formulations or a single pharmaceutical formulation, and sequential administration in any order. In some embodiments, the anti-CD73 antibody described herein (e.g., clone 10.3 antibody or clone 2C5 antibody) is administered in combination with another drug, where the antibody or its antigen-binding fragment, variant, or derivative and the therapeutic agent may be administered sequentially in any order, or simultaneously (i.e., at the same time or within the same timeframe).

[0407] Combination therapy can provide a "synergistic effect," meaning that the effect achieved when the active ingredients are used together may be greater than the sum of the effects obtained when those compounds are used individually. Synergistic effects can be achieved when the active ingredients are (1) co-formulated and administered, or delivered simultaneously in combined unit dose formulations; (2) delivered alternately or in parallel as separate formulations; or (3) by any other regimen. When delivered in alternating dosing therapy, synergistic effects can be achieved when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, in alternating dosing therapy, the effective dose of each active ingredient is administered sequentially, i.e., consecutively, whereas in combination therapy, the effective doses of two or more active ingredients are administered together.

[0408] In other embodiments, the CD73-conjugating molecules disclosed herein (e.g., clone 10.3 antibody or clone 2C5 antibody) may be administered in combination with a tyrosine kinase inhibitor. In any other specific embodiment, the CD73-conjugating molecules disclosed herein may be administered in combination with an inhibitor of EGFR and / or HER2 / neu-related tyrosine kinase activity, such as lapatinib. In some embodiments, the CD73-conjugating molecules disclosed herein may be administered in combination with an antimitotic agent. In some specific embodiments, the CD73-conjugating molecules disclosed herein may be administered in combination with an agent that stabilizes spindle microtubule assembly, such as paclitaxel or docetaxel. Further embodiments involve the use of anti-CD73-conjugating molecules, such as antibodies or their antigen-binding fragments, variants, or derivatives (e.g., clone 10.3 antibody or clone 2C5 antibody) for diagnostic monitoring of tissue protein levels as part of a clinical trial procedure, thereby determining, for example, the effectiveness of a given treatment regimen. Detection may be enhanced, for example, by coupling the antibody with a detectable substance.

[0409] Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Suitable enzyme examples include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; suitable fluorescent substances include umbelliferone, fluorescein, isothiocyanate fluorescein, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; a luminescent substance example is luminol; bioluminescent substances include luciferase, luciferin, and aequorin; and suitable radioactive substances include, 125 I, 131 I, 35 S, or 3 H can be mentioned.

[0410] VIII. Combination and co-therapy with anti-CD73 antibodies This disclosure provides a method for treating patients with cancer (including colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer) using a therapeutic combination comprising an anti-CD73 binding molecule, such as an antibody, such as its antigen-binding fragment, variant, and derivative (e.g., clone 10.3 antibody or clone 2C5 antibody).

[0411] The following discussion refers to therapeutic combinations featuring the CD73-binding molecules of this disclosure (e.g., clone 10.3 antibody or clone 2C5 antibody), but 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, for example, having the ability to specifically bind to CD73 and neutralize its 5'-nucleotidase activity. In some embodiments, the CD73-binding molecule is a non-human ADCC-mediated human or humanized antibody, or an anti-CD73 antibody modified not to mediate ADCC.

[0412] Treatment of patients with solid tumors using combinations of the present invention, such as anti-PD-1, anti-PD-L1, or anti-CTLA4 antibodies, or anti-CD73 antibodies or their antigen-binding fragments in combination, 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 effect of monotherapy (for example, the combination of an anti-CD73 antibody (e.g., MEDI9447) with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody).

[0413] The synergistic effect of combining therapies (for example, the synergistic effect of combining an anti-CD73 antibody (e.g., MEDI9447) with an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody) allows for the use of lower doses and / or less frequent administration of one or more therapeutic agents in patients with solid tumors. Because lower doses and / or less frequent administration of therapies are possible, the toxicity associated with the administration of therapies to the subject is reduced without compromising the efficacy of therapies in treating solid tumors. In addition, the synergistic effect may enhance the efficacy of the therapeutic agents in the management, treatment, or improvement of solid tumors. The synergistic effect of combining therapeutic agents may avoid or reduce the harmful or undesirable side effects associated with the use of any single therapy.

[0414] In co-therapy, the combination of an anti-CD73 antibody (e.g., MEDI9447) or its antigen-binding fragment and an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or its antigen-binding fragment may be included in the same pharmaceutical composition or in separate pharmaceutical compositions, at the discretion of the parties. In the latter case, the pharmaceutical composition containing the anti-CD73 antibody (e.g., MEDI9447) or its antigen-binding fragment is suitable for administration before, concurrently with, or after administration of the pharmaceutical composition containing the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody or its antigen-binding fragment. In some cases, the anti-CD73 antibody (e.g., MEDI9447) or its antigen-binding fragment may be administered in separate compositions at overlapping times with the anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody.

[0415] Anti-CD73 antibodies (e.g., MEDI9447) or their antigen-binding fragments, along with anti-PD-1, anti-PD-L1, or anti-CTLA4 antibodies or their antigen-binding fragments, may be administered only once or at infrequently while still providing benefits to the patient. In a further embodiment, the patient may receive additional follow-on doses. Follow-on doses may be administered at various time intervals depending on other factors, including the patient's age, weight, clinical assessment, tumor burden, and / or the physician's judgment.

[0416] The methods provided herein can reduce or delay tumor growth. In some embodiments, this reduction or delay may be statistically significant. The reduction in tumor growth can be measured by comparison to the patient's tumor growth at baseline, to predicted tumor growth, to predicted tumor growth based on a large patient population, or 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 in treating tumors. B7-H1, also known as PD-L1, is a type I transmembrane protein of approximately 53 kDa size. In humans, B7-H1 is expressed in several immune cell types, such as activated and anerious / depleted T cells, naive and activated B cells, as well as bone marrow dendritic cells (DCs), monocytes, and mast cells. B7-H1 is also expressed in non-immune cells, including pancreatic islets, hepatic Kupffer cells, vascular endothelium, and certain epitheliums, such as airway epithelium and renal tubular epithelium, and its expression is enhanced during inflammatory episodes. B7-H1 expression is also found at high 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), 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 initially identified in T cell lines that induce activation-induced apoptosis. PD-1 is expressed on activated T cells, B cells, and monocytes, as well as other immune system cells, and binds to both B7-H1 (PD-L1) and its associated B7-DC (PD-L2). The second is B7-1, a member of the B7 family, which is expressed on activated T cells, B cells, monocytes, and antigen-presenting cells.

[0419] Signaling via the PD-1 / B7-H1 axis is thought to play a crucial and 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 driving the depletion and anergy of tumor-infiltrating T lymphocytes, stimulating the secretion of immunosuppressive cytokines into the tumor microenvironment, stimulating inhibitory regulatory T cell function, and protecting 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 the binding of B7-H1 to PD-1 and CD80 receptors. MEDI4736 can reduce B7-H1-mediated suppression of human T cell activation in vitro and inhibit tumor growth via T cell-dependent mechanisms in xenograft models. Other possible agents include those that inhibit PD-L1 and / or PD-1 (e.g., AB).

[0421] Information regarding MEDI4736 (or its fragments) used in the methods provided herein can be found in U.S. Patent Application Publication No. 20130034559 / U.S. Patent No. 8779108 and U.S. Patent Application Publication No. 20140356353 (each of which disclosures is incorporated herein by reference). The crystallizable fragment (Fc) domain of MEDI4736 contains a triple mutation in the constant domain of the IgG1 heavy chain that reduces the binding of complement component C1q and the Fcγ receptor, which are involved in mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0422] MEDI4736 and its antigen-binding fragment used in the methods provided herein include 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 its antigen-binding fragment used in the methods provided herein includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 130 and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 131. In a specific embodiment, MEDI4736 or its antigen-binding fragment used in the methods provided herein includes a heavy chain variable region and a light chain variable region, where the heavy chain variable region includes the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 132-134, and the light chain variable region includes the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 135-137. Those skilled in the art will readily be able to identify the Chothia definition, the Abm definition, or other CDR definitions known to those skilled in the art. In a particular embodiment, MEDI4736 or its antigen-binding fragment used in the method provided herein comprises a variable heavy chain and variable light chain CDR sequence of a 2.14H9OPT antibody as disclosed in U.S. Patent Application Publication No. 20130034559 / U.S. Patent No. 8779108 and U.S. Patent Application Publication No. 20140356353 (each of which disclosures is incorporated herein by reference as a whole).

[0423] X. Anti-CTLA4 antibody Accordingly, in one embodiment, the therapeutic combination of the present invention includes a CTLA4 blocking antibody (e.g., tremelimumab) and / or an antibody that reduces PD1 / PD-L1 interaction. Two T cell regulatory pathways that have attracted considerable attention to date transmit signals via cytotoxic T lymphocyte 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, suppressing the T cell response after CD28-mediated T cell activation. CTLA4 is thought to be part of a central inhibitory pathway that modulates the amplitude of initial activation of naive and memory T cells after TCR association, influencing both anti-tumor and autoimmune responses. CTLA4 is primarily expressed on T cells, and the expression of its ligands, CD80 (B71) and CD86 (B7.2), is generally limited to antigen-presenting cells, T cells, and other immune-mediated cells. 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 at that time, it did not show a significant increase in overall survival compared to standard treatment (temozolomide or dacarbazine).

[0425] Information regarding tremelimumab (or its antigen-binding fragment) used in the methods provided herein can be found in U.S. Patent No. 6,682,736 (referred to herein as section 11.2.1) (this disclosure is incorporated herein by reference in its entirety). Tremelimumab (also known as CP-675,206, CP-675, CP-675206, and tisilimubab) 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 induce immune activation in vitro, and some patients treated with tremelimumab have shown tumor regression.

[0426] Tremelimumab used in the methods provided herein includes a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. In a specific embodiment, tremelimumab or its antigen-binding fragment used in the methods provided herein includes a light chain variable region and a heavy chain variable region. In a specific embodiment, tremelimumab or its antigen-binding fragment used in the methods provided herein includes a heavy chain variable region and a light chain variable region as specified herein. Those skilled in the art will readily be able to identify the Chothia definition, the Abm definition or other CDR definitions known to those skilled in the art. In a specific embodiment, tremelimumab or its antigen-binding fragment used in the methods provided herein includes the variable heavy chain and variable light chain CDR sequences of an antibody as disclosed in U.S. Patent No. 6,682,736 (which is incorporated herein by reference in whole).

[0427] XI.VII. Pharmaceutical Compositions and Methods of Administration Methods for preparing anti-CD73 conjugating molecules, such as antibodies, or their antigen-binding fragments, variants, or derivatives (e.g., clone 10.3 antibody or clone 2C5 antibody), and administering them to subjects in need are well known to those skilled in the art or readily determined by those skilled in the art. The routes of administration of anti-CD73 conjugating molecules, such as antibodies, or their antigen-binding fragments, variants, or derivatives, may be, for example, oral, parenteral, inhalation, or topical. Parenteral administration, as used herein, includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. However, other methods conforming to the teachings herein may be used to directly deliver the anti-CD73 conjugating molecules of this disclosure, such as antibodies, or their antigen-binding fragments, variants, or derivatives, to the site of a harmful cell population, thereby increasing the exposure of affected tissue to the therapeutic agent.

[0428] As discussed herein, the anti-CD73 binding molecules of this disclosure, such as antibodies, or their antigen-binding fragments, variants, or derivatives (e.g., clone 10.3 antibody or clone 2C5 antibody), can be administered in pharmaceutically effective amounts for in vivo treatment of CD73-expressing cell-mediated diseases, such as certain types of cancer.

[0429] Methods for preparing therapeutic combinations comprising anti-PD-1, anti-PD-L1, and / or anti-CTLA4 antibodies, or anti-CD73-binding molecules in combination with their antigen-binding fragments, such as antibodies, or their antigen-binding fragments, variants, or derivatives (e.g., clone 10.3 antibody or clone 2C5 antibody), and administering them to subjects in need are well known to those skilled in the art or readily determined by those skilled in the art. The routes of administration of these combinations may be, for example, oral, parenteral, inhalation, or topical. Parenteral administration as used herein includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. However, other methods conforming to the teachings herein may be used to directly deliver the combinations of this disclosure to the site of a harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent. As discussed herein, the combination of an anti-CD73 antibody (e.g., MEDI9447) with anti-PD-1, anti-PD-L1, and / or anti-CTLA4 antibodies can be administered in pharmaceutically effective doses for in vivo treatment of CD73-expressing cell-mediated diseases, such as certain types of cancer.

[0430] The pharmaceutical compositions used in this disclosure may include, for example, water, ion exchangers, proteins, buffers, and salts, and may also include a pharmaceutically acceptable carrier. Preservatives and other additives may also be present. The carrier may be a solvent or a dispersion medium. Formulations suitable for use in the therapies disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[0431] In any case, the therapeutic combination of the present invention, the required amount of the active compound (for example, an anti-CD73 antibody, or its antigen-binding fragment, variant, or derivative, such as clone 10.3 antibody or clone 2C5 antibody, alone or in combination with other active agents) can be combined in a suitable solvent and subsequently filtered sterile to prepare a sterile injectable solution. Furthermore, such preparations can be packaged and sold in the form of kits. Such products may have labels or accompanying documents indicating that the relevant compositions are useful in treating subjects suffering from or susceptible to a disease or disorder.

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

[0433] This composition may be administered as a single dose, multiple doses, or over a specified period in an infusion. The dosage regimen may also be adjusted to obtain the optimal desired response (e.g., therapeutic or prophylactic response).

[0434] The therapeutically effective dose of the compositions of this disclosure for the treatment of certain cancers, including those mediated by CD73-expressing cells, such as colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer, varies depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, other drug therapies being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can also be treated. The therapeutic dose can be titrated to optimize safety and efficacy using routine methods known to those skilled in the art.

[0435] The dosage of at least one anti-CD73 binding molecule of the present invention, e.g., an antibody or its binding fragment, variant, or derivative (e.g., clone 10.3 antibody or clone 2C5 antibody), or a therapeutic combination, can be readily determined by those skilled in the art without excessive experimentation, given the disclosures of this invention. Factors influencing the method of administration and the respective amounts of the at least one anti-CD73 binding molecule of the present invention, e.g., an antibody, its antigen-binding fragment, variant, or derivative, 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 the anti-CD73 binding molecule of the present invention, e.g., an antibody, its fragment, variant, or derivative, or a therapeutic combination, will depend on the method of administration and whether the subject receives a single dose or multiple doses of the drug.

[0436] This disclosure also provides the use of the anti-CD73 binding molecule of the present invention, such as an antibody or its antigen-binding fragment, variant, or derivative (e.g., clone 10.3 antibody or clone 2C5 antibody), or therapeutic combinations in the manufacture of pharmaceuticals for the treatment of certain cancers, including, for example, colon cancer, melanoma, breast cancer, lymphoma, non-small cell lung cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, and Burkitt lymphoma, ovarian cancer, breast cancer, head and neck cancer, and pancreatic cancer.

[0437] This disclosure also provides the use of anti-CD73 conjugating molecules, such as antibodies, or their antigen-binding fragments, variants, or derivatives (e.g., clone 10.3 antibody or clone 2C5 antibody) in the manufacture of a therapeutic pharmacopoeia for treating certain types of cancer. In certain embodiments, the pharmacopoeia is used in a subject that has been previously treated with at least one other therapy.

[0438] "Priorly treated" or "prior treatment" is intended to mean that the subject has received one or more other therapies (e.g., treatment with at least one other anticancer therapy) prior to receiving a drug containing an anti-CD73 binding molecule, e.g., an antibody or its antigen-binding fragment, variant, or derivative (e.g., clone 10.3 antibody or clone 2C5 antibody). The subject does not need to have been a responder to the preceding one or more therapies. Therefore, a subject receiving a drug containing an anti-CD73 binding molecule, e.g., an antibody or its antigen-binding fragment, variant, or derivative may or may not have responded to the preceding therapy, or, if the prior treatment included multiple therapies, to one or more of the preceding therapies.

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

[0440] VIII. Diagnosis This disclosure further provides a diagnostic method useful in diagnosing CD73-expressing cell-mediated diseases, such as certain types of cancer, which includes measuring the expression level of the CD73 protein in tissues or other cells or body fluids from an individual, and comparing the measured expression level to a standard CD73 expression level in normal tissue or body fluid, where an increase in the expression level compared to the standard serves as an indicator of the disorder.

[0441] The anti-CD73 antibodies disclosed herein, as well as their antigen-binding fragments, variants, and derivatives (e.g., clone 10.3 antibody or clone 2C5 antibody), can be used in assays of CD73 protein levels in biological samples using classical immunohistological methods known to those skilled in the art (see, for example, 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. Preferred assays are described in more detail in other parts of this specification.

[0442] "Assassinating the expression level of CD73 polypeptide" means to qualitatively or quantitatively measure or estimate the level of CD73 polypeptide in a first biological sample, either directly (e.g., by determining or estimating the absolute protein level) or relatively (e.g., by comparing it to the level of disease-related polypeptide in a second biological sample). The CD73 polypeptide expression level in the first biological sample can be measured or estimated and compared to a standard CD73 polypeptide level, which is determined either from a second biological sample obtained from a non-disabled individual or by averaging the levels of a non-disabled population. As will be understood in the art, once a "standard" CD73 polypeptide level is known, it can be repeatedly used as a standard for comparison.

[0443] "Biological sample" refers to 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 bodily fluids from mammals are well known in the art.

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

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

[0446] CD73-binding molecules, such as anti-CD73 antibodies or their antigen-binding fragments, and their variants or derivatives (e.g., clone 10.3 antibody or clone 2C5 antibody), can be histologically used 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 taking a tissue sample from a patient and applying a labeled CD73-binding molecule (e.g., antibody or fragment) to the biological sample, preferably by overlaying it with the labeled CD73-binding molecule, such as an anti-CD73 antibody or its antigen-binding fragment, its variant, or derivative, to the sample. Using such a procedure, it is possible to determine not only the presence of CD73 or its conserved variant or peptide fragment, but also its distribution in the test tissue. By using this disclosure, those skilled in the art will readily understand that any of the various histological methods (such as staining procedures) can be modified to achieve such in-situ detection.

[0447] The binding activity of a given lot of CD73-binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody), their antigen-binding fragments, variants, or derivatives, can be determined by well-known methods. Those skilled in the art will be able to determine effective and optimal assay conditions for each determination using routine experiments.

[0448] Suitable methods and reagents for determining the binding properties of isolated CD73-binding molecules, such as anti-CD73 antibodies (e.g., clone 10.3 antibody or clone 2C5 antibody) or their antigen-binding fragments, their variants, or modified / mutated derivatives, are known and / or commercially available in the art. Instruments and software designed for such kinetic analysis are commercially available (e.g., BIAcore, BIAevaluation software, GE Healthcare; KinExa software, Sapidyne Instruments).

[0449] Unless otherwise indicated, the implementation of this disclosure will utilize prior arts in cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art in those fields. Such techniques are fully described in the literature. For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DNGlover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. Synthesis; Mullis et al. U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press)(1986);Perbal(1984)A Practical Guide To Molecular Cloning;the treatise,Methods In Enzymology(Academic Press,Inc.,NY);Miller and Calos eds.(1987)Gene Transfer Vectors For Mammalian Cells,(Cold Spring Harbor Laboratory);Wu et al.,eds.,Methods In Enzymology,Vols.154 and 155;Mayer and Walker,eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). See also.

[0450] The general principles of antibody engineering are described in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). The general principles of protein engineering are described in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). The general principles of antibodies and antibody-hapten binding are described in Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.) and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, NY). In addition, standard immunological methods known in the relevant technical field, but not specifically described, are commonly used, as can be seen in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (WH Freeman and Co., NY).

[0451] Standard references describing the general principles of immunology include: Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) “Monoclonal Antibody Technology” in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) Kuby Immunology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al.(2005)Cellular and Molecular Immunology(5th ed.;Elsevier Health Sciences Division);Kontermann and Dubel(2001)Antibody Engineering(Springer Verlan);Sambrook and Russell(2001)Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Press);Lewin(2003)Genes VIII(Prentice Hall2003);Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[0452] All references cited above, as well as all references cited herein, are incorporated herein by reference in their entirety.

[0453] The following examples are provided as illustrations, not as limitations. [Examples]

[0454] The aspects of this disclosure may be further defined by reference to the following non-limiting examples, which describe in detail the preparation of the particular antibodies of this disclosure and the methods of using the antibodies of this disclosure. It will be apparent to those skilled in the art that many modifications can be made to both materials and methods without departing from the scope of this disclosure.

[0455] CD73 (differentiation cluster 73), also known as ecto-5'-nucleotidase (NT5E), is a transmembrane receptor found in tumor cells as well as normal stromal cells, such as endothelial cells and certain types of leukocytes. CD73 catalyzes the conversion of adenosine monophosphate to adenosine and organophosphates. Binding of the extracellular portion of the adenosine receptor transmits signals via cyclic AMP, inhibiting T cell receptor activation (reviewed by Linden and Cekic, 2012). CD73 is thought to play a role in mediating the inhibitory function of regulatory B and T lymphocytes (Saze et al, 2013) and in maintaining endothelial integrity (reviewed by Jalkanen and Salmi, 2008).

[0456] ...

Claims

1. Antibody V Ｌ An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, including the V Ｌ However, the amino acid sequence: []. １ ]SGSLSNIGRNX 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 This represents the framework domain, 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 An isolated and bound molecule or its antigen-binding fragment containing the amino acid residue leucine (L) or threonine (T).

2. FW １ if sequence number 25 or 26 is included, FW ２ This includes sequence number 27 or 28, FW ３ This includes sequence number 29, and FW ４ The isolated binding molecule or antigen-binding fragment thereof according to claim 1, wherein the molecule contains SEQ ID NO:

30.

3. An isolated binding molecule containing antibody VH that specifically binds to CD73 or an antigen-binding fragment thereof, wherein V Ｈ However, 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 area, where, 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 An isolated binding molecule or its antigen-binding fragment containing the amino acid residue tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

4. FW ５ This includes sequence number 31, FW ６ This includes sequence number 32, FW ７ This includes sequence number 33, and FW ８ The isolated binding molecule or antigen-binding fragment thereof according to claim 3, wherein the molecule contains SEQ ID NO:

34.

5. Antibody V Ｌ and antibody V Ｈ An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, including the V Ｌ However, the amino acid sequence: []. １ ]SGSLSNIGRNX 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 This represents the framework domain, and here, 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 includes an amino acid residue leucine (L) or threonine (T), and the V Ｈ has an 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 area, where, 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 An isolated binding molecule or its antigen-binding fragment containing the amino acid residue tyrosine (Y), lysine (K), methionine (M), leucine (L), or glutamic acid (E).

6. FW １ if sequence number 25 or 26 is included, FW ２ This includes sequence number 27 or 28, FW ３ This includes sequence number 29, FW ４ This includes sequence number 30, FW ５ This includes sequence number 31, FW ６ This includes sequence number 32, FW ７ This includes sequence number 33, and FW ８ The isolated binding molecule or antigen-binding fragment thereof according to claim 5, wherein the molecule contains SEQ ID NO:

34.

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

8. Antibody V Ｌ An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, including the V Ｌ However, each contains an isolated binding molecule or antigen-binding fragment thereof, which includes the amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3, which 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, or identical to, SEQ ID NOs: 46, 52, 2, or 1 in one or more VL-CDRs.

9. Antibody V Ｈ An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, including the V Ｈ However, each contains an isolated binding molecule or antigen-binding fragment thereof, which includes the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3, which 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, or identical to, SEQ ID NOs: 36, 40, and 45, except for 4, 3, 2, or 1 amino acid substitution in one or more VH-CDRs.

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

11. Sequence numbers 46, 49, 53, 35, 37, and 41; Sequence numbers 47, 49, 53, 35, 37, and 41; Sequence numbers 47, 49, 54, 36, 37, and 42; Sequence numbers 46, 50, 54, 36, 38, and 43; Sequence numbers 46, 51, 55, 36, 39, and 44; Sequence numbers 48, 52, 54, 36, 40, and 44; Sequence numbers 46, 49, 56, 35, 37, and 41; Sequence numbers 46, 49, 53, 35, 37, and 45; Sequence numbers 47, 49, 56, 36, 37, and V containing the amino acid sequences of VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3, which are identical to or identical to sequence numbers 46, 50, 56, 36, 38, and 45, or identical to them except for 4, 3, 2, or 1 amino acid substitution in one or more CDRs; V45; VL-CDR1, VL-CRD2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3; V45 Ｌ and V Ｈ An isolated antibody or its antigen-binding fragment that specifically binds to CD73, including [the specified component].

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

13. V containing sequence number 68 Ｌ and V including sequence number 82 Ｈ An isolated antibody or its antigen-binding fragment, comprising the above.

14. Essentially V from Sequence ID 68 Ｌ V essentially derived from sequence number 82. Ｈ An isolated antibody or its antigen-binding fragment, comprising the above.

15. V consisting of sequence number 68 Ｌ V consisting of sequence number 82 Ｈ An isolated antibody or its antigen-binding fragment, comprising the above.

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

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

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

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

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

21. 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 the cells according to claim 20, and (b) isolating the antibody or antigen-binding fragment thereof.

22. An isolated binding molecule or antigen-binding fragment thereof according to claim 7, which is an antagonist of CD73.

23. An isolated binding molecule or antigen-binding fragment thereof according to claim 10, which is an antagonist of CD73.

24. An isolated antibody or antigen-binding fragment thereof according to any one of claims 11 to 15, which is a CD73 antagonist.

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

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

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

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

29. The use according to 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 its antigen-binding fragment in the manufacture of a pharmaceutical product for treating a subject identified as having a tumor with increased CD73 expression compared to a reference.

31. The use according to claim 30, wherein the anti-CD73 antibody is MEDI9447 or Phen0203 IgG1.

32. The use according to claim 30 or 31, wherein the subject is currently receiving, has received, or is scheduled to receive anti-PD-1, anti-PD-L1, or anti-CTLA4 therapy.

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

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

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

37. A pharmaceutical preparation comprising an effective amount of anti-CD73 antibody or its antigen-binding fragment, and anti-PD-L1 antibody or its antigen-binding fragment.

38. The pharmaceutical preparation according to claim 37, wherein the anti-CD73 antibody is MEDI9447, Phen0203 IgG1, or an antigen-binding fragment thereof.

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

40. A pharmaceutical preparation comprising an effective amount of anti-CD73 antibody or its antigen-binding fragment, and anti-CTLA4 antibody or its antigen-binding fragment.

41. The pharmaceutical preparation according to claim 40, wherein the anti-CD73 antibody is MEDI9447, Phen0203 IgG1, or an antigen-binding fragment thereof.

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

43. Antibody V Ｌ and antibody V Ｈ An isolated binding molecule or antigen-binding fragment thereof that specifically binds to CD73, and specifically binds to an epitope of the CD73 protein containing one or more amino acids corresponding to Val144, Lys180, and Asn185.

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

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

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

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

48. An isolated binding molecule or antigen-binding fragment thereof according to claim 43 or 44, comprising the amino acid sequence in Tyr132 to Val144 and / or Lys180 to Asn187.

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