CD73 blocking antibodies

Abstract

The present invention relates to antibodies and fragments thereof that bind to and inhibit CD73. The present invention also relates to cells that produce such compounds; methods for preparing such compounds and antibodies, fragments, variants and derivatives thereof; pharmaceutical compositions comprising the compounds; and methods for diagnosing, treating or preventing diseases, such as cancer, using the compounds.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 837,214, filed on April 23, 2019, which is incorporated herein by reference in its entirety, including any drawings.

[0003] References to sequence listings

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a 62KB file named "CD73-6_ST25" created on April 1, 2020. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to antibodies and fragments thereof that bind to and inhibit CD73. The present invention also relates to cells that produce such compounds; methods for preparing such compounds and antibodies, fragments, variants and derivatives thereof; pharmaceutical compositions comprising the compounds; and methods for diagnosing, treating or preventing diseases, such as cancer, using the compounds. Background Art

[0006] CD73 (ecto-5'-nucleotidase) is a 70-kDa glycosylphosphatidylinositol (GPI)-anchored protein normally expressed on endothelial cells and hematopoietic cell subsets. CD73, together with CD39, regulates adenosine triphosphate (ATP) metabolism. CD39 (NTPDase-1) converts ATP to AMP, in which only trace amounts of ADP are released, while CD73 catalyzes the conversion of AMP to adenosine.

[0007] Adenosine triphosphate (ATP) and its metabolites AMP and adenosine play an important role in cell metabolism, signal transduction and immune homeostasis. The release of extracellular adenosine triphosphate (ATP) in response to cell death or cell stress plays a role in activating immune responses. However, its metabolite adenosine has immunosuppressive activity. Extracellular adenosine accumulates in cancer tissues and constitutes an important mechanism of tumor immune escape. Among other effects, tumor-derived adenosine significantly inhibits infiltrating effector T cells by activating A2A receptors via adenylate cyclase.

[0008] CD73 expression has been reported in a range of tumor cells, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer and breast cancer. CD73 expression is also associated with a pro-metastatic phenotype in melanoma and breast cancer. It has been reported that therapy with antibodies that bind to murine CD73 can inhibit breast tumor growth and metastasis in mice (Stagg et al. (2010) Proc. Natl. Acad. Sci. USA 104: 1547-1552). Genetic deletion of the A2A receptor has been shown to induce T cell-dependent tumor rejection (Ohta et al., (2006) Proc. Natl. Acad. Sci. USA 103: 13132–13137). Knockdown using siRNA or overexpression of CD73 on tumor cells can modulate tumor growth and metastasis (Beavis et al. (2013) Proc. Natl. Acad. Sci. USA 110:14711-716; Stagg et al. (2010), supra; Jin et al. (2010) Cancer Res. 70:2245-55). CD73- / - mice are protected from transplanted and spontaneous tumors (Stagg et al. (2010) Cancer Res. 71:2892-2900). In humans, high CD73 expression has been shown to be a negative prognosis for triple-negative breast cancer (Loi et al. (2013) Proc. Natl. Acad. Sci. USA 110:11091-11096). However, while CD73 is expressed on tumor cells, it is also expressed on different cells of the immune system, particularly CD4 and CD8 T cells and B cells. Furthermore, an additional complicating factor is that many of the antibodies described in the literature are typically of murine isotypes which are able to bind via Fcγ receptors, making it difficult to separate any potential blocking effects from Fc-mediated effects.

[0009] Despite the long-standing interest in CD73 as a therapeutic target, there remains a need for antibodies that are more potent in inhibiting the enzymatic activity of CD73 and that are suitable for human therapy. Summary of the invention

[0010] The present inventors provide antibodies that bind to epitopes present on CD73 expressed on the surface of cells, including tumor cells, and inhibit the enzymatic (extracellular-5' nucleotidase) activity of the CD73 enzyme. The antibody has a framework region of human origin, in which the non-human (e.g., mouse) sequence content is minimal. The antibody can bind to CD73 in an intradimer mode, and can inhibit the enzymatic activity of both soluble CD73 and membrane-bound CD73 proteins expressed at the surface of cells. Advantageously, these antibodies can be used as pure CD73 blocking antibodies, for example, they inhibit the enzymatic activity of membrane-bound CD73 proteins expressed at the surface of cells without substantially binding to Fcγ receptors and / or without substantially directing ADCC toward CD73-expressing cells. Optionally, the antibody retains the Fc domain and retains binding to human FcRn. The risk of immunogenicity of the antibody can be advantageously lower, for example, the likelihood of inducing a human anti-mouse antibody (HAMA) response when administered to humans is lower or reduced (compared to the mouse parent antibody).

[0011] The antibodies bind to an epitope present on a human CD73 polypeptide expressed at the surface of cells, including but not limited to tumor cells, and inhibit the enzymatic (ecto-5' nucleotidase) activity of the CD73 enzyme. It is particularly advantageously capable of inhibiting the inhibition of T cell proliferation mediated by CD73, and thus can cause biological activity of cytotoxic CD4 and / or CD8 T cells, including but not limited to increased proliferation, such as observed in T cell proliferation assays. In one embodiment, inhibition or neutralization of the enzymatic activity of CD73 is determined by evaluating the ability of the antibody fragment to increase T cell proliferation when T cell proliferation is induced in vitro (e.g., by TCR co-stimulation) in the presence of AMP (e.g., exogenously added AMP).

[0012] In one embodiment, an antibody or antibody fragment or an antigen-binding domain thereof is provided, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37, and the light chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 33, 34, 35 or 36. In one embodiment, an antibody or antibody fragment or an antigen-binding domain thereof is provided, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 33.

[0013] In one embodiment, an antibody or antibody fragment or an antigen-binding domain thereof is provided, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42, and the light chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 43, 44, 45 or 46. In one embodiment, an antibody or antibody fragment or an antigen-binding domain thereof is provided, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 43.

[0014] In one embodiment, an anti-CD73 antibody or antibody fragment is provided, which comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:38, and the light chain comprises the amino acid sequence of SEQ ID NO:39.

[0015] In one embodiment, an anti-CD73 antibody or antibody fragment is provided, which comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:47, and the light chain comprises the amino acid sequence of SEQ ID NO:48.

[0016] In one embodiment, an anti-CD73 antigen-binding domain or a protein comprising the antigen-binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, the antigen-binding domain comprising: a heavy chain variable region (VH), the VH comprising CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 2, 3 and 4, and a framework FR1, FR2 and FR3 amino acid sequence from a human IGHV1-3 gene (and optionally further a framework 4 (FR4) amino acid sequence from a human IGHJ4 gene); and a light chain variable region (VL), comprising CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 5, 6 and 7, and a framework FR1, FR2 and FR3 amino acid sequence from a human IGKV1-33 gene (and optionally further a framework 4 (FR4) amino acid sequence from a human IGKJ2 gene). Optionally, the residue at Kabat position 59 (HCDR2) present in the heavy chain variable region is a leucine or glutamine residue. Optionally, the residue at Kabat position 60 (HCDR2) present in the heavy chain variable region is a threonine or lysine residue. Optionally, the residue at Kabat position 97 (HCDR3) present in the heavy chain variable region is a glycine or asparagine residue. Optionally, the residue at Kabat position 30 (LCDR1) present in the light chain variable region is a serine or threonine residue. Optionally, the residue at Kabat position 53 (LCDR2) present in the light chain variable region is a threonine or asparagine residue.

[0017] In one embodiment, an anti-CD73 antigen-binding domain or a protein comprising the antigen-binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, the antigen-binding domain comprising: a heavy chain variable region (VH), the VH comprising CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 2, 8 and 9, and a framework FR1, FR2 and FR3 amino acid sequence from a human IGHV1-3 gene (and optionally further a framework 4 (FR4) amino acid sequence from a human IGHJ4 gene); and a light chain variable region (VL), comprising CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 10, 11 and 7, and a framework FR1, FR2 and FR3 amino acid sequence from a human IGKV1-33 gene (and optionally further a framework 4 (FR4) amino acid sequence from a human IGKJ2 gene). In one embodiment, VH further comprises one, two or three amino acid substitutions in the Kabat heavy chain CDRs at the amino acid substitutions at the Kabat heavy chain positions selected from the group consisting of 59 (HCDR2), 60 (HCDR2) and 97 (HCDR3). Optionally, the leucine residue at position 59 is replaced by a glutamine residue. Optionally, the threonine residue at position 60 is replaced by a lysine residue. Optionally, the glycine residue at position 97 is replaced by an asparagine residue. In one embodiment, the VL further comprises amino acid substitutions at Kabat light chain positions 30 (LCDR1) and / or 53 (LCDR2) in the Kabat light chain CDRs, optionally further wherein the serine residue at position 30 is replaced by a threonine residue, optionally further wherein the threonine residue at position 53 is replaced by an asparagine residue.

[0018] In one embodiment, an anti-CD73 antigen-binding domain or a protein comprising the antigen-binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, the antigen-binding domain comprising: a heavy chain variable region (VH), the VH comprising CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 2, 12 and 13, and a framework FR1, FR2 and FR3 amino acid sequence from a human IGHV1-3 gene (and optionally further a framework 4 (FR4) amino acid sequence from a human IGHJ4 gene); and a light chain variable region (VL), comprising CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 14, 15 and 7, and a framework FR1, FR2 and FR3 amino acid sequence from a human IGKV1-33 gene (and optionally further a framework 4 (FR4) amino acid sequence from a human IGKJ2 gene).

[0019] In one aspect of any embodiment herein, the heavy chain variable region (VH) may be characterized by including one, two, three, four or five amino acid substitutions at Kabat heavy chain positions selected from the group consisting of 2, 30, 48, 69 and 73, optionally wherein the residue at the specific position present in the human sequence is replaced by the residue at the specific position present in the mouse donor sequence; and the light chain variable region (VL) may be characterized by including an amino acid substitution at Kabat light chain position 67, optionally wherein the residue at the specific position present in the human sequence is replaced by the residue at the specific position present in the mouse donor sequence. In one embodiment, VL includes a substitution at Kabat light chain position 67, and optionally further one, two or three amino acid substitutions at Kabat light chain positions selected from the group consisting of 2, 67, 87, optionally wherein the residue at the specific position present in the human sequence is replaced by the residue at the specific position present in the mouse donor sequence. In one embodiment, the antibody or antibody binding domain comprises a heavy chain variable region comprising amino acid substitutions V2I, T30A, M48I, I69L and T73K and a light chain variable region comprising amino acid substitution S67Y, wherein numbering is according to Kabat.

[0020] In one aspect of any of the embodiments herein, the amino acid at position 2 of the Kabat heavy chain is isoleucine.

[0021] In one aspect of any of the embodiments herein, the amino acid at position 30 of the Kabat heavy chain is alanine.

[0022] In one aspect of any of the embodiments herein, the amino acid at position 48 of the Kabat heavy chain is isoleucine.

[0023] In one aspect of any of the embodiments herein, the amino acid at position 69 of the Kabat heavy chain is leucine.

[0024] In one aspect of any of the embodiments herein, the amino acid at Kabat heavy chain position 73 is lysine.

[0025] In one embodiment, the VH comprises an isoleucine residue at Kabat position 2, an alanine at position 30, an isoleucine at position 48, a leucine at position 69, and a lysine at position 73.

[0026] In one aspect of any of the embodiments herein, the amino acid at position 67 of the Kabat light chain is tyrosine.

[0027] In one aspect of any of the embodiments herein, the amino acid at position 60 of the Kabat light chain is aspartic acid.

[0028] In one aspect of any of the embodiments herein, the amino acid at position 2 of the Kabat light chain is isoleucine.

[0029] In one aspect of any of the embodiments herein, the amino acid at position 87 of the Kabat light chain is phenylalanine.

[0030] In one embodiment, the VL comprises a tyrosine residue at Kabat position 67; optionally, the VL comprises no substitution in the Kabat framework by a non-human residue and / or has a fully human VL Kabat framework other than a substitution at residue 67.

[0031] In one embodiment, the VL comprises a tyrosine residue at position 67 and an aspartic acid at position 60 of Kabat.

[0032] In one embodiment, the VL comprises a tyrosine residue at Kabat position 67, an aspartic acid at position 60, and an isoleucine at position 2.

[0033] In one embodiment, the VL comprises a tyrosine residue at position 67, an aspartic acid at position 60, an isoleucine at position 2, and a phenylalanine at position 87 of the Kabat sequence.

[0034] In one embodiment, an anti-CD73 antigen binding domain or a protein comprising the antigen binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, the antigen binding domain comprising a heavy chain variable region (VH), the VH comprising an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 37; and a light chain variable region (VL), the VL comprising an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO: 33, 34, 35 or 36. In one embodiment, the VH comprises CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NO: 2, 12 and 13, and the VL comprises CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NO: 14, 15 and 7. In one embodiment, the VH comprises an isoleucine residue at Kabat position 2, an alanine at position 30, an isoleucine at position 48, a leucine at position 69, and a lysine at position 73. In one embodiment, the VL comprises a tyrosine residue at Kabat position 67.

[0035] In one embodiment, an anti-CD73 antigen-binding domain or a protein comprising the antigen-binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, the antigen-binding domain comprising: a heavy chain variable region (VH), the VH comprising CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 2, 3 and 4 and a human framework (e.g., FR1, FR2, FR3 and FR4 of human origin); and a light chain variable region (VL) CDR1, CDR2 and CDR3, the VL comprising the corresponding amino acid sequences shown in SEQ ID NOs: 5, 6 and 7 (e.g., FR1, FR2, FR3 and FR4 of human origin), wherein the (VH) comprises an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 37 or 42, and the light chain variable region (VL) comprises an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 37 or 42. The amino acid sequence of any one of NO:33-36 or 43-46 is at least 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence. Optionally, the residue at Kabat position 59 (HCDR2) present in the heavy chain variable region is a leucine or glutamine residue. Optionally, the residue at Kabat position 60 (HCDR2) present in the heavy chain variable region is a threonine or lysine residue. Optionally, the residue at Kabat position 97 (HCDR3) present in the heavy chain variable region is a glycine or asparagine residue. Optionally, the residue at Kabat position 30 (LCDR1) present in the light chain variable region is a serine or threonine residue. Optionally, the residue at Kabat position 53 (LCDR2) present in the light chain variable region is a threonine or asparagine residue. In one embodiment, the VH comprises an isoleucine residue at Kabat position 2, an alanine at position 30, an isoleucine at position 48, a leucine at position 69, and a lysine at position 73. In one embodiment, the VL comprises a tyrosine residue at Kabat position 67.

[0036] In any embodiment, the VH may be characterized by including a human VH acceptor framework amino acid sequence, and the VL includes a human VL acceptor framework amino acid sequence. In one embodiment, the VH segment of the VH human acceptor framework is from the human IGHV1-3 gene segment, and the J segment is from the human IGHJ4 gene segment. In one embodiment, the VH human acceptor framework is from the human IGHV1-3*01 gene segment. In one embodiment, the VL domain human acceptor framework is from the human IGKV1-33 gene segment, and optionally the VL domain human acceptor framework is from the human IGKV1-33*01 gene segment. In one embodiment, the VL domain human acceptor framework includes a J segment from the human IGKJ2 gene segment.

[0037] In one embodiment, the antibody or antibody fragment comprises H4+VH domains and L1, L2, L3 or L4 VL domains. In one embodiment, the antibody is antibody H4+L1.

[0038] In one embodiment, an anti-CD73 antigen-binding domain or a protein comprising the antigen-binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, wherein the antigen-binding domain comprises: a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37; and a light chain variable region, the light chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 33, 34, 35 or 36.

[0039] In one embodiment, an antibody or antibody fragment is provided, which binds to human CD73 and neutralizes the ATPase activity of CD73, wherein the antibody or antibody fragment comprises the heavy chain variable region and the light chain variable region of antibody H4+L1.

[0040] In one embodiment, an antibody or antibody fragment is provided, which binds to a human CD73 polypeptide and neutralizes the ATPase activity of CD73, wherein the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 33.

[0041] In one embodiment, an antibody or antibody fragment is provided, which binds to a human CD73 polypeptide and neutralizes the ATPase activity of CD73, wherein the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 38, and the light chain comprises the amino acid sequence of SEQ ID NO: 39.

[0042] In one embodiment, the antibody or antibody fragment comprises a 2H4+VH domain and a 2L1, 2L2, 2L3 or 2L4 VL domain. In one embodiment, the antibody is antibody 2H4+2L1.

[0043] In one embodiment, an anti-CD73 antigen-binding domain or a protein comprising the antigen-binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, wherein the antigen-binding domain comprises: a heavy chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42; and a light chain variable region, the light chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 43, 44, 45 or 46.

[0044] In one embodiment, an antibody or antibody fragment is provided, which binds to human CD73 and neutralizes the ATPase activity of CD73, wherein the antibody or antibody fragment comprises the heavy chain variable region and the light chain variable region of antibody 2H4+2L1.

[0045] In one embodiment, an antibody or antibody fragment is provided, which binds to a human CD73 polypeptide and neutralizes the ATPase activity of CD73, wherein the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 43.

[0046] In one embodiment, an antibody or antibody fragment is provided, which binds to a human CD73 polypeptide and neutralizes the ATPase activity of CD73, wherein the antibody or antibody fragment comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 47, and the light chain comprises the amino acid sequence of SEQ ID NO: 48.

[0047] In one embodiment, the antibody is non-depleting and neutralizes the enzymatic activity of CD73 in the tumor environment.

[0048] In one embodiment, the antibody is a human IgG1, IgG2, IgG3 or IgG4 isotype antibody. For example, the antibody can be an antibody comprising an Fc domain of a human IgG4 isotype or comprising an Fc domain modified to reduce or lack the binding between a human Fcγ receptor (e.g., CD16), optionally modified to reduce the binding between a Fc domain and a plurality of human Fcγ receptors, such as CD16A, CD16B, CD32A, CD32B and CD64, any human IgG isotype (e.g., IgG1, IgG2, IgG3 or IgG4) of the Fc domain. In one embodiment, the antibody comprises an Fc domain of a human IgG subtype (e.g., IgG1), and the human IgG subtype comprises an amino acid modification that results in a reduction in binding to each of CD16A, CD16B, CD32A, CD32B and CD64 (compared to an antibody comprising a wild-type human IgG1 Fc domain) or substantially completely lost.

[0049] In any embodiment, the antibody heavy chain comprises a human CH1 constant domain and a modified human Fc domain, optionally of human IgG1 isotype, optionally further comprising the amino acid sequence of any one of SEQ ID NO: 16, 17, 18 or 19. In any embodiment, the antibody light chain comprises a human light chain constant domain, optionally wherein the constant domain is a human kappa domain.

[0050] In one aspect, the antibodies of the present disclosure do not induce or increase intracellular internalization or more generally downregulation of cell surface expressed CD73 polypeptides and / or are independent of their CD73 inhibitory activity. The antibodies of the present disclosure can provide greater inhibitory potency (ability to substantially neutralize CD73 enzymatic activity) than antibodies that inhibit CD73 by causing CD73 internalization. In contrast to antibodies that inhibit soluble CD73 by other mechanisms (e.g., causing CD73-antibody oligomer formation), the antibodies of the present disclosure are able to inhibit the enzymatic activity of CD73 at a high excess (e.g., 10-fold) of antibody:enzyme. Furthermore, unlike antibodies that bind to epitopes on recombinant CD73 that may be modified or may not be present on cell surface CD73 (e.g., antibody 7G2) or that bind to epitopes on recombinant CD73 with too low an affinity to translate into efficacy in CD73-expressing cells, the antibodies of the present invention bind with high affinity to epitopes present on cell surface CD73 and / or that remain intact on the cell surface, thereby providing antibodies that are capable of potently neutralizing the enzymatic activity of cellular CD73. The antibodies of the present invention inhibit CD73 enzymatic activity in cells, but may also optionally inhibit the ecto-5' nucleotidase activity of soluble recombinant CD73 (as observed in cell-free assays using soluble dimeric CD73 polypeptides).

[0051] In one aspect, the antibodies of the present disclosure are capable of inhibiting the activity of a human CD73 polypeptide without binding to the enzymatic active site of the CD73 polypeptide, and / or are non-competitive inhibitors of CD73, for example, they inhibit the activity of a human CD73 polypeptide without significantly reducing the binding between the CD73 polypeptide and its natural substrate.

[0052] In one aspect, the antibodies of the present disclosure lose binding to CD73 mutants having substitutions at residue K136. In one aspect, the antibodies of the present disclosure have reduced binding to CD73 mutants having substitutions at residue K136 (with reference to the sequence of SEQ ID NO: 1; and compared to wild-type CD73 including the amino acid sequence of SEQ ID NO: 1); optionally, the antibodies have reduced binding to CD73 mutants having substitutions at residues A99, E129, K133, E134, and A135 (with reference to the sequence of SEQ ID NO: 1; and compared to wild-type CD73 including the amino acid sequence of SEQ ID NO: 1); optionally further, the antibodies have reduced binding to CD73 mutants having substitutions at residues K97, E125, Q153, and K330 (with reference to the sequence of SEQ ID NO: 1; and compared to wild-type CD73 including the amino acid sequence of SEQ ID NO: 1).

[0053] In one embodiment, the antibody binds to an epitope on each CD73 polypeptide chain within a CD73 dimer (i.e., binds to CD73 in an intradimeric manner), optionally further wherein the epitope is present on the same face of the CD73 dimer. Thus, the antibody can bind bivalently to a CD73 dimer, particularly at a more "closed" position, in which the antibody binding sites are spatially spaced farther apart than in an "open" position. In view of binding to ligand-bound CD73, the antibodies described herein can be used to bind to CD73 when bound to AMP, for example, to treat individuals or cancers characterized by (e.g., known or suspected to be characterized by) a tumor environment with significant levels of upstream ADP and / or AMP prior to treatment. The antibodies can be used to treat individuals or cancers having (e.g., known or suspected of having) a tumor environment characterized by high levels of ADP (e.g., produced by dying cells and taken up by CD73 on the stroma and cellular infiltrates (e.g., TReg cells) to give high levels of AMP) and more generally AMP, adenosine, the presence or level of CD73 expression or CD73-expressing cells, a high (e.g., compared to healthy tissue) number or frequency of CD73-expressing cells and / or high levels of CD73 expression on cells (e.g., assessed by immunohistochemistry assay), high (e.g., compared to healthy tissue) soluble CD73 polypeptide (e.g., assessed by ELISA or generally any antibody-based detection assay), or the presence or level of adenosine receptor expression or adenosine receptor-expressing cells. The CD73 molecule in the tumor environment may be in a substrate-bound conformation, and the ability to bind to and inhibit substrate-bound cellular CD73 (e.g., cells expressing CD73 pre-incubated with a substrate such as AMP) in addition to non-substrate-bound CD73 may provide a greater ability to inhibit CD73 in vivo. Optionally, soluble CD73 protein, the number or frequency of CD73-expressing cells, and / or the level of CD73 expression on cells can be assessed in the tumor environment prior to treatment. The antibodies have particular advantages for the treatment of individuals with significant levels (e.g., high levels, compared to a reference) of soluble CD73 protein, a certain number or frequency of CD73-expressing cells, and / or a certain level of CD73 expression on cells in a tumor sample.

[0054] Therefore, in one aspect, the present disclosure provides a humanized antibody or antibody fragment that binds to a human CD73 polypeptide segment expressed at the surface of a cell and inhibits the enzymatic (ecto-5' nucleotidase) activity of the CD73 polypeptide, wherein the antibody is capable of bivalently binding to a single CD73 polypeptide dimer (a soluble CD73 polypeptide dimer or a CD73 polypeptide dimer expressed by a cell). Optionally, the antibody binds to a first CD73 polypeptide within the dimer using a first antigen binding domain and binds to a second CD73 polypeptide using a second antigen binding domain. In one aspect, the antibody is an allosteric inhibitor of a CD73 polypeptide.

[0055] The epitope on CD73 bound by the antibody is present on a CD73 polypeptide as expressed by a range of cells, e.g., cancer cells, CD4 T cells, CD8 T cells, B cells, transfected cells, and is bound with high affinity as determined by flow cytometry. For example, the antibody can be characterized by being resistant to an EC that binds to a cell expressing a CD73 polypeptide at its surface. 50 No more than 5 μg / ml, optionally no more than 2 μg / ml, no more than 1 μg / ml, no more than 0.5 μg / ml, no more than 0.1 μg / ml or no more than 0.05 μg / ml as determined by flow cytometry. In one embodiment, the cell is a cell that expresses CD73 at its surface. In one embodiment, the cell is a cell that endogenously expresses CD73 at its surface, such as a cancer cell, a leukemia cell, a bladder cancer cell, a glioma cell, a glioblastoma cell, an ovarian cancer cell, a melanoma cell, a prostate cancer cell, a thyroid cancer cell, an esophageal cancer cell, or a breast cancer cell.

[0056] In one embodiment, the CD73 neutralizing antibody may be characterized by being able to reduce the 5'-ectonucleotidase activity of CD73 of a cell by at least 60%, 75%, or 80%. In one embodiment, the CD73 neutralizing antibody may be characterized by inhibiting the 5'-ectonucleotidase activity of CD73 expressed by a cell by at least 60%, 75%, or 80%. 50 No more than 1 μg / ml, optionally no more than 0.5 μg / ml, optionally no more than 0.2 μg / ml.

[0057] Optionally, inhibition of 5'-ectonucleotidase activity of CD73 expressed by cells is determined by assessing neutralization of 5'-ectonucleotidase activity in CD73-expressing cells (e.g., MDA-MB-231 cells) by quantifying the hydrolysis of AMP to adenosine.

[0058] Optionally, inhibition of the 5'-ectonucleotidase activity of CD73 expressed by cells is determined by assessing the ability of the antibody fragment to increase the proliferation of T cells (e.g., from a healthy human donor) when inducing proliferation of said T cells (e.g., from a healthy human donor) in vitro in the presence of AMP (e.g., exogenously added AMP) (e.g., by, for example, stimulation of TCR co-stimulation, CD3 and CD28 signaling by contacting the T cells with beads functionalized with a CD3 agonist and a CD28 agonist). Optionally, T cell proliferation is assessed using an assay described in the Examples herein (see Example 5 and Methods).

[0059] In one aspect, the anti-CD73 antibody binds to a common antigenic determinant present on both soluble CD73 and CD73 expressed at the cell surface.

[0060] In one aspect, an anti-CD73 antibody binds to an antigenic determinant within each CD73 polypeptide chain within a CD73 dimer (in an intradimeric pattern), e.g., wherein the antigenic determinant is present on a common face of the CD73 dimers.

[0061] In one aspect, the anti-CD73 antibody binds to an epitope on CD73 that includes residue K136 (see SEQ ID NO: 1).

[0062] In one aspect, the anti-CD73 antibody binds to an epitope on CD73 that includes one, two, three or four residues selected from the group consisting of: K97, E125, Q153 and K330 (see SEQ ID NO: 1).

[0063] In one aspect, the anti-CD73 antibody binds to an epitope on CD73 that includes one, two, three, four or five residues selected from the group consisting of: A99, E129, K133, E134 and A135 (see SEQ ID NO: 1).

[0064] In one aspect, the anti-CD73 antibody binds at least partially to a domain or segment of amino acid residues on a human CD73 protein (e.g., a CD73 homodimeric protein) comprising amino acid residues K97, A99, E125, E129, K133, E134, A135, K136, Q153, and K330 (with reference to SEQ ID NO: 1). In one aspect, the anti-CD73 antibody binds to an epitope on CD73 comprising at least one, two, three, four, or five or more residues selected from the group consisting of K97, A99, E125, E129, K133, E134, A135, K136, Q153, and K330 (with reference to SEQ ID NO: 1).

[0065] In one aspect, the anti-CD73 antibody has reduced binding to a CD73 polypeptide having a mutation at residue K136 (with reference to SEQ ID NO: 1) compared to a wild-type CD73 polypeptide comprising the amino acid sequence of SEQ ID NO: 1, optionally, the mutant CD73 polypeptide has the mutation: K136A.

[0066] In one aspect, the anti-CD73 antibody has reduced binding to a CD73 polypeptide having a mutation at a residue selected from the group consisting of: K97, E125, Q153 and K330 (with reference to SEQ ID NO: 1), compared to a wild-type CD73 polypeptide comprising the amino acid sequence of SEQ ID NO: 1; optionally, the mutant CD73 polypeptide has mutations: K97A, E125A, Q153A and / or K330A (e.g., K97A, E125A and K330A; K97A, E125A and / or Q153A).

[0067] In one aspect, the binding of an anti-CD73 antibody to a CD73 polypeptide having a mutation at a residue selected from the group consisting of: A99, E129, K133, E134, and A135 (with reference to SEQ ID NO: 1) is reduced compared to a wild-type CD73 polypeptide comprising the amino acid sequence of SEQ ID NO: 1; optionally, the mutant CD73 polypeptide has the mutations: A99S, E129A, K133A, E134N, and A135S.

[0068] In one embodiment, a method of treating or preventing a disease, such as an infectious disease or cancer, using an antibody is provided. In one embodiment, the antibody is administered to an individual with cancer in an amount and frequency sufficient to neutralize the activity of CD73 in the tumor microenvironment. In one embodiment, the antibody is administered in an amount and frequency sufficient to reduce the production and / or concentration of adenosine in the tumor microenvironment. In one embodiment, the antibody is administered in an amount and frequency sufficient to increase the production and / or concentration of ATP in the tumor microenvironment. In one embodiment, the antibody is administered in an amount and frequency sufficient to neutralize the activity of CD73 expressed by tumor cells. In one embodiment, the antibody is administered in an amount and frequency sufficient to neutralize the activity of CD73 expressed by CD4 T cells, CD8 T cells and / or B cells.

[0069] The antibodies will be useful for inhibiting CD73-mediated catabolism of adenosine by AMP, for example, reducing the concentration of adenosine in the tumor microenvironment. Thus, these antibodies will be useful for reversing the immunosuppressive effects of CD73 and / or adenosine on T cells, B cells, and other cells expressing adenosine receptors, for example, for treating cancer. In one embodiment, the anti-CD73 antibodies neutralize adenosine-mediated inhibition of proliferation, cytokine production, cytotoxicity, and / or NFκB activity in T cells.

[0070] Because the CD73-mediated catabolism of AMP to adenosine is irreversible, while the catabolism of ATP to ADP and ADP to AMP by CD73 is reversible (by NDK kinase and adenylate kinase, respectively), antibodies that block the irreversible CD73-mediated catabolism will increase the pool of AMP, thereby increasing, for example, the concentration of ADP and ATP in the tumor microenvironment. The antibodies can be used to increase the formation of ADP from AMP and the formation of ATP from ADP. Since ATP has an immunoactivating effect, anti-CD73 antibodies can be used to activate T cells, for example, for the treatment of cancer.

[0071] The antibodies will be used to inhibit the production, amount and / or concentration of adenosine entering the tumor microenvironment.

[0072] Antibodies that neutralize the activity of soluble human CD73 polypeptide dimers can further neutralize CD73 in any other suitable context, such as in reporter cells made to express CD73, in T cells, etc.

[0073] A method for treating an individual is provided, the method comprising administering to an individual (e.g., an individual suffering from a disease, a tumor, etc.) a therapeutically active amount of any anti-CD73 antigen binding compound described herein. In one aspect, a method for treating an individual is provided, the method comprising, consisting essentially of, or consisting of administering to an individual (e.g., an individual suffering from a disease, a tumor, etc.) a therapeutically active amount of an antigen binding compound of the present disclosure.

[0074] In one aspect, a method for reducing adenosine produced by CD73-expressing cells (e.g., immune cells and / or tumor cells in an individual), or a method for neutralizing the enzymatic activity of cellular CD73 is provided, the method comprising, consisting essentially of, or consisting of contacting CD73-expressing cells with an antigen-binding compound of the present disclosure (e.g., an anti-CD73 antibody or antibody fragment, or a composition comprising such an antibody). In one embodiment, the step of contacting CD73-expressing cells with an antigen-binding compound of the present disclosure comprises administering a therapeutically active amount of an antigen-binding compound to an individual. In one embodiment, the individual suffers from cancer.

[0075] In one aspect, a method for reducing adenosine present in a tumor environment (e.g., in a subject) is provided, the method comprising, consisting essentially of, or consisting of administering to a subject a therapeutically active amount of a compound of the disclosure (e.g., an anti-CD73 antibody or antibody fragment or a composition comprising such an antibody). In one embodiment, the subject has cancer. Optionally, the subject is a human suffering from or susceptible to cancer.

[0076] Optionally the antibody is characterized by a binding affinity (K) for a human CD73 polypeptide (e.g., in the form of a CD73 dimer) as determined by SPR (e.g., according to the methods of the Examples). D ) is less than (better than) 10 -9 M, preferably less than 10 -10 M, or preferably less than 10 -11 M and / or characterized by binding to human CD73 polypeptide at an EC less than (better than) 1 μg / ml 50 Binding. Optionally, binding affinity can be specified as bivalent.

[0077] In one embodiment, the antibody pair binds to cells expressing human CD73 at the cell surface (e.g., tumor cells, MDA-MB-231 cells). 50 No more than 0.5 μg / ml, optionally no more than 0.2 μg / ml, optionally no more than 0.1 μg / ml.

[0078] Optionally, the antibody is characterized by neutralization of the enzymatic activity of CD73 in CD73-expressing cells (e.g., tumor cells, MDA-MB-231 cells). 50 Less than (better than) 1 μg / ml, optionally less than 0.5 μg / ml.

[0079] In one embodiment, the antibody is a monoclonal antibody or fragment thereof that retains binding specificity and the ability to neutralize the enzymatic activity of CD73 (e.g., determined by assessing the ability of the antibody or antibody fragment to increase the proliferation of T cells when the T cells are induced to proliferate in vitro in the presence of AMP (e.g., exogenously added AMP). In one embodiment, the antibody is substantially as effective or at least as effective as a parent antibody having the corresponding VH and VL amino acid sequences of SEQ ID NOs: 40 and 41 in neutralizing the enzymatic activity of CD73, optionally wherein the antibody has an EC of 100 for neutralizing the enzymatic activity of CD73. 50 EC of the parental antibody 50In one embodiment, the antibody is more effective than a parent antibody having the corresponding VH and VL amino acid sequences of SEQ ID NOs: 40 and 41 in neutralizing the enzymatic activity of CD73, optionally wherein the antibody has an EC of 1.0001 to ... 50 EC of the parent antibody having the corresponding VH and VL amino acid sequences of SEQ ID NOs: 40 and 41 50 Low.

[0080] In one embodiment, the antibody is substantially as effective or at least as effective as a parent antibody having the corresponding VH and VL amino acid sequences of SEQ ID NOs: 27 and 28 in neutralizing the enzymatic activity of CD73, optionally wherein the antibody has an EC of 1:100 for neutralizing the enzymatic activity of CD73. 50 EC of the parental antibody 50 In one embodiment, the antibody is more effective than a parent antibody having the corresponding VH and VL amino acid sequences of SEQ ID NOs: 27 and 28 in neutralizing the enzymatic activity of CD73, optionally wherein the antibody has an EC of 1.0001 to ... 50 EC of the parent antibody having the corresponding VH and VL amino acid sequences of SEQ ID NOs: 27 and 28 50 Small.

[0081] Also provided are isolated and / or recombinant nucleic acids (e.g., nucleic acid sets) encoding the antibodies (or their heavy or light chains) or VH and / or VL domains of the present disclosure, vectors or vector sets comprising such nucleic acids, cells comprising such vectors, and methods of producing human anti-CD73 antibodies or antibody fragments, the methods comprising culturing such cells under conditions suitable for expressing the anti-CD73 antibodies or antibody fragments. The present disclosure also relates to compositions, such as pharmaceutically acceptable compositions and kits, comprising such proteins, nucleic acids, vectors and / or cells, and one or more additional ingredients that may be active ingredients or inactive ingredients that generally facilitate the formulation, delivery, stability or other properties of the composition (e.g., various carriers). The present disclosure further relates to various new and useful methods for preparing such antibodies, nucleic acids, vectors, cells, organisms and / or compositions and using them, such as to modulate CD73-mediated biological activities, such as to treat diseases associated therewith, particularly cancer.

[0082] The present disclosure also provides methods for producing or testing antibodies or antibody fragments that bind to CD73 and neutralize the enzymatic activity of CD73.

[0083] The present disclosure also provides a method for enhancing the activity of lymphocytes (e.g., T cells) of a subject in need or for restoring the activity of lymphocytes (e.g., T cells), or a method for alleviating adenosine-mediated inhibition of lymphocyte activity (e.g., T cells), the method comprising administering an effective amount of any of the foregoing compositions to the subject. In one embodiment, the subject is a patient suffering from cancer. For example, the patient may suffer from a solid tumor, such as colorectal cancer, renal cancer, ovarian cancer, lung cancer, breast cancer, or malignant melanoma. Alternatively, the patient may suffer from a hematopoietic cancer, such as acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, or non-Hodgkin's lymphoma.

[0084] These aspects will be more fully described, and additional aspects, features, and advantages will be apparent, in the description provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1A-1B Shown are titration curves obtained with humanized variants having H0, H1, H2, H3 and H4 heavy chain variable regions, each in combination with each of the L0, L1, L2, L3 and L4 light chain variable regions and the parental murine antibody HPLP, as in expressing human ( Figure 1A ) or cynomolgus monkey ( Figure 1B ) tested on cell lines transfected with CD73 protein.

[0086] Figure 2 Blockade of the enzymatic activity of soluble recombinant CD73 is shown. The upper left panel (A) shows the high level of luminescence measured when ATP and CTG substrates are mixed. When AMP is added to the reaction mixture, the CTG reaction is inhibited, resulting in a decrease in luminescence. In the presence of rhCD73 protein that hydrolyzes AMP, the level of luminescence is restored. Blockade of CD73 enzymatic activity by different humanized variants is shown.

[0087] Figures 3A-3C 4A-4C show two series of experiments comparing the potency of humanized antibodies in reversing AMP-mediated inhibition of T cell proliferation. Figure 3A and 4A Control of T cell proliferation and its inhibition by AMPs are shown for each series of experiments. Figure 3B and 4B Demonstrating the efficacy of anti-CD73 antibodies in restoring CD4+, and Figure 3C and 4C The efficacy of anti-CD73 antibodies in restoring CD8+ T cell proliferation is shown. Cell proliferation was determined by dilution of Cell Trace Violet marker. Data are presented as mean + / - standard deviation of replicates.

[0088] Figures 5A-5C All anti-CD73 antibodies restored T cell proliferation. Figure 5A Control of T cell subset proliferation and its inhibition by AMPs is shown. Figure 5B The efficacy of H4+Lx antibody and parental antibody HPLP in restoring CD4+ and CD8+ T cell proliferation was shown. Figure 5C The efficacy of 2H4+2Lx antibodies (2H4+ chain in combination with 2L1, 2L2, 2L3 or 2L4 chain) and parental antibody 2HP2LP in restoring CD4+ and CD8+ T cell proliferation is shown. Data are presented as mean + / - standard deviation of replicates.

[0089] Fig. 6A and 6B It is shown that T cell proliferation restored by the humanized variants is reproducible in two representative human donors, respectively. The efficacy of the 2H4+2Lx anti-CD73 antibody variants in restoring CD4 and CD8 T cell proliferation is shown. The data are expressed as the mean + / - standard deviation of the replicate values.

[0090] Figure 7 CD4+T cell proliferation is shown to be inhibited by ATP and restored with 2H4+2Lx antibody variants. Control and inhibition of CD4+T cell proliferation by 100 μM ATP, as shown for two representative donors D795 (upper left) and D664 (lower left) tested. Data are expressed as mean + / - standard deviation of replicates. DETAILED DESCRIPTION

[0091] definition

[0092] As used in the specification, "a" or "an" may mean one or more. As used in one or more claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more.

[0093] When "comprising" is used, this may optionally be replaced by "consisting essentially of" or "consisting of.

[0094] Human CD73, encoded by the NT5E gene, is also known as ecto-5'-nucleotidase and 5-prime-ribonucleotide phosphohydrolase, EC 3.1.3.5 exhibits 5'-nucleotidase, in particular AMP-, NAD- and NMN-nucleosidase activity. CD73 catalyzes the conversion of purine 5-prime mononucleotides to nucleosides at neutral pH, preferably the substrate is AMP. The enzyme consists of a dimer of 2 identical 70-kD subunits bound to the outer surface of the plasma membrane by a glycosylphosphatidylinositol bond. The amino acid sequence of the human CD73 preprotein (monomer), including the signal sequence at amino acids 1-26, is shown in Genbank as accession number NP_002517 and is as follows, the entire disclosure of which is incorporated herein by reference:

[0095]

[0096] In the context of this article, "neutralizing the enzymatic activity of CD73" refers to the process of inhibiting the 5'-nucleotidase (5'-ectonucleotidase) activity of CD73. This includes specifically inhibiting the CD73-mediated generation of adenosine, i.e., inhibiting the CD73-mediated catabolism of adenosine by AMP. This can be measured, for example, in a cell-free assay that measures the capacity of a test compound to inhibit the direct or indirect conversion of AMP to adenosine. In one embodiment, the antibody preparation results in at least a 50% reduction in the conversion of AMP to adenosine, at least a 70% reduction in the conversion of AMP to adenosine, or at least an 80% reduction in the conversion of AMP to adenosine, for example, with reference to the assays described herein.

[0097] Whenever reference is made throughout the specification to an anti-CD73 binding agent (e.g., an antibody) to "treating cancer" or the like, this refers to: (a) a method of treating cancer, the method comprising the steps of administering (for at least one treatment) an anti-CD73 binding agent (preferably in a pharmaceutically acceptable carrier material) to an individual, mammal, especially a human in need of such treatment, in a dose (therapeutically effective amount) allowing for the treatment of cancer, preferably a dose (amount) as specified herein; (b) using an anti-CD73 binding agent for treating cancer or an anti-CD73 binding agent for use in said treatment (especially in a human); (c) using an anti-CD73 binding agent to manufacture a pharmaceutical preparation for treating cancer, a method of using an anti-CD73 binding agent to manufacture a pharmaceutical preparation for treating cancer comprising admixing an anti-CD73 binding agent with a pharmaceutically acceptable carrier or a pharmaceutical preparation comprising an effective dose of an anti-CD73 binding agent suitable for treating cancer; or (d) any combination of a), b) and c) of the subject matter of patents allowed in the country in which this application is filed.

[0098] As used herein, the term "antibody" refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are designated as one of the following five major classes: IgA, IgD, IgE, IgG and IgM. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25kDa) and a "heavy" chain (about 50-70kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (V H ) refer to these light chains and heavy chains, respectively. The heavy chain constant domains corresponding to different classes of immunoglobulins are called "α", "δ", "ε", "γ" and "μ", respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG is an exemplary antibody class used herein because it is the most common antibody in physiological situations and because it is easiest to prepare in a laboratory environment. Optionally, the antibody is a monoclonal antibody. Specific examples of antibodies are humanized, chimeric, human or other human suitable antibodies. "Antibody" also includes any fragment or derivative of any antibody described herein.

[0099] The term "specific binding" means that the antibody can preferentially bind to a binding partner, e.g., CD73, in a competitive binding assay as assessed using a recombinant form of the protein, an epitope therein, or the native protein present on the surface of an isolated target cell. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

[0100] When an antibody is said to "compete" with a particular monoclonal antibody, this means that the antibody competes with the monoclonal antibody in a binding assay using recombinant CD73 molecules or surface-expressed CD73 molecules. For example, if a test antibody reduces binding of the reference antibody to CD73 polypeptide or CD73-expressing cells in a binding assay, the antibody is said to "compete" with the reference antibody accordingly.

[0101] As used herein, the term "affinity" refers to the strength with which an antibody binds to an epitope. The affinity of an antibody is given by the dissociation constant, Kd, ​​defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant K aDefined by 1 / Kd. Methods for determining the affinity of mAbs can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988); Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Associates and Wiley Interscience, New York, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entirety. A standard method for determining the affinity of mAbs, well known in the art, is to use surface plasmon resonance (SPR) screening (e.g., by using BIAcore TM SPR analysis device analysis).

[0102] In the present context, a "determinant" refers to a site of interaction or binding with a polypeptide.

[0103] The term "epitope" refers to an antigenic determinant, and is a region or region on an antigen to which an antibody binds. Protein epitopes may include amino acid residues that are directly involved in binding and amino acid residues that are effectively blocked by specific antigen-binding antibodies or peptides, i.e., amino acid residues within the "footprint" of the antibody. It is the simplest form or smallest structural region on a complex antigen molecule, which can be combined with, for example, an antibody or a receptor. An epitope may be linear or conformational / structural. The term "linear epitope" is defined as an epitope consisting of continuous amino acid residues on a linear sequence of amino acids (primary structure). The term "conformational or structural epitope" is defined as an epitope consisting of incompletely continuous amino acid residues, and therefore represents a separate portion (secondary, tertiary and / or quaternary structure) of a linear sequence of amino acids that is close to each other by folding the molecule. Conformational epitopes depend on 3-dimensional structures. Therefore, the term 'conformation' is usually used interchangeably with 'structure'.

[0104] The term "deplete" or "depleting" with respect to CD73-expressing cells refers to a process, method or compound that results in killing, elimination, lysis or inducing such killing, elimination or lysis, thereby negatively affecting the number of such CD73-expressing cells present in a sample or in a subject.

[0105] The term "internalization" used interchangeably with "intracellular internalization" refers to the molecular, biochemical and cellular events associated with the process of transferring molecules from the extracellular surface of a cell to the intracellular surface of a cell. The processes responsible for the internalization of intracellular molecules are well known and may particularly involve the internalization of extracellular molecules (such as hormones, antibodies and small organic molecules); membrane-associated molecules (such as cell surface receptors); and complexes of membrane-associated molecules bound to extracellular molecules (e.g., ligands bound to transmembrane receptors or antibodies bound to membrane-associated molecules). Therefore, "inducing and / or increasing internalization" includes events in which intracellular internalization is triggered and / or the rate and / or degree of intracellular internalization is increased.

[0106] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, biological macromolecules, or extracts made from biological materials. The term "therapeutic agent" refers to an agent that has biological activity.

[0107] For the purposes of this article, "humanized" or "human" antibodies refer to antibodies in which the constant and variable framework regions of one or more human immunoglobulins are fused to the binding region of animal immunoglobulins, such as CDRs. Such antibodies are designed to maintain the binding specificity of the non-human antibodies derived from the binding region, but to avoid immune responses against non-human antibodies. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered" to produce specific human antibodies in response to antigenic attack (see, e.g., Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) International Immunopharmacology 6:579, all teachings of the document are incorporated herein by reference). Fully human antibodies can also be constructed by genetic or chromosome transfection methods and phage display technology, all of which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies can also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated by reference in their entireties).

[0108] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region generally includes amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991) and / or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol 1987; 196:901-917), or an analogous system for defining essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in this region is performed by the methods described above by Kabat et al., supra. Phrases such as "Kabat position," "variable domain residue numbering as in Kabat," and "according to Kabat" refer herein to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids, corresponding to shortening or insertion of a FR or CDR of a variable domain. For example, a heavy chain variable domain may include a single amino acid insert after residue 52 of CDR H2 (residue 52a according to Kabat) and an insertion residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat residue numbering of a given antibody may be determined by comparing the homologous region of an antibody sequence with a "standard" Kabat numbering sequence.

[0109] As used herein, "framework" or "FR" residues refer to regions of an antibody variable domain other than those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0110] The terms "Fc domain", "Fc portion" and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, e.g., about amino acids (aa) 230 to about aa 450 from a human gamma (γ) heavy chain, or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε and μ of a human antibody), or its naturally occurring allotypes. Unless otherwise indicated, the generally accepted Kabat amino acid numbering of immunoglobulins is used throughout this disclosure (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

[0111] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein is substantially purified if it is the predominant species present in a preparation.

[0112] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid.

[0113] The term "recombinant" when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, means that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all.

[0114] In the present context, the term "an antibody that binds" a polypeptide or epitope means that the antibody binds to said determinant with specificity and / or affinity.

[0115] The terms "identity" or "identical" when used in the context of a relationship between sequences of two or more polypeptides refer to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with gap alignments (if any) resolved by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., M. Stockton Press, New York, 1993. Press), 1991; and Carillo et al., Journal of the Society for Industrial and Applied Mathematics (SIAM J. Applied Math.), 48, 1073 (1988).

[0116] The method for determining identity is designed to give the maximum match between the test sequences. The method for determining identity is described in a publicly available computer program. The computer program method for determining the identity between two sequences includes the GCG program package, including GAP (Devereux et al., "Nucl. Acid. Res." 12,387 (1984); Genetics Computer Group of the University of Wisconsin in Madison, Wisconsin), BLASTP, BLASTN and FASTA (Altschul et al., "Journal of Molecular Biology" 215,403-410 (1990)). The BLASTX program can be publicly obtained from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., Bethesda, Maryland NCB / NLM / NIH, 20894; Altschul et al., the same). The well-known Smith-Waterman algorithm (Smith Waterman algorithm) can also be used to determine consistency.

[0117] Antibody production

[0118] The present invention is based in part on the discovery of modified human receptor framework sequences that can be incorporated into antibody CDRs so that the resulting anti-CD73 variable regions have high potency in neutralizing the enzymatic activity of human CD73. The advantage of the antibodies is low or reduced immunogenicity in humans, such as low ability or likelihood to cause an undesirable anti-CD73 antibody-directed immune response after administration to humans. Examples of such antibodies disclosed herein include antibodies comprising H4+ or 2H4+VH domains in combination with L1, L2, L3, L4, 2L1, 2L2, 2L3 or 2L4 VL domains. Examples of antibodies disclosed herein include antibodies comprising a VH and VL domain pair of any one of antibodies H4+L1, H4+L2, H4+L3, H4+L4, 2H4+L1, 2H4+L2, 2H4+L3 or 2H4+L4.

[0119] In one aspect, an antibody or antibody fragment that binds to a human CD73 polypeptide comprises VH and VL frameworks (e.g., FR1, FR2, FR3, and FR4) of human origin. In one aspect, the antibody or antibody fragment comprises: HCDR1 (heavy chain CDR1) comprising the amino acid sequence SYNMY as shown in SEQ ID NO:2; HCDR2 (heavy chain CDR2) comprising the amino acid sequence YIDPYNGGSSYNQKFKG as shown in SEQ ID NO:12, optionally further wherein the glutamine residue (Q) at position 13 of SEQ ID NO:12 can be substituted with a leucine residue (L), wherein the lysine residue (K) at position 14 of SEQ ID NO:12 can be optionally substituted with a threonine residue (T); HCDR3 (heavy chain CDR3) comprising the amino acid sequence GYNNYKAWFAY as shown in SEQ ID NO:13, optionally further wherein the asparagine residue (N) at position 3 of SEQ ID NO:13 can be substituted with a glycine residue (G); LCDR1 (light chain CDR1) comprising the amino acid sequence KASQSVTNDVA as shown in SEQ ID NO:14, optionally further wherein the asparagine residue (N) at position 3 of SEQ ID NO:13 can be substituted with a glycine residue (G); The threonine residue (T) at position 7 of NO:14 can be replaced by a serine residue (S); LCDR2 (light chain CDR2), which includes the amino acid sequence YASNRYT as shown in SEQ ID NO:15, optionally wherein the asparagine residue (N) at position 4 of SEQ ID NO:15 can be replaced by a threonine residue (T); and LCDR3 (light chain CDR3), which includes the amino acid sequence QQDYSSLT as shown in SEQ ID NO:7.

[0120] In one embodiment, HCDR2 comprises the amino acid sequence of Formula I:

[0121] YIDPYNGGSSYN-Xaa1-Xaa2-FKG (SEQ ID NO: 3), or a subsequence thereof, wherein Xaa1 is Q (Gln) or L (Leu), and wherein Xaa2 is K (Lys) or T (Thr).

[0122] In one embodiment, HCDR3 comprises the amino acid sequence of Formula II:

[0123] GY-Xaa1-NYKAWFAY (SEQ ID NO: 4) or a subsequence thereof, wherein Xaa1 is N (Asp) or G (Gly).

[0124] In one embodiment, LCDR1 comprises the amino acid sequence of Formula III:

[0125] KASQSV-Xaa1-NDVA (SEQ ID NO: 5)

[0126] or a subsequence thereof, wherein Xaa1 is T (Thr) or S (Ser).

[0127] In one embodiment, LCDR2 comprises the amino acid sequence of Formula IV:

[0128] YAS-Xaa1-RYT (SEQ ID NO: 6)

[0129] or a subsequence thereof, wherein Xaa1 is T (Thr) or N (Asn).

[0130] In one embodiment, the isolated antibody that binds to a human CD73 polypeptide comprises: a HCDR1 comprising the amino acid sequence SYNMY as shown in SEQ ID NO:2; a HCDR2 comprising the amino acid sequence YIDPYNGGSSYNLTFKG as shown in SEQ ID NO:8; a HCDR3 comprising the amino acid sequence GYGNYKAWFAY as shown in SEQ ID NO:9; a LCDR1 comprising the amino acid sequence KASQSVSNDVA as shown in SEQ ID NO:10; a LCDR2 comprising the amino acid sequence YASTRYT as shown in SEQ ID NO:11; and a LCDR3 comprising the amino acid sequence QQDYSSLT as shown in SEQ ID NO:7.

[0131] In one embodiment, the isolated antibody that binds to a human CD73 polypeptide comprises: a HCDR1 comprising the amino acid sequence SYNMY as shown in SEQ ID NO:2; a HCDR2 comprising the amino acid sequence YIDPYNGGSSYNQKFKG as shown in SEQ ID NO:12; a HCDR3 comprising the amino acid sequence GYNNYKAWFAY as shown in SEQ ID NO:13; a LCDR1 comprising the amino acid sequence KASQSVTNDVA as shown in SEQ ID NO:14; a LCDR2 comprising the amino acid sequence YASNRYT as shown in SEQ ID NO:15; and a LCDR3 comprising the amino acid sequence QQDYSSLT as shown in SEQ ID NO:7.

[0132] In one embodiment, the antibody comprises a heavy chain framework from human subgroup IGHV1-3 (optionally together with IGHJ4), optionally IGHV1-3 is IGHV1-3*01. In one embodiment, the humanized antibody comprises a light chain framework from human subgroup IGKV1-33 (optionally together with IGKJ2), optionally from IGKV1-33*01.

[0133] The antibody may further include one, two, three, four, five or more amino acid substitutions across the human heavy and / or light chain frameworks, for example to enhance affinity, stability or other properties of the antibody.

[0134] Examples of VH and VL amino acid sequences for anti-CD73 antibodies are shown in Example 6 (H4+ chain shown, and Table 5 for 2H4+2Lx antibodies) and in Table 1 for L1-L4 chains.

[0135] In one aspect, the present invention provides an antigen binding domain or an antibody or an antibody fragment that binds to a human CD73 polypeptide, the antigen binding domain or the antibody or the antibody fragment comprising:

[0136] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2;

[0137] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, 8 or 12;

[0138] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4, 9 or 13;

[0139] (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 5, 10 or 14;

[0140] (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 6, 11 or 15;

[0141] (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and

[0142] (g) Human heavy and light chain framework sequences.

[0143] In one embodiment, the antibody comprises a heavy chain framework from human subgroup IGHV1-3 together with IGHJ4, optionally the antibody comprises IGHV1-3*01 together with IGHJ4. In one embodiment, the humanized antibody comprises a light chain framework from human subgroup IGKV1-33, optionally IGKV1-33*01 together with IGKJ2.

[0144] Optionally, in any embodiment, the antibody can be designated as an antibody other than a murine parent antibody (e.g., a murine parent antibody having the corresponding VH and VL of SEQ ID NOs: 27 and 28, or a murine parent antibody having the corresponding VH and VL of SEQ ID NOs: 40 and 41).

[0145] Optionally, the human framework includes one or more mutations, such as back mutations, to introduce residues present at specific positions in a non-human mammal (eg, a mouse).

[0146] In one aspect of any of the embodiments herein, the amino acid at position 2 of the Kabat heavy chain is isoleucine.

[0147] In one aspect of any of the embodiments herein, the amino acid at position 30 of the Kabat heavy chain is alanine.

[0148] In one aspect of any of the embodiments herein, the amino acid at position 48 of the Kabat heavy chain is isoleucine.

[0149] In one aspect of any of the embodiments herein, the amino acid at position 69 of the Kabat heavy chain is leucine.

[0150] In one aspect of any of the embodiments herein, the amino acid at Kabat heavy chain position 73 is lysine.

[0151] In one embodiment, the VH comprises an isoleucine residue at Kabat position 2, an alanine at position 30, an isoleucine at position 48, a leucine at position 69, and a lysine at position 73.

[0152] In one aspect of any of the embodiments herein, the amino acid at position 67 of the Kabat light chain is tyrosine.

[0153] In one aspect of any of the embodiments herein, the amino acid at position 60 of the Kabat light chain is serine or aspartic acid.

[0154] In one aspect of any of the embodiments herein, the amino acid at position 2 of the Kabat light chain is valine or isoleucine.

[0155] In one aspect of any of the embodiments herein, the amino acid at position 87 of the Kabat light chain is tyrosine or phenylalanine.

[0156] In one embodiment, the VL comprises a tyrosine residue at position 67 of Kabat, a serine at position 60, an isoleucine at position 2, and a tyrosine at position 87.

[0157] In one aspect of any of the embodiments herein, the amino acid at position 28 of the Kabat heavy chain is threonine (T).

[0158] In one aspect of any of the embodiments herein, the amino acid at position 66 of the Kabat heavy chain is arginine (R).

[0159] In one aspect of any of the embodiments herein, the amino acid at position 67 of the Kabat heavy chain is valine (V).

[0160] In one aspect of any of the embodiments herein, the amino acid at position 71 of the Kabat heavy chain is arginine (R).

[0161] Positions within the VH and VL domains herein are described using the Kabat numbering system (Kabat et al. (1991) Protein Sequences of Immunological Significance, 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, MD).

[0162] In one aspect, the anti-CD73 antibody comprises a VH domain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the VH domain of SEQ ID NO: 37 or 42.

[0163] In one aspect, the anti-CD73 antibody or antibody fragment comprises a VL domain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the VL domain of any one of SEQ ID NOs: 33-36 or 43-46.

[0164] In one aspect, the anti-CD73 antibody or antibody fragment comprises a VH domain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the VH domain of SEQ ID NO: 42, and a VL domain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the VL domain of any one of SEQ ID NOs: 43-46.

[0165] In one aspect, the anti-CD73 antibody or antibody fragment comprises a heavy chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the heavy chain of SEQ ID NO: 38 of the H4+L1 antibody. In one embodiment, the antibody or antibody fragment comprises a light chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the light chain of SEQ ID NO: 39 of the H4+L1 antibody.

[0166] In one aspect, the anti-CD73 antibody or antibody fragment comprises a heavy chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98%, or 99% identity, or 100% identity) to the heavy chain of SEQ ID NO: 47 of the 2H4+2L1 antibody. In one aspect, the antibody or antibody fragment comprises a light chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98%, or 99% identity, or 100% identity) to the light chain of SEQ ID NO: 48 of the 2H4+2L1 antibody.

[0167] DNA encoding the antibody can be prepared and placed in an appropriate expression vector for transfection into an appropriate host. The host is then used to recombinantly produce the antibody or its variant, such as a humanized form of the monoclonal antibody, an active fragment of an antibody, a chimeric antibody comprising the antigen recognition portion of the antibody, or a form comprising a detectable portion.

[0168] DNA encoding the monoclonal antibodies of the present disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). In one aspect, nucleic acids encoding the heavy or light chains of the anti-CD73 antibodies of any of the embodiments herein are provided. Once isolated, the DNA can be placed in an expression vector and then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulins, to obtain the synthesis of monoclonal antibodies in a recombinant host cell. Such DNA sequences can be modified for any of a number of purposes, such as for humanizing the antibody, producing fragments or derivatives, or for modifying the sequence of the antibody, such as in an antigen binding site, to optimize the binding specificity of the antibody. In one embodiment, isolated nucleic acid sequences encoding the light and / or heavy chains of the antibody are provided, as well as recombinant host cells comprising (e.g., in their genome) such nucleic acids. Recombinant expression in bacteria of DNA encoding antibodies is well known in the art (see, e.g., Skerra et al., Curr. Opinion in Immunol., 5, 256 (1993); and Pluckthun, Immunol. 130, 151 (1992).

[0169] Fragments and derivatives of antibodies (which are covered by the terms "antibody" or "antibodies" as used in this application, unless otherwise specified or clearly contradictory in context) can be produced by techniques known in the art. "Fragments" include a portion of an intact antibody, typically the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment having a polypeptide having a primary structure consisting of one uninterrupted sequence of consecutive amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"); and multispecific (e.g., bispecific) antibodies formed from antibody fragments.

[0170] Typically, the anti-CD73 antibodies provided herein have an affinity for a CD73 polypeptide (e.g., a CD73 polypeptide produced in the Examples herein) of about 10 4 M -1 To about 10 11 M -1 (For example, about 10 8 M -1 To about 10 10 M -1 ). For example, in certain aspects, the present disclosure provides an average dissociation constant (K) for CD73.D ) is less than 1×10 -9 M anti-CD73 antibodies, such as by surface plasmon resonance (SPR) screening (such as by using BIAcore TM In more specific exemplary aspects, the present disclosure provides a K of CD73. D About 1×10 -8 M to about 1×10 -10 M, or about 1×10 -9 M to about 1×10 -11 Anti-CD73 antibody of M.

[0171] The antibody can be characterized, for example, by an average KD of no greater than about (i.e., affinity better than) 100 nanomolar, 60 nanomolar, 10 nanomolar, 5 nanomolar, or 1 nanomolar, preferably subnanomolar, or optionally no greater than about 500 picomolar, 200 picomolar, 100 picomolar, or 10 picomolar. The KD can be determined, for example, by immobilizing a recombinantly produced human CD73 protein on a chip surface, followed by application of the test antibody in solution. In one embodiment, the method further comprises the step (d): selecting an antibody from (b) that is capable of competing with the control antibody for binding to CD73.

[0172] In one embodiment, the anti-CD73 antibody can be prepared so that it does not substantially bind to any one or more of human Fcγ receptors, e.g., CD16A, CD16B, CD32A, CD32B, and / or CD64. Such antibodies may include constant regions of various heavy chains known to lack binding to Fcγ receptors or to bind low thereto. Alternatively, antibody fragments that do not include (or include part of) the constant region, such as F(ab')2 fragments, can be used to avoid Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including, for example, testing the binding of antibodies to Fc receptor proteins in a BIACORE assay. Moreover, any antibody IgG isotype can generally be used, in which the Fc portion is modified (e.g., by introducing 1, 2, 3, 4, 5 or more amino acid substitutions) to minimize or eliminate binding to Fc receptors (see, e.g., WO 03 / 101485, the disclosure of which is incorporated herein by reference). Assays such as for assessing Fc receptor binding are well known in the art and are described, for example, in WO 03 / 101485.

[0173] In one embodiment, the antibody may include one or more specific mutations in the Fc region, which results in an "Fc silent" antibody with minimal interaction with effector cells. The silent effector function can be obtained by mutations in the Fc region of the antibody, and has been described in the art: N297A mutation, LALA mutation (Strohl, W., 2009, "Curr. Opin. Biotechnol. " Vol. 20 (Issue 6): 685-691); and D265A (Baudino et al., 2008, "Journal of Immunology (J. Immunol.) " 181: 6664-69) also see Heusser et al., WO2012 / 065950, the disclosure of which is incorporated herein by reference. In one embodiment, the antibody includes one, two, three or more amino acid substitutions in the hinge region. In one embodiment, the antibody is IgG1 or IgG2, and includes one, two or three substitutions at residues 233-236, optionally 233-238 (EU numbering). In one embodiment, the antibody is IgG4 and includes one, two or three substitutions at residues 327, 330 and / or 331 (EU numbering). The example of a silent Fc IgG1 antibody is a LALA mutant including L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent Fc mutation is a mutation at residue D265 or D265 and P329, such as used as DAPA (D265A, P329A) mutation in IgG1 antibodies (US 6,737,056). Another silent IgG1 antibody includes a mutation at residue N297 (e.g., N297A, N297S mutations), which produces aglycosylated / aglycosylated antibody.Other silent mutations included: substitutions at residues L234 and G237 (L234A / G237A); substitutions at residues S228, L235, and R409 (S228P / L235E / R409K, T, M, L); substitutions at residues H268, V309, A330, and A331 (H268Q / V309L / A330S / A331S); substitutions at residues C220, C226, C229 and P238 (C220S / C226S / C229S / P238S); substitutions at residues C226, C229, E233, L234 and L235 (C226S / C229S / E233P / L234V / L235A); substitutions at residues K322, L235 and L2 substitutions at residues L234, L235 and P331 (L234F / L235E / P331S); substitutions at residues 234, 235 and 297; substitutions at residues E318, K320 and K322 (L235E / E318A / K320A / K322A); substitutions at residues (V234A, G237A, P238S); substitutions at residues 243 and 264; substitutions at residues 297 and 299; substitutions such that residues 233, 234, 235, 237 and 238, as defined by the EU numbering system, include a sequence selected from PAAAP, PAAAS and SAAAS (see WO2011 / 066501).

[0174] In one embodiment, the antibody may include one or more specific mutations in the Fc region. For example, such an antibody may include an Fc domain of human IgG1 origin, including one or more mutations at Kabat residues 234, 235, 237, 330 and / or 331. An example of such an Fc domain includes substitutions at Kabat residues L234, L235 and P331 (e.g., L234A / L235E / P331S or (L234F / L235E / P331S). Another example of such an Fc domain includes substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A / L235E / G237A / P331S). Such an Fc domain Another example of a domain includes substitutions at Kabat residues L234, L235, G237, A330, and P331 (e.g., L234A / L235E / G237A / A330S / P331S). In one embodiment, the antibody includes an Fc domain, optionally of the human IgG1 isotype, comprising: L234X1 substitution, L235X2 substitution, and P331X3 substitution, wherein X1 is any amino acid residue except leucine, X 2 is any amino acid residue except leucine, and X3 is any amino acid residue except proline; optionally, wherein X1 is alanine or phenylalanine or its conservative substitution; optionally, wherein X2 is glutamic acid or its conservative substitution; optionally, wherein X3 is serine or its conservative substitution. In another embodiment, the antibody includes an Fc domain of an optionally human IgG1 isotype, which includes: L234X1 substitution, L235X2 substitution, G237X4 substitution and P331X4 substitution, wherein X1 is any amino acid residue except leucine, X2 is any amino acid residue except leucine, X3 is any amino acid residue except glycine, and X4 is any amino acid residue except proline; optionally, wherein X1 is alanine or phenylalanine or its conservative substitution; optionally, wherein X2 is glutamic acid or its conservative substitution; optionally, X3 is alanine or its conservative substitution; optionally, X4 is serine or its conservative substitution.In another embodiment, the antibody comprises an Fc domain, optionally of the human IgG1 isotype, comprising: an L234X1 substitution, an L235X2 substitution, a G237X4 substitution, a G330X4 substitution, and a P331X5 substitution, wherein X1 is any amino acid residue except leucine, X2 is any amino acid residue except leucine, X3 is any amino acid residue except glycine, X4 is any amino acid residue except alanine, and X5 is any amino acid residue except proline; optionally, wherein X1 is alanine or phenylalanine or a conservative substitution thereof; optionally, wherein X2 is glutamic acid or a conservative substitution thereof; optionally, X3 is alanine or a conservative substitution thereof; optionally, X4 is serine or a conservative substitution thereof; optionally, X5 is serine or a conservative substitution thereof.

[0175] In the shorthand notation used herein, the format is: wild-type residue:position in polypeptide:mutated residue, where the residue position is indicated according to EU numbering according to Kabat.

[0176] In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence that is at least 90%, 95%, or 99% identical thereto but retains the amino acid residues at Kabat positions 234, 235, and 331 (underlined):

[0177] AS TK GPS VFPL AP SSKSTSGGT AAL GCLVKDYFPEPV TV SWN SG AL TSGVHTFP AV LQSSGLYSLSSVVTVPSSSLG TQ TYICNV NH KPSNTKVDKRVEPKSCDKTHT CPPCP AP E A E G GPS VFLFPP KP KDTLMISRTPEVTCVVVDVSH EDPEVK FN WYVDGVEVHN AKT KPREEQYNSTYRVVSVLTVLH QDWL NG KEYKCKVSNKALPA SIEKTISK AK GQPREPQVYTLPPS RE EMTKNQVSLTCLVKGF YP SD IAVEWESNGQPENNYKTT PP VLD SD GSFFLYSKLTVDKS RW QQGNVFSCSVMHEALHN HY TQKSLSLSPGK (SEQ ID NO: 16).

[0178] In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence that is at least 90%, 95%, or 99% identical thereto but retains the amino acid residues at Kabat positions 234, 235, and 331 (underlined):

[0179] ASTK GPS VFPLAPSSKSTSGGTAALGCL VKDYFPE PV TVSWN SG ALTSGVHTFPAVLQSSGLYSL SSVVTVPSS SL GTQTYICNV NH KPSNTKVDKRVEPKSCDKTHT CPPCP AP E F E G GPS VFLFPP KP KDTLMISRTPEVTCVVVDVSH ED PE VKFNWYVDGVEVHNA KT KPREEQYNSTYRVVSVLTVL HQ DWLNGKEYKCKVSNK AL PA SIEKTISK AK GQPREPQVY TL PPSREEMTKNQVSLTCLVKGF YP SD I AV EWESNGQPEN NY KTTPPVLD SD GSFFLYSKLTVDKS RW QQGNVFSCSVMH EAL HNHYTQKSLSLSPGK (SEQ ID NO: 17).

[0180] In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence that is at least 90%, 95%, or 99% identical thereto but retains the amino acid residues at Kabat positions 234, 235, 237, 330, and 331 (underlined):

[0181] AS TK GPS VFPL AP SSKSTSGGTAALGCLVKDYFP EP VT VSWN SG ALTSGVHTFPAVLQSSGLYSL SSVVTVPSSS LG TQTYICNV NH KPSNTKVDKRVEPKSCDKTHT CPPCPA PE A E G A PSVFLFPP KP KDTLMISRTPEVTCVVVDVSH ED PE VKFNWYVDGVEVHNA KT KPREEQYNSTYRVVSVLTVL HQ DWLNGKEYKCKVSNKALP SSIEKTISKAKGQPREPQV YT LP PSREEMTKNQVSLTCLVKGF YP SD I AV EWESNGQPEN NY KTTPPVLD SD GSFFLYSKLTVDKS RW QQGNVFSCSVMH EAL HNHYTQKSLSLSPGK (SEQ ID NO: 18).

[0182] In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence below, or a sequence that is at least 90%, 95%, or 99% identical thereto but retains the amino acid residues at Kabat positions 234, 235, 237, and 331 (underlined):

[0183] AS TK GPS VFPL AP SSKSTSGGT AAL GCLVKDYFPEPV TV SWN SG ALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSL GT QTYICNV NH KPSNTKVDKRVEPKSCDKTHT CPPCPAPE A E G A PSVFLFPP KP KDTLMISRTPEVTCVVVDVSH EDP EV KFNWYVDGVEVHNA KT KPREEQYNSTYRVVSVLTVLH QD WLNGKEYKCKVSNKALPA SIEKTISKAKGQPREPQVY TL PPSREEMTKNQVSLTCLVKGF YP SD I AV EWESNGQPEN NY KTTPPVLD SD GSFFLYSKLTVDKS RW QQGNVFSCSVMH EAL HNHYTQKSLSLSPGK (SEQ ID NO: 19).

[0184] Fc-silent antibodies result in no or low ADCC activity, which means that the Fc-silent antibodies exhibit ADCC activity of less than 50% specific cell lysis. Preferably, the antibody substantially lacks ADCC activity, for example, the Fc-silent antibodies exhibit less than 5% or less than 1% ADCC activity (specific cell lysis). Fc-silent antibodies can also result in the absence of FcγR-mediated cross-linking of CD73 at the surface of CD73-expressing cells.

[0185] In one embodiment, the antibody has a substitution in the heavy chain constant region at any one, two, three, four, five or more of the residues selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 243, 264, 268, 297, 298, 299, 309, 310, 318, 320, 322, 327, 330, 331 and 409 (the numbering of the residues in the heavy chain constant region is according to EU numbering of Kabat). In one embodiment, the antibody includes a substitution at residues 234, 235 and 322. In one embodiment, the antibody includes a substitution at residues 234, 235 and 331. In one embodiment, the antibody includes a substitution at residues 234, 235, 237 and 331. In one embodiment, the antibody includes a substitution at residues 234, 235, 237, 330 and 331. In one embodiment, the Fc domain is of human IgG1 subtype.Amino acid residues are indicated according to EU numbering according to Kabat.

[0186] In one embodiment, the antibody includes an Fc domain that includes amino acid substitutions that increase binding to human FcRn polypeptides to increase the in vivo half-life of the antibody. Exemplary mutations are described in Strohl, W., 2009, Current Reviews in Biotechnology, Vol. 20 (No. 6): 685-691, the disclosure of which is incorporated herein by reference. Examples of substitutions used in antibodies of the human IgG1 isotype are substitutions at Kabat residues M252, S254, and T256; substitutions at residues T250 and M428; substitutions at residue N434; substitutions at residues H433 and N434; substitutions at residues T307, E380, and N434; substitutions at residues T307, E380, and N434; substitutions at residues M252, S254, T256, H433, N434, and 436; substitutions at residue 1253; substitutions at residues P257, N434, D376, and N434. Antigen binding compounds can be evaluated at any desired stage for their ability to inhibit the enzymatic activity of CD73, in particular to block the 5'-nucleotidase activity of CD73 and reduce the production of adenosine by CD73-expressing cells, and thereby restore the activity of lymphocytes and / or relieve adenosine-mediated inhibition of lymphocytes.

[0187] The ability of an antibody to inhibit the enzymatic activity of CD73 can be tested in a cell-free assay using recombinant soluble human CD73 (in dimer form) and AMP, wherein the conversion of AMP to adenosine (and / or its inhibition) is detected directly (e.g., by measuring substrate and product, i.e., AMP, adenosine, and / or phosphate) or indirectly. In one example, AMP and / or adenosine are detected by HPLC before and after incubation of the test compound with recombinant CD73.

[0188] The CD73 inhibitory activity of an antibody can also be assessed in any of a variety of other ways. For example, in an indirect assay, using a luciferase-based reagent (e.g., luciferase-based reagent available from Promega) The luciferase reaction in the assay is inhibited by AMP. Adding the CD73 enzyme to the reaction degrades AMP and relieves the inhibition, resulting in a detectable signal.

[0189] Assays using soluble CD73 will involve testing under conditions where a significant molar excess (e.g., 10-fold, 20-fold, 50-fold, 100-fold, etc.) of antibody is provided compared to the CD73 polypeptide dimer. When provided to the enzyme in molar excess, the anti-CD73 antibody will no longer be able to form multimeric complexes of the antibody and CD73 dimer; antibodies that retain inhibition of the enzymatic activity of CD73 can then be selected.

[0190] The ability of the antibody to inhibit the 5'-ectonucleotidase enzymatic activity of CD73 may also be tested alternatively or additionally in a cell assay (using cells expressing CD73). Advantageously, the antibodies may be first tested or screened in a cell-free assay to identify antibodies that block enzymatic activity to reduce the possibility of selecting antibodies that inhibit CD73 by causing CD73 internalization, and then tested in a cell assay in the form of purified antibodies. The cell assay may be performed as shown in the Examples herein or as disclosed in PCT Publication No. WO2016 / 055609, the disclosure of which is incorporated herein by reference. For example, CD73-expressing cells (e.g., MDA-MB-231 cell line) are seeded in a flat-bottom 96-well plate and cultured in the presence of an anti-CD73 antibody, as described in detail in WO2016 / 055609. AMP is added to the cells and cultured at 4°C (to avoid CD73 downregulation). The plate is then centrifuged and the supernatant is transferred to a flat-bottom 96-well culture plate. The free phosphate generated by the hydrolysis of AMP to adenosine is then quantified. A reduction in the hydrolysis of AMP to adenosine in the presence of the antibody indicates that the antibody inhibits cellular CD73.

[0191] In one embodiment, the antibody preparation results in at least a 50% reduction in the enzymatic activity of a CD73 polypeptide, preferably at least a 60%, 70% or 80% reduction in the enzymatic activity of a CD73 polypeptide (e.g., a soluble homodimeric CD73 polypeptide; CD73 expressed by a cell).

[0192] The activity of the antibody can also be measured in an indirect assay for the ability of the antibody to modulate lymphocyte activity, such as to relieve adenosine-mediated inhibition of lymphocyte activity or to cause activation of lymphocyte activity. This can be addressed, for example, using a cytokine release assay. In another example, the ability of the antibody to modulate lymphocyte proliferation can be evaluated in an indirect assay.

[0193] In one embodiment, the antibody neutralizes the 5'-ectonucleotidase activity of a homodimeric human CD73 polypeptide in solution. In one embodiment, the antibody binds to a soluble human CD73 polypeptide, particularly an antibody that neutralizes CD73-mediated catabolism of AMP to adenosine, and inhibits the enzymatic activity of the polypeptide. In one embodiment, the antibody binds to CD73 in a bivalent manner. In one embodiment, the antibody is a non-depleting antibody, e.g., an Fc-silent antibody that substantially lacks binding to human Fcγ receptors. In one embodiment, the antibody neutralizes CD73 in solution without relying on the induction of CD73 polypeptide: anti-CD73 antibody oligomers.

[0194] In one embodiment, the antibody specifically binds to human CD73 at the surface of a cell and is capable of neutralizing the 5'-ectonucleotidase activity of a soluble human CD73 polypeptide. In one embodiment, the antibody does not induce oligomerization of soluble CD73.

[0195] In one embodiment, the antibody specifically binds to human CD73 at the surface of the cell and is capable of neutralizing the 5'-ectonucleotidase activity of cellular CD73 (CD73 expressed by the cell). In one embodiment, the antibody specifically binds to human CD73 at the surface of the cell and neutralizes the 5'-ectonucleotidase activity of the human CD73, and is not internalized into CD73-expressing cells when bound to CD73. The antibody does not cause multimerization and subsequent internalization of CD73. In one embodiment, the antibody binds to a recombinant human CD73 polypeptide in solution and is capable of inhibiting the enzymatic activity of the polypeptide, wherein the antibody is not internalized into CD73-expressing cells. In one embodiment, the non-internalizing antibody binds to CD73 in a bivalent manner. In one embodiment, the antibody is a non-depleting antibody, such as an Fc silent antibody. The antibody is capable of neutralizing the 5'-ectonucleotidase activity of a dimeric human CD73 polypeptide in solution, and is further independent of the induction of CD73 polypeptide: anti-CD73 antibody oligomers.

[0196] In one embodiment, the antibody specifically binds to a human CD73 polypeptide in a bivalent manner and inhibits the enzymatic activity of cellular human CD73 (and optionally further recombinant soluble human CD73), wherein the antibody does not induce or increase intracellular internalization of CD73 in CD73-expressing cells. Preferably, the antibody substantially lacks Fcγ receptor binding (e.g., via its Fc domain).

[0197] In one embodiment, the antibody specifically binds to human CD73 at the surface of cells pre-incubated with AMP and is capable of neutralizing its 5'-ectonucleotidase activity. Optionally, neutralization of 5'-ectonucleotidase activity is determined by assessing the internalization of 5'-ectonucleotidase activity in CD73-expressing cells (e.g., MDA-MB-231 cells) by quantifying the hydrolysis of AMP to adenosine.

[0198] In one aspect, neutralization of 5'-ectonucleotidase activity of CD73 expressed by cells is determined by assessing the ability to increase T cell proliferation when the proliferation of the T cells is induced in vitro (e.g., by TCR co-stimulation, stimulation of CD3 and CD28 signaling, e.g., by contacting the T cells with beads functionalized with a CD3 agonist and a CD28 agonist) in the presence of AMP (e.g., exogenously added AMP). In one aspect, the 5'-ectonucleotidase activity neutralization assay is assessed using the T cell proliferation assay described in the section entitled "Methods" in the Examples.

[0199] The antibody can be characterized by its ability to bind to cells transfected with a CD73 mutant compared to the ability of the anti-CD73 antibody to bind to a wild-type CD73 polypeptide (e.g., SEQ ID NO: 1). Reduced binding between an anti-CD73 antibody and a mutant CD73 polypeptide means a decrease in binding affinity (e.g., as measured by known methods, such as FACS assay of cells expressing a particular mutant, or by Biacore assay of binding to the mutant polypeptide) and / or a decrease in the overall binding capacity of the anti-CD73 antibody (e.g., as measured by the anti-CD73 antibody concentration versus polypeptide concentration in B in a graph). max The significant reduction in binding indicates that the mutated residue is directly involved in binding to the anti-CD73 antibody or is in close proximity to the binding protein when the anti-CD73 antibody binds to CD73.

[0200] In some embodiments, a significant reduction in binding means that the binding affinity and / or capacity between the anti-CD73 antibody and the mutant CD73 polypeptide is reduced by greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% relative to the binding between the antibody and the wild-type CD73 polypeptide. In certain embodiments, the binding is reduced to below the detectable limit. In some embodiments, a significant reduction in binding is demonstrated when the binding of the anti-CD73 antibody to the mutant CD73 polypeptide is less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%) of the binding observed between the anti-CD73 antibody and the wild-type CD73 polypeptide.

[0201] In one aspect, the binding of an anti-CD73 antibody to a CD73 polypeptide having a mutation at residue K136 (refer to SEQ ID NO: 1) is reduced, optionally, the mutant CD73 polypeptide has the mutation: K136A.

[0202] In one aspect, the binding of an anti-CD73 antibody to a CD73 polypeptide having a mutation at a residue selected from the group consisting of: K97, E125, Q153 and K330 (with reference to SEQ ID NO: 1); optionally, the mutant CD73 polypeptide has a mutation: K97A, E125A, Q153A and / or K330A (e.g., K97A, E125A and K330A; K97A, E125A and / or Q153A).

[0203] In one aspect, the binding of an anti-CD73 antibody to a CD73 polypeptide having a mutation at a residue selected from the group consisting of: A99, E129, K133, E134 and A135 (with reference to SEQ ID NO: 1) is reduced; optionally, the mutant CD73 polypeptide has the mutations: A99S, E129A, K133A, E134N and / or A135S.

[0204] Optionally, in one aspect, the binding of the anti-CD73 antibody to a CD73 polypeptide having a mutation at a residue selected from the group consisting of: Q70, R73, A74, A107 and R109 (with reference to SEQ ID NO: 1) is not reduced; optionally, the mutant CD73 polypeptide has a mutation: A99S, Q70S, R73A, A74E, A107I and / or R109G.

[0205] In one aspect, the anti-CD73 antibody binds to an epitope on CD73 that includes residue K136 (see SEQ ID NO: 1).

[0206] In one aspect, the anti-CD73 antibody binds to an epitope on CD73 that includes one, two, three or four residues selected from the group consisting of: K97, E125, Q153 and K330 (see SEQ ID NO: 1).

[0207] In one aspect, the anti-CD73 antibody binds to an epitope on CD73 that includes one, two, three, four or five residues selected from the group consisting of: A99, E129, K133, E134 and A135 (see SEQ ID NO: 1).

[0208] In one aspect, the anti-CD73 antibody binds to an epitope on CD73 that includes one, two, three, four, five or more residues selected from the group consisting of: K97, A99, E125, E129, K133, E134, A135, and K136 (with reference to SEQ ID NO: 1). In one aspect, the anti-CD73 antibody binds to an epitope on CD73 that includes one, two, three, four, five or more residues selected from the group consisting of: K97, A99, E125, E129, K133, E134, A135, K136, Q153, and K330 (with reference to SEQ ID NO: 1).

[0209] Optionally, in one aspect, the anti-CD73 antibody does not bind to an epitope on CD73 that includes one, two, three, four or five residues selected from the group consisting of: Q70, R73, A74, A107 and R109 (see SEQ ID NO: 1).

[0210] The anti-CD73 antibody can be incorporated into a pharmaceutical formulation comprising a concentration of 1 mg / ml to 500 mg / ml, wherein the pH of the formulation is 2.0 to 10.0. The formulation may further include a buffer system, one or more preservatives, one or more tonicity agents, one or more chelating agents, stabilizers, and surfactants. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation comprising water. Such formulations are typically solutions or suspensions. In further embodiments, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution comprising at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w water.

[0211] In another embodiment, the pharmaceutical formulation is a lyophilized formulation and the physician or patient adds solvents and / or diluents prior to use.

[0212] In another embodiment, the pharmaceutical formulation is a dry formulation (eg, freeze-dried or spray-dried) and is ready for use without any prior dissolution.

[0213] In further aspects, pharmaceutical formulations include aqueous solutions of such antibodies and buffer, wherein the antibody is present at a concentration of 1 mg / ml or greater, and wherein the pH of the formulation is from about 2.0 to about 10.0.

[0214] In another embodiment, the pH of the formulation is in a range selected from the list consisting of about 2.0 to about 10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, and about 5.5 to about 7.5.

[0215] In further embodiments, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate and tris(hydroxymethyl)-aminomethane, bicine, trimethylglycine, malic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment.

[0216] In other embodiments, the formulation further comprises a pharmaceutically acceptable preservative. In other embodiments, the formulation further comprises an isotonic agent. In other embodiments, the formulation further comprises a chelating agent. In other embodiments, the formulation further comprises a stabilizer. In other embodiments, the formulation further comprises a surfactant. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0217] Other ingredients may be present in the peptide drug formulation. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, fillers, tension modifiers, chelating agents, metal ions, oily vehicles, proteins (e.g., human serum albumin, gelatin or protein) and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine and histidine). Of course, such additional ingredients should not adversely affect the overall stability of the drug formulation.

[0218] Pharmaceutical compositions containing antibodies can be administered to patients in need of such treatment at several sites, such as local sites, such as skin and mucosal sites, sites of bypass absorption, such as in arteries, veins, heart, and sites involved in absorption, such as skin, subcutaneous, muscle, or abdomen. Administration of the pharmaceutical composition can be by several routes of administration, such as subcutaneous, intramuscular, intraperitoneal, intravenous, lingual, sublingual, buccal, oral, gastric and intestinal, nasal, pulmonary, such as through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, such as through the conjunctiva, urethra, and parenteral to patients in need of such treatment.

[0219] Diagnosis and treatment of malignant tumors

[0220] Also provided are methods of treating individuals, particularly human patients, using anti-CD73 antibodies or antibody fragments as described herein. In one embodiment, the disclosure provides the use of antibodies or antibody fragments as described herein in the preparation of a pharmaceutical composition for administration to a human patient. Typically, the patient suffers from cancer or an infectious disease (e.g., a viral or bacterial infection) or is at risk of cancer or an infectious disease.

[0221] For example, in one aspect, a method of restoring or enhancing the activity of immune cells (e.g., lymphocytes) in a patient in need thereof is provided, the method comprising the step of administering to the patient a neutralizing anti-CD73 antibody of the present disclosure. In one embodiment, the method involves increasing the activity of immune cells (e.g., T cells) in a patient suffering from a disease such as cancer or an infectious disease, in which an increase in immune cell activity and / or infiltration to a disease site (e.g., a tumor environment or an infection site) is beneficial, or the disease is caused or characterized by immunosuppression, immunosuppressive cells, or adenosine produced, for example, by CD4 T cells, CD8 T cells, B cells.

[0222] The methods will be particularly useful for treating individuals with tumors in which the suspected tumor microenvironment (and in which CD73-mediated adenosine production) may result in a lack of recognition by the immune system (immune escape). The tumor environment can, for example, be characterized by CD73-expressing immune cells, e.g., CD4 T cells, CD8 T cells, B cells, and / or CD73-expressing tumor cells.

[0223] The methods and anti-CD73 antibodies can be used to treat and / or prevent a variety of cancers and other proliferative diseases. Because these methods work by reducing adenosine, which inhibits the anti-tumor activity of lymphocytes, and possibly additionally by increasing ATP, which can increase the anti-tumor activity of lymphocytes, they are applicable to a wide range of cancers, particularly solid tumors in which adenosine in the tumor microenvironment may play an important role in suppressing anti-tumor immune responses. In one embodiment, the human patient treated with an anti-CD73 antibody of the present disclosure has liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, non-small cell lung cancer (NSCLC), castration-resistant prostate cancer (CRPC), melanoma, uterine cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney cancer or ureter cancer, renal pelvis cancer, central nervous system tumor (CNS), primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma (Kaposi's sarcoma), epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers, including those induced by asbestos, hematological malignancies, including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma and precursor T-lymphoblastic lymphoma, and any combination of the cancers. The present disclosure is also applicable to the treatment of metastatic cancers. One or more of the above clinical attributes of the patient may be tested or selected before, during or after treatment.

[0224] Because the methods and anti-CD73 antibodies work by reducing adenosine, which inhibits the activity of and / or infiltration of immune cells (e.g., lymphocytes), and possibly additionally by increasing ATP, which can increase the activity of lymphocytes, they are applicable to a wide range of infectious diseases, preferably including any infection caused by a viral, bacterial, protozoan, mold or fungal infection.

[0225] In one embodiment, the amount of the antigen binding compound is effective to achieve and / or maintain (e.g., for 1 week, 2 weeks, 3 weeks, 4 weeks, and / or until subsequent administration of an antigen binding compound) at least an EC for neutralizing the enzymatic activity of CD73 in an individual.50 , optionally EC 70 , optionally substantially EC 100 In one embodiment, the anti-CD73 antibody is administered in an amount effective to achieve an EC of neutralizing the enzymatic activity of CD73 in the extravascular tissue of the individual. 50 , optionally EC 70 , optionally substantially EC 100 In one embodiment, the active amount of the anti-CD73 antibody is an EC effective to achieve (or maintain) inhibition of the enzymatic activity of neutralizing CD73 in an individual. 50 , optionally EC 70 , optionally substantially EC 100 The amount.

[0226] In one embodiment, the amount of the anti-CD73 antibody administered is effective to achieve and / or maintain (e.g., for 1 week, 2 weeks, 3 weeks, 4 weeks, and / or until subsequent administration of an anti-CD73 antibody) at least an EC for inhibiting CD73-mediated catabolism of AMP to adenosine in an individual. 50 , optionally EC 70 , optionally substantially EC 100 The anti-CD73 antibody is administered in an amount effective to achieve (or maintain) an EC of inhibiting CD73-mediated catabolism of AMP to adenosine in the extravascular tissue of the individual. 50 , optionally EC 70 , optionally substantially EC 100 The amount.

[0227] In one embodiment, a method for treating or preventing cancer in an individual is provided, the method comprising administering to an individual with the disease an anti-CD73 antibody in an amount that achieves or maintains a concentration in the circulation, optionally in an extravascular tissue of interest (e.g., a tumor or tumor environment) above the concentration required for 50%, 70%, or all (e.g., 90%) receptor saturation of CD73-expressing cells in the circulation (e.g., as assessed in PBMCs) for a specified period of time. Optionally, the concentration achieved is at least 20%, 50%, or 100% higher than the concentration required for a specified receptor saturation.

[0228] In one embodiment, a method for treating or preventing cancer in an individual is provided, the method comprising: achieving or maintaining a concentration in the circulation, optionally in an extravascular tissue of interest (e.g., a tumor or tumor environment) above that of an EC that binds to a CD73-expressing cell for a specified period of time. 50 , optionally EC70 or optionally EC 100 The anti-CD73 antibody is administered to the individual in an amount that is greater than the EC binding to CD73 expressing cells (e.g., as assessed by titrating the anti-CD73 antibody on CD73 expressing cells, such as MDA-MB-231 cells). Optionally, the concentration achieved is relative to the EC binding to CD73 expressing cells. 50 , optionally EC 70 or optionally EC 100 At least 20%, 50% or 100% higher.

[0229] In any embodiment, the antibody is to EC binding to CD73 expressing cells in human PBMCs. 50 , EC optional 70 or optionally EC 100 It may be, for example, between 0.5-100 ng / ml, optionally 1-100 ng / ml, optionally 30-100 ng / ml, such as about 30-90 ng / ml (e.g., as assessed by titration of anti-CD73 antibodies on CD73 expressing cells, such as MDA-MB-231 cells).

[0230] ECs for neutralization of CD73 enzymatic activity with anti-CD73 antibodies 50 It may be, for example, between about 0.01 μg / ml and 1 μg / ml, optionally between 0.1 μg / ml and 10 μg / ml, optionally between 0.1 μg / ml and 1 μg / ml. For example, EC 50 It can be about 0.1 μg / ml, about 0.2 μg / ml or about 0.3 μg / ml. Thus, for example, an amount of this anti-CD73 antibody is administered to achieve and / or maintain a blood concentration of at least 0.1 μg / ml, optionally at least 0.2 μg / ml, optionally at least 1 μg / ml, or optionally at least 2 μg / ml.

[0231] When targeting tissues outside the vasculature (tumor environment, e.g., in the treatment of solid tumors), it is generally believed that a dose approximately 10-fold higher than that which provides a corresponding concentration in the circulation is required. The amount of anti-CD73 antibody administered to achieve (and / or maintain) a concentration of about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml in the circulation (blood) is expected to achieve (and / or maintain) an extravascular tissue (e.g., tumor tissue) concentration of about 0.1 μg / ml, 0.2 μg / ml, 1 μg / ml, 2 μg / ml, respectively.

[0232] In one embodiment, the anti-CD73 antibody is administered, for example, to achieve and / or maintain a tissue (e.g., tumor environment) concentration of at least 0.1 μg / ml, optionally at least 0.2 μg / ml, optionally at least 1 μg / ml, or optionally at least 2 μg / ml. The antibody can be administered, for example, to achieve and / or maintain a blood concentration of at least about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml, for example, between 1-100 μg / ml, 10-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml, or 1-10 μg / ml. The amount administered can be adjusted to provide a desired concentration maintained for a specified period of time after administration (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, etc.).

[0233] In some embodiments, an amount of anti-CD73 antibody is administered to obtain an enzymatic activity in the blood (serum) or extravascular tissue (e.g., tumor environment) corresponding to at least an EC for neutralizing CD73. 70 or EC 100 The antibody can be administered, for example, in an amount to achieve and / or maintain a blood concentration or extravascular tissue (e.g., a tumor environment) of at least about 1 μg / ml, 2 μg / ml, 10 μg / ml, or 20 μg / ml.

[0234] EC 50 ,EC 70 or EC 100 Neutralization of the enzymatic activity of CD73 can be assessed in a cellular assay (e.g., neutralization of 5' ectonucleotidase activity in MDA-MB-231 cells by hydrolysis of AMP to adenosine). With respect to neutralization of the enzymatic activity of CD73, "EC 50 ” refers to the effective concentration of an anti-CD73 antibody that produces 50% of its maximum response or efficacy with respect to neutralization of enzymatic activity. With respect to neutralization of the enzymatic activity of CD73, “EC 70 ” refers to the effective concentration of an anti-CD73 antibody that produces 70% of its maximal response or efficacy. With respect to neutralization of the enzymatic activity of CD73, “EC 100 ” refers to the effective concentration of an anti-CD73 antibody that produces a substantially maximal response or efficacy thereof relative to such neutralization of enzymatic activity.

[0235] In some embodiments, particularly for the treatment of solid tumors, the concentration achieved is designed to result in a concentration in the tissue (outside the vasculature, e.g., in a tumor or tumor environment) corresponding to at least an EC for neutralization of the enzymatic activity. 50 , optionally about or at least about EC 100 .

[0236] In one embodiment, the amount of anti-CD73 antibody is between 1 mg / kg and 20 mg / kg body weight. In one embodiment, the amount is administered to the individual once a week, once every two weeks, once a month, or once every two months.

[0237] In one embodiment, a method of treating a human subject is provided, the method comprising: administering to the subject an effective amount of an anti-CD73 antibody of the present disclosure for at least one administration cycle (optionally at least 2, 3, 4 or more administration cycles), wherein the cycle is a period of eight weeks or less, wherein for each of at least one cycle, one, two, three or four doses of the anti-CD73 antibody are administered at a dose of 1-20 mg / kg body weight. In one embodiment, the anti-CD73 antibody is administered by intravenous infusion.

[0238] Suitable treatment regimens for treating humans include, for example, administering to a patient an amount of an anti-CD73 antibody as disclosed herein, wherein the method comprises administering at least one administration cycle of at least one dose of the anti-CD73 antibody. Optionally, at least 2, 3, 4, 5, 6, 7, or 8 doses of the anti-CD73 antibody are administered. In one embodiment, the administration cycle is between 2 and 8 weeks.

[0239] Patients with cancer may be treated with anti-CD73 antibodies with or without a prior detection step to assess the expression of CD73 on cells in the tumor microenvironment (e.g., on tumor cells, CD4 T cells, CD8 T cells, B cells). Optionally, the method of treatment may include a step of detecting CD73 nucleic acid or polypeptide in a biological sample from or from a tumor of the individual (e.g., cancer tissue, tissue near or at the periphery of the cancer, cancer adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue). Determining that the biological sample includes cells expressing CD73 (e.g., expressing CD73 at a high level, high intensity of staining with an anti-CD73 antibody compared to a reference, such as healthy tissue) indicates that the patient has a cancer that may benefit greatly from treatment with an anti-CD73 antibody. In one embodiment, the method includes determining the expression level of a CD73 nucleic acid or polypeptide in a biological sample and comparing the level to a reference level corresponding to a healthy individual. Determining that the biological sample includes cells expressing CD73 nucleic acid or polypeptide at an elevated level compared to a reference level (e.g., healthy tissue) indicates that the patient suffers from a cancer that can be advantageously treated with an anti-CD73 antibody of the present disclosure. In one embodiment, detecting a CD73 polypeptide in a biological sample comprises detecting a CD73 polypeptide expressed on the surface of a malignant cell, a CD4 T cell, a CD8 T cell, or a B cell.

[0240] Determining whether an individual has a cancer characterized by cells expressing a CD73 polypeptide can, for example, comprise obtaining a biological sample (e.g., by performing a biopsy) from an individual that includes cells from a cancer environment (e.g., a tumor or tumor adjacent tissue), contacting the cells with an antibody that binds a CD73 polypeptide, and detecting whether the cells express CD73 on their surface. Optionally, determining whether an individual has cells that express CD73 comprises performing an immunohistochemistry assay.

[0241] The antibody composition can be advantageously used in combination with one or more other therapeutic agents, including agents typically used for the specific therapeutic purpose for which the antibody is administered. The additional therapeutic agent will typically be administered in a monotherapy for the specific disease or condition being treated, in an amount and treatment regimen typically used for the agent. Such therapeutic agents include, but are not limited to, anticancer agents and chemotherapeutic agents.

[0242] In one embodiment, the anti-CD73 antibody enhances the efficacy of an agent that neutralizes the inhibitory activity of human PD-1, such as inhibiting the interaction between PD-1 and PD-L1. Thus, in one embodiment, the second or additional second therapeutic agent is an antibody or other agent that neutralizes the inhibitory activity of human PD-1.

[0243] Programmed death 1 (PD-1) (also known as "programmed cell death 1") is an inhibitory member of the CD28 receptor family. The complete human PD-1 sequence can be found under GenBank accession number U64863. Inhibition or neutralization of the inhibitory activity of PD-1 can involve the use of polypeptide agents (e.g., antibodies, polypeptides fused to an Fc domain, immunoadhesins, etc.) that block PD-1 signaling induced by PD-L1. There are currently at least six agents that block the PD-1 / PD-L1 pathway, which are sold on the market or in clinical evaluation. One agent is BMS-936558 (Nivolumab / ONO-4538, Bristol-Myers Squibb; formerly MDX-1106). Nivolumab (trade name ) is an FDA-approved fully human IgG4 anti-PD-L1 mAb that inhibits the binding of the PD-L1 ligand to both PD-1 and CD80, and is described as antibody 5C4 in WO 2006 / 121168, the disclosure of which is incorporated herein by reference. For melanoma patients, the most significant OR was observed at a dose of 3 mg / kg, while for other cancer types, it can be observed at 10 mg / kg. Nivolumab is usually administered at 10 mg / kg every 3 weeks until cancer progresses. The terms "reducing the inhibitory activity of human PD-1", "neutralizing PD-1" or "neutralizing the inhibitory activity of human PD-1" refer to the process in which the signal transduction ability of PD-1 is inhibited due to the interaction of PD-1 with one or more of its binding partners, such as PD-L1 or PD-L2. Agents that neutralize the inhibitory activity of PD-1 reduce, block, inhibit, eliminate or interfere with signal transduction caused by the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and PD-L2. Therefore, such agents can reduce negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes, thereby enhancing T cell effector functions such as proliferation, cytokine production and / or cytotoxicity.

[0244] MK-3475 (human IgG4 anti-PD1 mAb from Merck), also known as lambrolizumab or pembrolizumab (trade name ) has been approved by the FDA for the treatment of melanoma and is being tested in other cancers. Pembrolizumab is tested at 2 mg / kg or 10 mg / kg every 2 or 3 weeks until disease progression. MK-3475, also known as Merck 3745 or SCH-900475, is also described in WO2009 / 114335.

[0245] MPDL3280A / RG7446 (atezolizumab from Roche / Genentech, trade name Tecentriq TM MPDL3280A (MPDL3280A, anti-PD-L1) is a human anti-PD-L1 mAb containing an engineered Fc domain designed to optimize efficacy and safety by minimizing FcγR binding and subsequent antibody-dependent cellular cytotoxicity (ADCC). Doses of ≤1 mg / kg, 10 mg / kg, 15 mg / kg, and 25 mg / kg MPDL3280A were administered every 3 weeks for up to 1 year. In a Phase 3 trial, 1200 mg of MPDL3280A was administered by intravenous infusion every three weeks in NSCLC.

[0246] AMP-224 (Amplimmune and GSK) is an immunoadhesin comprising the extracellular domain of PD-L2 fused to an Fc domain. Other examples of agents that neutralize PD-1 may include antibodies that bind to PD-L2 (anti-PD-L2 antibodies) and block the interaction between PD-1 and PD-L2.

[0247] Pidlizumab (CT-011; CureTech) (a humanized IgG1 anti-PD1 mAb from CureTech / Teva), Pidlizumab (CT-011; CureTech) (see, e.g., WO2009 / 101611) is another example; the agent was tested in thirty patients with rituximab-sensitive relapsed FL, treated with 3 mg / kg intravenous CT-011 every 4 weeks in combination with weekly dosing of rituximab at 375 mg / m2 for 4 infusions for 4 weeks, starting 2 weeks after the first infusion of CT-011.

[0248] Other known PD-1 antibodies and other PD-1 inhibitors include AMP-224 (B7-DC / IgG1 fusion protein licensed from GSK), AMP-514 described in WO 2012 / 145493, and the antibody MEDI-4736 described in WO 2011 / 066389 and US 2013 / 034559 (durvalumab, trade name Imfinzi TM, anti-PD-L1 developed by AstraZeneca / Medimmune), antibody YW243.55.S70 (anti-PD-L1) described in WO2010 / 077634, MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody developed by Bristol-Myers Squibb described in WO2007 / 005874, and antibodies and inhibitors described in WO2006 / 121168, WO2009 / 014708, WO2009 / 114335 and WO2013 / 019906, the disclosures of which are incorporated herein by reference. Additional examples of anti-PD1 antibodies are disclosed in WO2015 / 085847 (Shanghai Hengrui Pharmaceutical Co. Ltd.), for example, antibodies having light chain variable domain CDR1, 2 and 3 of SEQ ID NO: 6, SEQ ID NO: 7 and / or SEQ ID NO: 8, respectively, and antibodies having heavy chain variable domain CDR1, 2 and 3 of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, respectively, wherein SEQ ID NO references are numbered according to WO2015 / 085847, the disclosure of which is incorporated herein by reference. Antibodies that compete with any of these antibodies for binding to PD-1 or PD-L1 can also be used. In some embodiments, the PD-1 neutralizer is an anti-PD-L1 antibody that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 neutralizer is an anti-PD1 antibody that inhibits the binding of PD-1 to PD-L1. An exemplary anti-PD-1 antibody is pembrolizumab (commercialized as Keytruda by Merck & Co. TM , see also WO2009 / 114335, the disclosure of which is incorporated herein by reference). An exemplary anti-PD-L1 antibody is the antibody MEDI-4736 (dubamibulin, trade name Imfinzi TM , anti-PD-L1 developed by AstraZeneca / Medimmune).

[0249] In the treatment method, the CD73 binding compound and the second therapeutic agent can be administered separately, together or sequentially, or in a combined administration form. In some embodiments, the antigen binding compound is administered before the second therapeutic agent is administered. For example, the CD73 binding compound can be administered from about 0 days to 30 days before the second therapeutic agent is administered. In some embodiments, the CD73 binding compound is administered from about 30 minutes to about 2 weeks, about 30 minutes to about 1 week, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 1 day, or about 1 day to 5 days before the second therapeutic agent is administered. In some embodiments, the CD73 binding compound is administered simultaneously with the administration of the therapeutic agent. In some embodiments, the CD73 binding compound is administered after the second therapeutic agent is administered. For example, the CD73 binding compound can be administered from about 0 days to 30 days after the second therapeutic agent is administered. In some embodiments, the CD73-binding compound is administered about 30 minutes to about 2 weeks, about 30 minutes to about 1 week, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to 1 day, or about 1 day to 5 days after administration of the second therapeutic agent.

[0250] Examples

[0251] method

[0252] Cloning, production and purification of recombinant huCD73

[0253] Molecular Biology

[0254] The huCD73 protein was cloned from MIAPACA-2 cDNA using the following primers: TACGACTCACAAGCTTGCCGCCACCATGTGTCCCCGAGCCGCGCG (SEQ ID NO: 20) (forward) and CCGCCCCGACTCTAGAtcaGTGATGGTGATGATGGTGcttgat ccgaccttcaactg (SEQ ID NO: 21) (reverse). The purified PCR product was then cloned into an expression vector using the InFusion cloning system. A 6xHis tag was added to the C-terminal portion of the protein for the purification step.

[0255] Amino acid sequence of cloned huCD73:

[0256] MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVAR

[0257] LFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEG

[0258] LIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGT

[0259] NLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGN

[0260] PPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIP

[0261] EDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDE

[0262] MFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKKAFEHSVHR

[0263] YGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANG

[0264] GDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKHHHHHH (SEQ ID NO: 22).

[0265] Expression and purification of huCD73 protein

[0266] After verifying the sequence of the clones, the cells were nucleofected and the resulting pool was then subcloned to obtain cell clones producing huCD73 protein. The supernatant from the huCD73 clones grown in drums was harvested and purified using a Ni-NTA column and eluted using 250 mM imidazole. The purified protein was then loaded onto a S200 size exclusion chromatography column. The purified protein corresponding to the dimer was formulated in Tris 20 mM pH 7.5, NaCl 120 mM and CaCl2 4 mM buffer for enzyme activity assays, while the formulation buffer was supplemented with 20% glycerol.

[0267] SPR analysis to evaluate Ab KD on recombinant CD73 protein

[0268] At 25 °C in Biacore TM SPR measurements were performed on a T200 device. Protein-A (GE Healthcare) was immobilized on a sensor chip CM5 (GE Healthcare). EDC / NHS (N-ethyl-N'-(3-dimethylaminopropyl) carbodiamide hydrochloride) and N-hydroxysuccinimide (Biacore, GE Healthcare) were used to analyze the protein. TM ) to activate the chip surface. Protein A was diluted to 10 μg / mL in coupling buffer (10 mM acetate, pH 5.6) and injected until an appropriate immobilization level (i.e., 2000 RU) was achieved. 100 mM ethanolamine pH 8 (Biacore GE Healthcare) was used. TM ) to perform deactivation of the remaining activated groups. The standard capture kinetics protocol recommended by the manufacturer (GE Healthcare Kinetics Wizard Biacore TM ) Affinity studies were performed. Serial dilutions of human recombinant soluble CD73 protein ranging from 1.23 to 300 nM were sequentially injected over the captured anti-CD73 antibody and allowed to dissociate for 10 minutes before regeneration. A 1:1 kinetic binding model was fitted to the entire sensor set. Bivalent affinity and kinetic association and dissociation rate constants were calculated.

[0269] Flow cytometric analysis to evaluate the recognition of antibodies against CD73

[0270] Resuspend the 10 5 MDA-MB-231 cells are distributed into round-bottom 96W microplates (50 μL per well).Anti-CD73 mAb of a certain dose range is added to the plate, and the cells are cultivated for 1 hour at +5 ± 3 ° C. The cells are washed three times in FCSB by rotating the plate at 400 g for 3 minutes at 4 ° C. The PE-conjugated goat F (ab') 2 anti-human IgG (H + L) secondary Ab diluted in FCSB is added to the cells, and the plate is cultivated for another 30 minutes at +5 ± 3 ° C. The cells are washed three times as described above and analyzed on a flow cytometer.

[0271] The median fluorescence intensity versus mAb concentration was plotted on a graph and analyzed using GraphPad Prism TM Program to calculate EC 50 .

[0272] In vitro enzymatic assays using recombinant human or cynomolgus monkey CD73

[0273] Briefly, recombinant human or cynomolgus CD73 was incubated in white 96W flat-bottom microplates in the presence of a dose range of anti-CD73 or isotype control mAbs. The plates were incubated for 1 hour at +37±1°C. ATP (12.5 μM) and AMP (125 μM) were added to each well and the plates were incubated for another 30 minutes at +37±1°C. CellTiter-Glo containing luciferase / luciferin was added to the plates. TM (Promega) was added to the wells, the plates were incubated in the dark at RT for 5 min, and the Enspire TM The emitted light was measured with a Spectroscopy® instrument (Perkin Elmer).

[0274] ·condition:

[0275] ATP+AMP: Maximum luciferase inhibition (100%)

[0276] CD73+ATP+AMP: No luciferase inhibition (0%)

[0277] Using GraphPad Prism7 TM The software plots residual enzyme activity versus CD73 Ab concentration on a graph.

[0278] T cell proliferation assay

[0279] Peripheral blood was obtained from healthy donors and mononuclear cells were isolated on a Ficol gradient. Lymphocytes were further enriched on 52% Percoll. TM Gradient (cell pellet) and 2 μM CellTrace TM Violet (Thermofisher) was used for staining. 4 to 1×10 5 The stained cells were distributed in 96w round bottom plates, incubated with anti-CD73 Ab for 1 hour at 37°C and activated for 3 to 5 days by adding anti-CD3 / anti-CD28 coated beads. Inhibition of T cell proliferation was achieved by adding 200 μM AMP. T cell proliferation and the ability of Ab to block the immunosuppressive effect of AMP were assessed by flow cytometry by quantifying the dye dilution on proliferating T cells. The results were analyzed using GraphPad Prism TM The software plots the percentage of proliferating T cells against the CD73 Ab concentration on a graph.Some experiments were performed on whole PBMCs from healthy donors or cancer patients; the protocol was as described above except that T cell inhibition was achieved by adding 0.5 to 1 mM ATP.

[0280] For comparison between donors or patients, T cell proliferation was normalized using the following formula:

[0281]

[0282] Allogeneic mixed lymphocyte reaction (MLR) assay

[0283] Monocytes from healthy donors were separated on a Ficoll gradient and purified by immunomagnetic selection using CD14 microbeads (Miltenyi Biotec). Monocytes were differentiated into dendritic cells (MoDCs) by culturing for 5-7 days in the presence of GM-CSF (400 ng / ml) and IL-4 (20 ng / ml). On the day of DC recovery, CD4+ T cells from allogeneic donors were purified by immunomagnetic depletion of non-CD4+ T cells (Miltenyi Biotec) and purified by Cell Trace TM DCs (10 4 cells / well) and T cells (5×10 4 T cell proliferation and the ability of Ab to reverse ATP-mediated inhibition were assessed as described for the T cell proliferation assay.

[0284] Example 1: Modeling and generation of anti-huCD73 antibodies with human framework sequences

[0285] The parent antibody 3C12 described in PCT Publication WO2016 / 055609 having the VH and VL amino acid sequences of SEQ ID NOs: 27 and 28, respectively, was modified by introducing into the VH a heavy chain framework (FR1, FR2, FR3) from human subgroup IGHV1-3*01 together with IGHJ4*01 (FR4) and into the VL a light chain framework (FR1, FR2, FR3) from human subgroup IGKV1-33*01 together with IGKJ2*01 (FR4).

[0286] Three-dimensional models based on different human VH and VL gene segments were superimposed and all amino acid differences were examined one by one. The 3D models of both the parent chimeric (HPLP) and humanized (H0L0) antibodies were used to stimulate computer molecular design. Intra-chain and extra-chain connections between residues were also evaluated to identify and avoid the destruction of any important low-energy bonds.

[0287] The VH and VL sequences of each designed domain are cloned into vectors containing huIgG1 derived constant domains and huCk constant domains with L234A / L235E / G237A / A330S / P331S substitutions, respectively. The vectors obtained by both are co-transfected into a CHO cell line. The cell pool established is used to produce antibodies in CHO culture medium. The antibodies are then purified using protein A. The heavy and light chain sequences for modeling a chimeric Fab version of 3C12 comprising an Fc domain including L234A / L235E / G237A / A330S / P331S substitutions (retaining the glycosylation of N297 connections) with a human IgG1 constant region are as follows:

[0288] HP Fab heavy chain (variable domain underlined)

[0289] QIQLQQSGPELVKPGASVKVSCKASGYAFASYNMNWVKQSHGKSLDWIGYIDPYNGGSSYNLT

[0290] FKGKATLTVDKSSTTAYMHLNSLTSEDSAVYYCARGYGNYKAWFAYWGQGTLVTVSA ASTKGP

[0291] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

[0292] TVPSSSLGTQTYICNVNHKPSNTKVDK (SEQ ID NO: 23).

[0293] LP Fab light chain (variable domain underlined)

[0294] DVVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASTRYTGVPDRFT

[0295] GSGYGTDFTFTISTVQAEDLAVYFCQQDYSSLTFGAGTKLELK RTVAAPSVFIFPPSDEQLKS

[0296] GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24).

[0297] H0 Fab heavy chain (variable domain underlined)

[0298] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMYWVRQAPGQRLEWMGYIDPYNGGSSYNLT

[0299] FKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARGYGNYKAWFAYWGQGTLVTVSS ASTKGP

[0300] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK (SEQ ID NO: 25).

[0301] L0 Fab light chain (variable domain underlined)

[0302] DIQMTQSPSSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYASTRYTGVPSRFS

[0303] GSGSGTDFTFTISSLQPEDIATYYCQQDYSSLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKS

[0304] GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26).

[0305] The heavy chain variable region and light chain variable region sequences of the HPLP parent antibody are shown below:

[0306] HP VH

[0307] QIQLQQSGPELVKPGASVKVSCKASGYAFASYNMNWVKQSHGKSLDWIGYIDPYNGGSSYNLTFKGKATLTVDKSSSTTAYMHLNSLTSEDSAVYYCARGYGNYKAWFAYWGQGTLVTVSA (SEQ ID NO: 27).

[0308] LP VL

[0309] DVVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYASTRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSLTFGAGTKLELK (SEQ ID NO: 28).

[0310] Heavy chain design

[0311] In order to study whether residue I2 (also position 2 according to Kabat numbering) forms a hydrophobic bond with CDR_H3-Y109 and plays a role in CDR_H3 positioning or flexibility, the valine residue at position 2 was replaced by isoleucine. In two models, the side chains of V2 and I2 are inconsistent. V2 also forms a hydrophobic bond with H0-Y109 (Kabat Y108), but the Y109 position changes slightly, and H0-Y109 forms a h bond with LC-Y55 (Kabat Y54). Y55 is a CDR_L2 residue. V2 substitution may affect the position of both CDR_H3 and CDR_L2.

[0312] To investigate whether the residues at positions 28 and 30 (framework 1, also positions 28 and 30 according to Kabat) affect the binding to the antigen, these positions were replaced by alanine. Compared to the YTFT of the H0L0 antibody, the parent antibody has the sequence YAFA. Residues A28 and A30 are exposed to the molecular surface close to the para position. It seems possible that the threonine at position 28 would negatively affect the binding to the antigen. On the other hand, A30 does not seem to be crucial.

[0313] To investigate whether the residue at position 44 (also Kabat 44) plays a role in the VH / VL interface, this residue was backmutated to retain the serine present in the parental antibody. The parental antibody has the sequence GKS compared to GQR of the H0L0 antibody. R44 is located at the VH / VL interface, very close to the L45 side chain.

[0314] To investigate whether the residue at position 48 (also Kabat 48) is important, a back mutation was performed on this residue, thereby retaining the residue present in the parental antibody. The parental antibody has the sequence WIG compared to WMG of the H0L0 antibody. I48 and M48 appear to occupy a key position just below CDR_H2 and are involved in a complex hydrophobic network.

[0315] To investigate the importance of the residues at positions 67 (Kabat position 66) and 68 (Kabat position 67), these residues were backmutated, retaining the residues present in the parental antibody. The parental antibody had the sequence KAT compared to the RVT of the H0L0 antibody. The loop between F64 (Kabat F63) and K67 / R67 (Kabat K66 / R66) showed very different conformations in the two models. A68 (Kabat 67) is a Vernier residue.

[0316] To investigate the role of the residue at Abnum position 70 (Kabat position 69), a back mutation was performed on this residue, thereby retaining the leucine present in the parental antibody. Compared with the TIT of the H0L0 antibody, the parental antibody has the sequence TLT. L70 and I70 (Kabat L69 and I69) involve a complex hydrophobic network.

[0317] In order to study the effect of the residue at position 72 (Kabat 71), a back mutation was performed on this residue, thereby retaining the serine present in the parent antibody. Compared with the TRD of the H0L0 antibody, the parent antibody has the sequence TVD. V72 and R72 (Kabat V71 and R71) occupy the key position just below CDR_H2. The distal hydrophilic side chain of residue V72 is prominent at the molecular surface and forms an h-key with N55 (Kabat N54). N55 occupies the same position in both models. However, the h-key with R72 (Kabat R71) may reduce the flexibility for the ring. Therefore, it is impossible to predict the impact of replacing the parent valine with arginine on antigen binding in H0L0.

[0318] To investigate whether the residue at position 74 (Kabat 73) affects the binding to the antigen, a back mutation was performed on this residue, thereby retaining the serine present in the parent antibody. Compared with the DTS of the H0L0 antibody, the parent antibody has the sequence DKS. K74 and T74 (Kabat K73 and T73) are exposed on the molecular surface near the para position, and they may be involved in antigen binding.

[0319] The first heavy chain variant (H1) having the amino acid sequence shown in SEQ ID NO:29 has V2I and T73K substitutions. The second heavy chain (H2) variant having the amino acid sequence shown in SEQ ID NO:30 has substitutions V2I, T28A, R71V and T73K. The third heavy chain variant (H3) having the amino acid sequence shown in SEQ ID NO:31 has substitutions V2I, T28A, M48I, R66K, V67A, R71V and T73K. The fourth heavy chain variant (H4) having the amino acid sequence shown in SEQ ID NO:32 has substitutions V2I, T28A, T30A, M48I, R66K, V67A, I69L, R71V and T73K. The numbering of the substitutions is according to Kabat.

[0320] Light chain design

[0321] Compared to DIQM of the H0L0 antibody, the parent antibody has the sequence DVVM. Residue V2 forms a hydrophobic bond with A25. Residue I2 may be exposed at the para surface, but the effect on the para organization cannot be predicted. I2 does not form a hydrophobic bond with A25, and this may slightly change the position of CDR-L1. Therefore, the isoleucine at residue 2 is replaced by valine.

[0322] Compared with the PSR of H0L0 antibody, the parent antibody has sequence PDR. Residue R61 occupies a rotamer position. The loops appear to be oriented in the same way, but do not overlap (lateral translation corresponding to the global translation of the entire domain). In the parent antibody, D60 forms a salt bridge and h-bond with CDR-L2-R54. In the H0L0 antibody, residue S60 does not contact R54. R54 occupies a rotamer position. Contact with D60 may be crucial for applying a specific and appropriate position to CDR-L2 residues. Therefore, the aspartic acid residue at position 60 in the H0L0 antibody is replaced by serine.

[0323] The parent antibody had the sequence GYG compared to GSG of the H0L0 antibody.To investigate whether residue Y67 contributes to paraposition, the serine in the H0L0 antibody was replaced by tyrosine.

[0324] The parent antibody had the sequence YFC compared to YYC of the H0L0 antibody. Although Y87 did not appear to form h-bonds with adjacent residues, the role of this residue remains to be investigated, and the tyrosine at position 87 in H0L0 was substituted with phenylalanine.

[0325] The first light chain variant (L1) having the amino acid sequence shown in SEQ ID NO: 33 has the substitution S67Y. The second light chain (L2) variant having the amino acid sequence shown in SEQ ID NO: 34 has the substitutions S60D and S67Y. The third light chain variant (L3) having the amino acid sequence shown in SEQ ID NO: 35 has the substitutions I2V, S60D and S67Y. The fourth light chain variant (L4) having the amino acid sequence shown in SEQ ID NO: 36 has the substitutions I2V, S60D, S67Y and Y87F.

[0326] The amino acid sequences of the corresponding heavy ("H" chain in Table 1) and light ("L" chain in Table 1) chain variable regions are shown in Table 1 below.

[0327] Table 1

[0328]

[0329] Antibodies were generated having the heavy and light chain combinations shown in Table 2 below.

[0330] Table 2

[0331]

[0332] Example 2: Binding to soluble CD73 protein by SPR

[0333] The binding of the antibodies in Table 2 of Example 1 was tested for their binding to human soluble dimeric CD73 protein and they were cloned, produced and purified. The affinity and association and dissociation constants of the humanized variants were evaluated by SPR analysis. Table 3 summarizes all calculated constants.

[0334] The H0L0 antibody with the entire human IGHV1-3*01 and IGHJ4*01 heavy chain framework and the entire human IGKV1-33*01 and IGKJ2*01 light chain framework resulted in a KD of 65 nM. As shown in Table 3, the humanized variants HxL0 (H1L0, H2L0, H3L0 and H4L0) exhibited equivalent association constants to the other variants, but had faster dissociation curves. Therefore, the KD of the HxL0 variant was higher than those observed for all other variants (Table 3). The introduction of the complete human IGKV1-33*01 and IGKJ2*01 light chain amino acid sequences in the humanized variants seemed to be detrimental to the affinity of the humanized Ab for CD73.

[0335] However, all other humanized variants had similar affinity for CD73 as the chimeric parental 3C12 antibody (0.68 nM). Notably, all heavy chains, including fully human unmodified IGHV1-3*01 and IGHJ4*01 heavy chain frameworks, provided high affinity binding to CD73.

[0336] Table 3

[0337] Humanized variants KD(nM) ka(1 / Ms) kd(1 / s) H0L0 65 1.36E+05 8.82E-03 H0L1 0.9 1.44E+05 1.27E-04 H0L2 0.7 1.68E+05 1.19E-04 H0L3 0.6 1.70E+05 9.49E-05 H0L4 0.6 1.37E+05 8.65E-05 H1L0 28.7 1.03E+05 2.96E-03 H1L1 0.8 1.00E+05 8.43E-05 H1L2 0.6 1.35E+05 8.38E-05 H1L3 0.8 9.26E+04 7.20E-05 H1L4 0.7 9.82E+04 6.77E-05 H2L0 5.6 2.38E+05 3.20E-03 H2L1 1.4 8.37E+04 1.18E-04 H2L2 1.3 1.18E+05 1.55E-04 H2L3 1 1.20E+05 1.23E-04 H2L4 0.9 1.20E+05 1.06E-04 H3L0 29.4 2.71E+05 7.97E-03 H3L1 1.8 7.89E+04 1.40E-04 H3L2 1.4 8.66E+04 1.22E-04 H3L3 1.1 9.57E+04 1.05E-04 H3L4 1 9.04E+04 8.91E-05 H4L0 7.8 1.58E+05 1.23E-03 H4L1 1.7 1.06E+05 1.79E-04 H4L2 0.9 7.75E+04 7.12E-05 H4L3 0.7 1.10E+05 7.69E-05 H4L4 1.2 6.01E+04 7.14E-05

[0338] Example 3: Binding to cells expressing human CD73 by flow cytometry

[0339] The binding of the humanized antibodies in Table 2 was tested for binding to CD73 expressing cells by flow cytometry. As shown in Figure 1, all Abs tested recognized human CD73 protein. The binding efficacy to huCD73 was similar between the humanized variants and the chimeric format, except for H0L0, H2L0 and H3L0, which were not as efficient as the parent antibody. These observations are consistent with the SPR data, which showed lower affinity for the HxL0 variants (H1L0, H2L0, H3L0 and H4L0). Therefore, all heavy chains, including unmodified fully human IGHV1-3*01 and IGHJ4*01 heavy chain frameworks, provided excellent binding to CD73 expressing cells comparable to the parent antibodies.

[0340] Example 4: In vitro enzymatic assay using recombinant soluble human CD73 protein

[0341] The efficacy of the humanized variants was compared with the parent antibody to inhibit the enzymatic activity of the recombinant CD73 protein ( Figure 2 ).

[0342] like Figure 2 As shown in (Panel A), high levels of luminescence were measured when ATP and CTG substrate were mixed. When AMP was added to the reaction mixture, the CTG reaction was inhibited, resulting in a decrease in luminescence. In the presence of rhCD73 protein, which hydrolyzes AMP, the level of luminescence was restored. Figure 2 Blockade of CD73 enzymatic activity by humanized variants is shown in .

[0343] The results show that, except for the L0 variants (H0L0, H1L0, H2L0, H3L0 and H4L0) whose activity was reduced compared to the parental antibody, all humanized variants showed strong inhibition of the enzymatic activity of soluble CD73 protein. However, surprisingly, for most antibodies, their potency was somewhat lower than that of the parental antibody. Only H1L4 and all H4 variants blocked enzymatic activity with potency similar to that of the parental antibody.

[0344] These experiments showed that, independent of the number of back mutations on the heavy chain, the absence of mutations on the light chain resulted in a significant reduction in the ability to inhibit the activity of soluble CD73 protein when using IGKV1-33*01 and IGKJ2*01 sequences as acceptor frameworks.

[0345] The best efficacy in blocking CD73 protein activity was achieved with H1L4 and H4Lx variants, but not H4L0 (i.e., H4L1, H4L2, H4L3, and H4L4).

[0346] Example 5: T cell proliferation assay

[0347] AMP concentrations capable of inhibiting T cell proliferation were tested in two series of experiments to establish AMP concentrations that induce high inhibition and allow recovery of proliferation in the presence of the parental 3C12 antibody. The enriched lymphocyte fraction was incubated for 3 days in the presence of anti-CD3 / anti-CD28 beads and a range of doses of AMP (50 μM to 1.6 mM to 50 μM). In two series of experiments, the best recovery percentages of CD4+ and CD8+ T cells were observed at 0.2 to 0.8 mM and 0.2 to 1 mM AMP, respectively.

[0348] The ability of the humanized variants and the parental antibody to restore T cell proliferation inhibited by 0.8 mM AMP was evaluated in two series of experiments.

[0349] As shown in Figure 3, except for the HxL0 variant, chimeric Ab and humanized Ab were able to restore T cell proliferation (for both CD4+ and CD8+ subsets). Despite their ability to restore proliferation, most humanized variants were less potent than the parental antibody. The H0L4, H1L4, and H4L4 variants exhibited similar potency levels to the parental antibody, but did not reach the full potency of the parental antibody.

[0350] A second experiment was performed using the same experimental conditions except that the L0 variant was replaced by an isotype control. The results are presented in FIG4 .

[0351] Similar to what is shown in Figure 3, chimeric and humanized Abs, except for the HxL0 variant, were able to restore T cell proliferation (for both CD4+ and CD8+ subsets). However, despite their ability to restore proliferation, the potency of most humanized variants was again somewhat lower compared to the parental antibody. The H0L4, H1L4, and H4L4 variants were closest to the efficiency of the parental antibody.

[0352] Example 6: Design of new framework and CDR variants

[0353] Surprisingly, the foregoing examples show no direct correlation between flow cytometry titration and potency in inhibiting the enzymatic activity of CD73. Interestingly, while the H1, H2, H3, and H4 variants were essentially indistinguishable based on binding affinity to CD73 recombinant protein in SPR assays and flow cytometry on CD73-expressing cells, the H2 and H3 variants were less effective than the H4 variants in the functional assay in terms of their ability to restore T cell proliferation in Example 5. One possibility is that mutations introduced into the H4 variant frameworks compensate for mutations introduced into the H2 and H3 frameworks that are detrimental to the potency of inhibiting CD73 (but not to the binding affinity of CD73). Therefore, new variants with substitutions in the heavy chain framework and / or CDR residues were designed. Based on these observations, substitutions made in the H2, H3, and H4 chains were classified as residues considered critical for function, residues that may not affect function, residues that negatively affect function, and residues that restore function.

[0354] A new heavy chain variant called "H4+" with the amino acid sequence shown below (SEQ ID NO:37; amino acid substitutions are in bold, Kabat CDRs are underlined) was designed and combined with the L1, L2, L3 and L4 light chains (SEQ ID NO:33, 34, 35 and 36, respectively) to produce four new antibodies H4+L1, H4+L2, H4+L3 and H4+L4.

[0355] H4+VH:

[0356]

[0357] The amino acid sequence of the full H4+ heavy chain including a human IgG1 Fc domain with L234A / L235E / G237A / A330S / P331S substitutions is shown below:

[0358] QIQLVQSGAEVKKPGASVKVSCKASGYTFASYNMYWVRQAPGQRLEWIGYIDPYNGGSSYNLT

[0359] FKGRVTLTRDKSASTAYMELSSLRSEDTAVYYCARGYGNYKAWFAYWGQGTLVTVSSASTKGP

[0360] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

[0361] TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKD

[0362] TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0363] WLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS

[0364] DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ

[0365] KSLSLSPGK

[0366] (SEQ ID NO:38).

[0367] The H4+L1 antibody contains an L1 light chain including a human Cκ domain, the complete sequence of which is shown below:

[0368] DIQMTQSPSSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYASTRYTGVPSR

[0369] FSGSGYGTDFTFTISSLQPEDIATYYCQQDYSSLTFGQGTKLEIKRTVAAPSVFIFPPSDEQL

[0370] KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 39).

[0371] To investigate how the same framework substitutions would affect antibodies with differences in their heavy and light chain CDRs, an additional series of four antibodies with a common heavy chain and one of four different light chains was generated. The common heavy chain, designated "2H4+," had a glutamine at Kabat position 59 (Q59) in HCDR2 (i.e., an L59Q substitution, compared to the H4+ chain), a lysine (K60) at Kabat residue 60 in HCDR2 (i.e., a T60K substitution, compared to the H4+ chain), and an asparagine at Kabat position 97 (N97) in HCDR3 (i.e., a G97N substitution, compared to the H4+ chain). The four light chains designated as 2L1, 2L2, 2L3 and 2L4 have the same framework residues as L1, L2, L3 and L4 chains, respectively, and their CDRs differ from the L1-L4 chains by the presence of a threonine at Kabat position 30 (T30) in LCDR1 (i.e., an S30T substitution compared to the L1-L4 chains), and an asparagine at Kabat residue 53 (N53) in LCDR2 (i.e., a T53N substitution compared to the L1-L4 chains). The four new antibodies generated were designated as 2H4+2L1, 2H4+2L2, 2H4+2L3 and 2H4+2L4 (see Table 4 below). Parent antibodies sharing CDRs in the mouse framework were generated, with heavy and light chain variable regions designated as 2HP and 2LP (amino acid sequences are shown in Table 5 below).

[0372] As in Example 1, all antibodies were produced as human IgG1 isotype with L234A / L235E / G237A / A330S / P331S substitutions to reduce Fc receptor binding.

[0373] Table 4

[0374]

[0375] Table 5

[0376]

[0377] The amino acid sequence of the full 2H4+ heavy chain including a human IgG1 Fc domain with L234A / L235E / G237A / A330S / P331S substitutions is shown below:

[0378] QIQLVQSGAEVKKPGASVKVSCKASGYTFASYNMYWVRQAPGQRLEWIGYIDPYNGGSSYNQK

[0379] FKGRVTLTRDKSASTAYMELSSLRSEDTAVYYCARGYNNYKAWFAYWGQGTLVTVSSASTKGP

[0380] SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

[0381] TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKD

[0382] TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0383] WLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS

[0384] DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47).

[0385] The amino acid sequence of the complete 2L1 light chain of the antibody including the human CK domain is shown below:

[0386] DIQMTQSPSSSLSASVGDRVTITCKASQSVTNDVAWYQQKPGKAPKLLIYYASNRYTGVPSRFS

[0387] GSGYGTDFTFTISSLQPEDIATYYCQQDYSSLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS

[0388] GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 48).

[0389] Example 7: Studies using novel variants of recombinant CD73 protein in in vitro enzymatic assays

[0390] The efficacy of the humanized variants was compared to the parent antibody to inhibit the enzymatic activity of the recombinant CD73 protein using the methods described in Example 4. All of the new humanized variants generated in Example 6 potently inhibited the activity of the CD73 protein.

[0391] The H4+Lx antibody was as efficient as its parental counterpart. In addition, all 2H4+2Lx (2H4+L1, 2H4+L2, 2H4+L3, and 2H4+L4) variants showed increased potency of CD73 inhibition compared to the H4+Lx variant. The 2H4+2Lx variants inhibited the activity of CD73 with efficacy similar to that of the parental 2HP2LP antibody.

[0392] Additional experiments were performed using different concentrations of CD73 protein (50, 100, 200, 400 ng / mL). The purpose of this experiment was to study the ability of the humanized antibody to block the activity of large amounts of CD73 protein. Again, the 2H4+2Lx variant was as effective as the parental 2HP2LP antibody.

[0393] Example 8: Study of the efficacy of new variants against cynomolgus monkey CD73 protein

[0394] Experiments were performed on recombinant rec cyCD73 protein using 2H1Lx and 2H4+2Lx variants. The experimental conditions were the same as for the human protein experiments, except for the concentration of cyCD73 protein used (400 ng / mL).

[0395] The efficacy of the humanized variants in blocking the enzymatic activity of the cyCD73 protein was the same as that of the parent (chimeric) antibody.

[0396] Example 9: Study of new variants in T cell proliferation assay

[0397] Humanized variants of different antibodies were tested for their ability to restore T cell proliferation inhibited by AMP.

[0398] Figure 5A The inhibitory effect of AMP on T cell proliferation is shown. All humanized variants strongly blocked the inhibitory effect of AMP on T cell proliferation ( Figure 5Band C). While most humanized variants generally appeared to be as efficient as their chimeric parental counterparts in reversing AMP-mediated T cell inhibition, the 2H4+2Lx variant was the most potent of all and, surprisingly, blocked the inhibitory effects of AMP even more effectively than the parental 2HP2LP antibody.

[0399] Example 10: Comparative study of a novel variant in two human donors in a T cell proliferation assay

[0400] Based on the results obtained previously, the 2H4+2Lx humanized variant was further characterized in an additional series of T cell proliferation experiments.

[0401] Fig. 6A and 6B Results obtained using cells from two healthy donors in a T cell proliferation assay are shown separately. As previously observed, each of the 2H4+2Lx variants restored T cell proliferation more efficiently than the parental 2HP2LP ( Fig. 6A A and B, right). No significant differences were observed between the different 2H4+2Lx variants. Thus, once again, the 2H4+2Lx variants blocked the inhibitory effects of AMP more effectively than the parental 2HP2LP antibody.

[0402] In summary, the results obtained in the T cell proliferation assays indicate that the humanized variants with frameworks of both H4+ and H4+ chains are the most potent antibody variants and that the 2H4+2Lx variant overall has the greatest potency among all antibodies.

[0403] Example 11: Investigation of novel variants in the allogeneic mixed lymphocyte reaction (MLR) assay

[0404] The 2H4+2Lx antibody variants were tested in an allogeneic MLR to confirm the results previously obtained in a T cell proliferation assay. T cell proliferation was inhibited by the addition of 100 μM ATP (degraded to ADP and AMP by CD39), Figure 7 Two human donor samples are shown.

[0405] like Figure 7 , as shown in the left figure, T cell proliferation was inhibited by adding ATP. T cell proliferation was restored in the presence of anti-CD73 antibody. All 2H4+2Lx humanized variants were able to restore T cell proliferation with efficacy comparable to or better than the parental 2HP2LP antibody. In these cases, all 2H4+2Lx variants restored T cell proliferation more effectively than the parental 2HP2LP. These results are consistent with the results obtained using AMP as an inhibitor in T cell proliferation assays.

[0406] In summary, the substitutions introduced in the H4+ and 2H4+ variable regions together with the substitutions introduced into the L1 and 2L1 chains (as well as the L2, L3, L4, 2L2, 2L3, 2L4 chains) appear to restore (and even improve) the important functional properties of their parent murine antibodies, but with human framework regions and thus a reduced risk of human immunogenicity. The 2H4+2Lx antibody is the most potent of all.

[0407] One possible explanation for the surprising finding that binding affinity for CD73 is not directly related to the efficacy of functional inhibition of CD73 activity may be related to the fact that these antibodies that bind to CD73 act as allosteric inhibitors. The antibodies bind to CD73 in an intradimer binding mode with a 1:1 stoichiometry between the intact full-length antibody and the CD73 dimer. Previous structural studies of CD73 have shown that its enzymatic activity requires extensive N-terminal domain rotation to define the open (inactive) and closed (active, substrate-bound) states of the enzyme (Knapp et al., 2012 Structure 20(12):2161-73). Based on the crystal structure of the parent anti-CD73 F(ab) complexed with the extracellular domain of CD73, and given the 1:1 stoichiometry between the intact antibody and the CD73 dimer, it is expected that steric hindrance will make it unlikely that the intact antibody will bind to the CD73 open conformer. Instead, the intact antibody confines CD73 to an intermediate state where AMP cannot be hydrolyzed. Since CD73 is thus bound by the antibody while in an energetically unstable conformation, small changes that alter the rigidity of the antibody structure here may affect the ability of the antibody to adapt to conformational changes of the CD73 dimer. These changes may therefore affect the ability of the antibody to inhibit CD73 activity in the presence of substrate. However, these small changes in antibody structure are not observed in SPR or other binding assays performed in the absence of substrate. The heavy and light chain variable regions described herein appear to be able to capture (and even improve upon) the full CD73 interaction kinetics of the parent antibody while being incorporated into a human framework.

[0408] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of whether incorporation of a specific document referenced elsewhere herein is provided separately.

[0409] The use of the terms "a," "an," and "the" and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0410] Unless otherwise indicated, all exact values ​​provided herein represent corresponding approximate values ​​(eg, all exact exemplary values ​​provided for a particular factor or measurement may be considered to also provide the corresponding approximate measurement, modified by "about," where appropriate).

[0411] The description of any aspect or embodiment herein using terms such as "comprising," "having," "including," or "containing" is intended to provide support for similar aspects or embodiments herein "consisting of," "consisting essentially of," or "consisting essentially of the one or more specific elements," unless otherwise stated or clearly contradicted by context (for example, a composition described herein comprising a particular element should be understood to also describe a composition consisting of the element, unless otherwise stated or clearly contradicted by context).

[0412] Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. Any language in this specification should not be construed as indicating any unclaimed element as necessary to practice the present invention. Sequence Listing <110> Innate Pharmaceuticals <120> CD73 blocking antibodies <130> CD73-6 <150> US 62 / 837,214 <151> 2019-04-23 <160> 48 <170> PatentIn Version 3.5 <210> 1 <211> 574 <212> PRT <213> Homo sapiens <400> 1 Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu 1 5 10 15 Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu 20 25 30 His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser 35 40 45 Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu 50 55 60 Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu 65 70 75 80 Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr 85 90 95 Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala 100 105 110 Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile 115 120 125 Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro Ile Leu Ser Ala Asn Ile 130 135 140 Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro 145 150 155 160 Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr 165 170 175 Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val 180 185 190 Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu Val Asp Lys Leu Lys 195 200 205 Thr Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu 210 215 220 Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val 225 230 235 240 Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys 245 250 255 Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile Val Thr Ser Asp Asp Gly 260 265 270 Arg Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly 275 280 285 Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His 290 295 300 Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile 305 310 315 320 Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr 325 330 335 Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser 340 345 350 Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met 355 360 365 Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val 370 375 380 Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu 385 390 395 400 Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro 405 410 415 Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys 420 425 430 Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu 435 440 445 Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys 450 455 460 Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg 465 470 475 480 Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile 485 490 495 Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys 500 505 510 Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val 515 520 525 Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly 530 535 540 Arg Ile Lys Phe Ser Thr Gly Ser His Cys His Gly Ser Phe Ser Leu 545 550 555 560 Ile Phe Leu Ser Leu Trp Ala Val Ile Phe Val Leu Tyr Gln 565 570 <210> 2 <211> 5 <212> PRT <213> House mouse <400> 2 Ser Tyr Asn Met Tyr 1 5 <210> 3 <211> 17 <212> PRT <213> House mouse <220> <221> X <222> (13) <223> X can be Gln or Leu <220> <221> X <222> (14)..(14) <223> X can be Lys or Thr <400> 3 Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Xaa Xaa Phe Lys 1 5 10 15 Gly <210> 4 <211> 11 <212> PRT <213> House mouse <220> <221> X <222> (3) <223> X can be Asp or Gly <400> 4 Gly Tyr Xaa Asn Tyr Lys Ala Trp Phe Ala Tyr 1 5 10 <210> 5 <211> 11 <212> PRT <213> House mouse <220> <221> X <222> (7) <223> X can be Thr or Ser <400> 5 Lys Ala Ser Gln Ser Val Xaa Asn Asp Val Ala 1 5 10 <210> 6 <211> 7 <212> PRT <213> House mouse <220> <221> X <222> (4) <223> X can be Thr or Asn <400> 6 Tyr Ala Ser Xaa Arg Tyr Thr 1 5 <210> 7 <211> 8 <212> PRT <213> House mouse <400> 7 Gln Gln Asp Tyr Ser Ser Leu Thr 1 5 <210> 8 <211> 17 <212> PRT <213> House mouse <400> 8 Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe Lys 1 5 10 15 Gly <210> 9 <211> 11 <212> PRT <213> House mouse <400> 9 Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> House mouse <400> 10 Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala 1 5 10 <210> 11 <211> 7 <212> PRT <213> House mouse <400> 11 Tyr Ala Ser Thr Arg Tyr Thr 1 5 <210> 12 <211> 17 <212> PRT <213> House mouse <400> 12 Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 13 <211> 11 <212> PRT <213> House mouse <400> 13 Gly Tyr Asn Asn Tyr Lys Ala Trp Phe Ala Tyr 1 5 10 <210> 14 <211> 11 <212> PRT <213> Mus musculus <400> 14 Lys Ala Ser Gln Ser Val Thr Asn Asp Val Ala 1 5 10 <210> 15 <211> 7 <212> PRT <213> Mus musculus <400> 15 Tyr Ala Ser Asn Arg Tyr Thr 1 5 <210> 16 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 16 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 17 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 17 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 18 <211> 330 <212> PRT <213> Synthesis <400> 18 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 19 <211> 330 <212> PRT <213> Synthetic <400> 19 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 20 <211> 45 <212> DNA <213> Homo sapiens <400> 20 tacgactcac aagcttgccg ccaccatgtg tccccgagcc gcgcg 45 <210> 21 <211> 57 <212> DNA <213> Homo sapiens <400> 21 ccgccccgac tctagatcag tgatggtgat gatggtgctt gatccgacct tcaactg 57 <210> 22 <211> 553 <212> PRT <213> Artificial <220> <223> Synthetic <400> 22 Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu 1 5 10 15 Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu 20 25 30 His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser 35 40 45 Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu 50 55 60 Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu 65 70 75 80 Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr 85 90 95 Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala 100 105 110 Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile 115 120 125 Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro Ile Leu Ser Ala Asn Ile 130 135 140 Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro 145 150 155 160 Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr 165 170 175 Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val 180 185 190 Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu Val Asp Lys Leu Lys 195 200 205 Thr Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu 210 215 220 Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val 225 230 235 240 Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys 245 250 255 Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile Val Thr Ser Asp Asp Gly 260 265 270 Arg Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly 275 280 285 Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His 290 295 300 Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile 305 310 315 320 Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr 325 330 335 Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser 340 345 350 Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met 355 360 365 Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val 370 375 380 Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu 385 390 395 400 Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro 405 410 415 Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys 420 425 430 Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu 435 440 445 Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys 450 455 460 Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg 465 470 475 480 Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile 485 490 495 Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys 500 505 510 Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val 515 520 525 Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly 530 535 540 Arg Ile Lys His His His His His His 545 550 <210> 23 <211> 216 <212> PRT <213> Artificial <220> <223> Synthetic <400> 23 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ala Ser Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Asp Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys 210 215 <210> 24 <211> 213 <212> PRT <213> Artificial <220> <223> Synthetic <400> 24 Asp Val Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 25 <211> 216 <212> PRT <213> artificial <220> <223> synthesis <400> 25 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys 210 215 <210> 26 <211> 213 <212> PRT <213> artificial <220> <223> synthesis <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 27 <211> 120 <212> PRT <213> artificial <220> <223> synthesis <400> 27 Gln Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ala Ser Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Asp Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 28 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 28 Asp Val Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 29 <211> 120 <212> PRT <213> artificial <220> <223> synthesis <400> 29 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 30 <211> 120 <212> PRT <213> artificial <220> <223> synthesis <400> 30 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 31 <211> 120 <212> PRT <213> artificial <220> <223> synthesis <400> 31 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Lys Ala Thr Ile Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 32 <211> 120 <212> PRT <213> artificial <220> <223> synthesis <400> 32 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ala Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 33 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 33 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 34 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 35 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 35 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 36 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 36 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 37 <211> 120 <212> PRT <213> artificial <220> <223> synthesis <400> 37 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 450 <212> PRT <213> artificial <220> <223> synthesis <400> 38 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Leu Thr Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 39 <211> 213 <212> PRT <213> Artificial <220> <223> Synthetic <400> 39 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Thr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 40 <211> 120 <212> PRT <213> artificial <220> <223> synthesis <400> 40 Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr 20 25 30 Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met His Leu Asn Asn Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Asn Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 41 <211> 106 <212> PRT <213> Artificial <220> <223> Synthetic <400> 41 Asp Val Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Met Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 42 <211> 120 <212> PRT <213> artificial <220> <223> synthesis <400> 42 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Asn Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 43 <211> 106 <212> PRT <213> synthesis <400> 43 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 44 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 44 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 45 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 45 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 46 <211> 106 <212> PRT <213> artificial <220> <223> synthesis <400> 46 Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 47 <211> 450 <212> PRT <213> artificial <220> <223> synthesis <400> 47 Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Asn Asn Tyr Lys Ala Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Light 450 <210> 48 <211> 213 <212> PRT <213> artificial <220> <223> synthesis <400> 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Thr Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Tyr Ser Ser Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210

Claims

1. An antibody that is capable of specifically binding to a human CD73 polypeptide, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO: 42 and a light chain variable region as shown in any one of SEQ ID NOs: 43, 44, 45 and 46.

2. The antibody according to claim 1, wherein the antibody comprises a heavy chain variable region as shown in SEQ ID NO:42 and a light chain variable region as shown in SEQ ID NO:

43.

3. The antibody of claim 1, wherein the antibody comprises a heavy chain as shown in SEQ ID NO:47 and a light chain as shown in SEQ ID NO:

48.

4. The antibody according to any one of the preceding claims, wherein the antibody is capable of neutralizing the 5'-ectonucleotidase activity of the CD73 polypeptide.

5. The antibody according to any one of the preceding claims 1 to 3, wherein the antibody is selected from Fab, Fab', Fab'-SH, F(ab')2, Fv fragment, diabody or multispecific antibody.

6. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. A nucleic acid or a set of nucleic acids encoding the heavy chain and the light chain of the antibody according to any one of claims 1 to 5.

8. A recombinant host cell which produces the antibody according to any one of claims 1 to 5.

Images