RU2819204C2 - Cd73 blocking antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are antibodies and fragments thereof, which bind and inhibit human CD73. Also disclosed are nucleic acids coding said antibodies, pharmaceutical compositions containing antibodies, use of said antibodies for preparing a drug for treating cancer.

EFFECT: invention provides more effective inhibition of CD73 enzymatic activity.

9 cl, 15 dwg, 5 tbl, 11 ex

Description

LINK TO RELATED APPLICATIONS

This application claims benefit from U.S. Provisional Patent Application No. US 62/837214, filed April 23, 2019, which is incorporated herein by reference in its entirety, including all drawings.

LINK TO LIST OF SEQUENCES

This application is filed together with the Sequence Listing in electronic format. The sequence listing is provided as a file named "CD73-6_ST25", created on April 1, 2020, which is 62 kilobytes in size. The electronic format information on the Sequence Listing is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention provides antibodies and fragments thereof that bind and inhibit CD73. The present invention also provides cells that produce such compounds; methods for producing such compounds and antibodies, fragments, variants and derivatives thereof; pharmaceutical compositions containing them; methods of using the compounds for the diagnosis, treatment or prevention of diseases, such as cancer.

BACKGROUND OF THE ART

CD73 (ecto-5'-nucleotidase) is a 70 kDa glycosylphosphatidylinositol (GPI)-anchored protein that is typically expressed on endothelial cells and subsets of hematopoietic cells. CD73, together with CD39, regulates adenosine triphosphate (ATP) metabolism. CD39 (NTPDase-1) converts ATP to AMP, releasing only trace amounts of ADP, while CD73 catalyzes the conversion of AMP to adenosine.

Adenosine triphosphate (ATP) and its metabolites AMP and adenosine play important roles in cellular metabolism, signal transduction, and immune homeostasis. The release of extracellular adenosine triphosphates (ATP) in response to cell death or cellular stress leads to the activation of immune responses. However, their metabolite adenosine exhibits immunosuppressive activity. Extracellular adenosine accumulates in cancer tissues and is part of an important tumor immune evasion mechanism. Among other effects, tumor-derived adenosine potently inhibits tumor-infiltrating effector T cells via A2A receptors that activate adenylate cyclase.

CD73 expression has been reported in various tumor cells, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, and breast cancer. CD73 expression has also been associated with a prometastatic phenotype in melanoma and breast cancer. It has been reported that therapy with an antibody that binds mouse CD73 can inhibit mammary tumor growth and metastasis in mice (Stagg, et al. (2010) Proc. Natl. Acad. Sci. USA 104:1547-1552). It has been shown that genetic deletion of A2A receptors can cause T cell-dependent tumor rejection (Ohta, et al., (2006) Proc Natl Acad Sci USA 103:13132-13137). SiRNA knockdown 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 against 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 prognostic factor in triple-negative breast cancer (Loi et al., 2013, Proc. Natl. Acad. Sci. USA 110: 11091-11096). However, although CD73 is expressed on tumor cells, it is also expressed on various cells of the immune system, particularly CD4 and CD8 T cells, as well as B cells. Additionally, another complicating factor is that many of the antibodies described in the literature tended to be of mouse isotypes that Fcγ receptors are able to bind, making it difficult to distinguish any potential blocking effect from Fc-mediated effects.

Despite the long-standing interest in CD73 as a therapeutic target, there remains a need for antibodies with higher potency in inhibiting CD73 enzymatic activity that are suitable for use in human therapy.

BRIEF DESCRIPTION OF THE INVENTION

The present inventors provide antibodies that bind to an epitope present on CD73 expressed on the surface of cells, including tumor cells, and that inhibit the enzymatic (ecto-5'nucleotidase) activity of the CD73 enzyme. These antibodies contain framework regions of human origin with minimal sequence of non-human origin (eg, mouse). Antibodies can bind CD73 in an intradimeric manner and can inhibit the enzymatic activity of both soluble CD73 and membrane-associated CD73 protein expressed on the cell surface. Preferably, these antibodies can be used as purely CD73 blocking antibodies, for example, they inhibit the enzymatic activity of the membrane associated CD73 protein expressed on the surface of cells without substantially binding Fcγ receptors and/or without substantially targeting ADCC to the CD73 expressing cell. Optionally, the antibodies retain the Fc domain and retain binding to human FcRn. Preferably, said antibodies may have a low risk of immunogenicity, eg, a low or reduced likelihood (compared to the parent mouse antibody) of eliciting a human anti-mouse antibody (HAMA) response when administered to a human.

The antibodies bind to an epitope present on the human CD73 polypeptide expressed on the surface of cells, including but not limited to tumor cells, and inhibit the enzymatic (ecto-5'-nucleotidase) activity of the CD73 enzyme. They have a particularly advantageous ability to inhibit CD73-mediated suppression of T cell proliferation and may therefore cause an increase in biological activity, including, but not limited to, proliferation of cytotoxic CD4 and/or CD8 T cells, for example, as observed in the T proliferation assay -cells. In one embodiment, inhibition or neutralization of CD73 enzymatic activity is determined by assessing the ability of an antibody or antibody fragment to enhance T cell proliferation when said T cells are induced to proliferate in vitro (e.g., through TCR costimulation) in the presence of AMP (e.g., exogenously added AMP) .

In one embodiment, there is provided an antibody or antibody fragment, or an antigen binding domain thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a 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, there is provided an antibody or antibody fragment, or an antigen binding domain thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 ID NO: 33.

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

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

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

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

In one embodiment, there is provided an anti-CD73 antigen-binding domain or a protein that contains it (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) containing a heavy chain variable region (VH) containing CDR1, CDR2 and CDR3 with the corresponding amino acid sequences shown in SEQ ID NOs: 2, 8 and 9, and the amino acid sequences of framework regions FR1, FR2 and FR3 from the human IGHV1-3 gene (and, optionally, additional amino acid sequences of framework region 4 (FR4) from human IGHJ4 gene); and CDR1, CDR2 and CDR3 of the variable light chain (VL) region with the corresponding amino acid sequences set forth in SEQ ID NOs: 10, 11 and 7, and the amino acid sequences of the framework regions FR1, FR2 and FR3 from the human IGKV1-33 gene (and, optionally , additional amino acid sequences of framework region 4 (FR4) from the human IGKJ2 gene). In one embodiment, VH further comprises one, two or three amino acid substitutions in the Kabat heavy chain CDR at 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, VL further comprises an amino acid substitution in the Kabat light chain CDR at light chain Kabat positions 30 (LCDR1) and/or 53 (LCDR2), optionally further wherein the serine residue at position 30 is replaced by a threonine residue, optionally additionally at In this case, the threonine residue at position 53 is replaced by an asparagine residue.

In one embodiment, there is provided an anti-CD73 antigen-binding domain or a protein that contains it (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) containing a heavy chain variable region (VH) containing CDR1, CDR2 and CDR3 with the corresponding amino acid sequences shown in SEQ ID NOs: 2, 12 and 13, and the amino acid sequences of framework regions FR1, FR2 and FR3 from the human IGHV1-3 gene (and, optionally, additional amino acid sequences of framework region 4 (FR4) from human IGHJ4 gene); and CDR1, CDR2 and CDR3 of the variable light chain (VL) region with the corresponding amino acid sequences set forth in SEQ ID NOs: 14, 15 and 7, and the amino acid sequences of the framework regions FR1, FR2 and FR3 from the human IGKV1-33 gene (and, optionally , additional amino acid sequences of framework region 4 (FR4) from the human IGKJ2 gene).

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

In one aspect of any embodiment described herein, the amino acid at position 2 of the Kabat heavy chain is isoleucine.

In one aspect of any embodiment described herein, the amino acid at position 30 of the Kabat heavy chain is alanine.

In one aspect of any embodiment described herein, the amino acid at position 48 of the Kabat heavy chain is isoleucine.

In one aspect of any embodiment described herein, the amino acid at position 69 of the Kabat heavy chain is leucine.

In one aspect of any embodiment described herein, the amino acid at position 73 of the Kabat heavy chain is lysine.

In one embodiment, VH contains an isoleucine residue at the 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 aspect of any embodiment described herein, the amino acid at Kabat position 67 of the light chain is tyrosine.

In one aspect of any embodiment described herein, the amino acid at position 60 of the Kabat light chain is aspartic acid.

In one aspect of any embodiment described herein, the amino acid at position 2 of the Kabat light chain is isoleucine.

In one aspect of any embodiment described herein, the amino acid at position 87 of the Kabat light chain is phenylalanine.

In one embodiment, VL contains a tyrosine residue at Kabat position 67; optionally, VL contains no other substitutions in the Kabat framework region for non-human residues, and/or contains a Kabat framework region VL that is entirely human, other than the substitution at residue 67.

In one embodiment, VL contains a tyrosine residue at Kabat position 67 and an aspartic acid residue at position 60.

In one embodiment, VL contains a tyrosine residue at Kabat position 67, aspartic acid at position 60, and isoleucine at position 2.

In one embodiment, VL contains a tyrosine residue at Kabat position 67, aspartic acid at position 60, isoleucine at position 2, and phenylalanine at position 87.

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

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

In any embodiment, VH can be characterized as comprising amino acid sequences of a human VH acceptor framework region, and VL comprises amino acid sequences of a human VL acceptor framework region. In one embodiment, the VH segment of the human VH acceptor framework region is derived from the human IGHV1-3 gene segment, and the J segment is derived from the human IGHJ4 gene segment. In one embodiment, the human VH acceptor framework region is derived from the human IGHV1-3*01 gene segment. In one embodiment, the human VL domain acceptor framework region is derived from the human IGKV1-33 gene segment, optionally the human VL domain acceptor framework region is derived from the human IGKV1-33*01 gene segment. In one embodiment, the human VL domain acceptor framework region comprises a J segment from a human IGKJ2 gene segment.

In one embodiment, the antibody or antibody fragment comprises an H4+VH domain and an L1, L2, L3, or L4 VL domain. In one embodiment, the antibody is an H4+L1 antibody.

In one embodiment, a CD73-directed antigen binding domain or protein is provided that comprises said antigen binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37, and a 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 is provided that binds human CD73 and that neutralizes the ATPase activity of CD73, wherein the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region of an H4+L1 antibody.

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

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

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 a 2H4+2L1 antibody.

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

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

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

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

In one embodiment, the antibodies are non-depleting and neutralize CD73 enzymatic activity in the tumor environment.

In one embodiment, the antibody is an antibody of the human IgG1, IgG2, IgG3, or IgG4 isotype. For example, the antibody may be an antibody containing the Fc domain of a human IgG4 isotype, or an antibody containing the Fc domain of any human IgG isotype (e.g., IgG1, IgG2, IgG3, or IgG4) modified to reduce or lose binding between the Fc domain and the human Fcγ receptor (eg CD16), optionally modified to reduce binding between the Fc domain and a variety of human Fcγ receptors, eg CD16A, CD16B, CD32A, CD32B and CD64). In one embodiment, the antibodies comprise an Fc domain of a human IgG subtype (e.g., IgG1) containing an amino acid modification that results in reduced (compared to an antibody containing a wild-type human IgG1 Fc domain) or substantially complete loss of binding to each of the CD16A , CD16B, CD32A, CD32B and CD64.

In any embodiment, the antibody heavy chain comprises a human CH1 constant domain and a modified human Fc domain, optionally of the human IgG1 isotype, optionally further comprising an amino acid sequence set forth in any of SEQ ID NO: 16, 17, 18, or 19. In any embodiment the light chain of the antibody comprises a human light chain constant domain, optionally the constant domain being a human kappa domain.

In one aspect, antibodies in accordance with the present invention do not cause or increase intracellular internalization, or more generally, down-modulation, of a cell surface expressed CD73 polypeptide and/or do not depend on it to exert their CD73 inhibitory activity. Antibodies of the present invention may have greater inhibitory activity (the ability to substantially neutralize the enzymatic activity of CD73) than antibodies that inhibit CD73 by causing internalization of CD73. Unlike antibodies that inhibit soluble CD73 through other mechanisms (eg, causing the formation of a CD73-antibody oligomer), the antibodies of the present invention are capable of inhibiting CD73 enzymatic activity at high (eg, 10-fold) excess antibody:enzyme. Additionally, in contrast to antibodies that bind an epitope on recombinant CD73 that may be modified or may be absent from CD73 on the cell surface (e.g., antibody 7G2), or bind it with an affinity that is too low to result in efficacy in expressing CD73 cells, antibodies in accordance with the present invention bind with high affinity to an epitope that is present and/or remains intact on CD73 on the cell surface, which gives the antibodies the ability to effectively neutralize the enzymatic activity of cellular CD73. Antibodies of the present invention inhibit the enzymatic activity of CD73 in cells, but may optionally also inhibit the ecto-5'-nucleotidase activity of soluble recombinant CD73 (as observed in a cell-free assay using soluble dimeric CD73 polypeptide).

In one aspect, the antibodies of the present invention are capable of inhibiting the activity of a human CD73 polypeptide without binding to the active site of the CD73 polypeptide enzyme, and/or are non-competitive inhibitors of CD73, e.g., they inhibit the activity of a human CD73 polypeptide without detectably reducing the binding between the CD73 polypeptide and its natural substrate.

In one aspect, antibodies of the present invention lose binding to CD73 mutants containing a substitution at residue K136. In one aspect, there is reduced binding of antibodies in accordance with the present invention to CD73 mutants containing a substitution at residues K136 (based on the sequence of SEQ ID NO: 1; and compared to wild-type CD73 containing the amino acid sequence of SEQ ID NO: 1); optionally there is also a decrease in antibody binding to CD73 mutants containing substitutions at residues A99, E129, K133, E134 and A135 (in the sequence SEQ ID NO: 1; and compared to wild-type CD73 containing the amino acid sequence SEQ ID NO: 1; optional Additionally, there is a decrease in antibody binding to CD73 mutants containing substitutions at residues K97, E125, Q153 and K330 (based on the sequence SEQ ID NO: 1; and compared to wild-type CD73 containing the amino acid sequence SEQ ID NO: 1.

In one embodiment, the antibodies bind to an epitope on each CD73 polypeptide chain in a CD73 dimer (ie, bind to CD73 in an intradimeric manner), optionally further wherein the epitope is present on the same surface of the CD73 dimer. Thus, antibodies can bind bivalently to one CD73 dimer, that is, in a position that is more “closed,” in which the antibody binding sites are spatially further apart than in the “open” position. In view of binding to ligand-bound CD73, the antibodies described herein may be useful for binding to CD73 when bound to AMP, for example, to treat an individual or cancer characterized (e.g., known or suspected to be characterized by) a tumor environment , in which the precursor ADP and/or AMP are present at significant levels before treatment. The antibodies may be useful for treating an individual or cancer in which (eg, known or suspected) the tumor environment is characterized by high levels of ADP (eg, produced by dying cells, which hijacks CD73 on the stromal and cellular infiltrate (eg, on TReg cells) with production of high levels of AMP), and also, more generally, AMP, adenosine, the presence or levels of expression of CD73 or CD73-expressing cells, high (e.g., compared to healthy tissue) numbers or frequencies of CD73-expressing cells, and/or high levels expression of CD73 on cells (eg, as assessed by immunohistochemical analysis), high (eg, compared with healthy tissue) levels of soluble CD73 polypeptides (eg, as assessed by ELISA or, in general, any antibody-based detection assay), or the presence or expression levels of adenosine receptor or adenosine receptor-expressing cells. CD73 molecules in the tumor environment may be in a substrate-bound conformation, and the ability to bind and inhibit substrate-bound cellular CD73 (e.g., cells expressing CD73 preincubated with a substrate such as AMP), in addition to non-substrate CD73, may cause greater ability to inhibit CD73 in vivo. Optionally, the level of soluble CD73 protein, the number or frequency of CD73-expressing cells, and/or the levels of CD73 expression on cells surrounding the tumor may be assessed prior to treatment. The antibodies may be particularly useful for treating an individual who has significant levels (eg, high levels compared to reference levels) of soluble CD73 protein, the number or frequency of CD73 expressing cells, and/or levels of CD73 expression on cells in a tumor sample.

Accordingly, in one aspect of the present invention there is provided a humanized antibody or antibody fragment that binds a human CD73 polypeptide expressed on the surface of cells and inhibits the enzymatic (ecto-5'nucleotidase) activity of the CD73 polypeptide, wherein the antibody is capable of bivalently binding to one dimer of CD73 polypeptides ( with a soluble dimer of CD73 polypeptides or with a dimer of CD73 polypeptides expressed by the cell). Optionally, the antibody binds the first antigen binding domain to the first CD73 polypeptide in the dimer and the second antigen binding domain to the second CD73 polypeptide. In one aspect, the antibody is an allosteric inhibitor of a CD73 polypeptide.

The epitope on CD73 to which these antibodies bind is present on CD73 polypeptides expressed by a number of cells, for example, cancer cells, CD4 T cells, CD8 T cells, B cells, transfected cells, and binding occurs with high affinity, as determined by using flow cytometry. For example, an antibody may have an _EC50 determined by flow cytometry of 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 0.1 µg/ml or no more than 0.05 µg/ml for binding to cells that express CD73 polypeptide on the surface. In one embodiment, the cells are cells that are caused to express CD73 on their surface. In one embodiment, the cells are cells that endogenously express CD73 on the surface, e.g., cancer cells, leukemia cells, bladder cancer cells, glioma cells, glioblastoma cells, ovarian cancer cells, melanoma cells, prostate cancer cells, thyroid cancer cells glands, esophageal cancer cells or breast cancer cells.

In one embodiment, CD73 neutralizing antibodies can be characterized as being capable of causing a reduction in CD73 5'-ectonucleotidase activity in cells by at least 60%, 75%, or 80%. In one embodiment, CD73 neutralizing antibodies may have an EC ₅₀ for inhibiting the 5'-ectonucleotidase activity of CD73 expressed by the cell of no more than 1 μg/ml, optionally no more than 0.5 μg/ml, optionally no more than 0.2 μg /ml.

Optionally, inhibition of the 5'-ectonucleotidase activity of cell-expressed CD73 is determined by assessing the neutralization of 5'-ectonucleotidase activity in CD73-expressing cells (eg, MDA-MB-231 cells) by quantitating the hydrolysis of AMP to adenosine.

Optionally, inhibition of the 5'-ectonucleotidase activity of CD73 expressed by a cell is determined by assessing the ability of the antibody or antibody fragment to enhance T cell proliferation when said T cells (e.g., from a healthy human donor) are induced to proliferate in vitro (e.g., through TCR costimulation , stimulating CD3 and CD28 signaling, for example by contacting T cells with beads functionalized with CD3 agonists and CD28 agonists) in the presence of AMP (eg, exogenously added AMP). Optionally, T cell proliferation is assessed using the assay described in the Examples section herein (see Example 5 and Methods).

In one aspect, an anti-CD73 antibody binds a common antigenic determinant present on both soluble CD73 and CD73 expressed on the surface of cells.

In one aspect, an anti-CD73 antibody binds an antigenic determinant on each CD73 polypeptide chain in a CD73 dimer (in an intradimeric manner), for example, when the antigenic determinants are present on the common surface of the CD73 dimer.

In one aspect, an anti-CD73 antibody binds an epitope on CD73 containing residue K136 (at SEQ ID NO: 1).

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

In one aspect, an anti-CD73 antibody binds an epitope on CD73 containing one, two, three, four, or five residues selected from the group consisting of A99, E129, K133, E134, and A135 (in the sequence of SEQ ID NO: 1).

In one aspect, the anti-CD73 antibody binds at least partially within a domain or fragment of amino acid residues on a human CD73 protein (e.g., CD73 homodimeric protein) comprising amino acid residues K97, A99, E125, E129, K133, E134, A135, K136, Q153 and K330 (in sequence SEQ ID NO: 1). In one aspect, an anti-CD73 antibody binds an epitope on CD73 containing at least 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 (in sequence SEQ ID NO: 1).

In one aspect, an anti-CD73 antibody exhibits reduced binding to a CD73 polypeptide containing a mutation at residue K136 (at SEQ ID NO: 1) compared to a wild-type CD73 polypeptide containing the amino acid sequence SEQ ID NO: 1; optionally the mutant CD73 polypeptide contains the K136A mutation.

In one aspect, an anti-CD73 antibody exhibits reduced binding to a CD73 polypeptide containing a mutation at a residue selected from the group consisting of: K97, E125, Q153, and K330 (in SEQ ID NO: 1), compared to a wild-type CD73 polypeptide. containing the amino acid sequence SEQ ID NO: 1; optionally, the mutant CD73 polypeptide contains the following mutations: K97A, E125A, Q153A and/or K330A (eg, K97A, E125A and K330A; K97A, E125A and/or Q153A).

In one aspect, an anti-CD73 antibody exhibits reduced binding to a CD73 polypeptide containing a mutation at a residue selected from the group consisting of: A99, E129, K133, E134, and A135 (in SEQ ID NO: 1), compared to wild-type CD73 polypeptide type containing the amino acid sequence SEQ ID NO: 1; optionally, the mutant CD73 polypeptide contains the following mutations: A99S, E129A, K133A, E134N and A135S.

In one embodiment, methods are provided for using antibodies to treat or prevent a disease, such as an infectious disease or cancer. In one aspect, the antibodies are administered to an individual suffering from cancer in an amount and frequency sufficient to neutralize CD73 activity in the tumor microenvironment. In an aqueous embodiment, the antibodies are administered in an amount and frequency sufficient to reduce the formation and/or concentration of adenosine in the tumor microenvironment. In an aqueous embodiment, the antibodies are administered in an amount and frequency sufficient to increase the production and/or concentration of ATP in the tumor microenvironment. In one embodiment, the antibodies are administered in an amount and frequency sufficient to neutralize the activity of CD73 expressed by the tumor cells. In one embodiment, the antibodies are 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.

Antibodies will be useful for inhibiting CD73-mediated catabolism of AMP to adenosine, for example, to reduce adenosine concentrations in the tumor microenvironment. These antibodies will therefore be useful in reversing the immunosuppressive effects of CD73 and/or adenosine on T cells, B cells and other cells that express adenosine receptors, for example in the treatment of cancer. In one embodiment, the anti-CD73 antibody neutralizes adenosine-mediated inhibition of proliferation, cytokine production, cytotoxicity, and/or NFkB activity in T cells.

Because CD73-mediated catabolism of AMP to adenosine is irreversible, while CD73-mediated catabolism of ATP to ADP and ADP to AMP is reversible (via NDK kinase and adenylate kinase, respectively), antibodies that block CD73-mediated irreversible catabolism will increase the supply of AMP and thus be useful for increasing ADP and ATP concentrations, for example, in the tumor microenvironment. These antibodies may be useful in increasing the formation of ADP from AMP and the formation of ATP from ADP. Since ATP plays an immunostimulatory role, antibodies against CD73 may be useful in activating T cells, for example in the treatment of cancer.

Antibodies will be useful for inhibiting the production, amounts and/or concentrations of adenosine in the tumor microenvironment.

Antibodies that neutralize the activity of a soluble human CD73 polypeptide dimer may further neutralize CD73 in any other suitable context, for example, in a reporter cell that has been induced to express CD73, a T cell, etc.

A method of treating an individual is provided, the method comprising administering to an individual (eg, an individual suffering from a disease, tumor, etc.) a therapeutically active amount of any of the anti-CD73 antigen-binding compounds described herein. In one aspect, a method of treating an individual is provided, the method comprising, consisting essentially of, or consisting of: administering to an individual (eg, an individual suffering from a disease, tumor, etc.) a therapeutically active amount of an antigen-binding compound in accordance with the present invention.

In one aspect, there is provided a method of reducing the amount of adenosine produced by a CD73 expressing cell (e.g., an immune cell and/or a tumor cell in an individual), or a method of neutralizing the enzymatic activity of a cellular CD73, the method comprising, consisting essentially of, or consisting of: bringing the CD73 expressing cells into contact with an antigen-binding compound in accordance with the present invention (eg, an anti-CD73 antibody or antibody fragment, or a composition containing the same). In one embodiment, the step of contacting a CD73-expressing cell with an antigen-binding compound in accordance with the present invention comprises administering to a subject a therapeutically active amount of the antigen-binding compound. In one embodiment, the individual has cancer.

In one aspect, a method is provided for reducing the amount of adenosine present in the environment of a tumor (e.g., in the body of an individual), said method comprising, consisting essentially of, or consisting of: administering to the individual a therapeutically active amount of a compound of the present invention (e.g., directed against CD73 antibody or antibody fragment, or a composition containing the same). In one embodiment, the individual has cancer. The individual is not necessarily a person who has cancer or is susceptible to cancer.

Antibodies do not necessarily have a binding affinity (K _D ) for human CD73 polypeptide (e.g., as a CD73 dimer), as determined by surface plasmon resonance (SPR, e.g., according to the methods described in the Examples section), less (better) 10 ^-9 M, preferably less than 10 ^-10 M or preferably less than 10 ^-11 M, and/or binding to a human CD73 polypeptide with an EC ₅₀ of less (best binding) 1 μg/ml. In some cases, it can be specified that the binding affinity is a bivalent binding affinity.

In one embodiment, the EC ₅₀ of said antibody is no more than 0.5 μg/ml, in some cases no more than 0.2 μg/ml, in some cases no more than 0.1 μg/ml for binding to cells (e.g., tumor cells , MDA-MB-231 cells) expressing human CD73 on their surface.

Antibodies optionally have an EC ₅₀ for neutralizing CD73 enzymatic activity in CD73 expressing cells (eg, tumor cells, MDA-MB-231 cells) of less than (better) 1 μg/ml, optionally less than 0.5 μg/ml.

In one embodiment, the antibody is a monoclonal antibody or fragment thereof that retains binding specificity and ability to neutralize CD73 enzymatic activity (e.g., as determined by assessing the ability of the antibody or antibody fragment to enhance T cell proliferation when said T cells are induced to proliferate in vitro in the presence of AMP (eg, exogenously added AMP). In one embodiment, the antibody is substantially as effective or at least as effective as the parent antibody containing the corresponding VH and VL amino acid sequences set forth in SEQ ID NO: 40. and 41, for neutralizing CD73 enzymatic activity, optionally wherein the EC ₅₀ of the antibody to neutralize CD73 enzymatic activity is within 1-log, 0.5-log EC ₅₀ of the parent antibody. In one embodiment, said antibody is more effective than the parent antibody. containing the corresponding VH and VL amino acid sequences set forth in SEQ ID NOs: 40 and 41 for neutralizing CD73 enzymatic activity, wherein the EC ₅₀ of the antibody for neutralizing CD73 enzymatic activity is optionally lower than that of the parent antibody containing the corresponding VH and VL amino acid sequences , presented in SEQ ID NO: 40 and 41.

In one embodiment, the antibody is substantially as effective or at least as effective as the parent antibody comprising the corresponding VH and VL amino acid sequences set forth in SEQ ID NOs: 27 and 28 in neutralizing CD73 enzymatic activity, optionally at This antibody's EC ₅₀ to neutralize CD73 enzymatic activity is within 1-log or 0.5-log of the parent antibody's EC ₅₀ . In one embodiment, the antibody is more effective than the parent antibody comprising the corresponding VH and VL amino acid sequences set forth in SEQ ID NOs: 27 and 28 at neutralizing CD73 enzymatic activity, optionally the EC ₅₀ of the antibody for neutralizing CD73 enzymatic activity being less. than the parent antibody having the corresponding amino acid sequences VH and VL SEQ ID NO: 27 and 28.

Also provided are isolated and/or recombinant nucleic acids (for example, sets of nucleic acids) encoding an antibody (or a heavy or light chain thereof) or a VH and/or VL domain according to the present invention, a vector or a set of vectors containing such nucleic acid(s) acid(s), a cell containing such a vector, and a method of producing a human anti-CD73 antibody or antibody fragment, comprising culturing such cell under conditions suitable for expression of the anti-CD73 antibody or antibody fragment. The present invention also provides compositions, such as pharmaceutically acceptable compositions, and kits containing such proteins, nucleic acids, vectors and/or cells and typically one or more additional ingredients, which may be active ingredients or inactive ingredients that contribute to formulation, delivery, stability, or other characteristics of the composition (eg, various media). The present invention further provides various new and useful methods for producing and using such antibodies, nucleic acids, vectors, cells, organisms and/or compositions, for example, to modulate CD73-mediated biological activity, for example, to treat diseases associated therewith, in particular cancer.

The present invention also provides methods for producing or testing an antibody or antibody fragment that binds and neutralizes the enzymatic activity of CD73.

The present invention also provides a method of enhancing the activity of lymphocytes (eg, T cells) in a subject in need thereof or restoring the activity of lymphocytes (eg, T cells), or a method of attenuating adenosine-mediated inhibition of lymphocyte activity (eg, T cells), such as the method includes administering to the subject an effective amount of any of the above compositions. 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, kidney 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.

These aspects are set forth more fully in the description provided herein, and upon examination thereof additional aspects, features and advantages will become apparent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B show the titration curves obtained for humanized variants containing the heavy chain variable regions H0, H1, H2, H3 and H4, each of which is in combination with each of the light chain variable regions L0, L1, L2, L3 and L4, and the parent mouse HPLP antibody, tested in transfected cell lines expressing human (Figure 1A) or cynomolgus (Figure 1B) CD73 protein.

Figure 2 shows the blocking of the enzymatic activity of soluble recombinant CD73. The top left panel (A) shows that high levels of luminescence were measured when the substrates ATP and CTG were mixed. When AMF was added to the reaction mixture, the CTG reaction was inhibited, resulting in a decrease in luminescence. In the presence of the rhCD73 protein, which hydrolyzes AMP, the level of luminescence was restored. Blocking of CD73 enzymatic activity by various humanized variants has been shown.

Figures 3A-3C and 4A-4C show two sets of experiments performed to compare humanized antibodies for their effectiveness in reversing AMP-mediated inhibition of T cell proliferation. Figures 3A and 4A show the control of T cell proliferation and its inhibition by AMP for each set of experiments. Figures 3B and 4B show the effectiveness of anti-CD73 antibodies in restoring CD4+ T cell proliferation, and Figures 3C and 4C show the effectiveness of anti-CD73 antibodies in restoring CD8+ T cell proliferation. Cell proliferation was determined by dilution of the Cell Trace Violet marker. Data are expressed as the mean of two replicates +/- standard deviation.

Figures 5A-5C show that T cell proliferation was restored by all anti-CD73 antibodies. In fig. 5A shows the control of proliferation of a subset of T cells and its inhibition by AMP. Figure 5B shows the effectiveness of the H4+Lx antibody and the parent HPLP antibody in restoring CD4+ and CD8+ T cell proliferation. Figure 5C shows the effectiveness of 2H4+2Lx antibodies (2H4+ chains in combination with 2L1, 2L2, 2L3 or 2L4 chains) and the parent antibody 2HP2LP in restoring CD4+ and CD8+ T cell proliferation. Data are expressed as the mean of two replicates +/- standard deviation.

Figures 6A and 6B show that T cell proliferation restored by the humanized variants is reproducible, respectively, in two representative human donors. The 2H4+2Lx variants of the anti-CD73 antibody have been shown to be effective in restoring the proliferation of CD4 and CD8 T cells. Data are expressed as the mean of two replicates +/- standard deviation.

Figure 7 shows that CD4+ T cell proliferation is inhibited by ATP and restored by the 2H4+2Lx antibody variants. Control of CD4+ T cell proliferation and its inhibition by 100 μM ATP demonstrated in two representative donors D795 (Fig. 7A) and D664 (Fig. 7B). Data are expressed as the mean of two replicates +/- standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, singular forms may mean one or more. In claim(s), when used in conjunction with the term “including,” the singular forms may mean one or more than one. As used herein, “other” may mean at least a second or more.

If the term "comprising" is used, it may not necessarily be replaced by "consisting essentially of" or "consisting of".

Human CD73, also known as ecto-5'-nucleotidase and as 5'-ribonucleotide phosphohydrolase, EC 3.1.3.5, encoded by the NT5E gene, exhibits 5'-nucleotidase, namely AMP, NAD and HMN nucleosidase activities. At neutral pH, CD73 catalyzes the conversion of purine 5'-mononucleotides to nucleosides, the preferred substrate being AMP. This enzyme consists of a dimer of 2 identical subunits with a molecular weight of 70 kDa, linked by a glycosylphosphatidylinositol bond to the outer surface of the plasma membrane. The amino acid sequence of the human CD73 preprotein (monomer), including the signal sequence (amino acids 1-26), is presented in Genbank under accession number NP_002517, details of which are incorporated herein by reference in their entirety and are set forth below:

As used herein, “neutralize the enzymatic activity of CD73” refers to the process in which the 5'-nucleotidase (5'-ectonucleotidase) activity of CD73 is inhibited. This includes, in particular, inhibition of CD73-mediated adenosine formation, ie, inhibition of CD73-mediated catabolism of AMP to adenosine. This can be measured, for example, in a cell-free assay that measures the ability of a test compound to inhibit the conversion of AMP to adenosine, either directly or indirectly. In one embodiment, the antibody preparation causes at least a 50% reduction in the conversion of AMP to adenosine, an at least 70% reduction in the conversion of AMP to adenosine, or an at least 80% reduction in the conversion of AMP to adenosine, as determined, for example, in the methods analysis described in this application.

Whenever "treating cancer" or the like is referred to herein with respect to an anti-CD73 binding agent (e.g., an antibody), it means: (a) a method of treating cancer, wherein said method includes the step of administering (for at least one treatment) of an anti-CD73 binding agent (preferably in a pharmaceutically acceptable carrier material) to an individual, mammal, in particular a human, in need of such treatment, at a dose that treats the cancer (therapeutically effective amount), preferably at a dose (amount) specified in this document; (b) the use of an anti-CD73 binding agent for the treatment of cancer, or an anti-CD73 binding agent for use in such treatment (particularly in humans); (c) using an anti-CD73 binding agent for the production of a pharmaceutical composition for the treatment of cancer, a method of using an anti-CD73 binding agent for the production of a pharmaceutical composition for the treatment of cancer, comprising mixing the anti-CD73 binding agent with a pharmaceutically acceptable carrier, or a pharmaceutical composition containing an effective a dose of an anti-CD73 binding agent suitable for the treatment of cancer; or (d) any combination of a), b) and c), in accordance with the subject matter of the invention, which is eligible for patenting in the country in which this application is filed.

The term “antibody” as used herein refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are classified into one of five main classes: IgA, IgD, IgE, IgG and IgM. Some of them are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4 and similar isotypes. A typical immunoglobulin (antibody) structural unit includes a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair containing one "light" (approximately 25 kDa) and one "heavy" (approximately 50-70 kDa) chain. The N-terminus of each chain contains a variable region of approximately 100-110 or more amino acids, which is primarily responsible for antigen recognition. The terms variable light chain ( _VL ) and variable heavy chain ( _VH ) refer to these light and heavy chains, respectively. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called "alpha", "delta", "epsilon", "gamma" and "mu", respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. IgG are examples of antibody classes used in this application because they are the most abundant antibodies under physiological conditions and because they are the easiest to obtain in vitro. Optionally, the antibody is a monoclonal antibody. Specific examples of antibodies include humanized, chimeric, human or otherwise human antibodies. The term "antibodies" also includes any fragment or derivative of any of the antibodies described in this application.

The term “specifically binds” means that the antibody may preferentially bind in a competitive binding assay to a binding partner, such as CD73, as assessed using either recombinant forms of the proteins, or epitopes thereof, or native proteins present on the surface of isolated target cells . Competitive binding assay methods and other methods for determining specific binding are further described below and are well known in the art.

When an antibody is stated to “compete with” a particular monoclonal antibody, this means that the antibody competes with the monoclonal antibody in a binding assay using either recombinant CD73 molecules or surface expressed CD73 molecules. For example, if a test antibody reduces the binding of a reference antibody to a CD73 polypeptide or CD73-expressing cell in a binding assay, the antibody is said to “compete,” respectively, with the reference antibody.

The term “affinity” as used herein refers to the strength of binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, which is defined as [AT]x[AG]/[AT-AG], where [AT-AG] is the molar concentration of the antibody-antigen complex, [AT] is the molar concentration of unbound antibody, and [ AG] is the molar concentration of unbound antigen. The affinity constant K _a is defined as 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 Assoc. and Wiley Interscience, New York, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), these materials are incorporated herein by reference in their entirety. One standard method well known in the art for determining mAb affinity is the use of surface plasmon resonance (SPR) screening (such as the BIAcore™ SPR assay).

As used herein, “determinant” refers to an interaction or binding site on a polypeptide.

The term "epitope" refers to an antigenic determinant and is the region or region on an antigen to which an antibody binds. A protein epitope may contain amino acid residues directly involved in binding, as well as amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide, i.e., amino acid residues within the “recognition region” of the antibody. It is the simplest form or smallest structural region on a complex antigen molecule that can bind to, for example, an antibody or receptor. Epitopes can be linear or conformational/structural. The term "linear epitope" is an epitope consisting of amino acid residues that are contiguous on a linear amino acid sequence (primary structure). The term "conformational or structural epitope" is an epitope consisting of amino acid residues, not all of which are contiguous and therefore represent distinct parts of a linear sequence of amino acids that are brought into close proximity to each other by molecular folding (secondary, tertiary and/or quaternary structures). The conformational epitope depends on the 3-dimensional structure. The term "conformational" is therefore often used interchangeably with the term "structural".

The term “deplete” or “depletion”, with respect to CD73 expressing cells, means a process, method or compound that results in the destruction, elimination, lysis or induction of such destruction, elimination or lysis so as to adversely affect the number of such CD73 expressing cells present in the sample or in the subject's body.

The term "internalization", used interchangeably with the term "intracellular internalization", refers to the molecular, biochemical and cellular events associated with the process of moving a molecule from the extracellular surface of a cell to the intracellular surface of a cell. The processes responsible for the intracellular internalization of molecules are well known and may include, among others, 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 associated with extracellular molecules (eg, a ligand associated with a transmembrane receptor or an antibody associated with a membrane-associated molecule). Thus, the phrase “cause and/or enhance internalization” includes events in which intracellular internalization is caused and/or the rate and/or extent of intracellular internalization is increased.

The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract derived from biological materials. The term "therapeutic agent" refers to an agent that exhibits biological activity.

For purposes described herein, a “humanized” or “human” antibody refers to an antibody in which a constant and variable region of a framework region of one or more human immunoglobulins is linked to a binding region, such as a complementarity determining region (CDR), of an animal immunoglobulin. Such antibodies are designed to retain the binding specificities of the non-human antibody from which the binding regions are derived, but to avoid an immune system reaction against the non-human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered" to produce specific human antibodies in response to antigen challenge (see, for example, Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al (1994) Int Immun 6:579, the information of which is incorporated herein by reference in its entirety. A fully human antibody can also be constructed using genetic or chromosomal transfection techniques, as well as phage display technology, all of which are known in the art (see, for example, McCafferty et al. (1990) Nature 348:552-553). Human antibodies can also be produced by in vitro activated B cells (see, for example, US Pat. Nos. 5,567,610 and 5,229,275, the contents of which are incorporated herein by reference in their entirety).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for binding to an antigen. The hypervariable region typically contains amino acid residues from the “complementarity determining region” or “CDR” (for example, 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 residues from the “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 see a similar system for determining the essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in a given region is carried out using the method described in Kabat et al., above. Phrases such as “Kabat position,” “Kabat variable domain residue numbering,” and “Kabat numbering” as used herein refer to this numbering system for heavy chain variable domains or light chain variable domains. When using the Kabat numbering system, the actual linear amino acid sequence of the peptide may contain fewer amino acids, corresponding to a shortening, or additional amino acids, corresponding to an insertion into the FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 in the H2 CDR and inserted residues (e.g., residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 of the heavy chain FR . Kabat numbering of residues can be determined for a given antibody by aligning regions of homology between the antibody sequence and a “standard” Kabat numbering sequence.

The term “framework” residues or “FR” as used herein means the variable domain region of an antibody excluding regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions separated by CDR regions (FR1, FR2, FR3 and FR4).

The terms “Fc domain,” “Fc portion,” and “Fc region” refer to the C-terminal portion of an antibody heavy chain, for example, from about amino acid (AA) 230 to about AA 450 of human γ (gamma) heavy chain or similar sequence in another type of antibody heavy chain (eg, α, δ, ε, and μ for a human antibody), or a naturally occurring allotype thereof. Unless otherwise noted, Kabat's generally accepted amino acid numbering for immunoglobulins is used herein (see Kabat et al. (1991), Sequences of Protein of Immunological Interest, 5th ed., United States Department of Health, National Institutes of Health, Bethesda, Maryland).

The terms “isolated,” “purified,” or “biologically pure” refer to a material that is virtually or substantially free of the components that would normally accompany it in its natural state. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, which is the predominant species present in the preparation, is essentially purified.

The terms "polypeptide", "peptide" and "protein" are used interchangeably in this application to refer to a polymer of amino acid residues. These terms include amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "recombinant", used for example in relation to a cell, or nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or by alteration of a native nucleic acid or protein, or that the cell originated from a cell modified in this way. Therefore, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, expressed at a reduced level, or not expressed at all.

As used herein, the term antibody that “binds” a polypeptide or epitope means an antibody that binds the determinant with specificity and/or affinity.

The term "identity" or "identical", as used in relation to the relatedness between the sequences of two or more polypeptides, refers to the degree of relatedness between polypeptide sequences, which is determined by the number of matches between chains of two or more amino acid residues. “Identity” is a measure of the percentage of exact matches of the smaller of two or more sequences to gap-aligned (if necessary) sequences, as calculated using a specific mathematical model or computer program (i.e., “algorithms”). The identity of related polypeptides can be easily calculated using known methods. Such methods include, but are not limited to those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

Identity determination methods are designed to determine the closest match between sequences of interest. Methods for determining identity are described in publicly available computer programs. Methods for determining identity between two sequences using computer programs include the GCG software package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, MD 20894; Altschul et al., supra). The well-known Smith-Waterman algorithm can also be used to determine identity.

Obtaining antibodies.

The present invention is based, in part, on the discovery of modified human acceptor framework sequences into which antibody CDRs can be inserted such that the resulting anti-CD73 variable region exhibits high potency in neutralizing human CD73 enzymatic activity. These antibodies have the advantage of low or reduced immunogenicity in humans, eg, a lower ability or likelihood of causing an undesirable anti-CD73 antibody-directed immune response when administered to a human. Examples of such antibodies according to the present invention include antibodies containing an H4+ or 2H4+VH domain in combination with an L1, L2, L3, L4, 2L1, 2L2, 2L3 or 2L4 VL domain. Examples of antibodies in accordance with the present invention include antibodies containing pairs of VH and VL domains of any of the antibodies H4+L1, H4+L2, H4+L3, H4+L4, 2H4+L1, 2H4+L2, 2H4+L3 or 2H4+L4 .

In one aspect, an antibody or antibody fragment that binds a human CD73 polypeptide comprises VH and VL scaffolds (eg, 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 set forth in SEQ ID NO: 2; HCDR2 (heavy chain CDR2) containing the amino acid sequence YIDPYNGGSSYNQKFKG presented in SEQ ID NO: 12, optionally additionally, the glutamine residue (Q) at position 13 of SEQ ID NO: 12 can be replaced by a leucine residue (L), and a lysine residue (K) at position 14 of SEQ ID NO: 12 may optionally be replaced by a threonine residue (T); HCDR3 (heavy chain CDR3) containing the amino acid sequence GYNNYKAWFAY presented in SEQ ID NO: 13, optionally further, the asparagine (N) residue at position 3 of SEQ ID NO: 13 may be replaced by a glycine (G) residue; LCDR1 (light chain CDR1) containing the amino acid sequence KASQSVTNDVA presented in SEQ ID NO: 14, optionally further, the threonine (T) residue at position 7 of SEQ ID NO: 14 can be replaced by a serine (S) residue; LCDR2 (light chain CDR2) containing the amino acid sequence YASNRYT shown in SEQ ID NO: 15, optionally the asparagine (N) residue at position 4 of SEQ ID NO: 15 may be replaced by a threonine (T) residue; and LCDR3 (light chain CDR3) containing the amino acid sequence QQDYSSLT shown in SEQ ID NO: 7.

In one embodiment, HCDR2 comprises a Formula I amino acid sequence:

Y - I - D - P - Y - N - G - G - S - S - Y - N - Xaa ₁ - Xaa ₂ - F - K - G (SEQ ID NO: 3) or a subsequence thereof, where Xaa ₁ represents is Q (Gln) or L (Leu), and where Xaa ₂ is K (Lys) or T (Thr).

In one embodiment, HCDR3 comprises a Formula II amino acid sequence:

G - Y - Xaa ₁ - N - Y - K - A - W - F - A - Y (SEQ ID NO: 4) or a subsequence thereof, where Xaa ₁ is N (Asp) or G (Gly).

In one embodiment, LCDR1 comprises a Formula III amino acid sequence:

K - A - S - Q - S - V - Xaa ₁ - N - D - V - A (SEQ ID NO: 5),

or a subsequence thereof, where Xaa ₁ represents T (Thr) or S (Ser).

In one embodiment, LCDR2 comprises a Formula IV amino acid sequence:

Y - A - S - Xaa ₁ - R - Y - T (SEQ ID NO: 6),

or a subsequence thereof, where Xaa ₁ represents T (Thr) or N (Asn).

In one aspect, an isolated antibody that binds a human CD73 polypeptide comprises: HCDR1 comprising the amino acid sequence SYNMY set forth in SEQ ID NO: 2; HCDR2 containing the amino acid sequence YIDPYNGGSSYNLTFKG shown in SEQ ID NO: 8; HCDR3 containing the amino acid sequence GYGNYKAWFAY shown in SEQ ID NO: 9; LCDR1 containing the amino acid sequence KASQSVSNDVA shown in SEQ ID NO: 10; LCDR2 containing the amino acid sequence YASTRYT presented in SEQ ID NO: 11; and LCDR3 containing the amino acid sequence QQDYSSLT shown in SEQ ID NO: 7.

In one aspect, an isolated antibody that binds a human CD73 polypeptide comprises: HCDR1 comprising the amino acid sequence SYNMY set forth in SEQ ID NO: 2; HCDR2 containing the amino acid sequence YIDPYNGGSSYNQKFKG shown in SEQ ID NO: 12; HCDR3 containing the amino acid sequence GYNNYKAWFAY presented in SEQ ID NO: 13; LCDR1 containing the amino acid sequence KASQSVTNDVA shown in SEQ ID NO: 14; LCDR2 containing the amino acid sequence YASNRYT shown in SEQ ID NO: 15; and LCDR3 containing the amino acid sequence QQDYSSLT shown in SEQ ID NO: 7.

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

The antibody may further contain one, two, three, four, five or more amino acid substitutions in the human heavy and/or light chain framework regions, for example, to improve the affinity, stability or other properties of the antibody.

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

In one aspect of the present invention there is provided an antigen binding domain or antibody or antibody fragment that binds a human CD73 polypeptide comprising:

(a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 2;

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

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

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

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

(f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 7; And

(g) sequences of human heavy and light chain framework regions.

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

Optionally, any embodiment may specify that the antibody may be an antibody other than the parent mouse antibody, for example, a parent mouse antibody comprising VH and VL with the corresponding sequences of SEQ ID NOs: 27 and 28, or containing VH and VL with the corresponding sequences SEQ ID NO: 40 and 41.

Optionally, the human framework region contains one or more mutations, for example, back mutations to introduce a residue present at a particular position in a non-human mammal (eg, mouse).

In one aspect of any embodiment described herein, the amino acid at position 2 of the Kabat heavy chain is isoleucine.

In one aspect of any embodiment described herein, the amino acid at position 30 of the Kabat heavy chain is alanine.

In one aspect of any embodiment described herein, the amino acid at position 48 of the Kabat heavy chain is isoleucine.

In one aspect of any embodiment described herein, the amino acid at position 69 of the Kabat heavy chain is leucine.

In one aspect of any embodiment described herein, the amino acid at position 73 of the Kabat heavy chain is lysine.

In one embodiment, VH contains an isoleucine residue at the 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 aspect of any embodiment described herein, the amino acid at Kabat position 67 of the light chain is tyrosine.

In one aspect of any embodiment described herein, the amino acid at position 60 of the Kabat light chain is serine or aspartic acid.

In one aspect of any embodiment described herein, the amino acid at position 2 of the Kabat light chain is valine or isoleucine.

In one aspect of any embodiment described herein, the amino acid at position 87 of the Kabat light chain is tyrosine or phenylalanine.

In one embodiment, VL contains a tyrosine residue at Kabat position 67, a series at position 60, an isoleucine at position 2, and a tyrosine at position 87.

In one aspect of any embodiment described herein, the amino acid at position 28 of the Kabat heavy chain is threonine (T).

In one aspect of any embodiment described herein, the amino acid at position 66 of the Kabat heavy chain is arginine (R).

In one aspect of any embodiment described herein, the amino acid at position 67 of the Kabat heavy chain is valine (V).

In one aspect of any embodiment described herein, the Kabat heavy chain amino acid position 71 is arginine (R).

Positions in the VH and VL domains are described herein using the Kabat numbering system (Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ed., United States Department of Health, National Institutes of Health, Bethesda, MD) .

In one aspect, the anti-CD73 antibody comprises a VH domain that is at least about 80% sequence identical (e.g., at least about 85%, 90%, 95%, 97%, 98%, or 99% identical, or 100% identical to ) VH domain sequence shown in SEQ ID NO: 37 or 42.

In one aspect, an anti-CD73 antibody or antibody fragment comprises a VL domain that is at least about 80% sequence identical (e.g., at least about 85%, 90%, 95%, 97%, 98%, or 99% identical, or is 100%) identical to the VL domain sequence presented in any of SEQ ID NO: 33-36 or 43-46.

In one aspect, an anti-CD73 antibody or antibody fragment comprises a VH domain that is at least about 80% sequence identical (e.g., at least about 85%, 90%, 95%, 97%, 98%, or 99% identical, or is 100% identical to the sequence of a VH domain set forth in SEQ ID NO: 42, and a VL domain that is at least about 80% sequence identical (e.g., at least about 85%, 90%, 95% identical, 97 %, 98% or 99% or 100% identical to the VL domain sequence presented in any of SEQ ID NO: 43 -46.

In one aspect, an anti-CD73 antibody or antibody fragment comprises a heavy chain that is at least about 80% sequence identical (e.g., at least about 85%, 90%, 95%, 97%, 98%, or 99% identical, or is 100% identical to the heavy chain sequence of the H4+L1 antibody set forth in SEQ ID NO: 38. In one embodiment, the antibody or antibody fragment comprises a light chain that is at least about 80% identical in sequence (e.g., identical to at least approximately 85%, 90%, 95%, 97%, 98% or 99% identical or 100%) identical to the light chain sequence of the H4+L1 antibody shown in SEQ ID NO: 39.

In one aspect, an anti-CD73 antibody or antibody fragment comprises a heavy chain that is at least about 80% sequence identical (e.g., at least about 85%, 90%, 95%, 97%, 98%, or 99% identical, or is 100% identical to the heavy chain sequence of the 2H4+2L1 antibody set forth in SEQ ID NO: 47. In one embodiment, the antibody or antibody fragment comprises a light chain that is at least about 80% identical in sequence (e.g., identical to at least approximately 85%, 90%, 95%, 97%, 98% or 99% identical or 100% identical to the light chain sequence of the 2H4+2L1 antibody shown in SEQ ID NO: 48.

The DNA encoding the antibody can be prepared and placed into a suitable expression vector for transfection into a suitable host. The host is then used to recombinantly produce the antibody, or variants thereof, such as a humanized version of the monoclonal antibody, active antibody fragments, chimeric antibodies containing an antigen recognition site from the antibody, or variants containing a detection molecule.

DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of mouse antibodies). In one aspect, a nucleic acid encoding the heavy chain or light chain of an anti-CD73 antibody according to any embodiment described herein is provided. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, COS monkey cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to achieve synthesis monoclonal antibodies in recombinant host cells. Such DNA sequences can be modified for any of a variety of purposes, for example, to humanize antibodies, produce fragments or derivatives, or to modify the antibody sequence, for example, at the antigen binding site, to optimize the binding specificity of the antibody. In one embodiment, an isolated nucleic acid sequence encoding an antibody light chain and/or a heavy chain is provided, as well as a recombinant host cell containing (eg, in its genome) such nucleic acid. Recombinant expression in bacteria of DNA encoding an antibody is well known in the art (see, for example, Skerra et al., Curr. Opinion in Immunol., 5, p. 256 (1993); and Pluckthun, Immunol. 130, p. 151 (1992).

Antibody fragments and derivatives (which are included within the scope of the term “antibody” or “antibodies” as used herein unless otherwise indicated or the context clearly requires) can be prepared using techniques known in the art. "Fragments" contain part of an intact antibody, typically an 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 that is a polypeptide having a primary structure consisting of one contiguous sequence of contiguous amino acid residues (referred to herein as a “single chain antibody fragment” or “single chain polypeptide”); and multispecific (eg, bispecific) antibodies formed from antibody fragments.

Typically, the affinity of the anti-CD73 antibody provided herein for a CD73 polypeptide (e.g., a CD73 polypeptide prepared as described in the Examples section herein) ranges from about 10 ⁴ to about 10 ¹¹ M ^-1 (e.g., approximately 10 ⁸ to approximately 10 ¹⁰ M ^-1 ). For example, a particular aspect of the present invention provides an anti-CD73 antibody with an average dissociation constant (K _D ) from CD73 of less than 1x10 ⁹ M, as determined by, for example, surface plasmon resonance (SPR) screening (for example, by assay for PPR BIAcore™). In a more specific exemplary aspect of the present invention, anti-CD73 antibodies are provided with a CD73 K _D of from about 1x10 ^-8 M to about 1x10 ^-10 M, or from about 1x10 ^-9 M to about 1x10 ^-11 M.

Antibodies can be characterized, for example, by an average KD of no more than about (i.e., best affinity) 100, 60, 10, 5, or 1 nanomolar, preferably subnanomolar, or in some cases no more than about 500, 200, 100, or 10 picomolar. KD can be determined, for example, by immobilizing recombinantly produced human CD73 proteins on the surface of a microchip and then applying the antibody to be tested in solution. In one embodiment, the method further includes the step of (d) selecting antibodies from (b) that are capable of competing with the control antibody for binding to CD73.

In one embodiment, anti-CD73 antibodies can be prepared such that they do not exhibit significant binding to human Fcγ receptors, for example, any one or more of CD16A, CD16B, CD32A, CD32B and/or CD64). Such antibodies may contain constant regions of various heavy chains that are known to exhibit no or reduced binding to Fcγ receptors. Alternatively, antibody fragments that do not contain (or contain parts of) constant regions, such as F(ab')2 fragments, can be used to prevent binding to the Fc receptor. Fc receptor binding can be assessed according to methods known in the art, including, for example, testing antibody binding to Fc receptor protein in the BIACORE assay. In addition, any isotype of IgG antibody 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 can generally be used (see eg WO 03/101485, the disclosure of which is incorporated herein by reference). Assays that are used to assess Fc receptor binding are well known in the art and are described, for example, in WO 03/101485.

In one embodiment, the antibody may contain one or more specified mutations in the Fc region that result in the formation of "Fc-silent" antibodies that interact minimally with effector cells. Silencing of effector functions can be achieved by introducing mutations into the Fc region of antibodies, and such mutations have been described in the art: N297A mutation, LALA mutations (Strohl, W., 2009, Curr. Opin. Biotechnol. Vol. 20(6): 685-691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69) see also Heusser et al., WO 2012/065950, the disclosures of which are incorporated herein by reference. In one embodiment, the antibody contains one, two, three, or more amino acid substitutions in the hinge region. In one embodiment, the antibody is IgG1 or IgG2 and contains one, two or three substitutions at residues 233-236, optionally 233-238 (EU numbering). In one embodiment, the antibody is IgG4 and contains one, two or three substitutions at residues 327, 330 and/or 331 (EU numbering). Examples of Fc-silencing antibodies against IgG1 are the LALA mutant, which contains the L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of an Fc silencing mutation is a mutation at residue D265 or at D265 and P329, such as that used in an IgG1 antibody called the DAPA mutation (D265A, P329A) (US Pat. No. 6,737,056). Another silent IgG1 antibody contains a mutation at residue N297 (eg, N297A, N297S mutation), which results in the formation of aglycosylated/non-glycosylated antibodies. Other silent mutations include: substitutions at residues L234 and G237 (L234A/G237A); substitutions in residues S228, L235 and R409 (S228P/L235E/R409K,T,M,L); substitutions in residues H268, V309, A330 and A331 (H268Q/V309L/A330S/A331S); substitutions in residues C220, C226, C229 and P238 (C220S/C226S/C229S/P238S); replacements in residues C226, C229, E233, L234 and L235 (C226S/C229S/E233P/L234V/L235A; replacements in residues K322, L235 and L235 (K322A/L234A/L235A); replacements in residues L234, L235 and P331 (L234F / L235S/P331S); replacements in residues 234, 235 and 297; replacements in residues E318 and K320 and K322 (L235E/E318A/K320A/K322A); replacements in residues 243 and 264; substitutions at residues 297 and 299; substitutions such as substitutions at residues 233, 234, 235, 237 and 238, defined by the EU numbering system, including a sequence selected from PAAAP, PAAAS and SAAAS (see WO 2011/066501).

In one embodiment, the antibody may contain one or more specified mutations in the Fc region. For example, such an antibody may contain an Fc domain derived from human IgG1 containing a mutation at Kabat residue(s) 234, 235, 237, 330 and/or 331. One example of such an Fc domain contains substitutions at residues L234, L235 and P331 Kabat (e.g. L234A/L235E/P331S or (L234F/L235E/P331S). Another example of such an Fc domain contains substitutions at Kabat residues L234, L235, G237 and P331 (e.g. L234A/L235E/G237A/P331S). Other an example of such an Fc domain contains substitutions at Kabat residues L234, L235, G237, A330, and P331 (e.g., L234A/L235E/G237A/A330S/P331S) In one embodiment, the antibody comprises an Fc domain, optionally of the human IgG1 isotype, comprising: a substitution). L234X ₁ , the L235X ₂ substitution, and the P331X ₃ substitution, wherein X ₁ is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, and X ₃ is optionally any amino acid residue other than proline; wherein X ₁ represents alanine or phenylalanine or a conservative replacement thereof; optionally, X ₂ represents glutamic acid or a conservative replacement thereof; optionally, X ₃ represents a series or a conservative replacement thereof. In another embodiment, the antibody contains an Fc domain, optionally of the human IgG1 isotype, containing: an L234X ₁ substitution, a L235X ₂ substitution, a G237X ₃ substitution, and a P331X ₄ substitution, where X ₁ represents any amino acid residue other than leucine, X ₂ represents any an amino acid residue other than leucine, X ₃ is any amino acid residue other than glycine, and X ₄ is any amino acid residue other than proline; optionally, X ₁ represents alanine or phenylalanine or a conservative replacement thereof; optionally, X ₂ represents glutamic acid or a conservative replacement thereof; optionally X ₃ represents alanine or a conservative substitute thereof; optionally X ₄ is a series or a conservative replacement thereof. In another embodiment, the antibody comprises an Fc domain, optionally of the human IgG1 isotype, comprising: an L234X ₁ substitution, a L235X ₂ substitution, a G237X ₃ substitution, a G330X ₄ substitution, and a P331X ₅ substitution, where X ₁ is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, X ₃ is any amino acid residue other than glycine, X ₄ is any amino acid residue other than alanine, and X ₅ is any amino acid residue other than proline; optionally, X ₁ represents alanine or phenylalanine or a conservative replacement thereof; optionally, X ₂ represents glutamic acid or a conservative replacement thereof; optionally X ₃ represents alanine or a conservative substitute thereof; optionally X ₄ is a series or a conservative replacement thereof; optionally X ₅ is a series or a conservative replacement thereof.

The abbreviated notation used herein adopts the following format: Wild-type residue: Position in polypeptide: Mutant residues, with residue positions indicated according to EU Kabat numbering.

In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence set forth below, or an amino acid sequence at least 90%, 95%, or 99% identical thereto but retaining amino acid residues at positions 234, 235, and 331 according to Kabat (underlined):

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

In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence set forth below, or an amino acid sequence at least 90%, 95%, or 99% identical thereto but retaining amino acid residues at positions 234, 235, 237 , 330 and 331 according to Kabat (underlined):

In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence set forth below, or a sequence at least 90%, 95%, or 99% identical thereto, but retaining amino acid residues at positions 234, 235, 237, and 331 according to Kabat (underlined):

Fc-silenced antibodies have no or low ADCC activity, which means that the Fc-silenced antibody exhibits ADCC activity that is below 50% specific cell lysis. Preferably, the antibody has substantially no ADCC activity, for example, an Fc-silenced antibody exhibits ADCC (specific cell lysis) activity that is below 5% or below 1%. Fc-silencing antibodies may also lack FcγR-mediated cross-linking of CD73 on the surface of CD73-expressing cells.

In one embodiment, the antibody contains a substitution in the heavy chain constant region at any one, two, three, four, five or more 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 (residue numbering in the heavy chain constant region corresponds to Kabat's EU numbering). In one embodiment, the antibody contains a substitution at residues 234, 235, and 322. In one embodiment, the antibody contains a substitution at residues 234, 235, and 331. In one embodiment, the antibody contains a substitution at residues 234, 235, 237, and 331. In one embodiment implementation, the antibody contains a substitution at residues 234, 235, 237, 330, and 331. In one embodiment, the Fc domain is of the human IgG1 subtype. Amino acid residues are indicated according to Kabat's EU numbering.

In one embodiment, the antibody comprises an Fc domain containing an amino acid substitution that enhances binding to human FcRn polypeptides to increase the half-life of the antibody in vivo. Typical mutations are described in Strohl, W., 2009, Curr. Opin. Biotechnol. Vol. 20(6):685-691, the description of which is incorporated herein by reference. Examples of substitutions used in human IgG1 isotype antibodies include substitutions at Kabat residues M252, S254 and T256; replacements in the remnants of T250 and M428; substitutions in residue N434; substitutions in residues H433 and N434; substitutions in residues T307, E380 and N434; substitutions in residues T307, E380 and N434; substitutions in residues M252, S254, T256, H433, N434 and 436; substitutions in residue I253; substitutions in residues P257, N434, D376 and N434. At any desired step, the ability of the antigen binding compound to inhibit CD73 enzymatic activity, particularly to block CD73 5'-nucleotidase activity and reduce adenosine production by a CD73 expressing cell, and in turn restore activity and/or reverse adenosine-mediated inhibition of lymphocytes, can be assessed.

The ability of an antibody to inhibit CD73 enzymatic activity can be examined in a cell-free assay using recombinant soluble human CD73 (as dimers) and AMP, in which the conversion of AMP to adenosine (and/or its inhibition) is detected directly (for example, by measuring substrates and products , 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.

The inhibitory activity of an antibody against CD73 can also be assessed by any of a variety of other methods. For example, an indirect assay uses a luciferase-based reagent (eg, the CellTiter-Glo® system, available from Promega) to detect the disappearance of AMP. The luciferase reaction in this assay is inhibited by AMP. Addition of the CD73 enzyme to the reaction mixture degrades AMP and relieves inhibition, causing a detectable signal.

Assay methods using soluble CD73 will involve testing under conditions in which the antibodies are present in a significant molar excess (eg, 10-fold, 20-fold, 50-fold, 100-fold, etc.) over CD73 polypeptide dimers. When present in molar excess over the enzyme, anti-CD73 antibodies will no longer be able to form multimeric complexes of antibodies and CD73 dimers; antibodies that retain the ability to inhibit CD73 enzymatic activity can then be selected.

The ability of an antibody to inhibit the 5'-ectonucleotidase enzymatic activity of CD73 can alternatively or in addition also be tested in a cell-based assay (using cells that express CD73). Preferably, the antibodies can be first tested or screened in an assay in a cell-free system to identify antibodies that block enzyme activity to reduce the likelihood of selecting antibodies that inhibit CD73 by causing internalization of CD73, and then tested as a purified antibody in a cell-based assay. Cellular analysis can be performed as shown in the Examples section of this document, or as described in PCT Publication No. WO 2016/055609, the description of which is incorporated herein by reference. For example, CD73-expressing cells (eg, MDA-MB-231 cell line) are seeded in 96-well flat-bottom plates in the presence of anti-CD73 antibodies and incubated as described in WO 2016/055609. AMP is added to the cells and incubated at 4°C (to avoid CD73 down-modulation). The plates are then centrifuged and the supernatant is transferred to a 96-well culture plate with a flat bottom well. Free phosphate resulting from the hydrolysis of AMP to form adenosine is then quantitated. The reduction in AMP hydrolysis to adenosine in the presence of the antibody indicates that the antibody inhibits cellular CD73.

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

Antibody activity can also be measured in an indirect assay for its ability to modulate lymphocyte activity, for example, to reverse adenosine-mediated inhibition of lymphocyte activity or to cause activation of lymphocyte activity. This can be done, for example, using a cytokine release assay. In another example, an antibody can be assessed in an indirect assay for its ability to modulate lymphocyte proliferation.

In one embodiment, the antibody neutralizes the 5'-ectonucleotidase activity of a human CD73 homodimeric polypeptide in solution. In one embodiment, the antibody binds to and inhibits the enzymatic activity of a soluble human CD73 polypeptide, particularly an antibody that neutralizes CD73-mediated catabolism of AMP to adenosine. In one embodiment, the antibody binds CD73 in a bivalent manner. In one embodiment, the antibody is a non-depleting antibody, such as an Fc-silencing antibody, that does not substantially bind to human Fcγ receptors. In one embodiment, the antibody neutralizes CD73 in solution without the need to induce the formation of CD73 polypeptide:anti-CD73 antibody oligomers.

In one embodiment, the antibody specifically binds human CD73 on 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 cause oligomerization of soluble CD73.

In one embodiment, the antibody specifically binds human CD73 on the surface of a cell and is capable of neutralizing the 5'-ectonucleotidase activity of cellular CD73 (cell-expressed CD73). In one embodiment, the antibody specifically binds and neutralizes the 5'-ectonucleotidase activity of human CD73 on the cell surface, and is not internalized into CD73-expressing cells upon binding to CD73. The antibody does not induce multimerization and subsequent internalization of CD73. In one embodiment, the antibody binds to and is capable of inhibiting the enzymatic activity of recombinant human CD73 polypeptide in solution, wherein the antibody is not internalized into CD73 expressing cells. In one embodiment, the non-internalizing antibody binds CD73 in a bivalent manner. In one embodiment, the antibody is a non-depleting antibody, such as an Fc-silencing antibody. The antibody is capable of neutralizing the 5'-ectonucleotidase activity of dimeric human CD73 polypeptide in solution, furthermore, without the need to induce the formation of CD73 polypeptide:anti-CD73 polypeptide oligomers.

In one embodiment, the antibody specifically binds divalently to human CD73 polypeptides and inhibits the enzymatic activity of cellular human CD73 (and optionally additionally recombinant soluble human CD73), wherein the antibody does not cause or enhance intracellular internalization of CD73 in CD73-expressing cells. Preferably, the antibody does not substantially bind to the Fcγ receptor (eg, through its Fc domain).

In one aspect, the antibody specifically binds human CD73 on the surface of a cell pre-incubated with AMP and is capable of neutralizing its 5'-ectonucleotidase activity. Optionally, neutralization of 5'-ectonucleotidase activity is determined by assessing neutralization of 5'-ectonucleotidase activity in CD73-expressing cells (eg, MDA-MB-231 cells) by quantitating the hydrolysis of AMP to adenosine.

In one aspect, neutralization of the 5'-ectonucleotidase activity of CD73 expressed by a cell is determined by assessing the ability of the antibody to enhance T cell proliferation when said T cells are induced to proliferate in vitro (e.g., through TCR co-stimulation, stimulation of CD3 and CD28 signaling, e.g. by contacting T cells with beads functionalized with CD3 agonists and CD28 agonists) in the presence of AMP (eg, exogenously added AMP). In one aspect, neutralization of 5'-ectonucleotidase activity is assessed using the T cell proliferation assay described in the Methods section of the Examples section.

Antibodies can be characterized by their ability to bind to cells transfected with CD73 mutants, compared with the ability of an anti-CD73 antibody to bind wild-type CD73 polypeptide (eg, SEQ ID NO: 1). A decrease in binding between an anti-CD73 antibody and a mutant CD73 polypeptide means that there is a decrease in binding affinity (e.g., as measured by known methods such as FACS testing of cells expressing a particular mutant or by analyzing binding to mutant polypeptides using the Biacore method) and/or a decrease in the overall binding capacity of the anti-CD73 antibody (eg, as evidenced by a decrease in B _max in a graph of anti-CD73 antibody concentration versus polypeptide concentration). 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 is bound to CD73.

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

In one aspect, anti-CD73 antibodies exhibit reduced binding to a CD73 polypeptide containing a mutation at residue K136 (in SEQ ID NO: 1); optionally the mutant CD73 polypeptide contains the mutation: K136A.

In one aspect, anti-CD73 antibodies exhibit reduced binding to a CD73 polypeptide containing a mutation at a residue selected from the group consisting of: K97, E125, Q153 and K330 (in the sequence of SEQ ID NO: 1); optionally, the mutant CD73 polypeptide contains the following mutations: K97A, E125A, Q153A and/or K330A (eg, K97A, E125A and K330A; K97A, E125A and/or Q153A).

In one aspect, anti-CD73 antibodies exhibit reduced binding to a CD73 polypeptide containing a mutation at a residue selected from the group consisting of: A99, E129, K133, E134 and A135 (in the sequence of SEQ ID NO: 1); optionally, the mutant CD73 polypeptide contains the following mutations: A99S, E129A, K133A, E134N and/or A135S.

Optionally, in one aspect, anti-CD73 antibodies do not exhibit reduced binding to a CD73 polypeptide containing a mutation at a residue selected from the group consisting of: Q70, R73, A74, A107 and R109 (in the sequence of SEQ ID NO: 1); optionally, the mutant CD73 polypeptide contains the following mutations: A99S, Q70S, R73A, A74E, A107I and/or R109G.

In one aspect, anti-CD73 antibodies bind an epitope on CD73 containing residue K136 (at SEQ ID NO: 1).

In one aspect, anti-CD73 antibodies bind an epitope on CD73 containing one, two, three or four residues selected from the group consisting of K97, E125, Q153 and K330 (in SEQ ID NO: 1).

In one aspect, anti-CD73 antibodies bind an epitope on CD73 containing one, two, three, four or five residues selected from the group consisting of A99, E129, K133, E134 and A135 (in the sequence of SEQ ID NO: 1).

In one aspect, anti-CD73 antibodies bind an epitope on CD73 containing one, two, three, four, five or more residues selected from the group consisting of K97, A99, E125, E129, K133, E134, A135 and K136 (in SEQ ID NO: 1). In one aspect, anti-CD73 antibodies bind an epitope on CD73 containing 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 (in sequence SEQ ID NO: 1).

Optionally, in one aspect, anti-CD73 antibodies do not bind an epitope on CD73 containing one, two, three, four or five residues selected from the group consisting of Q70, R73, A74, A107 and R109 (in the sequence SEQ ID NO: 1 ).

The anti-CD73 antibody may be included in a pharmaceutical formulation at a concentration including from 1 mg/ml to 500 mg/ml, the pH of the composition being from 2.0 to 10.0. Said composition may further contain a buffer system, preservative(s), tonicity control agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment, the pharmaceutical composition is an aqueous composition, i.e., a composition containing water. This composition is usually a solution or suspension. In a further embodiment, the pharmaceutical composition is an aqueous solution. The term "aqueous composition" describes a composition containing at least 50% by weight water. Likewise, the term “aqueous solution” describes a solution containing at least 50% by weight water, and the term “aqueous suspension” describes a suspension containing at least 50% by weight water.

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

In another embodiment, the pharmaceutical composition is a dried composition (eg, lyophilized or spray dried) ready for use without any prior dissolution.

In a further aspect, the pharmaceutical composition contains an aqueous solution of such antibody and a buffer, wherein the antibody is present at a concentration of 1 mg/ml or higher, and wherein the pH of the composition is from about 2.0 to about 10.0.

In another embodiment, the pH of the formulation is within a range selected from: about 2.0 to about 10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, from about 5.0 to about 8.0 and from about 5.5 to about 7.5.

In a further embodiment, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, monosodium phosphate, disodium phosphate, sodium phosphate and tris(hydroxymethyl)aminomethane, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffers represents an alternative implementation.

In a further embodiment, the composition further comprises a pharmaceutically acceptable preservative. In a further embodiment, the composition further comprises an isotonic agent. In a further embodiment, the composition also contains a chelating agent. In a further embodiment, the composition further comprises a stabilizer. In a further embodiment, the composition further comprises a surfactant. For convenience, reference may be made to Remington: The Science and Practice of Pharmacy, ^19th edition, 1995.

It is possible that other ingredients may be present in the peptide pharmaceutical formulation. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, fillers, tonicity modifiers, chelating agents, metal ions, oily media, proteins (for example, human serum albumin, gelatin or proteins) and zwitterion (for example, an amino acid such as betaine , taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not undesirably affect the overall stability of the pharmaceutical composition.

Pharmaceutical compositions containing the antibody can be administered to a patient in need of such treatment at multiple sites, for example, at topical sites, such as skin and mucosal sites, at sites that do not require absorption, such as injection into an artery , into a vein, into the heart, and into sites where absorption occurs, such as injection into the skin, subcutaneously, into a muscle, or into the abdominal cavity. Administration of pharmaceutical compositions can be accomplished by several routes of administration, for example, subcutaneous, intramuscular, intraperitoneal, intravenous, lingual, sublingual, buccal, oral, oral, gastric and intestinal, nasal, pulmonary, for example, bronchioles and alveoli, or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ophthalmological routes, for example, through the conjunctiva, through the urethra and parenterally, to patients requiring such treatment.

Diagnosis and treatment of malignant neoplasms

Also provided are methods of treating an individual, particularly a human patient, using an anti-CD73 antibody or antibody fragment described herein. In one embodiment, the present invention provides the use of an antibody or antibody fragment described herein to prepare a pharmaceutical composition for administration to a human patient. Typically, the patient has or is at risk of developing cancer or an infectious disease (such as a viral or bacterial infection).

For example, in one aspect, there is provided a method of restoring or enhancing the activity of immune cells (eg, lymphocytes) in a patient in need thereof, comprising the step of administering to said patient an anti-CD73 neutralizing antibody in accordance with the present invention. In one embodiment, the method is directed to increasing the activity of immune cells (e.g., T cells) in patients suffering from a disease, such as cancer or an infectious disease, in which increased activity and/or infiltration of immune cells at the site of the disease is preferred (e.g., in surrounding a tumor or site of infection), or which is caused or characterized by immunosuppression, immunosuppressive cells or, for example, adenosine produced by CD4 T cells, CD8 T cells, B cells).

These methods will be particularly useful for the treatment of individuals with a tumor in which it is suspected that the tumor microenvironment (and CD73-mediated production of adenosine therein) may contribute to a lack of recognition by the immune system (immune evasion). The tumor environment, for example, may be characterized by CD73-expressing immune cells, eg, CD4 T cells, CD8 T cells, B cells and/or CD73-expressing tumor cells.

These methods and anti-CD73 antibodies can be used to treat and/or prevent various types of cancer and other proliferative diseases. Because these methods act by reducing the amount of adenosine, which inhibits the antitumor activity of lymphocytes, and possibly additionally by increasing ATP, which can enhance the antitumor activity of lymphocytes, they are applicable to a wide range of cancers, including, in particular, solid tumors in which adenosine in the tumor microenvironment may play a significant role in suppressing the antitumor immune response. In one embodiment of the present invention, the human patient receiving anti-CD73 antibody treatment has liver cancer, bone cancer, pancreatic cancer, skin cancer, head or 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, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, urinary cancer bladder, kidney or ureteral cancer, renal carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced carcinoma, including asbestos-related cancers, hematological malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, B-lymphoblastic progenitor cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma and T-lymphoblastic lymphoma from progenitor cells, and any combination of these cancers. The present invention can also be used to treat metastatic cancers. Patients may be tested or selected for one or more of the clinical signs described above before, during or after treatment.

Because anti-CD73 methods and antibodies act by reducing the amount of adenosine, which inhibits the activity and/or infiltration of immune cells (eg, lymphocytes), and possibly additionally by increasing ATP, which can increase the activity of lymphocytes, they are applicable to a wide range of infectious diseases, including, preferably, any infections caused by infection with viruses, bacteria, protozoa, mold or fungi.

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

In one embodiment, the anti-CD73 antibody is administered in an amount effective to achieve and/or maintain (e.g., for 1, 2, 3, 4 weeks and/or until subsequent administration of the anti-CD73 antibody) a blood concentration in the individual of at least at least EC ₅₀ , optionally EC ₇₀ , optionally substantially EC ₁₀₀ , to inhibit CD73-mediated catabolism of AMP to adenosine (e.g., as determined by assessing neutralization of 5'-ectonucleotidase activity in MDA-MB-231 cells by quantitating AMP hydrolysis to adenosine). In one embodiment, the amount of anti-CD73 antibody is an amount effective to achieve (or maintain) EC ₅₀ , optionally EC ₇₀ , optionally substantially EC ₁₀₀ , to inhibit CD73-mediated catabolism of AMP to adenosine in extravascular tissue of an individual.

In one embodiment, a method of treating or preventing cancer in an individual is provided, the method comprising administering to the individual suffering from the disease an anti-CD73 antibody in an amount that achieves or maintains for a specified period of time a concentration in the bloodstream, optionally in the extravascular tissue of interest (eg , in the tumor or in the tumor environment) that is higher than the concentration required to achieve 50%, 70%, or complete (eg, 90%) receptor saturation of CD73-expressing cells in the circulation (eg, as assessed by PBMC ). Optionally, the concentration achieved is at least 20%, 50% or 100% higher than the concentration required to saturate certain receptors.

In one embodiment, a method of treating or preventing cancer in an individual is provided, the method comprising administering to the individual an anti-CD73 antibody in an amount that achieves or maintains for a specified period of time a concentration in the bloodstream, optionally in the extravascular tissue of interest (e.g., tumor or tumor environment) that is higher than EC ₅₀ , optionally EC ₇₀ or optionally EC ₁₀₀ , to bind to CD73-expressing cells (e.g., as assessed by titrating anti-CD73 antibody on CD73-expressing cells, e.g., MDA-MB-231 cells ). Optionally, the achieved concentration is at least 20%, 50% or 100% higher than EC ₅₀ , optionally EC ₇₀ or optionally EC ₁₀₀ for binding to cells expressing CD73.

In any embodiment, the antibody may, for example, have an _EC50 , in some cases an _EC70 , or in some cases an _EC100 for binding to CD73-expressing cells among human PBMCs of 0.5-100 ng/ml, in some cases 1- 100 ng/ml, in some cases 30-100 ng/ml, eg about 30-90 ng/ml (eg, as assessed by titrating anti-CD73 antibody on cells expressing CD73, eg MDA-MB-231 cells).

The EC ₅₀ for neutralizing CD73 enzymatic activity with an anti-CD73 antibody may be, for example, from about 0.01 μg/ml to 1 μg/ml, optionally from 0.1 μg/ml to 10 μg/ml, optionally from 0.1 µg/ml to 1 µg/ml. For example, the EC ₅₀ may be about 0.1 μg/ml, about 0.2 μg/ml, or about 0.3 μg/ml. Thus, an amount of said anti-CD73 antibody is, for example, administered to achieve and/or maintain a blood concentration of at least 0.1 μg/ml, in some cases at least 0.2 μg/ml, in some cases at least 1 μg/ml or in some cases at least 2 μg/ml.

When tissues outside the vasculature (tumor surroundings, such as in the treatment of solid tumors) are targeted, it is generally believed that a dose approximately 10 times higher than the dose that results in the corresponding concentration in the bloodstream is required. An amount of anti-CD73 antibody administered to achieve (and/or maintain) a circulating (blood) concentration of approximately 1 μg/mL, 2 μg/mL, 10 μg/mL, or 20 μg/mL is expected to achieve ( and/or maintaining) a concentration in extravascular tissue (eg, tumor tissue) of approximately 0.1 μg/ml, 0.2 μg/ml, 1 μg/ml, 2 μg/ml, respectively.

In one embodiment, the anti-CD73 antibody is, for example, administered in an amount sufficient to achieve and/or maintain a concentration in tissue (eg, surrounding a tumor) 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 may, for example, be administered in an amount sufficient 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, in the range of 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 ensure that the required concentration is maintained for a specified period of time after administration (eg, 1, 2, 3, 4 weeks, etc.).

In some embodiments, an amount of anti-CD73 antibody is administered to obtain a concentration in the blood (serum) or extravascular tissue (eg, surrounding a tumor) that is at least EC ₇₀ or EC ₁₀₀ to neutralize CD73 enzymatic activity. The antibody may, for example, be administered in an amount sufficient to achieve and/or maintain a concentration in the blood or in extravascular tissue (eg, surrounding a tumor) of at least about 1 μg/ml, 2 μg/ml, 10 μg/ml or 20 mcg/ml.

EC ₅₀ , EC ₇₀ or EC ₁₀₀ can be assessed, for example, in a cell-based assay for neutralizing CD73 enzymatic activity (eg, neutralizing 5'-ectonucleotidase activity in MDA-MB-231 cells by quantitating the hydrolysis of AMP to form adenosine). "EC ₅₀ " for neutralizing CD73 enzymatic activity refers to the effective concentration of anti-CD73 antibody that produces 50% of the maximum response or effect in neutralizing enzymatic activity). "EC ₇₀ " with respect to neutralizing CD73 enzymatic activity refers to the effective concentration of anti-CD73 antibody that produces 70% of the maximum response or effect. "EC ₁₀₀ " with respect to neutralizing CD73 enzymatic activity refers to the effective concentration of anti-CD73 antibody that produces substantially maximum response or effect with respect to such neutralizing enzymatic activity.

In some embodiments, particularly for the treatment of solid tumors, it is expected that the concentration achieved will result in a tissue concentration (outside the vasculature, e.g., in the tumor or tumor surroundings) that corresponds to at least EC ₅₀ to neutralize enzymatic activity. and optionally approximately or at least approximately equal to EC ₁₀₀ .

In one embodiment, the amount of anti-CD73 antibody is from 1 to 20 mg/kg body weight. In one embodiment, said amount is administered to a subject weekly, every two weeks, monthly, or every two months.

In one embodiment, there is provided a method of treating a human subject, comprising administering to said subject an effective amount of an anti-CD73 antibody according to the present invention over at least one administration cycle (optionally at least 2, 3, 4 or more administration cycles), wherein said cycle is a period of eight weeks or less, with one, two, three or four doses of 1-20 mg/kg body weight of anti-CD73 antibody administered in each of at least one cycle. In one embodiment, the anti-CD73 antibody is administered by intravenous infusion.

Suitable treatment protocols for treating a human include, for example, administering to a patient an amount described herein of an anti-CD73 antibody, the method comprising at least one dosing cycle in which at least one dose of an anti-CD73 antibody is administered. Optionally, at least 2, 3, 4, 5, 6, 7, or 8 doses of anti-CD73 antibody are administered. In one embodiment, the administration cycle lasts from 2 weeks to 8 weeks.

A patient suffering from cancer can be treated with an anti-CD73 antibody, with or without a pre-detection step, to assess CD73 expression on cells in the tumor microenvironment (eg, tumor cells, CD4 T cells, CD8 T cells, B cells) . Optionally, the treatment methods may include the step of detecting a CD73 nucleic acid or polypeptide in a biological sample from a tumor from an individual (eg, cancer tissue or tissue adjacent to or distant from cancer, adjacent tissue to cancer, adjacent non-tumor tissue, or adjacent normal tissue). Determining that a biological specimen contains cells expressing CD73 (eg, expressing CD73 at high levels, detecting high staining intensity with anti-CD73 antibody compared to a reference, e.g., healthy tissue) indicates that the patient has cancer that can be very successfully treated with 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 with a reference level corresponding to a healthy individual. Determining that a biological sample contains cells expressing CD73 nucleic acid or polypeptide at a level that is elevated relative to a reference level (eg, healthy tissue) indicates that the patient has a cancer that can be successfully treated with an anti-CD73 antibody in in accordance with the present invention. Optionally, detecting a CD73 polypeptide in a biological sample includes detecting a CD73 polypeptide expressed on the surface of a cancer cell or a CD4 T cell, CD8 T cell or B cell.

Determining whether an individual has a cancer characterized by cells expressing the CD73 polypeptide may, for example, involve obtaining a biological sample (eg, by performing a biopsy) from the individual that contains cells from the environment of the cancer (eg, the tumor or adjacent to the tumor). tissue), contacting said cells with an antibody that binds the CD73 polypeptide, and detecting whether the cells express CD73 on their surface. Optionally, determining whether an individual has cells that express CD73 involves performing an immunohistochemical analysis.

Antibody compositions can be advantageously used in combination with one or more other therapeutic agents, including agents typically used for the particular therapeutic purpose for which the antibody is administered. The additional therapeutic agent is typically administered in amounts and using treatment regimens typically used for that agent in monotherapy for the particular disease or condition being treated. Such therapeutic agents include, but are not limited to, anticancer agents and chemotherapeutic agents.

In one embodiment, anti-CD73 antibodies enhance the effectiveness of agents that neutralize human PD-1 inhibitory activity, for example, those that inhibit the interaction between PD-1 and PD-L1. Accordingly, in one embodiment, the second or additional second therapeutic agent is an antibody or other agent that neutralizes human PD-1 inhibitory activity.

Programmed death protein 1 (PD-1) (also called “programmed cell death protein 1”) is an inhibitory member of the CD28 receptor family. The full-length sequence of human PD-1 can be found under GenBank accession number U64863. Inhibition or neutralization of PD-1 inhibitory activity may involve the use of a polypeptide agent (eg, antibody, Fc domain fusion polypeptide, immunoadhesin, etc.) that prevents PD-L1-induced PD-1 signaling. There are currently at least six agents that block the PD-1/PD-L1 signaling pathway that are commercially available or undergoing clinical evaluation. One agent is BMS-936558 (nivolumab/ONO-4538, Bristol-Myers Squibb; formerly MDX-1106). Nivolumab (trade name Opdivo®) is an FDA-approved anti-PD-L1 fully human IgG4 MAT that inhibits PD-L1 ligand binding to both PD-1 and CD80 and is described as antibody 5C4 in WO 2006/121168, the description of which is included into this application by reference. For patients with melanoma, the most significant overall response (OR) was observed at a dose of 3 mg/kg, while for other types of cancer it was observed at a dose of 10 mg/kg. Nivolumab was typically administered at a dose of 10 mg/kg every 3 weeks until cancer progression occurred. The terms “reduces human PD-1 inhibitory activity,” “neutralizes PD-1,” or “neutralizes human PD-1 inhibitory activity” refers to a process in which the ability of PD-1 to transduce a signal is inhibited by the interaction of PD-1 with one or more its binding partners such as PD-L1 or PD-L2. An agent that neutralizes the inhibitory activity of PD-1 reduces, blocks, inhibits, terminates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. Thus, such an agent may reduce the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes to enhance T cell effector functions such as proliferation, cytokine production and/or cytotoxicity.

MK-3475 (human IgG4 anti-PD1 MAT from Merck), also called lambrolizumab or pembrolizumab (trade name Keytruda®), has been approved by the FDA for the treatment of melanoma and is being tested in other types of cancer. Pembrolizumab was studied at a dose of 2 mg/kg or 10 mg/kg every 2 or 3 weeks until disease progression occurred. MK-3475, also known as Merck 3745 or SCH-900475, is also described in WO 2009/114335.

MPDL3280A/RG7446 (atezolizumab, brand name Tecentriq™, anti-PD-L1 antibody from Roche/Genentech) is a human anti-PD-L1 mAb that contains an engineered Fc domain designed to optimize efficacy and safety by minimizing FcγR binding and subsequent antibody-dependent cell death. cytotoxicity (ADCC). Doses ≤ 1, 10, 15, and 25 mg/kg MPDL3280A were administered every 3 weeks for up to 1 year. In a phase 3 clinical trial, MPDL3280A is administered at a dose of 1200 mg by intravenous infusion every three weeks for NSCLC.

AMP-224 (Amplimmune and GSK) is an immunoadhesin containing an extracellular PD-L2 domain fused to an Fc domain. Other examples of agents that neutralize PD-1 may include an antibody that binds PD-L2 (anti-PD-L2 antibody) and blocks the interaction between PD-1 and PD-L2.

Another example is pidlizumab (CT-011; CureTech) (humanized anti-PD1 IgG1 MAT from CureTech/Teva), pidlizumab (CT-011; CureTech) (see, for example, WO 2009/101611); This agent was tested in thirty patients with rituximab-sensitive relapsed follicular lymphoma (FL) who were administered intravenously 3 mg/kg ST-011 every 4 weeks for a total of 4 infusions, in combination with rituximab at a dose of 375 mg/m ² weekly for 4 weeks, starting 2 weeks after the first infusion of ST-011.

Additional known anti-PD-1 antibodies and other PD-1 inhibitors include AMP-224 (a B7-DC/IgG1 fusion protein proprietary to GSK), AMP-514 described in WO 2012/145493, MEDI-4736 antibody (durvalumab, trade name Imfinzi™, anti-PD-L1 antibody developed by AstraZeneca/Medimmune), described in WO 2011/066389 and US 2013/034559, antibody YW243.55.S70 (anti-PD-L1 antibody), described in WO 2010/077634, MDX- 1105, also known as BMS-936559, is an anti-PD-L1 antibody developed by Bristol-Myers Squibb described in WO 2007/005874, and antibodies and inhibitors described in WO 2006/121168, WO 2009/014708, WO 2009/ 114335 and WO 2013/019906, the disclosures of which are incorporated herein by reference. Additional examples of anti-PD-1 antibodies are described in WO 2015/085847 (Shanghai Hengrui Pharmaceutical Co. Ltd.), for example, antibodies containing CDR1, 2 and 3 of the light chain variable domain with the sequences SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 8, respectively, and CDR1, 2 and 3 of the variable domain of the heavy chain of the antibody with the sequences SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, respectively, and in the mentioned SEQ ID NO The numbering used is according to WO 2015/085847, the description of which is incorporated into this application 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 neutralizing agent is an anti-PD-L1 antibody that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 neutralizing agent is an anti-PD-1 antibody that inhibits the binding of PD-1 to PD-L1. An example of an anti-PD-1 antibody is pembrolizumab (marketed by Merck & Co. as Keytruda™, see also WO 2009/114335, the disclosure of which is incorporated herein by reference). An example of an anti-PD-L1 antibody is MEDI-4736 (durvalumab, trade name Imfinzi™, an anti-PD-L1 antibody developed by AstraZeneca/Medimmune).

In these methods of treatment, the CD73 binding compound and the second therapeutic agent can be administered separately, together or sequentially, or as a cocktail. In some embodiments, the antigen binding compound is administered prior to administration of the second therapeutic agent. For example, the CD73 binding compound can be administered approximately 0-30 days prior to administration of the second therapeutic agent. In some embodiments, the CD73 binding compound is administered over 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 to 5 days before administration of the second therapeutic agent. In some embodiments, the CD73 binding compound is administered concomitantly with the administration of the therapeutic agents. In some embodiments, the CD73 binding compound is administered after administration of the second therapeutic agent. For example, the CD73 binding compound can be administered approximately 0-30 days after administration of the second therapeutic agent. In some embodiments, the CD73 binding compound is administered over 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 to 5 days after administration of the second therapeutic agent.

Examples

Methods

Cloning, production and purification of recombinant huCD73 Molecular Biology

The huCD73 protein was cloned from the MIAPACA-2 cDNA using the following primers TACGACTCACAAGCTTGCCGCCACCATGTGTCCCCGAGCCGCGCG (SEQ ID NO: 20) (forward) and CCGCCCCGACTCTAGAtcaGTGATGGTGATGATGGTGcttgatccgaccttcaactg 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.

Amino acid sequence of cloned huCD73:

Expression and purification of huCD73 proteins

Once the cloned sequence was confirmed, the cells were nucleofected and the production cell pool was then subcloned to produce a huCD73 protein-producing cell clone. The supernatant of the huCD73 clone grown in a roller culture apparatus was collected and purified using a Ni-NTA column and eluting with 250 mM imidazole. The purified proteins were then loaded onto an S200 size exclusion chromatography column. The purified protein corresponding to the dimer was placed in a buffer solution containing Tris 20 mM, pH 7.5, NaCl 120 mM and CaCl2 4 mM for enzyme activity assay, while 20% glycerol was added to the formulation buffer.

SPR assay to evaluate KD AT on recombinant CD73 protein

SPR measurements were carried out on a Biacore™ T200 device at 25°C. Protein A (GE Healthcare) was immobilized on a CM5 sensor chip (GE Healthcare). The chip surface was activated with EDC/NHS (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (Biacore™, GE Healthcare). Protein A was diluted to 10 μg/ml in binding buffer (10 mM acetate, pH 5.6) and introduced into the system until the appropriate immobilization level was achieved (i.e. 2000 RU). Deactivation of the remaining activated groups was carried out using 100 mM ethanolamine pH 8 (Biacore™, GE Healthcare). According to the standard binding kinetics assay protocol recommended by the manufacturer (Biacore™, GE Healthcare kinetic wizard), serial dilutions of human recombinant soluble CD73 protein ranging from 1.23 to 300 nM were sequentially injected onto the captured anti-CD73 antibodies and allowed to dissociate for 10. minutes before regeneration. The entire set of sensorograms was fitted using a 1:1 binding kinetic model. Bivalent affinities and kinetic rate constants of association and dissociation were calculated.

Flow cytometry analysis to assess CD73 antibody recognition

10 ⁵ MDA-MB-231 cells resuspended in FCSB buffer were distributed into 96-well round-bottom microplates (50 μl per well). A range of doses of anti-CD73 mAbs were added to the plates and the cells were incubated for 1 hour at +5±3°C. Cells were washed three times in FCSB by centrifuging the plates at 400 g for 3 min at 4°C. PE-conjugated goat anti-human IgG (H+L) secondary AT F(ab')2 diluted in FCSB was added to the cells and the plates were incubated for an additional 30 minutes at +5±3°C. Cells were washed three times as described above and analyzed on a flow cytometer.

The dependence of the median fluorescence intensity on the MAT concentration was plotted and EC ₅₀ was calculated using the GraphPad Prism™ program.

In Vitro Enzymatic Assay with Recombinant Human or Cynomolgus CD73 Briefly, recombinant human or cynomolgus CD73 was incubated in white 96-well flat-bottomed microplates in the presence of a range of doses of anti-CD73 mAb or isotype control mAb. 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 at +37 ± 1°C for an additional 30 minutes. Luciferase/luciferin-containing CellTiter-Glo™ (Promega) was added to the wells, the plates were incubated for 5 minutes at room temperature in the dark, and the emitted light was measured using an Enspire™ device (Perkin Elmer).

• Conditions:

• ATP+AMP: maximum luciferase inhibition (100%)

• CD73+ATP+AMP: no luciferase inhibition (0%)

Graphs of residual enzyme activity versus anti-CD73 AT concentration were plotted using GraphPad Prism7™ software.

T cell proliferation assay.

Peripheral blood was obtained from healthy donors and mononuclear cells were isolated in a Ficoll gradient. Lymphocytes were further enriched on a 52% Percoll™ gradient (cell pellets) and stained with 2 µM CellTrace™ Violet (Thermofisher). 5x10 ⁴ -1x10 ⁵ stained cells were distributed into 96-well round-bottom plates, incubated for 1 hour at 37°C with anti-CD73 antibodies and activated for 3 to 5 days by adding anti-CD3/anti-CD3 antibody-coated beads. CD28. Inhibition of T cell proliferation was achieved by adding 200 μM AMP. T cell proliferation and the ability of AT to block the immunosuppressive effects of AMP were assessed using flow cytometry by quantitatively analyzing dye dilution on proliferating T cells. The percentage of proliferating T cells was plotted against the concentration of anti-CD73 AT using GraphPad Prism™ software. Some experiments were performed on whole PBMCs from healthy donors or patients with cancer; the protocol was as described above, except that T cell suppression was achieved by adding 0.5 to 1 mM ATP.

For donor or patient comparisons, T cell proliferation was normalized using the following formula:

Analysis of the allogeneic reaction of a mixed culture of lymphocytes (MSC)

Mononuclear cells from healthy donors were isolated on a Ficoll gradient, and monocytes were 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 collection, CD4+ T cells from allogeneic donors were purified by immunomagnetic depletion of non-CD4+ T cells (Miltenyi Biotec) and stained with Cell Trace™ Violet. DCs (10 ⁴ cells/well) and T cells (5x10 ⁴ cells/well) were mixed in 96-well round-bottom plates in the presence of a range of doses of anti-huCD73 mAb and a constant dose of ATP. T cell proliferation and the ability of AT to reverse ATP-mediated suppression were assessed as described for the T cell proliferation assay.

Example 1: Modeling and production of anti-huCD73 antibodies with human framework sequences

The original antibody 3C12, described in PCT publication WO 2016/055609, containing the amino acid sequences VH and VL presented in SEQ ID NO: 27 and 28, respectively, was modified by inserting heavy chain framework regions (FR1, FR2, FR3) from the subgroup into VH human IGHV1-3*01 together with IGHJ4*01 (FR4) and insertions into VL light chain framework regions (FR1, FR2, FR3) from the human IGKV1-33*01 subgroup together with IGKJ2*01 (FR4).

3D models based on different segments of the human VH and VL genes were superimposed and all amino acid differences were carefully examined one at a time. The in silico molecular design was validated using 3D models of both the original chimeric (HPLP) and humanized (H0L0) antibodies. Intra- and extra-chain connections between residues were also assessed to identify and avoid disruption of any important low-energy connections.

The VH and VL sequences of each designed domain were cloned into vectors containing huIgG1-derived constant domains containing the L234A/L235E/G237A/A330S/P331S substitutions and the huCk constant domain, respectively. The CHO cell line was cotransfected with the two resulting vectors. The created pool of cells was used to obtain antibodies in CHO medium. The antibody was then purified using Protein A. The heavy and light chain sequences used to model the chimeric Fab 3C12 variant with human IgG1 constant regions, including the Fc domain containing the substitutions L234A/L235E/G237A/A330S/P331S (retaining N297-linked glycosylation), were as follows:

HP Fab heavy chain (variable domain underlined)

LP Fab light chain (variable domain underlined)

H0 Fab heavy chain (variable domain underlined)

L0 Fab light chain (variable domain underlined)

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

HP VH

LP VL

Heavy chain design

To investigate whether residue 12 (also position 2 according to Kabat numbering) forms a hydrophobic bond with CDR_H3-Y109 and whether it plays a role in the positioning or flexibility of CDR_H3, the valine residue at position 2 was replaced by isoleucine. Sidechains V2 and 12 are not compatible between the two models. V2 also forms a hydrophobic bond with H0-Y109 (Kabat's Y108), but the position of Y109 is slightly modified and H0-Y109 forms a hydrogen bond with LC-Y55 (Kabat's Y54). Y55 is the residue of CDR_L2. Substitution of V2 may affect the position of both CDR_H3 and CDR_L2.

In order to investigate whether residues at positions 28 and 30 (framework region 1, positions 28 and 30 also according to Kabat) affect antigen binding, these positions were replaced by alanine. The original antibody contains the YAFA sequence compared to the YTFT in the H0L0 antibody. Residues A28 and A30 are exposed on the molecular surface near the paratope. It seems likely that the threonine at position 28 is likely to negatively affect antigen binding. On the other hand, the A30 appears unlikely to be decisive.

In order to investigate whether the residue at position 44 (also Kabat 44) plays any role in the VH/VL interface, this residue was backmutated, thereby retaining the series present in the original antibody. The parent antibody contains the GKS sequence compared to the GQR of the H0L0 antibody. R44 is located at the VH/VL contact surface, very close to L45 of the side chain.

In order to investigate whether the residue at position 48 (also Kabat 48) is important, this residue was reverse-mutated, thereby retaining the residue present in the original antibody. The parent antibody contains the WIG sequence compared to the WMG of the H0L0 antibody. I48 and M48 appear to occupy important positions directly downstream of CDR_H2 and participate in a complex hydrophobic network.

To examine whether residues at positions 67 (Kabat position 66) and 68 (Kabat position 67) are important, these residues were backmutated, thereby retaining the residues present in the original antibody. The parent antibody contains the KAT sequence compared to the RVT in the H0L0 antibody. We found very different loop conformations between F64 (Kabat's F63) and K67/R67 (Kabat's K66/R66) in the two models. A68 (67 according to Kabat) is a Vernier remainder.

In order to investigate the role of the residue at position Abnum 70 (position 69 according to Kabat), this residue was reverse-mutated, thereby retaining the leucine present in the original antibody. The parent antibody contains the TLT sequence compared to the TIT of the H0L0 antibody. L70 and 170 (Kabat's L69 and 169) participate in a complex hydrophobic network.

In order to investigate the role of the residue at position 72 (Kabat 71), this residue was reverse-mutated, thereby retaining the series present in the original antibody. The parent antibody contains the TVD sequence compared to the TRD of the H0L0 antibody. V72 and R72 (V71 and R71 according to Kabat) occupy important positions directly below CDR_H2. The distant hydrophilic side chain of residue V72 protrudes onto the surface of the molecule and forms a hydrogen bond with N55 (Kabat's N54). The N55 occupies the same position in both models. However, hydrogen bonding with R72 (Kabat's R71) may reduce the flexibility of the corresponding loop. Therefore, the effect of replacing the original valine with arginine in H0L0 on antigen binding cannot be predicted.

In order to investigate whether the residue at position 74 (Kabat 73) affects antigen binding, this residue was reverse-mutated, thereby retaining the series present in the original antibody. The original antibody contains the DKS sequence compared to the DTS in the H0L0 antibody. K74 and T74 (K73 and T73 according to Kabat) are exposed on the surface of the molecule near the paratope and can participate in antigen binding.

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

Light chain design

The parent antibody contains the DWM sequence compared to the DIQM of the H0L0 antibody. Residue V2 forms a hydrophobic bond with A25. Residue 12 may be exposed to the surface of the paratope, but the effect on paratope organization cannot be predicted. 12 does not form a hydrophobic bond with A25, and this may slightly change the position of CDR-L1. Thus, the isoleucine in residue 2 was replaced by valine.

The original antibody contains a PDR sequence compared to the PSR in the H0L0 antibody. Residue R61 occupies rotameric positions. The loops appear to be oriented in the same way, but do not overlap (lateral displacement corresponding to the overall displacement of the entire domain). In the parent antibody, D60 forms a salt bridge and hydrogen bond with CDR-L2-R54. In the H0L0 antibody, the S60 residue does not contact R54. R54 occupies rotameric positions. Contact with D60 may be important for the CDR-L2 remnants to occupy a specific and appropriate position. Therefore, the aspartic acid residue at position 60 in the H0L0 antibody was replaced by a series.

The original antibody contains the sequence GYG compared to the GSG in the H0L0 antibody. To examine whether residue Y67 contributes to the paratope, the serine in the H0L0 antibody was replaced by tyrosine.

The parent antibody contains the YFC sequence compared to the YYC in the H0L0 antibody. Although Y87 does not appear to form a hydrogen bond with adjacent residues, the role of this residue remains to be studied, and the tyrosine at position 87 in H0L0 was replaced by phenylalanine.

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

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

Antibodies were produced containing combinations of heavy and light chains presented in Table 2 below.

Example 2: Binding to Soluble Protein CD73/ Investigated by SPR

The antibodies shown in Table 2 of Example 1 were tested for binding to soluble human CD73 dimeric proteins and cloned, produced and purified. The affinity and association and dissociation constants of the humanized variants were assessed using SPR analysis. Table 3 summarizes all calculated constants.

For antibody H0L0 with the fully human heavy chain framework region IGHV1-3*01 and IGHJ4*01 and the fully human light chain framework region IGKV1-33*01 and IGKJ2*01, a KD of 65 nM was detected. Table 3 shows that the humanized HxL0 variants (H1L0, H2L0, H3L0, and H4L0) exhibited an association constant equivalent to the other variants but a faster dissociation profile. The KDs of the HxL0 variants were correspondingly higher than those observed for all other variants (Table 3). Therefore, the introduction of fully human light chain amino acid sequences IGKV1-33*01 and IGKJ2*01 into humanized variants appears to adversely affect the affinity of the humanized AT against CD73.

However, all other humanized variants showed an affinity for CD73 similar to that of the chimeric parent antibody 3C12 (0.68 nM). Notably, all heavy chains containing the fully human unmodified IGHV1-3*01 and IGHJ4*01 heavy chain framework regions provided high-affinity binding to CD73.

Example 3 Binding to Cells Expressing Human CD73 Measured by Flow Cytometry

The binding of the humanized antibodies shown in Table 2 to CD73-expressing cells was examined by flow cytometry. Figure 1 shows that all ATs tested recognized human CD73 proteins. The binding efficiency of huCD73 was similar among the humanized variants and the chimeric format, with the exception of H0L0, H2L0 and H3L0, which were less effective than the parent antibody. These observations are consistent with SPR data that revealed lower affinity for HxL0 variants (H1L0, H2L0, H3L0, and H4L0). Consequently, all heavy chains containing the unmodified fully human heavy chain framework regions of IGHV1-3*01 and IGHJ4*01 provided excellent binding to CD73-expressing cells, comparable to that of the parent antibody.

Example 4 In Vitro Enzymatic Assay with Recombinant Soluble Human CD73 Protein

The efficiency of inhibition of the enzymatic activity of the recombinant CD73 protein by the humanized variants was compared with that of the parent antibody (Figure 2).

Figure 2 (panel A) shows that measured luminescence levels were high when ATP and CTG substrates were mixed. When AMF was added to the reaction mixture, the CTG reaction was inhibited, resulting in a decrease in luminescence. In the presence of the rhCD73 protein, which hydrolyzes AMP, the level of luminescence was restored. The blocking of CD73 enzymatic activity by humanized variants is shown in Figure 2.

The results showed that with the exception of the L0 variants (H0L0, H1L0, H2L0, H3L0 and H4L0), which had reduced activity compared to the parent antibody, all humanized variants showed strong inhibition of the enzymatic activity of soluble CD73 protein. However, unexpectedly, most antibodies had slightly lower activity than the parent antibody. Only H1L4 and all H4 variants blocked enzymatic activity with potency similar to that of the parent antibody.

These experiments showed that regardless of the number of back mutations in the heavy chain, the absence of mutation in the light chain using the sequences IGKV1-33*01 and IGKJ2*01 as the acceptor framework region resulted in a significant reduction in the ability to inhibit the activity of soluble CD73 protein.

The best efficacy in blocking CD73 protein activity was achieved with non-H4L0 variants H1L4 and H4Lx (i.e., H4L1, H4L2, H4L3, and H4L4).

Example 5 T Cell Proliferation Assay.

The concentration of AMP capable of inhibiting T cell proliferation was tested in two sets of experiments to determine the concentration of AMP that produces a strong suppressive effect and that allows proliferation to be restored in the presence of the parent antibody 3C12. The enriched lymphocyte fraction was incubated for 3 days in the presence of anti-CD3/anti-CD28 beads and a range of AMP doses (1.6 mM to 50 μM). The best percentage of recovery of CD4+ and CD8+ T cells was observed with 0.2-0.8 mM and 0.2-1 mM AMP, respectively, in two sets of experiments.

The humanized variants and the parent antibody were evaluated in two sets of experiments to compare their ability to restore T cell proliferation inhibited by 0.8 mM AMP.

Figure 3 shows that, with the exception of HxL0 variants, chimeric and humanized ATs were able to restore T cell proliferation (in both CD4+ and CD8+ subsets). Despite their ability to restore proliferation, the potency of most humanized variants was lower than that of the parent antibody. For the variants, H0L4, H1L4, and H4L4 showed levels of activity similar to that of the parent antibody, although they did not achieve the full activity of the parent antibody.

The second experiment was carried out under the same experimental conditions, except that the L0 variants were replaced by an isotype control. The results are presented in Figure 4.

Similar to that demonstrated in Figure 3, with the exception of the HxL0 variants, chimeric and humanized ATs were able to restore T cell proliferation (in both CD4+ and CD8+ subsets). However, despite their ability to restore proliferation, the activity of most humanized variants was again slightly lower compared to that of the parent antibody. The effectiveness of the H0L4, H1L4 and H4L4 variants most closely approximated the effectiveness of the parent antibody.

Example 6: Design of new wireframe and CDR options

The previous examples showed, surprisingly, that there was no direct correlation between the titration in the flow cytometry assay and the effectiveness of inhibition of CD73 enzymatic activity. Interestingly, while variants H1, H2, H3 and H4 were essentially unable to be distinguished based on binding affinities to recombinant CD73 protein in SPR assays and to CD73-expressing cells in flow cytometry analyses, variants H2 and H3 were less effective than the H4 variants in the functional assay for the ability to restore T cell proliferation described in Example 5. One likely reason is that mutations introduced into the H2 and H3 framework regions, which negatively affected the effectiveness of CD73 inhibition (but did not negative effect on CD73 binding affinity) were compensated by mutations introduced into the framework regions of the H4 variant. Consequently, new variants have been developed with substitutions in the framework region and/or heavy chain CDR residues. Based on these observations, substitutions introduced in chains H2, H3, and H4 were classified as either residues that are believed to be essential for function, residues that are not likely to affect function, or residues that negatively affect function. , and residues that restore function.

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

H4+VH:

The amino acid sequence of the full-length H4+ heavy chain containing the human IgG1 Fc domain with substitutions L234A/L235E/G237A/A330S/P331S is presented below:

Antibody H4+L1 contained the human C-kappa domain-containing L1 light chain, the full length sequence of which is shown below:

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

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

The amino acid sequence of the full-length 2H4+ heavy chain containing the human IgG1 Fc domain with substitutions L234A/L235E/G237A/A330S/P331S is presented below:

The amino acid sequence of the full-length light chain of the human C-kappa domain-containing antibody 2L1 is given below:

Example 7: Investigation of new variants in an in vitro enzymatic assay with recombinant CD73 protein

The effectiveness of the humanized variants in inhibiting the enzymatic activity of the recombinant CD73 protein was compared with that of the parent antibody using the methods described in Example 4. All of the new humanized variants prepared as described in Example 6 effectively inhibited the activity of the CD73 protein.

Antibodies H4+Lx were as effective as their original counterpart. Additionally, all 2H4+2Lx variants (2H4+L1, 2H4+L2, 2H4+L3, and 2H4+L4) showed increased CD73 inhibition potency compared to H4+Lx variants. The 2H4+2Lx variants inhibited CD73 activity with potency similar to that of the parent antibody 2HP2LP.

An additional experiment was carried out with different concentrations of CD73 protein (50, 100, 200, 400 ng/ml). The purpose of this experiment was to study the ability of humanized antibodies to block the activity of large quantities of the CD73 protein. Again, the 2H4+2Lx variants were as effective as the parent antibody 2HP2LP.

Example 8: Study of the effectiveness of new variants using the cynomolgus CD73 protein

An experiment on the recombinant protein rec.cyCD73 was carried out with the 2H1Lx and 2H4+2Lx variants. The experimental conditions were the same as those for the human protein experiments, except for the concentration of cyCD73 protein used (400 ng/ml).

The effectiveness of blocking the enzymatic activity of the cyCD73 protein by humanized variants was the same as that of the original (chimeric) antibody.

Example 9: Exploration of New Variants in T Cell Proliferation Assay.

The ability of humanized variants of various antibodies to restore T cell proliferation inhibited by AMP was examined.

Figure 5A shows the inhibitory effect of AMP on T cell proliferation. All humanized variants effectively blocked the inhibitory effect of AMP on T cell proliferation (Figures 5B and C). While most humanized variants were generally as effective as their chimeric parental counterpart in reversing AMP-mediated T cell suppression, the 2H4+2Lx variants were the most effective of all variants, and surprisingly, they were even more effectively blocked the suppressive effect of AMP than the parent antibody 2HP2LP.

Example 10: Comparative Study of New Variants in T Cell Proliferation Assay from Two Human Donors.

Consistent with previous results, humanized 2H4+2Lx variants were further examined in an additional series of T cell proliferation experiments.

In FIG. 6A and 6B show the results obtained in a T cell proliferation assay using cells from two healthy donors, respectively. As previously observed, each of the 2H4+2Lx variants was more effective in restoring T cell proliferation than the parent 2HP2LP (Fig. 6A and B, right panels). No obvious differences were observed between the different 2H4+2Lx variants. Therefore, again, the 2H4+2Lx variants blocked the suppressive effects of AMP more effectively than the parent antibody 2HP2LP.

Taken together, the results obtained from the T cell proliferation assay suggest that humanized variants containing H4+ frameworks and H4+ chains are the most effective antibody variants, and that the 2H4+2Lx variants are overall the most effective of all antibodies.

Example 11. Study of new options in the analysis of allogeneic reaction of mixed culture of lymphocytes (RMCL)

The 2H4+2Lx antibody variant was tested in allogeneic RSCL to confirm the results obtained previously in the T cell proliferation assay. T cell proliferation was inhibited by adding 100 μM ATP (which was cleaved into ADP and AMP by CD39) as shown in Figure 7 for samples from two human donors.

Figure 7 left panel shows that T cell proliferation was inhibited by the addition of ATP. T cell proliferation was restored in the presence of anti-CD73 antibodies (middle and right panels). All humanized 2H4+2Lx variants were able to restore T cell proliferation with efficacy comparable to or better than the parent 2HP2LP antibody. All 2H4+2Lx variants were more effective than the parent 2HP2LP in restoring T cell proliferation under these conditions. These results were consistent with those obtained in a T cell proliferation assay using AMP as an inhibitor.

In conclusion, substitutions introduced into the H4+ and 2H4+ variable regions, together with substitutions introduced into chains L1 and 2L1 (and chains L2, L3, L4, 2L2, 2L3, 2L4), appear to restore (and even improve) important functional properties of the original murine antibodies, which also contain human framework regions and therefore have a reduced risk of immunogenicity in humans. Antibodies 2H4+2Lx are the most effective of all.

One possible explanation for the unexpected finding that CD73 binding affinity does not directly correlate with the effectiveness of functional inhibition of CD73 activity may be due to the fact that these CD73-binding antibodies act as allosteric inhibitors. Antibodies bind to CD73 by an intradimer binding mode in 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 significant rotation of the N-terminal domain, which defines 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) in complex with the CD73 ectodomain and taking into account the 1:1 stoichiometry between the intact antibody and the CD73 dimer, it is suggested that, as a result of steric hindrance, it is unlikely that the intact antibody would bind to open conformational isomers of CD73 . Instead, the intact antibody binds CD73 in an intermediate state in which AMP cannot be hydrolyzed. Because antibodies bind CD73 thus while in an energetically unstable conformation, small changes that alter the rigidity of the antibody structure described here may affect the ability of the antibody to accommodate the changing conformation of the CD73 dimer. Therefore, these changes may affect the ability of the antibody to inhibit CD73 activity in the presence of substrate. However, these small changes in antibody structure will not be observed in SPR assays or other binding assays that are also performed in the absence of substrate. The heavy and light chain variable regions described herein appear to be able to maintain (and even improve upon) the full CD73 interaction dynamics of the parent antibodies while containing human framework regions.

All referenced material, including publications, patent applications and patents cited in this application, are hereby incorporated by reference in their entirety to the same extent as if each referenced material had been separately and specifically identified as being incorporated by reference and were set forth in their entirety in this application (to the maximum extent permitted by law), notwithstanding any specifically cited inclusion of specific documents made elsewhere in this application.

The use of the singular form should be construed to include both the singular and plural unless otherwise indicated herein or unless clearly inconsistent with the context.

Unless otherwise indicated, all precise values provided in this application are examples of the corresponding approximate values (for example, all precise exemplary values proposed with respect to a particular indicator or measure may be considered to also include the corresponding approximate measure, modified by the term "about" when this is appropriate).

It is intended herein that the description of any aspect or embodiment herein by use of terms such as “including,” “having,” “comprising,” or “comprising” with respect to some element or elements includes a similar aspect or embodiment in this application, which “consists of,” “consists essentially of,” or “essentially includes” that specific element or elements, unless otherwise indicated or the context clearly contradicts (for example, reference to a composition described herein as containing a specific element, should be understood to also describe the composition consisting of such element, unless otherwise indicated or there is a clear conflict with the context).

The use of any and all examples or example language (eg, “such as”) provided in this application is intended only to better illuminate the present invention and does not constitute a limitation on the scope of the present invention unless otherwise stated. Nothing in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present invention.

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LIST OF SEQUENCES

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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> Protein

<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> Protein

<213> Synthetic

<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> Protein

<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> Protein

<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

545 550

<210> 23

<211> 216

<212> Protein

<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> Protein

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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> Protein

<213> Artificial

<220>

<223> Synthetic

<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 Lys

450

<210> 48

<211> 213

<212> Protein

<213> Artificial

<220>

<223> Synthetic

<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 (9)

1. An antibody or antibody fragment capable of specifically binding to a human CD73 polypeptide, wherein said antibody or antibody fragment comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (VL) containing the sequence amino acids selected from the group consisting of SEQ ID NOs: 43, 44, 45 and 46. 2. The antibody or antibody fragment according to claim 1, characterized in that the antibody or antibody fragment contains a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 43. 3. The antibody or antibody fragment of claim 1, wherein said antibody or antibody fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 47 and a light chain containing the amino acid sequence of SEQ ID NO: 48. 4. An antibody or antibody fragment according to any one of the above, characterized in that the antibody or antibody fragment is capable of neutralizing the 5'-ectonucleotidase activity of the CD73 polypeptide. 5. An antibody or antibody fragment capable of specifically binding to a human CD73 polypeptide on a cell surface and neutralizing the 5'-ectonucleotidase activity of a CD73 polypeptide on a cell surface, wherein the antibody or antibody fragment contains a heavy chain containing an amino acid sequence that is at least 95% identical to the sequence amino acids SEQ ID NO: 47, and a light chain containing an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 48, wherein said antibody or antibody fragment comprises a heavy chain variable region (VH ), containing the amino acid sequence of SEQ ID NO: 42, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 43. 6. A pharmaceutical composition for the treatment of cancer, comprising an antibody or antibody fragment according to any one of the claims above and a pharmaceutically acceptable carrier. 7. Nucleic acid encoding an antibody or antibody fragment according to any one of paragraphs. 1-5. 8. Use of an antibody or antibody fragment according to any one of paragraphs. 1-5 for the manufacture of a drug for the treatment of cancer. 9. Use according to claim 8, characterized in that the cancer is leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, gastric cancer, esophageal cancer, pancreatic cancer or breast cancer glands.

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