Compositions and methods for treating cancer

Abstract

The present invention relates to antigen-binding compounds that inhibit the enzymatic activity of soluble human CD39. The present invention also relates to cells that produce such compounds; methods for preparing such compounds and antibodies, fragments, variants and derivatives thereof; pharmaceutical compositions comprising the compounds; and methods for using the compounds to diagnose, treat or prevent diseases, such as cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 686,165, filed on June 18, 2018, which is incorporated herein by reference in its entirety including any drawings.

[0003] Sequence Listing Reference

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named "CD39-9_ST25" with a size of 72KB created on May 27, 2019. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to antigen-binding compounds (e.g., antibodies) that inhibit the enzymatic activity of soluble human CD39. The present invention also relates to cells that produce such compounds; methods for preparing such compounds and antibodies, fragments, variants, and derivatives thereof; pharmaceutical compositions comprising the compounds; and methods for using the compounds to diagnose, treat, or prevent diseases, such as cancer. Background Art

[0006] Eight different ENTPD genes encode members of the NTPDase protein family. The individual NTPDase isoforms differ in their cellular location and functional properties. Plasma membrane-bound nucleoside triphosphate diphosphohydrolases control nucleotide levels on the cell surface by hydrolyzing the c- and b-phosphates of nucleotides.

[0007] NTPDase 1 (extracellular nucleoside triphosphate diphosphohydrolase 1), also known as CD39 / ENTPD1 or vascular CD39, works together with another enzyme, CD73 (ecto-5'-nucleotidase), to hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds to adenosine receptors and inhibits T cell and natural killer (NK) cell responses, thereby suppressing the immune system. Adenosine generation via the CD73 / CD39 pathway is thought to be the primary mechanism of the immunosuppressive function of regulatory T cells (Tregs). CD39 + Treg numbers are increased in some human cancers, and CD39 + The importance of Tregs in promoting tumor growth and metastasis has been demonstrated using several in vivo models. However, CD39 is also expressed by tumor cells and CD39 + Tumor cells can mediate immunosuppression through the adenosine pathway. CD39 in cancer cells shows ATPase activity and produces adenosine together with CD73. + CD39 +Cancer cells inhibit the proliferation of CD4 and CD8 T cells and the generation of cytotoxic effector CD8 T cells (CTLs) in a CD39-dependent and adenosine-dependent manner. CD39 has been reported to increase in several solid tumors (colorectal cancer, head and neck cancer, pancreatic cancer) and chronic lymphocytic leukemia. Antibodies that bind to and inhibit CD39 in CD39-expressing cells are disclosed in WO2009 / 095478. Antibody "A1" (eBiosciences, Inc.) is used for staining applications and does not show the ability to neutralize CD39 activity in cells. Hayes et al., (2015) Journal of American Translational Research (Am. J. Transl. Res.) 7 (6): 1181-1188 utilizes anti-CD39 that binds to FcγR and has effector function, but the anti-CD39 is also allegedly blocked. The expression of CD39 on different cell types, including leukocytes and tumor cells, combined with the use of antibodies that do not actually block CD39 or are not pure blockers, creates a complex environment for evaluating the potential activity of antibodies. To date, the only reported inhibitors of the CD39 active site are still small molecule non-hydrolyzable ATP analogs, such as ARL67156, indicating that direct inhibition of the active site is required. However, ARL67156 is not specific for CD39 and also inhibits other NTPDases, such as NTPDase1, NTPDase3, NPP1 or mouse NTPDase8, and in addition only acts as a weak competitive inhibitor (Levesque et al. (2007) Br. J. Pharmacol. 152: 141-150).

[0008] CD39 has two transmembrane domains near the N- and C-termini, short cytoplasmic N- and C-terminal segments, and a large extracellular domain containing an active site. However, although CD39 is usually anchored to the membrane via two transmembrane domains at both ends of the molecule, it has recently been reported that a soluble catalytically active form of CD39 can be found in the circulation of humans and mice (Yegutkin et al., (2012) Journal of the Federation of American Societies for Experimental Biology (FASEB J.) 26(9): 3875–3883). Although various anti-CD39 antibodies have been described, no antibody has been reported to inhibit the ATPase activity of soluble CD39 protein. Summary of the invention

[0009] The inventors have obtained antibodies that inhibit the enzyme (ATPase activity) activity of soluble (extracellular domain) human CD39 proteins. The antibody additionally binds to an epitope on a human CD39 protein expressed on the surface of a cell containing a tumor cell, and effectively inhibits the enzyme (ATPase activity) activity of a cell membrane-bound CD39 enzyme (such as CD39 expressed on the cell surface). The antibody can be advantageously used to achieve greater neutralization of CD39 activity in an individual by neutralizing both membrane-bound and soluble CD39 proteins (including soluble CD39 released or shed from tumor cells), thereby reducing immunosuppression, for example, for the treatment of cancer and / or infectious diseases. Although other anti-CD39 antibodies that inhibit the enzyme (ATPase activity) activity of membrane-bound CD39 enzymes have been previously described, those antibodies do not inhibit soluble CD39 proteins that are not bound to cell membranes.

[0010] In one embodiment, an anti-CD39 antigen-binding domain or a protein comprising the antigen-binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, wherein the antigen-binding domain comprises a heavy chain variable region (VH), wherein the VH comprises CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 8, 9 and 10, and framework FR1, FR2 and FR3 amino acid sequences from human IGHV1-3 genes, such as IGHV1-3*01 (and optionally from human IGHJ1 genes, such as additional framework 4 (FR4) amino acid sequences of IGHJ1*01); and a light chain variable region (VL), wherein the VL comprises CDR1, CDR2 and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 8, 9 and 10, and framework FR1, FR2 and FR3 amino acid sequences from human IGHV1-3 genes, such as IGHV1-3*01; and NO: CDR1, CDR2 and CDR3 of the corresponding amino acid sequences shown in 11, 12 and 13, and framework FR1, FR2 and FR3 amino acid sequences from human IGKV4-1 gene (e.g., IGK4-1 * 01), and optionally from additional framework 4 (FR4) amino acid sequences from human IGKJ4 gene (e.g., IGKJ4 * 01). In one embodiment, the VH further includes one or more amino acid substitutions of residues present in the human framework sequence by different residues (e.g., residues present in a non-human framework) at Kabat positions selected from the group consisting of: 48, 67, 71 and 76. In one embodiment, the VH includes one or more amino acid substitutions in the heavy chain CDR2, for example, at Kabat positions 60 and / or 64. Optionally, the residue at position 60 is serine (e.g., CDR2 includes an N60S substitution). Optionally, the residue present at Kabat position 64 is glutamine (e.g., CDR2 includes a K64Q substitution). In one embodiment, the residue in the VL present at Kabat position 24 is lysine (eg, CDR1 comprises an R24K substitution). Optionally wherein a phenylalanine is present in the VL at Kabat position 36.

[0011] In one embodiment, an anti-CD39 antigen binding domain or a protein comprising the antigen binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, wherein the antigen binding domain or the protein comprising the antigen binding domain comprises a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 27-34, and optionally further comprises one or more amino acid substitutions of residues present in the human framework sequence by different residues (e.g., residues present in a non-human framework) at a Kabat position selected from the group consisting of: 48, 67, 71 and 76; and a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 27-34. NO:35-37 of any one of the amino acid sequence at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, optionally wherein phenylalanine is present at Kabat position 36. In one embodiment, the VH further comprises one or more amino acid substitutions in the heavy chain CDR2 at Kabat position 60 and / or 64. Optionally, the residue in the heavy chain present at Kabat position 60 is a serine residue. Optionally, the residue in the heavy chain present at Kabat position 64 is a glutamine residue. In one embodiment, the VL further comprises an amino acid substitution in the light chain CDR2 at Kabat light chain position 24, optionally further wherein the residue in the light chain present at position 24 is a lysine residue.

[0012] Optionally, the amino acid at position 48 of the Kabat heavy chain is isoleucine. Optionally, the amino acid at position 67 of the Kabat heavy chain is alanine. Optionally, the amino acid at position 71 of the Kabat heavy chain is valine. Optionally, the amino acid at position 76 of the Kabat heavy chain is arginine.

[0013] In one embodiment, VH comprises an alanine residue at position 67 and a valine at position 71 of Kabat.

[0014] In one embodiment, VH includes an isoleucine residue at Kabat position 48, an alanine residue at Kabat position 67, a valine at Kabat position 71, and an arginine at Kabat position 76.

[0015] In one embodiment, the VL comprises a phenylalanine at Kabat position 36 (FR2). In one embodiment, the VL comprises a lysine at Kabat position 24 (CDR1).

[0016] In one embodiment, the anti-CD39 antigen binding domain or a protein comprising the antigen binding domain (e.g., a monoclonal antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) can be characterized as comprising a heavy chain variable region (VH), wherein the VH comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31; and a light chain variable region (VL), wherein the VL comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 36 or 37.

[0017] In one embodiment, an anti-CD39 antigen-binding domain or a protein comprising the antigen-binding domain (e.g., a monoclonal antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) is provided, wherein the antigen-binding domain comprises a heavy chain variable region (VH) and a human framework (e.g., FR1, FR2, FR3, and FR4 of human origin), wherein the VH comprises CDR1, CDR2, and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 8, 9, and 10; and a light chain variable region (VL) and a human framework (e.g., FR1, FR2, FR3, and FR4 of human origin), wherein the VL comprises CDR1, CDR2, and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 11 (or 17 or 18), 12, and 13, wherein the (VH) comprises CDR1, CDR2, and CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 11, 17, or 18), 12, and 13. NO:31 or 6 is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of NO:31 or 6, and the light chain variable region (VL) includes an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO:36 or 37.

[0018] In any embodiment, VH can be characterized as including substitutions at one, two, three or all of Kabat positions 48, 67, 71 and 76. In one embodiment, the residue at position 48 is isoleucine (e.g., M48I substitution). In one embodiment, the residue at position 67 is alanine (e.g., V67A substitution). In one embodiment, the residue at position 71 is valine (e.g., R71V substitution). In one embodiment, the residue at position 76 is arginine (e.g., S76R substitution). In any embodiment, VL can be characterized as including substitutions at Kabat position 36. In one embodiment, the residue at position 36 is phenylalanine (e.g., Y36F substitution).

[0019] In one embodiment, VH includes a human VH framework amino acid sequence, and VL includes a human VL framework amino acid sequence. In one embodiment, the VH segment of the VH human acceptor framework is from a human IGHV1-3 gene segment, optionally further wherein the J segment is from a human IGHJ1 gene segment. In one embodiment, the VH human framework is from a human IGHV1-3*01 gene segment. In one embodiment, the VL domain human acceptor framework is from a human IGKV4-1 gene segment, optionally further wherein the J segment is from a human IGKJ4 gene segment.

[0020] In one embodiment, an anti-CD39 antigen binding domain or a protein comprising the antigen binding domain (e.g., a monoclonal antibody, a multispecific binding protein, a bispecific antibody, etc.) is provided, wherein the antigen binding domain is selected from the group consisting of:

[0021] (a) an antibody binding domain comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 36, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33 or 34; and

[0022] (b) an antibody binding domain, comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 37, and the heavy chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33 or 34.

[0023] In any embodiment, the antibody heavy chain comprises a human CH1 constant domain and a human Fc domain, optionally of the human IgG1 isotype, optionally further comprising the amino acid sequence of any one of SEQ ID NO: 23, 24, 25 or 26. In any embodiment, the antibody light chain comprises a human light chain constant domain, optionally wherein the constant domain is a human kappa domain.

[0024] In one embodiment, an anti-CD39 antibody is provided, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:38, and the light chain comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:39.

[0025] In one embodiment, an anti-CD39 antibody is provided, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:38, and the light chain comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:40.

[0026] In one embodiment, an anti-CD39 antibody or antibody fragment is provided, which comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 38, and the light chain comprises the amino acid sequence of SEQ ID NO: 39.

[0027] In one embodiment, an anti-CD39 antibody or antibody fragment is provided, which comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 38, and the light chain comprises the amino acid sequence of SEQ ID NO: 40.

[0028] In any embodiment, the antigen binding domain or a protein comprising the antigen binding domain, optionally an antibody or antibody fragment, can be characterized as binding to a soluble CD39 protein (sCD39) and inhibiting or neutralizing the ATPase activity of the soluble CD39 protein. In one embodiment, the sCD39 protein lacks the two transmembrane domains found in membrane-bound CD39 (i.e., transmembrane domains near the N- and C-termini). In one embodiment, sCD39 is a non-membrane-bound sCD39 protein found in circulation, such as in a human individual. In one embodiment, sCD39 includes or consists of the amino acid sequence of SEQ ID NO: 43, optionally further including a C-terminal tag or another non-CD39 derived amino acid sequence; optionally wherein the amino acid sequence of SEQ ID NO: 43 further lacks residues 1 to 37 of the sequence of SEQ ID NO: 1 at its N-terminus. The sCD39 protein is characterized as comprising or consisting of the Thr38-Val478 fragment of CD39. Thr38-Val478 protein with a C-terminal His tag is commercially available from R&D Systems, Inc. (Product No. 4397-EN). In one embodiment, when incubated with sCD39 in solution, the protein, antibody or antibody fragment inhibits the ATPase activity of sCD39, for example according to a method or assay performed in the absence of cells disclosed herein (see, e.g., Examples, Methods), for example in tumor cell supernatant. In one embodiment, the protein, antibody or antibody fragment specifically binds to human CD39 protein in both soluble (extracellular domain protein) and membrane-bound forms.

[0029] It is not wished to be bound by theory, some antibodies can neutralize membrane-bound CD39 (memCD39) by inhibiting the domain motion of membrane-bound CD39, but will not similarly affect the activity of soluble CD39 protein (sCD39). It is reported that memCD39 occurs as a homopolymer, and sCD39 is a monomer, and in addition the transmembrane domain in memCD39 undergoes dynamic motion, which is the basis of the functional relationship with the active site. Therefore, unlike sCD39, memCD39 can present the setting that makes antibody-mediated neutralization possible. One possibility is that functional neutralization needs to use a bivalent antibody that simultaneously combines two memCD39 molecules (e.g., in memCD39 homopolymers).

[0030] In addition to being used as a bivalent binder, the antibodies of the present invention that neutralize the activity of sCD39 (and memCD39) can also be effective as monovalent binders, whether or not they target memCD39 in addition to sCD39. Therefore, in one embodiment, an antigen binding protein is provided, which monovalently binds to human CD39 protein (sCD39 and / or memCD39), and neutralizes its enzyme (ATPase) activity. The antigen binding protein can optionally be specified as binding to a single CD39 protein and / or having a single antigen binding domain that can bind to CD39 protein. In one embodiment, an antibody fragment is provided, optionally a F (ab) fragment, a single-chain antibody, a scFv, a multispecific antibody, which monovalently binds to human CD39 protein (sCD39 and / or memCD39) and neutralizes its enzyme (ATPase) activity. In one embodiment, the CD39 neutralizing antigen binding protein monovalently binds to human CD39 protein is a multispecific antigen binding protein, such as a multispecific antibody, a bispecific antibody, a trispecific antibody, etc. In one embodiment, the CD39 neutralizing antigen binding protein that monovalently binds to human CD39 protein includes a first (or single) antigen binding domain that binds CD39 (sCD39 and / or memCD39) and a second antigen binding domain that binds a protein other than CD39.

[0031] Advantageously, in one embodiment, the antibody comprises a human Fc domain that is modified to have reduced or substantially lack binding to human Fcγ receptors, such as one or more (or all) of human CD16, CD32a, CD32b, and CD64. On the one hand, the antibody is independent of toxin-mediated depletion of CD39 inhibitory activity of ADCC-, CDC-, or CD39-expressing cells. These antibodies can be used as "pure" CD39 blockers, allowing for immunomodulatory activity.

[0032] In alternative embodiments, binding molecules can be produced such that they retain and / or mediate effector functions through their Fc domains. In one embodiment, the antibody comprises a human Fc domain that binds to a human Fcγ receptor, e.g., one or more (or all) of human CD16, CD32a, CD32b, and CD64.

[0033] In another embodiment, the Fc domain can be modified to reduce Fcγ receptor binding, optionally by maintaining binding to one or more human Fcγ receptors, but having reduced binding to one or more other human Fcγ receptors.

[0034] In one aspect, the antibody specifically binds to vascular CD39, e.g., the antibody binds to a polypeptide having a sequence of SEQ ID NO: 1, but does not bind to secreted CD39 isoform polypeptides, e.g., CD39-L2 and / or -L4 polypeptides. Optionally, the anti-CD39 antibody specifically binds to vascular CD39, e.g., the antibody binds to a polypeptide having a sequence of SEQ ID NO: 1, but does not bind to membrane-bound CD39 isoforms, e.g., CD39-L1 and / or -L3 polypeptides.

[0035] The antibodies of the present disclosure can inhibit the enzymatic activity of membrane-bound CD39 protein expressed on the cell surface.

[0036] On the one hand, the antibody downregulates its CD39 inhibitory activity independently of CD39.

[0037] In addition to inhibiting soluble CD39, the antibodies of the present disclosure may be able to inhibit the enzymatic activity of membrane-bound CD39 proteins expressed on the cell surface, with or without induction of CD39 internalization, and with or without binding of CD16 (FcγIII receptor), and / or with or without substantially directing ADCC and / or CDC to CD39-expressing cells. Optionally, the antibody retains an Fc domain and maintains binding to human FcRn.

[0038] Although antibodies that act by inducing ADCC and / or CDC may be effective even if they do not completely neutralize / inhibit the ATPase activity of CD39, neutralizing non-depleting antibodies may require stronger inhibition of the enzymatic activity of the ATPase, as long as sufficient antibody binds to CD39-expressing cells to induce ADCC. In one embodiment, the non-depleting antibody will provide a reduction in the ATPase activity of soluble CD39 protein by at least 50%, 60%, 70%, 80% or 90% (e.g., as assessed by the methods disclosed herein), optionally further at a concentration compatible with administering the antibody to a human. In one embodiment, the non-depleting antibody will provide a reduction in the ATPase activity of CD39-expressing cells by at least 70%, 80%, 90% (e.g., as assessed by a reduction in AMP generated by CD39+ cells, such as B cells, Ramos cells, as measured by the methods disclosed herein), optionally further at a concentration compatible with administering the antibody to a human.

[0039] The epitope on CD39 bound by the antibody is present on the CD39 polypeptide as expressed by a range of cells, e.g., cancer cells, CD4 T cells, CD8 T cells, B cells, transfected cells, and is bound with high affinity as determined by flow cytometry.

[0040] The antibodies can optionally be assayed by flow cytometry for EC 50Characterization, it is no more than 2 μg / ml, no more than 1 μg / ml, no more than 0.5 μg / ml, no more than 0.1 μg / ml or no more than 0.05 μg / ml, for binding to cells expressing CD39 polypeptides on their surfaces. In one embodiment, the cell is a cell expressing CD39 on its surface. In one embodiment, the cell is a cell endogenously expressing CD39 on its surface, such as regulatory T (TReg) cells, B cells, cancer cells, lymphoma cells (e.g., Ramos cells), leukemia cells, bladder cancer cells, glioma cells, glioblastoma cells, ovarian cancer cells, melanoma cells, prostate cancer cells, thyroid cancer cells, esophageal cancer cells or breast cancer cells.

[0041] On the one hand, anti-CD39 antibodies are capable of: (a) inhibiting the enzymatic activity of membrane-bound CD39 proteins (e.g., comprising the amino acid sequence of SEQ ID NO: 1) expressed on the cell surface, and (b) inhibiting the enzymatic activity of soluble CD39 proteins. In one embodiment, the antibody does not substantially bind (e.g., through its Fc domain) human Fcγ receptors (e.g., CD16, CD32a, CD32b, CD64) and / or C1q, and / or does not substantially direct ADCC and / or CDC toward CD39-expressing cells. Optionally, the antibody retains the Fc domain and remains bound to human FcRn.

[0042] In one embodiment, the antibody is administered in an amount effective to neutralize the enzymatic activity of sCD39 and / or memCD39 for a desired period of time, such as 1 week, 2 weeks, a month, until the next consecutive administration of the anti-CD39 antibody.

[0043] In one embodiment, the antibody is prepared to provide at least an EC 50 ,EC 70或 EC 100 The dosage and / or frequency of administration is to provide an antibody blood concentration for inhibiting the ATPase activity of the sCD39 protein, optionally wherein the concentration is maintained for at least 1 week, 2 weeks, a month or until the next continuous administration of the anti-CD39 antibody.

[0044] In one aspect, the antibody binds to an epitope on CD39 that includes amino acid residues (eg, one, two, or three residues) selected from the group consisting of: R138, M139, and E142 (see SEQ ID NO: 1).

[0045] In one aspect, the anti-CD39 antibody exhibits reduced binding (e.g., substantially complete loss of binding) to a CD39 polypeptide compared to a wild-type CD39 polypeptide (a CD39 polypeptide of SEQ ID NO: 1), wherein the CD39 polypeptide has a mutation at one, two or three residues selected from the group consisting of: R138, M139 and E142 (with reference to SEQ ID NO: 1); optionally, the mutant CD39 polypeptide has mutations: R138A, M139A and E142K. In an optional aspect, the antibody does not have loss of binding to any of the mutant CD39 polypeptides of Table 1 except mutant 19.

[0046] In one embodiment, the CD39 neutralizing antibody can be characterized by being able to cause a decrease in the ATPase activity of the sCD39 protein in a purified form in a cell-free assay (e.g., sCD39 from a tumor cell culture supernatant), optionally resulting in a decrease in AMP production by sCD39 of at least 70%, 80%, or 90%; optionally causing an increase in the ATP present (compared to a negative control), e.g., as assessed in an assay disclosed herein. For example, sCD39 inhibition can be assessed by quantifying luminescent units proportional to the amount of ATP present, followed by incubation with an anti-CD39 antibody. In one embodiment, the CD39 neutralizing antibody is characterized by an EC for inhibiting the ATPase activity of no more than 1 μg / ml, optionally no more than 0.5 μg / ml, optionally no more than 0.1 μg / ml of sCD39 protein. 50 .

[0047] Optionally, by using Cell Titer Glo in a cell-free version of the assay TM (Promega) was used to quantify the luminescence units to determine the inhibition of ATPase activity of sCD39 protein, wherein a dose range of the test antibody was incubated with soluble recombinant human CD39 protein as described in the Example Methods for 1 hour at 37°C, wherein Cell TiterGlo TM Before adding the (CTG) reagent, 20 μM ATP was added to the plate for another 30 min at 37°C and after incubation in the dark for 5 min, the plate was stained with Enspire TM The emitted light is quantified by a photometer (see, e.g., Methods, Examples).

[0048] In one embodiment, the sCD39 protein is shed sCD39 protein found in or obtained from human tumor cell culture supernatant, optionally from a tumor cell line expressing CD39 at high levels, optionally from Ramos tumor cells.

[0049] Optionally, the CD39 neutralizing antibody is further characterized by being able to cause a decrease in the cellular ATPase activity of CD39 in a purified form, optionally resulting in a reduction of AMP generated by CD39 expressing cells by at least 70%, 80% or 90%. In one embodiment, the CD39 neutralizing antibody is characterized by an EC for inhibiting the ATPase activity of CD39 expressed by cells at no more than 1 μg / ml, optionally no more than 0.5 μg / ml, optionally no more than 0.1 μg / ml. 50 (For example, ECs for inhibiting AMP production by CD39-expressing cells 50 ).

[0050] Optionally, inhibition of ATPase activity of CD39 expressed by the cells is determined by assessing neutralization of ATPase activity in Ramos cells by quantifying AMP generated from ATP hydrolysis (see, e.g., Methods, Examples).

[0051] In one aspect, the neutralization of the ATPase activity of CD39-expressing cells is determined by contacting CD39-expressing cells (e.g., Ramos lymphoma cells as used herein, e.g., available from ATCC, reference CRL-1596) with an antibody and assessing the production of AMP, e.g., by mass spectrometry, wherein a reduction in the generated AMP indicates neutralization of the ATPase activity. Optionally, the antibody causes a reduction of at least 70%, 80%, or 90% in the AMP generated in this assay. Optionally, the antibody causes a reduction of at least 70%, 80%, or 90% in the extracellular ATPase activity of B cells.

[0052] In one aspect, the neutralizing anti-CD39 antibody binds to an antigenic determinant (memCD39) present on both sCD39 and CD39 expressed on the surface of cells.

[0053] In one aspect, the neutralizing anti-CD39 antibody competes for binding to an epitope on CD39 bound by antibodies mAb 20, mAb 21 (or their parental I-394 antibodies), (e.g., an antibody that competes for binding to an epitope on a CD39 polypeptide with an antibody having the heavy and light chain CDRs or variable regions of any of mAb 20, mAb 21, or I-394).

[0054] In one aspect of any embodiment herein, the antigen binding compound binds to the same epitope as monoclonal antibody mAb 20, mAb 21 (or its parent I-394 antibody) and / or competes with it for binding to CD39 polypeptide (e.g., an antibody that binds to the same epitope and / or competes for binding to CD39 polypeptide of an antibody having heavy and light chain CDRs or variable regions of mAb 20, mAb 21 (or I-394)). In one embodiment, the antigen binding compound binds to the same epitope and / or competes for binding to CD39 polypeptide with an antibody having VH and VL regions of SEQ ID NOs: 38 and 39, respectively.

[0055] In one embodiment, the anti-CD39 antibody binds to an epitope comprising one, two or three amino acid residues selected from the group consisting of amino acid residues on CD39 to which mAb 20, mAb 21 (I-394) bind.

[0056] In any embodiment, the binding molecule (e.g., antibody or antibody fragment) includes a variable heavy chain domain (V H ), the V H The heavy chain CDR1, 2 and 3 of antibody I-394 (e.g., as described herein) and the variable light chain domain (V L ), the V L The light chain CDRs 1, 2 and 3 of the corresponding I-394 antibody (e.g., as described herein), or an amino acid sequence wherein the CDRs (or heavy chain and / or light chain CDR sets) have at least 70%, 80%, 90% or 95% amino acid identity with the CDRs (or the heavy chain and / or light chain CDR sets), wherein the VH and VL each comprise a framework domain of human origin (e.g., the VH and VL are different from the corresponding VH and VL of SEQ ID NOs: 6 and 7). Optionally, the CDRs are determined according to the Kabat or IMGT numbering scheme.

[0057] In one aspect, an antibody or antibody fragment comprising a protein comprises an Fc domain that is modified (compared to a wild-type Fc domain of the same isotype) to reduce binding between the Fc domain and a human CD16A, CD16B, CD32A, CD32B and / or CD64 polypeptide, wherein the antibody comprises: (i) a heavy chain comprising CDR1, 2 and 3 of the heavy chain variable region of SEQ ID NO: 31; and (ii) a light chain comprising CDR1, 2 and 3 of the light chain variable region of SEQ ID NO: 36 or 37. In one aspect, the Fc domain is modified (compared to a wild-type Fc domain of the same isotype) to reduce binding between the Fc domain and a human C1q polypeptide. In one embodiment, the antibody comprises amino acid substitutions in the heavy chain constant region of any one, two, three, four, five or more of the residues selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 243, 264, 268, 297, 298, 299, 309, 310, 318, 320, 322, 327, 330 and 331 (Kabat EU numbering). In one embodiment, the antibody has amino acid substitutions in the heavy chain constant region of any three, four, five or more of the residues selected from the group consisting of: 234, 235, 237, 322, 330 and 331.

[0058] In one embodiment, the antibody is administered to an individual with cancer in an amount and frequency sufficient to neutralize the activity of sCD39 in the tumor microenvironment and / or circulation. In one embodiment, the antibody is administered in an amount and frequency sufficient to reduce the generation and / or concentration of adenosine in the tumor microenvironment. In one embodiment, the antibody is administered in an amount and frequency sufficient to reduce the generation and / or concentration of AMP in the tumor microenvironment. In one embodiment, the antibody is administered in an amount and frequency sufficient to neutralize the activity of CD39 expressed by tumor cells. In one embodiment, the antibody is administered in an amount and frequency sufficient to neutralize the activity of CD39 expressed by leukocytes or lymphocytes, such as CD4 T cells, CD8 T cells, TReg cells and / or B cells.

[0059] The antibodies can be used to inhibit CD39-mediated ATP hydrolysis, for example, thereby resulting in a decrease in the concentration of adenosine in the tumor microenvironment and / or circulation. Therefore, these antibodies can be used to reverse the immunosuppressive effects of CD39 and / or adenosine on T cells, B cells, and other cells expressing adenosine receptors (A2A receptors), for example, for the treatment of cancer. In one embodiment, the anti-CD39 antibody neutralizes adenosine-mediated inhibition of proliferation, cytokine production, cytotoxicity, and / or NFκB activity in T cells.

[0060] In another aspect, a method for treating an individual is provided, the method comprising administering to the individual (eg, an individual suffering from a disease, a tumor, etc.) a therapeutically active amount of any anti-CD39 antigen binding compound described herein.

[0061] The antibodies will be useful for inhibiting the production, amount and / or concentration of adenosine into the tumor microenvironment and / or circulation, including but not limited to tumors characterized by detectable, significant, increased or elevated adenosine production, ATP catabolism, or catabolic activity of the CD39 / CD73 axis (e.g., compared to a reference value). In addition, at increased concentrations, antibodies that neutralize soluble CD39 provide substantially complete inhibition of the catabolic activity of the CD39 / CD73 axis. In one embodiment, the antibodies will be useful for inhibiting the production, amount and / or concentration of adenosine into the tumor microenvironment characterized by the presence of CD73 protein (e.g., tumors with soluble CD73 and / or CD73-expressing cells; CD73-positive tumors).

[0062] In one embodiment, antibodies of the present disclosure that neutralize soluble CD39 protein may be advantageously used in combination with CD73 blockade, for example, antibodies of the present disclosure may be administered to an individual with cancer in combination with an agent that inhibits CD73 activity.

[0063] In one aspect, a method for treating an individual is provided, the method comprising, consisting essentially of, or consisting of administering to an individual (e.g., an individual suffering from a disease, a tumor, etc.) a therapeutically active amount of an antigen-binding compound of the present disclosure that inhibits a CD39 polypeptide. In one embodiment, an anti-CD39 antigen-binding compound (e.g., an antibody) is administered to an individual in combination with a second therapeutic agent. In one embodiment, the second therapeutic agent is an agent (e.g., an antibody) that neutralizes the 5'-ectonucleotidase activity of human CD73. In one embodiment, the second therapeutic agent is an agent (e.g., an antibody) that neutralizes the inhibitory activity of human PD-1, optionally an anti-PD-1 antibody, optionally an anti-PD-L1 antibody. In one embodiment, the second therapeutic agent comprises an agent or treatment that induces extracellular release of ATP from tumor cells and / or induces tumor cell death (e.g., a chemotherapeutic agent, a taxane, an anthracycline, a camptothecin, an epothilone, mytomycin, combretastatin, a vinca alkaloid, a nitrogen mustard, a maytansinoid, a calicheamycin, a duocarmycin, a tubulysin, a dolastatin or auristatin, an enediyne, an amatoxin, a pyrrolobenzodiazepine, an ethyleneimine, a radioisotope, a therapeutic protein or peptide, or a toxin).

[0064] In one embodiment, an anti-CD39 antigen binding compound (e.g., an antibody) is administered to an individual who suffers from cancer and has a poor response or prognosis to treatment with an agent that neutralizes the inhibitory activity of human PD-1. In one embodiment, the antibody inhibits CD39 polypeptides in a cell assay. In one embodiment, the compound is a non-consumable antibody (an antibody that does not consume the cells it binds to, such as an Fc silent antibody). Optionally, the compound binds to CD39 polypeptides in a bivalent manner. Optionally, the antibody is a chimeric, humanized or human antibody. Optionally, the antibody includes a heavy chain constant region of an IgG (e.g., IgG1) isotype that is modified to eliminate binding to human Fcγ receptors (e.g., CD16A, CD16B, CD32A, CD32B and / or CD64).

[0065] On the one hand, a method for reducing ATP hydrolysis of CD39-expressing cells (e.g., leukocytes and / or tumor cells in an individual) or a method for neutralizing the enzymatic activity of cell CD39 is provided, the method comprising: contacting CD39-expressing cells with an antigen-binding compound (e.g., an antibody or antibody fragment) of the present disclosure that inhibits CD39. In one embodiment, the step of contacting CD39-expressing cells with an antigen-binding compound of the present disclosure comprises administering to an individual a therapeutically active amount of an antigen-binding compound that inhibits CD39. In one embodiment, the individual suffers from cancer.

[0066] In one aspect, a method for reducing adenosine present in a tumor environment (e.g., in an individual) is provided, the method comprising, consisting essentially of, or consisting of administering to the individual a therapeutically active amount of an antigen-binding compound (e.g., an antibody or antibody fragment) of the present disclosure that inhibits a CD39 polypeptide. In one embodiment, the individual suffers from cancer.

[0067] In one embodiment, the active amount of the antibody that inhibits the CD39 polypeptide is effective to achieve and / or maintain (e.g., until subsequent administration of an antigen binding compound) a blood concentration of at least EC 50 , optionally EC 70 , optionally substantially EC 100 , an amount for inhibiting CD39-mediated catabolism of ATP to AMP in an individual. In one embodiment, an active amount of an antigen binding compound that inhibits CD39 polypeptide is effective to achieve EC 50 , optionally EC 70 , optionally substantially EC 100 , an amount for inhibiting CD39-mediated catabolism of ATP to AMP in an extravascular tissue of an individual. In one embodiment, an active amount of an antigen binding compound that inhibits CD39 polypeptide is effective to achieve EC 50 , optionally EC 70, optionally substantially EC 100 , used to inhibit the catabolism of AMP by ATP mediated by CD39 in an individual. In one embodiment, the active amount of the antigen binding compound that inhibits the CD39 polypeptide is between 1 mg / kg body weight and 20 mg / kg body weight. In one embodiment, the active amount is administered to an individual weekly, biweekly, monthly, or bimonthly.

[0068] Optionally, the individual is a person suffering from or susceptible to cancer. Optionally, the individual is a person suffering from or susceptible to cancer, characterized by malignant cells expressing CD39 and / or the presence (secretion or shedding) or soluble CD39 protein. Optionally, the individual is a person suffering from or susceptible to cancer, and has detectable levels of circulating soluble extracellular CD39 protein or tumor infiltrating leukocytes expressing CD39.

[0069] Optionally, the antibody is characterized by a binding affinity (K) for human CD39 polypeptide. D ) is less than (better than) 10 -9 M, preferably less than 10 -10 M, or preferably less than 10 -11 M, and / or at an EC lower than (better than) 1 μg / ml 50 Binds to human CD39, preferably wherein the EC of the antibody 50 No more than 0.5 μg / ml, optionally no more than 0.2 μg / ml, optionally no more than 0.1 μg / ml, for binding to cells (eg, tumor cells) expressing human CD39 on their cell surface.

[0070] The antibody is optionally a chimeric, human or humanized antibody.

[0071] Optionally, the antibody is characterized by EC 50 , which is used to neutralize the enzymatic activity of CD39 in CD39-expressing cells (e.g., Ramos tumor cells) at less than (better than) 1 μg / ml, optionally less than 0.5 μg / ml.

[0072] In one embodiment, the antibody is a monoclonal antibody or a fragment thereof, which retains binding specificity and the ability to neutralize the enzymatic activity of CD39. In one embodiment, the antibody is an IgG1 antibody. For example, the antibody can be an antibody comprising an Fc domain of a human IgG1 isotype, which is modified to reduce the binding between the Fc domain and an Fcγ receptor (e.g., CD16). In one embodiment, the antibody or fragment lacks an Fc domain or comprises an Fc domain that does not induce antibody-mediated cytotoxicity (ADCC) and / or CDC; optionally, the antibody or fragment thereof comprises an Fc domain that does not bind to an FcγRIIIA (CD16) polypeptide. In one embodiment, compared to a wild-type Fc domain, (e.g., human IgG1, IgG2, IgG3, or IgG4 isotypes) Fc domains include amino acid modifications (e.g., substitutions), wherein the substitutions reduce the ability of the Fc domain (or an antibody containing it) to bind to Fcγ receptors (e.g., CD16) and / or to bind to supplements. In one embodiment, an antibody or fragment thereof is not connected to a toxic portion.

[0073] Also provided are nucleic acids encoding humanized antibodies or antibody fragments having any of the foregoing properties, vectors comprising such nucleic acids, cells comprising such vectors, and methods for producing human anti-CD39 antibodies, the methods comprising culturing such cells under conditions suitable for expressing the anti-CD39 antibodies, and optionally recovering or purifying the antibodies produced. The present disclosure also relates to compositions, such as pharmaceutically acceptable compositions and kits, comprising such proteins, nucleic acids, vectors and / or cells, and one or more additional ingredients that may be active ingredients or inactive ingredients that typically promote formulation, delivery, stability, or other properties of the composition (e.g., various carriers). The present disclosure further relates to various new and useful methods for preparing and using such antibodies, nucleic acids, vectors, cells, organisms and / or compositions, such as in the regulation of CD39-mediated biological activity, for example in the treatment of diseases associated therewith, particularly cancer.

[0074] The present disclosure also provides a method for enhancing the activity of lymphocytes (e.g., T cells) in a subject in need, or for restoring the activity of lymphocytes (e.g., T cells), or for alleviating adenosine-mediated inhibition of lymphocytes (e.g., T cells), the method comprising administering an effective amount of any of the foregoing compositions to the subject. In one embodiment, the subject is a patient suffering from cancer. For example, the patient may suffer from a solid tumor, such as colorectal cancer, renal cancer, ovarian cancer, lung cancer, breast cancer, or malignant melanoma. Alternatively, the patient may suffer from a hematopoietic cancer, such as acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, or non-Hodgkin's lymphoma.

[0075] The present disclosure also provides a method for treating a disease in an individual, the treatment comprising administering to the individual an anti-CD39 antibody that neutralizes the enzymatic activity of CD39 in at least one administration cycle, wherein the concentration in the blood (serum) or extravascular tissue (e.g., tumor environment) between two consecutive administrations of the anti-CD39 antibody is effective to achieve and / or maintain a concentration corresponding to at least an EC 50 (For example, EC between 0.01 μg / ml and 0.5 μg / ml 50 ), optionally EC 70 or optionally EC 100 , for neutralizing the enzymatic activity of CD39 (e.g., between 0.05 μg / ml and 1 μg / ml, between 0.1 μg / ml and 1 μg / ml EC 100 ) is administered at least once, optionally at least twice, in an amount of 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, or 2 μg / ml. For example, the antibody can be administered in an amount that achieves and / or maintains a concentration in the circulation or extravascular tissue (e.g., a tumor environment) of at least about 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, or 2 μg / ml. For example, in order to achieve a concentration in extravascular tissue between 0.05 μg / ml and 1 μg / ml, or between 0.1 μg / ml and 1 μg / ml, the anti-CD39 antibody is administered in an amount that effectively achieves a concentration of the circulation of the anti-CD39 antibody between 0.5 μg / ml and 10 μg / ml, or between 1 μg / ml and 10 μg / ml. Optionally, between two consecutive administrations of the anti-CD39 antibody and / or the entire administration cycle, the anti-CD39 antibody is administered at least twice, and is administered for at least 1 week, 2 weeks, 3 weeks, or 4 weeks in an amount that effectively maintains the concentration of the anti-CD39 antibody at least in the above concentration.

[0076] The present disclosure also provides a method for treating a disease in an individual, the treatment comprising administering to the individual an anti-CD39 antibody that neutralizes the enzymatic activity of CD39 in at least one administration cycle, wherein the anti-CD39 antibody is administered at least once, optionally at least twice, in an amount effective to achieve and / or maintain a blood or tissue concentration of the anti-CD39 antibody of at least 1 μg / ml, optionally at least 10 μg / ml, optionally between 1 μg / ml and 100 μg / ml between two consecutive administrations of the anti-CD39 antibody. Optionally, between two consecutive administrations of the anti-CD39 antibody and / or the entire administration cycle, the anti-CD39 antibody is administered at least twice, and in an amount effective to maintain a continuous blood or tissue concentration of the anti-CD39 antibody of at least 1 μg / ml, optionally at least 10 μg / ml, optionally between 1 μg / ml and 100 μg / ml for at least 1 week, 2 weeks, 3 weeks, 4 weeks.

[0077] These aspects will be more fully described, and additional aspects, features, and advantages will be apparent, in the description provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Representative screening results are shown showing antibodies 1-397, 1-398 and 1-399 compared to the positive control 1-394 antibody.

[0079] Figure 2A Antibodies BY40, 1-394, 1-395 and 1-396 were shown to inhibit cell membrane bound CD39, with both 1-394 and 1-395 showing higher potency at all concentrations and greater maximal inhibition of cellular CD39 compared to BY40. Figure 2B It is shown that antibodies 1-395 and 1-396 both inhibit soluble CD39 compared to negative control (BY40) and positive control (1-394) antibodies.

[0080] Figure 3A The positions of the residues mutated in mutants 5 (M5), 15 (M15), and 19 (M19) on the surface of the CD39 protein are shown. Figure 3B Results for binding to mutants 5, 15 and 19 are shown for the different antibodies.

[0081] Figure 4 The binding of antibody 1-394 to cells expressing human CD39 as assessed by flow cytometry is shown. 1-394 binds to cells expressing human CD39 (CHO-huCD39), cells expressing cynomolgus CD39 (CHO-cyCD39), and Ramos lymphoma cells, but not to cells expressing murine CD39 (CHO-moCD39).

[0082] Figure 5 It was shown that antibody 1-394 was highly effective in blocking CD39 enzymatic activity in tumor (Ramos) cells, cells expressing human CD39 (CHO-huCD39), and cells expressing cynomolgus CD39 (CHO-cyCD39), as assessed by quantification of luminescence units that are proportional to the amount of ATP present.

[0083] Figure 6 Antibody 1-394 was shown to be highly effective in blocking the enzymatic activity of soluble recombinant human CD39 protein as assessed by quantification of luminescence units that are proportional to the amount of ATP present.

[0084] Figure 7 Antibody 1-394 is shown to bind to human CD39, but not to any of the human isoforms CD39-L1, -L2, -L3 or -L4 as assessed in an ELISA assay.

[0085] Figure 8The experimental procedure for evaluating the effect of ATP-mediated DC activation on CD4 T cell activation is shown. ATP-activated DCs were washed and then incubated with allogeneic CD4 T cells (ratio 1 MoDC / 4 T cells) for 5 days for mixed lymphocyte reactions (MLR). T cell activation and proliferation were analyzed by CD25 expression and Cell Trace Violet dilution by flow cytometry.

[0086] Fig. 9 HLA-DR expression on moDCs is shown, and Fig.10 CD83 expression on moDC is shown. These figures show that anti-CD39 blocking antibody I-394 and chemical inhibitors of CD39 lead to moDC activation at each of 0.125mM, 0.25mM or 0.5mM. However, anti-CD39 antibody BY40 or anti-CD73 antibody cannot support ATP-induced dendritic cell (DC) activation, which indicates that the antibody cannot fully block enzyme activity to avoid ATP catabolism. The legend from top to bottom corresponds to the bar chart from left to right in the chart.

[0087] Fig.11 CD25 expression is shown, showing that MoDC activated in the presence of ATP can induce T cell activation and proliferation in the MLR assay; enhancement of ATP-mediated MoDC activation by anti-CD39 blocking antibody I-394 leads to higher T cell proliferation and activation. The legend from top to bottom corresponds to the bars from left to right in the graph.

[0088] Fig. 12A The dose range of anti-CD73 antibodies on CD4 T cell proliferation in the presence of added ATP at 3 different doses of anti-sCD39 antibodies (0.01 μg / ml, 0.1 μg / ml and 1 μg / ml) is shown. Anti-CD39 antibodies that are able to neutralize soluble human CD39 showed strong enhancement of anti-CD73 antibodies in restoring CD4 T cell proliferation. Fig. 12B The dose range of anti-CD73 antibodies on CD8 T cell proliferation is shown, and anti-sCD39 antibodies show a strong enhancement of anti-CD73 antibodies in restoring CD8 T cell proliferation in the presence of added ATP.

[0089] Fig.13 Antibodies titrated by flow cytometry on Ramos lymphoma cells are shown. Antibodies H2L1, H2L1*, H4L1 and H4L1* antibodies (mAbs 8, 9, 20 and 21 respectively) showed the best binding.

[0090] Fig.14Inhibition of ATPase activity in Ramos and Mino tumor cell lines expressing membrane-bound CD39 is shown. H4L1 and H4L1* antibodies (mAb 20 and mAb 21) were the most effective in blocking CD39 enzymatic activity.

[0091] Fig.15A It is shown that antibody I-394 (parental light and heavy chains) has a higher aggregation temperature (TAgg) and improved stability compared to antibody BY40. Fig. 15B It was shown that the humanized variant antibodies of the I-394 antibody having the variable regions H2L1 (mAb 8), H2L1* (mAb 9), H4L1 (mAb 20), and H4L1* (mAb 21) all had high aggregation temperatures (TAgg) and good stability. DETAILED DESCRIPTION

[0092] definition

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

[0094] Human CD39, also known as "vascular" CD39, NTPdase1, ENTPD1, ​​ATPDase, and vascular ATP diphosphohydrolase, exhibits ATPase activity. CD39 hydrolyzes extracellular ATP and ADP into AMP, which is further converted to adenosine by another enzyme, 5' nucleotidase. The amino acid sequence of the mature polypeptide chain of "vascular" human CD39 is shown in GenBank as accession number P49961, the entire disclosure of which is incorporated herein by reference, and is as follows:

[0095] 1MEDTKESNVK TFCSKNILAI LGFSSIIAVI ALLAVGLTQN KALPENVKYG IVLDAGSSHT

[0096] 61SLYIYKWPAE KENDTGVVHQ VEECRVKGPG ISKFVQKVNE IGIYLTDCME RAREVIPRSQ

[0097] 121HQETPVYLGA TAGMRLLRME SEELADRVLD VVERSLSNYP FDFQGARIIT GQEEGAYGWI

[0098] 181TINYLLGKFS QKTRWFSIVP YETNNQETFG ALDLGGASTQ VTFVPQNQTI ESPDNALQFR

[0099] 241LYGKDYNVYT HSFLCYGKDQ ALWQKLAKDI QVASNEILRD PCFHPGYKKV VNVSDLYKTP

[0100] 301CTKRFEMTLP FQQFEIQGIG NYQQCHQSIL ELFNTSYCPY SQCAFNGIFL PPLQGDFGAF

[0101] 361SAFYFVMKFL NLTSEKVSQE KVTEMMKKFC AQPWEEIKTS YAGVKEKYLS EYCFSGTYIL

[0102] 421SLLLQGYHFT ADSWEHIHFI GKIQGSDAGW TLGYMLNLTN MIPAEQPLST PLSHSTYVFL

[0103] 481MVLFSLVLFT VAIIGLLIFH KPSYFWKDMV (SEQ ID NO: 1).

[0104] In the present context, "neutralize" or "neutralizing" (e.g., "neutralize CD39", "neutralize the activity of CD39" or "neutralize the enzymatic activity of CD39"), when referring to a CD39 polypeptide, refers to a process in which the ATP hydrolysis (ATPase) activity of CD39 is inhibited. This particularly includes inhibition of CD39-mediated generation of AMP and / or ADP, i.e. inhibition of CD39-mediated catabolism of ATP to AMP and / or ADP. For membrane-bound CD39, this can be measured, for example, in a cell assay that directly or indirectly measures the ability of a test compound to inhibit the conversion of ATP to AMP and / or ADP. For soluble CD39, this can be measured by incubating a recombinant soluble CD39 as described herein with a test compound and measuring, directly or indirectly, the conversion of ATP to AMP and / or ADP. For example, the disappearance of ATP and / or the generation of AMP can be assessed as described herein, for example by quantifying luminescence units that are proportional to the amount of ATP present. In one embodiment, the antibody formulation results in at least a 60% decrease in the conversion of ATP to AMP, at least a 70% decrease in the conversion of ATP to AMP, or at least an 80% or 90% decrease in the conversion of ATP to AMP, e.g., with reference to an assay described herein (e.g., disappearance of ATP and / or generation of AMP).

[0105] Whenever reference is made to an anti-CD39 binding agent (e.g., an antibody) for the treatment of cancer, "treating cancer" or the like, this may include: (a) a method of treating cancer, the method comprising the steps of administering an anti-CD39 binding agent (for at least one treatment) (preferably in a pharmaceutically acceptable carrier material) to an individual, mammal, especially a human, in need of such treatment, in a dose (therapeutically effective amount) allowing for the treatment of cancer, preferably a dose (amount) as specified herein; (b) using an anti-CD39 binding agent for the treatment of cancer or an anti-CD39 binding agent for said treatment (especially a human); (c) using an anti-CD39 binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer, the method of using an anti-CD39 binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer optionally comprising mixing the anti-CD39 binding agent with a pharmaceutically acceptable carrier or a pharmaceutical preparation comprising an effective dose of an anti-CD39 binding agent suitable for the treatment of cancer; or (d) any combination of a), b) and c), according to the subject matter for which a patent application is allowed in the country in which this application is filed.

[0106] As used herein, the term "antigen binding domain" refers to a domain comprising a three-dimensional structure capable of immunospecific binding to an epitope. Thus, in one embodiment, the domain may include a hypervariable region, optionally a VH and / or VL domain of an antibody chain, optionally at least a VH domain. In another embodiment, the binding domain may include at least one complementarity determining region (CDR) of an antibody chain. In another embodiment, the binding domain may include a polypeptide domain from a non-immunoglobulin scaffold.

[0107] As used herein, the term "antibody" may include polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are designated as one of the following five major classes: IgA, IgD, IgE, IgG and IgM. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light chains and heavy chains, respectively. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as "α", "δ", "ε", "γ" and "μ", respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known. IgG is an exemplary antibody class used herein because it is the most common antibody in physiological situations and because it is most easily prepared in a laboratory setting. Optionally, the antibody is a monoclonal antibody. Specific examples of antibodies are humanized, chimeric, human or other human suitable antibodies. "Antibody" also includes any fragment or derivative of any antibody described herein.

[0108] The term "specific binding" means that the antibody can preferably bind to a binding partner, such as CD39, in a competitive binding assay as assessed using a recombinant form of the protein, an epitope therein, or the native protein present on the surface of an isolated target cell. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

[0109] When an antibody is said to "compete" with a particular monoclonal antibody (e.g., antibody I-394), this means that the antibody competes with the monoclonal antibody in a binding assay using recombinant CD39 molecules or surface-expressed CD39 molecules. For example, if the test antibody reduces the binding of the reference antibody to CD39 polypeptide or CD39-expressing cells in a binding assay, the antibody is said to "compete" with the reference antibody accordingly.

[0110] As used herein, the term "affinity" refers to the strength with which an antibody binds to an epitope. The affinity of an antibody is given by the dissociation constant, Kd, ​​defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant K aDefined by 1 / Kd. Methods for determining the affinity of mAbs can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988); Coligan et al., eds., Current Protocols in Immunology, Green Publishing Associates and Wiley Interscience, New York, (1992, 1993) and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entirety. A standard method for determining the affinity of mAbs, well known in the art, is to use surface plasmon resonance (SPR) screening (e.g., by using BIAcore TM SPR analysis device analysis).

[0111] In the present context, a "determinant" refers to a site of interaction or binding with a polypeptide.

[0112] The term "epitope" refers to an antigenic determinant, and is a region or region on an antigen to which an antibody binds. A protein epitope may include amino acid residues that are directly involved in binding and amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide, i.e., the amino acid residues within the "footprint" of the antibody. It is the simplest form or smallest structural region on a complex antigen molecule that can be combined with, for example, an antibody or a receptor. An epitope may be linear or conformational / structural. The term "linear epitope" is defined as an epitope consisting of continuous amino acid residues on a linear sequence of amino acids (primary structure). The term "conformational or structural epitope" is defined as an epitope consisting of incompletely continuous amino acid residues, and therefore represents a separate portion (secondary, tertiary and / or quaternary structure) of a linear sequence of amino acids that is close to each other by folding the molecule. Conformational epitopes depend on 3-dimensional structures. Therefore, the term 'conformation' is often used interchangeably with 'structure'.

[0113] The term "internalization" used interchangeably with "intracellular internalization" refers to the molecular, biochemical and cellular events associated with the process of transferring molecules from the extracellular surface of a cell to the intracellular surface of a cell. The processes responsible for the internalization of intracellular molecules are well known and may particularly involve the internalization of extracellular molecules (such as hormones, antibodies and small organic molecules); membrane-associated molecules (such as cell surface receptors); and complexes of membrane-associated molecules bound to extracellular molecules (e.g., ligands bound to transmembrane receptors or antibodies bound to membrane-associated molecules). Therefore, "inducing and / or increasing internalization" includes events in which intracellular internalization is triggered and / or the rate and / or degree of intracellular internalization is increased.

[0114] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, biological macromolecules, or extracts made from biological materials. The term "therapeutic agent" refers to an agent that has biological activity.

[0115] For the purposes of this article, a "humanized" antibody refers to an antibody in which the constant and variable framework regions of one or more human immunoglobulins are fused to the binding regions, e.g., CDRs, of an animal immunoglobulin. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding region was derived, but to avoid immune responses against the non-human antibody.

[0116] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally includes amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991) and / or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol 1987; 196:901-917), or an analogous system for defining essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in this region is performed by the methods described above by Kabat et al., supra. Phrases such as "Kabat position," "variable domain residue numbering as in Kabat," and "according to Kabat" refer herein to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids, corresponding to shortening or insertion of a FR or CDR of a variable domain. For example, a heavy chain variable domain may include a single amino acid insert after residue 52 of CDR H2 (residue 52a according to Kabat) and an insertion residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). By comparing the homologous region of an antibody sequence with a "standard" Kabat numbering sequence, the Kabat residue numbering for a given antibody may be determined.

[0117] As used herein, "framework" or "FR" residues refer to regions of an antibody variable domain other than those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0118] The terms "Fc domain", "Fc portion" and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, e.g., from about amino acids (aa) 230 to about aa 450 of a human gamma (γ) heavy chain, or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε and μ of human antibodies), or its naturally occurring allotypes. Unless otherwise indicated, the generally accepted Kabat amino acid numbering of immunoglobulins is used throughout this disclosure (see Kabat et al. (1991) Protein Sequences of Immunological Significance, 5th Ed., United States Public Health Service, National Institute of Health, Bethesda, Maryland).

[0119] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein is substantially purified if it is the predominant species present in a preparation.

[0120] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid.

[0121] The term "recombinant" when used with reference to, for example, a cell or nucleic acid, protein (e.g., an antibody or antibody fragment), or vector, means that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell or expresses native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all.

[0122] In the present context, the term "an antibody that binds" a polypeptide or epitope refers to an antibody that binds to said determinant with specificity and / or affinity.

[0123] The terms "identity" or "identical" when used in the context of a relationship between sequences of two or more polypeptides refer to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with gap alignments (if any) resolved by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, 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, 1993. Press), New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0124] The method for determining identity is designed to give the maximum match between the test sequences. The method for determining identity is described in a publicly available computer program. The computer program method for determining the identity between two sequences includes the GCG program package, including GAP (Devereux et al., "Nucl. Acid. Res." 12,387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wisconsin), BLASTP, BLASTN and FASTA (Altschul et al., "Journal of Molecular Biology (J. Mol. Biol.)" 215,403-410 (1990)). The BLASTX program can be publicly obtained from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIHBethesda, Md.20894; Altschul et al., supra). The well-known Smith Waterman algorithm can also be used to determine identity.

[0125] Antibody production

[0126] The anti-CD39 antigen binding domain or a protein (e.g., an antibody or antibody fragment) comprising such a domain binds to and neutralizes a soluble human CD39 polypeptide, such as a human CD39 polypeptide lacking the two transmembrane domains near the N- and C-termini found in membrane-bound CD39. In one embodiment, the agent inhibits the ATPase activity of CD39. In one embodiment, the antibody inhibits the generation of CD39-mediated adenosine. In one embodiment, the antibody inhibits the CD39-mediated catabolism of ATP to AMP. In one embodiment, the antibody inhibits the inhibition of adenosine-mediated lymphocyte activity (e.g., T cells). On the one hand, the antibody is selected from a full-length antibody, an antibody fragment, and a synthetic or semi-synthetic antibody-derived molecule.

[0127] In one embodiment, an antibody that effectively inhibits the enzymatic (ATPase activity) activity of a soluble (and optionally membrane-bound) CD39 protein can immobilize or restrict the domain movement of the soluble (and optionally membrane-bound) CD39 protein in one of its conformations, thereby preventing it from hydrolyzing its substrate. The antibody can achieve this by simultaneously binding to the C-terminal and N-terminal domains of the soluble (and optionally membrane-bound) CD39.

[0128] In one embodiment, the anti-CD39 antigen binding domain or an antigen binding protein (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) comprising the antigen binding domain includes a complementarity determining region (CDR) and a framework region (FR). The antigen binding domain can be designed or modified to provide desired and / or improved properties.

[0129] In one embodiment, the anti-CD39 antigen binding protein is capable of binding to and inhibiting the activity of a human CD39 polypeptide, the antigen binding protein comprising VH and VL, each comprising a framework (e.g., a framework having a human amino acid sequence) and CDR1, CDR2, and CDR3. In one embodiment, the antigen binding protein, when bound to CD39, restricts the movement of the domain of CD39. Optionally, the VH and / or VL framework (e.g., FR1, FR2, FR3, and / or FR4) is human.

[0130] In certain embodiments, binding molecules and domains can be derived from immunoglobulin variable domains, for example, in the form of related VL and VH domains found on two polypeptide chains or a single-chain antigen binding domain, such as a scFv, a VH domain, a VL domain, a dAb, a V-NAR domain, or a VHH domain.

[0131] In one aspect, the CD39 binding agent is an antibody selected from the group consisting of a fully human antibody, a humanized antibody, and a chimeric antibody.

[0132] In one aspect, the agent is an antibody fragment comprising a constant or Fc domain derived from a human IgG1 constant region or Fc domain, e.g., modified as further disclosed herein.

[0133] In one aspect, the reagent comprises an antibody fragment selected from the group consisting of a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, a Fv fragment, a heavy chain Ig (llama or camel Ig), a V HH Fragments, single domain FV and single chain antibody fragments. In one aspect, the reagent includes a synthetic or semisynthetic antibody-derived molecule selected from scFV, dsFV, miniantibodies, diabodies, triabodies, κ bodies, IgNARs; and multispecific (e.g., bispecific) antibodies. The reagent may optionally further include an Fc domain.

[0134] In one aspect, the antibody is in at least partially purified form.

[0135] In one aspect, the antibody is in substantially isolated form.

[0136] Antibodies can be produced by a variety of techniques known in the art. In one embodiment, antibodies of the present disclosure are produced by selecting from an antibody library (e.g., generated by a phage display library). In another embodiment, antibodies are produced by immunizing non-human animals, preferably mice, with an immunogen including a CD39 polypeptide, preferably a soluble human CD39 extracellular domain polypeptide. The CD39 polypeptide may optionally be or include a fragment or derivative of a full-length CD39 polypeptide, typically an immunogenic fragment, i.e., a portion of a polypeptide including an epitope exposed on the surface of a cell expressing the CD39 polypeptide. Such fragments typically contain at least about 7 consecutive amino acids of a mature polypeptide sequence, and even more preferably at least about 10 consecutive amino acids. Fragments are typically derived substantially from the extracellular domain of a receptor. In one embodiment, the immunogen includes a wild-type human CD39 polypeptide in a lipid membrane, typically on a cell surface. In a specific embodiment, the immunogen includes intact cells, particularly intact human cells, that are optionally processed or cracked. In another embodiment, the polypeptide is a recombinant CD39 polypeptide.

[0137] The step of immunizing a non-human mammal with an antigen can be performed in any manner well known in the art for stimulating the production of antibodies in mice (see, e.g., E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988), the entire disclosure of which is incorporated herein by reference). The isolation of antibody-producing hybridomas is well known and can be performed in any manner well known in the art.

[0138] Antibodies can also be produced by selecting combinatorial libraries of immunoglobulins, for example as disclosed in (Ward et al., Nature, 341 (1989) p. 544, the entire disclosure of which is incorporated herein by reference).

[0139] The identification of one or more antibodies that bind to CD39, particularly to a region on CD39 that is substantially or essentially the same as monoclonal antibody mAb 20 or mAb 21, can be readily determined using any of a variety of immunological screening assays in which antibody competition can be assessed. Many such assays are routinely performed and are well known in the art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997, which is expressly incorporated herein by reference).

[0140] For example, where the test antibody to be detected is obtained from a different source animal, or even a different Ig isotype, a simple competition assay can be used in which a control (e.g., mAb 20 or mAb 21) and test antibody are mixed (or pre-adsorbed) and applied to a sample containing a CD39 polypeptide, such as disclosed in PCT Publication No. WO2018 / 167267, the disclosure of which is incorporated herein by reference. The use of Western blot-based protocols and BIACORE analysis is suitable for such competition studies.

[0141] Determine whether antibody combines in epitope region and can be carried out in a manner known to those skilled in the art.As an example of such mapping / characterization method, the epitope region of anti-CD39 antibody can be determined by using the chemically modified epitope "footprint" of the amine / carboxyl group exposed in CD39 protein.A specific example of such footprint technology is to use HXMS (hydrogen-deuterium exchange detected by mass spectrometry), wherein hydrogen / deuterium exchange, combination and reverse exchange of receptor and ligand protein amide protons occur, wherein the main chain amide group participating in protein binding is protected from reverse exchange, and therefore deuteration will be maintained.At this point, the relevant region can be identified by digestion protein hydrolysis, rapid micropore high performance liquid chromatography separation and / or electrospray ionization mass spectrometry. See, for example, Ehring H, "Analytical Biochemistry", Vol. 267 (No. 2) pp. 252-259 (1999); Engen, JR and Smith, DL (2001) "Analytical Chemistry (Anal. Chem.)" 73, 256A-265A. Another example of a suitable epitope tagging technique is nuclear magnetic resonance epitope mapping (NMR), in which free antigen and antigens in complex with antigen-binding peptides, such as the position of signals in a two-dimensional NMR spectrum of an antibody, are usually compared. Antigens are usually selectively isotopically labeled with 15N so that only signals corresponding to the antigen are seen in the NMR spectrum, and signals from the antigen-binding peptide are not seen. Antigen signals derived from amino acids that participate in the interaction with the antigen-binding peptide will generally be shifted in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids involved in the binding can be identified in the described manner. See, e.g., Ernst Schering Res Found Workshop. 2004;(44):149-67; Huang et al., Journal of Molecular Biology, Vol. 281 (No. 1), pp. 61-67 (1998); and Saito and Patterson, Methods, Jun. 1996;9(3):516-24.

[0142] Epitope mapping / characterization can also be performed using mass spectrometry. See, e.g., Downard, J Mass Spectrom. April 2000; 35(4):493-503; and Kiselar and Downard, Analytical Chemistry, May 1, 1999; 71(9):1792-1801. Protease digestion techniques can also be used for epitope mapping and identification. Antigenic determinant-associated regions / sequences can be determined by protease digestion, for example, by digesting o / n with trypsin at a ratio of about 1:50 with CD39 or at pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. Peptides protected from trypsin cleavage by anti-CD39 binders (thereby revealing the footprint of the binder) can then be identified by incubating the sample with trypsin digestion with the antibody and then comparing the sample with trypsin digestion, for example. Other enzymes such as chymotrypsin, pepsin, etc. can also or alternatively be used in similar epitope characterization methods. Furthermore, enzymatic digestion can provide a rapid method for analyzing whether potential antigenic determinant sequences are within regions of the CD39 polypeptide that are not surface exposed and, therefore, are most likely not relevant in terms of immunogenicity / antigenicity.

[0143] Site-directed mutagenesis is another technique that can be used to elucidate binding epitopes. For example, in "alanine scanning", each residue within a protein segment is replaced with an alanine residue, and the results of binding affinity are measured. If the mutation results in a significant decrease in binding affinity, it is likely involved in binding. Monoclonal antibodies specific for structural epitopes (i.e., antibodies that do not bind to unfolded proteins) can be used to verify that alanine substitutions do not affect the overall folding of the protein. See, for example, Clackson and Wells, Science 1995; 267: 383–386; and Wells, Proc Natl Acad Sci USA 1996; 93: 1–6.

[0144] Electron microscopy can also be used for epitope "footprinting." For example, Wang et al., Nature 1992; 355: 275-278 used a coordinated application of cryo-electron microscopy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of Fab fragments on the capsid surface of native cowpea mosaic virus.

[0145] Other forms of "label-free" assays for epitope assessment include surface plasmon resonance (SPR, BIACORE) and reflectance interferometry spectroscopy (RifS). See, e.g., et al., Journal of Molecular Recognition 1990; 3:208-14; Nice et al., J. Chromatography 1993; 646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37:3308-3311; et al., Biosensors and Bioelectronics 2002;17:937-944.

[0146] Typically, the anti-CD39 antibodies provided herein have an affinity for a CD39 polypeptide (e.g., a monomeric CD39 polypeptide as produced in the Examples herein) in the range of about 10 4 M -1 To about 10 11 M -1 (For example, about 10 8 M -1 To about 10 10 M -1 For example, the average dissociation constant (KD) of the anti-CD39 antibody for CD39 may be less than 1×10 -9 M, such as by, for example, surface plasmon resonance (SPR) screening (e.g., by using BIAcore TM In a more specific exemplary aspect, the present disclosure provides an anti-CD39 antibody having a KD of about 1×10 -8 M to about 1×10 -10 M, or about 1×10 -9 M to about 1×10 -11 M.

[0147] The antibody can be characterized by, for example, an average K D The K can be determined, for example, by immobilizing recombinantly produced human CD39 protein on a chip surface and then applying the test antibody in solution. D In one embodiment, the method further comprises step (d) of selecting an antibody from (b) that is capable of competing with antibody 1-394 or any of, for example, mAb 1-24 for binding to CD39.

[0148] DNA encoding the monoclonal antibodies of the present disclosure, such as antibodies mAb 20 or mAb 21, can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of murine antibodies). In one aspect, nucleic acids encoding the heavy or light chains of the anti-CD39 antibodies of any embodiment herein are provided. After separation, the DNA can be placed in an expression vector and then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulins, to obtain the synthesis of monoclonal antibodies in a recombinant host cell. As described elsewhere in this specification, such DNA sequences can be modified for any number of purposes, such as for humanized antibodies, for producing fragments or derivatives, or for modifying sequences of antibodies, such as in an antigen binding site, to optimize the binding specificity of the antibody. In one embodiment, isolated nucleic acid sequences encoding the light and / or heavy chains of an antibody (e.g., mAb 20 or mAb 21) and recombinant host cells comprising (e.g., in their genome) such nucleic acids are provided. Recombinant expression of antibody-encoding DNA in bacteria is well known in the art (see, e.g., Skerra et al., Curr. Opinion in Immunol. 5, 256 (1993); and Pluckthun, Immunol. 130, 151 (1992).

[0149] Once an antibody is identified that is capable of binding to sCD39 and / or memCD39 and / or has other desired properties, it will generally also be evaluated for its ability to bind to other polypeptides, including unrelated polypeptides, using methods such as those described herein. Ideally, the antibody will only bind to CD39 with substantial affinity, and will not bind to unrelated polypeptides, or other polypeptides of the NTPDase family, particularly CD39-L1, L2, L3 and L4 or NTPDase 8, at significant levels. However, it will be appreciated that as long as the affinity for CD39 is substantially greater (e.g., 10-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold or more) than the affinity for other unrelated polypeptides, then the antibody is suitable for use in the present method.

[0150] In one embodiment, the anti-CD39 antibody can be prepared so that it has no substantial specific binding to human Fcγ receptors, such as any one or more of CD16A, CD16B, CD32A, CD32B and / or CD64). Such antibodies may include constant regions of various heavy chains known to lack or have low binding to Fcγ receptors. Alternatively, antibody fragments that do not include (or include part of) constant regions, such as F(ab')2 fragments, can be used to avoid Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including, for example, testing the binding of antibodies to Fc receptor proteins in a BIACORE assay. Moreover, any antibody IgG isotype can generally be used, in which the Fc portion is modified (e.g., by introducing 1, 2, 3, 4, 5 or more amino acid substitutions) to minimize or eliminate binding to Fc receptors (see, e.g., WO 03 / 101485, the disclosure of which is incorporated herein by reference). Assays for assessing Fc receptor binding, such as cell-based assays, are well known in the art and are described, for example, in WO 03 / 101485.

[0151] In one embodiment, the antibody may include one or more specific mutations in the Fc region, which results in an "Fc silent" antibody with minimal interaction with effector cells. The silent effector function can be obtained by mutations in the Fc region of the antibody, and has been described in the art: N297A mutation, LALA mutation (Strohl, W., 2009, "Curr. Opin. Biotechnol. " Vol. 20 (Issue 6): 685-691); and D265A (Baudino et al., 2008, "Journal of Immunology (J. Immunol.) " 181: 6664-69) also see Heusser et al., WO2012 / 065950, the disclosure of which is incorporated herein by reference. In one embodiment, the antibody includes one, two, three or more amino acid substitutions in the hinge region. In one embodiment, the antibody is IgG1 or IgG2, and includes one, two or three substitutions at residues 233-236, optionally 233-238 (EU numbering). In one embodiment, the antibody is IgG4, and includes one, two or three substitutions at residues 327, 330 and / or 331 (EU numbering). The example of silent Fc IgG1 antibody is the LALA mutant including L234A and L235A mutations in the IgG1Fc amino acid sequence. Another example of Fc silent mutation is the mutation at residue D265 or D265 and P329, for example, as DAPA (D265A, P329A) mutation (US 6,737,056) in IgG1 antibody. Another silent IgG1 antibody includes a mutation at residue N297 (e.g., N297A, N297S mutations), which produces aglycosylated / aglycosylated antibody.Other silent mutations included: substitutions at residues L234 and G237 (L234A / G237A); substitutions at residues S228, L235 and R409 (S228P / L235E / R409K, T, M, L); substitutions at residues H268, V309, A330 and A331 (H268Q / V309L / A330S / A331S); substitutions at residues C220, C226, C229 and P238 (C220S / C226S / C229S / P238S); substitutions at residues C226, C229, E233, L234 and L235 (C226S / C229S / E233P / L234V / L235A); substitutions at residues K322, L235 and L substitutions at residues E318, K320 and K322 (L235E / E318A / K320A / K322A); substitutions at residues (V234A, G237A, P238S); substitutions at residues 243 and 264; substitutions at residues 297 and 299; substitutions such that residues 233, 234, 235, 237 and 238 defined by the EU numbering system include a sequence selected from PAAAP, PAAAS and SAAAS (see WO2011 / 066501).

[0152] In one embodiment, the antibody may include one or more specific mutations in the Fc region. For example, such an antibody may include an Fc domain of human IgG1 origin, including one or more mutations at Kabat residues 234, 235, 237, 330 and / or 331. An example of such an Fc domain includes substitutions at Kabat residues L234, L235 and P331 (e.g., L234A / L235E / P331S or (L234F / L235E / P331S). Another example of such an Fc domain includes substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A / L235E / G237A / P331S). Such an Fc domain Another example of a domain includes substitutions at Kabat residues L234, L235, G237, A330, and P331 (e.g., L234A / L235E / G237A / A330S / P331S). In one embodiment, the antibody includes an Fc domain, optionally of the human IgG1 isotype, comprising: L234X1 substitution, L235X2 substitution, and P331X3 substitution, wherein X1 is any amino acid residue except leucine, X 2 is any amino acid residue except leucine, and X3 is any amino acid residue except proline; optionally, wherein X1 is alanine or phenylalanine or its conservative substitution; optionally, wherein X2 is glutamic acid or its conservative substitution; optionally, wherein X3 is serine or its conservative substitution. In another embodiment, the antibody includes an Fc domain of an optionally human IgG1 isotype, which includes: L234X1 substitution, L235X2 substitution, G237X4 substitution and P331X4 substitution, wherein X1 is any amino acid residue except leucine, X2 is any amino acid residue except leucine, X3 is any amino acid residue except glycine, and X4 is any amino acid residue except proline; optionally, wherein X1 is alanine or phenylalanine or its conservative substitution; optionally, wherein X2 is glutamic acid or its conservative substitution; optionally, X3 is alanine or its conservative substitution; optionally, X4 is serine or its conservative substitution.In another embodiment, the antibody comprises an Fc domain, optionally of the human IgG1 isotype, comprising: L234X1 substitution, L235X2 substitution, G237X4 substitution, G330X4 substitution, and P331X5 substitution, wherein X1 is any amino acid residue except leucine, X2 is any amino acid residue except leucine, X3 is any amino acid residue except glycine, X4 is any amino acid residue except alanine, and X5 is any amino acid residue except proline; optionally, wherein X1 is alanine or phenylalanine or a conservative substitution thereof; optionally, wherein X2 is glutamic acid or a conservative substitution thereof; optionally, X3 is alanine or a conservative substitution thereof; optionally, X4 is serine or a conservative substitution thereof; optionally, X5 is serine or a conservative substitution thereof. In the abbreviated notation used herein, the format is: wild-type residue: position in polypeptide: mutant residue, wherein the residue position is represented according to EU numbering according to Kabat.

[0153] In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence that is at least 90%, 95%, or 99% identical thereto but retains the amino acid residues at Kabat positions 234, 235, and 331 (underlined):

[0154] ASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFP EP VTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPS SS LGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPC PA PE A E GGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSH ED PEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLT VL HQDWLNGKEYKCKVSNKALPA SIEKTISKAKGQPREP QV YTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNG QP ENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23).

[0155] In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence that is at least 90%, 95%, or 99% identical thereto but retains the amino acid residues at Kabat positions 234, 235, and 331 (underlined):

[0156] ASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFP EP VTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPS SS LGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPC PA PE F E GGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSH ED PEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLT VL HQDWLNGKEYKCKVSNKALPA SIEKTISKAKGQPREP QV YTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNG QP ENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24).

[0157] In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence that is at least 90%, 95%, or 99% identical thereto but retains the amino acid residues at Kabat positions 234, 235, 237, 330, and 331 (underlined):

[0158] ASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFP EP VTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPS SS LGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPC PA PE A E G A PSVFLFPPKPKDTLMISRTPE VTCVVVDVSH ED PEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLT VL HQDWLNGKEYKCKVSNKALP S SIEKTISKAKGQPREP QV YTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNG QP ENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25).

[0159] In one embodiment, the antibody comprises a heavy chain constant region comprising the amino acid sequence below, or a sequence that is at least 90%, 95%, or 99% identical thereto but retains the amino acid residues at Kabat positions 234, 235, 237, and 331 (underlined):

[0160] ASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFP EP VTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPS SS LGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPC PA PE A E G A PSVFLFPPKPKDTLMISRTPE VTCVVVDVSH ED PEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLT VL HQDWLNGKEYKCKVSNKALPA SIEKTISKAKGQPREP QV YTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNG QP ENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26).

[0161] Fc silent antibodies mediate no or low ADCC activity, which means that Fc silent antibodies exhibit ADCC activity below 50% specific cell lysis. Preferably, the antibody substantially lacks ADCC activity, for example, Fc silent antibodies exhibit ADCC activity (specific cell lysis) below 5% or below 1%. Fc silent antibodies can also result in a lack of FcγR-mediated cross-linking of CD39 at the surface of CD39-expressing cells.

[0162] In one embodiment, the antibody has a substitution in the heavy chain constant region at any one, two, three, four, five or more of the residues selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 243, 264, 268, 297, 298, 299, 309, 310, 318, 320, 322, 327, 330, 331 and 409 (the numbering of the residues in the heavy chain constant region is according to EU numbering of Kabat). In one embodiment, the antibody comprises a substitution at residues 234, 235 and 322. In one embodiment, the antibody has a substitution at residues 234, 235 and 331. In one embodiment, the antibody has a substitution at residues 234, 235, 237 and 331. In one embodiment, the antibody has a substitution at residues 234, 235, 237, 330 and 331. In one embodiment, the Fc domain is composed of human IgG1 subtype.Amino acid residues are indicated according to EU numbering according to Kabat.

[0163] In one embodiment, the antibody includes an Fc domain that includes amino acid substitutions that increase binding to a human FcRn polypeptide so as to increase the in vivo half-life of the antibody. Exemplary mutations are described in Strohl, W., 2009, Current Reviews in Biotechnology, Vol. 20 (No. 6): 685-691, the disclosure of which is incorporated herein by reference. Examples of substitutions used in antibodies of the human IgG1 isotype are substitutions at Kabat residues M252, S254, and T256; substitutions at residues T250 and M428; substitutions at residue N434; substitutions at residues H433 and N434; substitutions at residues T307, E380, and N434; substitutions at residues T307, E380, and N434; substitutions at residues M252, S254, T256, H433, N434, and 436; substitutions at residue 1253; substitutions at residues P257, N434, D376, and N434.

[0164] In one embodiment, the antibody includes an Fc domain, and the Fc domain includes an amino acid substitution that gives a reduced sensitivity to protease cutting. Matrix metalloproteinases (MMPs) represent the most significant protease family associated with tumorigenesis. Although cancer cells can express MMPs, most extracellular MMPs are provided by different types of stromal cells that infiltrate tumors, and each stromal cell produces a group of specific proteases and protease inhibitors, which are released into the extracellular space and particularly change the environment around the tumor. The MMP present in the tumor microenvironment can cut the antibody in the hinge region, and therefore can cause the inactivation of therapeutic antibodies designed to work in the tumor site. In one embodiment, the Fc domain including amino acid substitutions is selected from any one, two, three or more (or all) of the proteases in the group consisting of the following to reduce the cutting sensitivity: GluV8, IdeS, gelatinase A (MMP2), gelatinase B (MMP-9), matrix metalloproteinase-7 (MMP-7), stromelysin (MMP-3) and macrophage elastase (MMP-12). In one embodiment, the antibody with reduced sensitivity to cutting includes an Fc domain including amino acid replacements at residues E233-L234 and / or L235. In one embodiment, the antibody includes an Fc domain including amino acid replacements at residues E233, L234, L235 and G236. In one embodiment, the antibody includes an Fc domain including amino acid replacements at residues E233, L234, L235 and G236. In one embodiment, the antibody includes an Fc domain including amino acid replacements at one or more residues 233-238, such as making the E233-L234-L235-G236 sequence replaced by P233-V234-A235 (G236 missing). See, for example, WO99 / 58572 and WO 2012087746, the disclosures of which are incorporated herein by reference.

[0165] Antigen binding compounds can be evaluated at any desired stage for their ability to inhibit the enzymatic activity of CD39, in particular to block the ATPase activity of sCD39 and reduce the production of ADP and AMP (and together with CD73, adenosine), and thereby restore adenosine-mediated lymphocyte suppression activity and / or alleviate adenosine-mediated lymphocyte suppression, via soluble CD39 protein and optionally further via CD39-expressing cells.

[0166] The inhibitory activity (eg, immunopotentiating potential) of an antibody can be assessed in, for example, assays that detect the disappearance (hydrolysis) of ATP and / or the generation of AMP.

[0167] The ability of an antibody to inhibit soluble recombinant human CD39 protein can be tested by measuring ATP after incubation of the test antibody with soluble CD39 protein. Briefly, ATP can be measured using the Cell Titer Glo TM Quantification was performed in an assay (Promega) in which a dose range of the test antibody was incubated with soluble recombinant human CD39 protein described in Example 1 for 1 hour at 37°C. 20 μM ATP was added to the plate for an additional 30 minutes at 37°C before adding the CTG reagent. After a brief incubation of 5 minutes in the dark, the plate was quantified using Enspire TM A luminometer quantifies the emitted light.

[0168] The ability of an antibody to inhibit cells expressing CD39 protein can be tested by detecting ATP after incubating the test antibody with cells (e.g., Ramos cells, cells transfected with CD39, etc.). See, e.g., Examples, Methods. Cells can be incubated with the test antibody for 1 hour at 37°C. The cells are then incubated with 20 μM ATP for another hour at 37°C. The plate is centrifuged at 400 g for 2 minutes, and the cell supernatant is transferred in a luminescent microplate (white wells). CTG is added to the supernatant, and after incubation in the dark for 5 minutes, the supernatant is measured using Enspire 500. TM The luminometer quantifies the emitted light. Anti-CD39 antibody efficacy was determined by comparing the emitted light in the presence of the antibody to ATP alone (maximum light emission) and ATP together with cells (minimum light emission).

[0169] In the presence of the antibody, a decrease in ATP to AMP hydrolysis and / or an increase in ATP and / or a decrease in AMP generation indicates that the antibody inhibits CD39. In one embodiment, the antibody preparation is capable of reducing the enzymatic activity of a CD39 polypeptide expressed by a cell by at least 60%, preferably the antibody reduces the enzymatic activity of a CD39 polypeptide in a cell by at least 70%, 80% or 90%, such as after incubating cells expressing a CD39 polypeptide (e.g., Ramos cells) with the test antibody, by using Cell Titer Glo TM (Promega) for ATP detection, e.g., as described in the Example Methods.

[0170] In one embodiment, the antibody preparation is capable of reducing the enzymatic activity of a soluble recombinant CD39 polypeptide by at least 60% (e.g., in the absence of cells), preferably reducing the enzymatic activity of a soluble recombinant CD39 polypeptide by at least 70%, 80% or 90%, such as by using Cell Titer Glo TM (Promega) for ATP detection, e.g., as described in the Example Methods.

[0171] The ability of the antibody to modulate the activity of immune cells (e.g., adenosine receptor-expressing immune cells; A2A receptor-expressing cells) can also be measured in an indirect assay, for example to alleviate adenosine-mediated inhibition of lymphocyte activity, or to cause activation of lymphocyte activity. This can be addressed, for example, using a cytokine release assay. In another example, the ability of an antibody to modulate the proliferation of lymphocytes can be assessed in an indirect assay.

[0172] Epitopes on CD39

[0173] In one aspect, the antibody binds to an antigenic determinant present on CD39 expressed on the surface of a cell.

[0174] In one aspect, the antibody binds to an epitope substantially the same as an antibody having the VH and VL of mAb 1-24 (or 1-394). In one embodiment, the antibody binds to an epitope of CD39 that at least partially overlaps with or includes at least one residue in the epitope to which antibody mAb 1-24 (or 1-394) binds. The residue to which the antibody binds can be specified as being present on the surface of a CD39 polypeptide, such as a CD39 polypeptide expressed on a cell surface.

[0175] The binding of anti-CD39 antibodies to cells transfected with CD39 mutants can be measured and compared with the ability of the anti-CD39 antibodies to bind to wild-type CD39 polypeptides (e.g., SEQ ID NO: 1). Reduced binding between anti-CD39 antibodies and mutant CD39 polypeptides (e.g., mutants of Table 1) means reduced binding affinity (e.g., as measured by known methods, such as FACS testing of cells expressing specific mutants, or by Biacore testing of binding mutant polypeptides) and / or a reduction in the total binding capacity of the anti-CD39 antibody (e.g., as demonstrated by a reduction in Bmax in a graph of anti-CD39 antibody concentration versus polypeptide concentration). A significant reduction in binding indicates that the mutated residue is directly involved in the binding of the anti-CD39 antibody or is in close proximity to the binding protein when the anti-CD39 antibody binds to CD39.

[0176] In some embodiments, a significant reduction in binding means that the binding affinity and / or capacity between the anti-CD39 antibody and the mutant CD39 polypeptide is reduced by greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% relative to the binding between the antibody and the wild-type CD39 polypeptide. In certain embodiments, the binding is reduced below the detectable limit. In some embodiments, a significant reduction in binding is demonstrated when the binding of the anti-CD39 antibody to the mutant CD39 polypeptide is less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%) of the binding observed between the anti-CD39 antibody and the wild-type CD39 polypeptide.

[0177] In some embodiments, anti-CD39 antibodies are provided that exhibit significantly lower binding to a mutant CD39 polypeptide in which residues in a segment including amino acid residues bound by antibody mAb 1-24 (or 1-394) are substituted with different amino acids as compared to the binding of a wild-type CD39 polypeptide (e.g., a polypeptide of SEQ ID NO: 1) that does not include such one or more substitutions.

[0178] In one embodiment, the binding of the antibody to a mutant CD39 polypeptide comprising a mutation at one or more (or all) residues selected from the group consisting of: R138, M139 and E142 (with reference to SEQ ID NO: 1) is reduced relative to the binding between the antibody and a wild-type CD39 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 in each case.

[0179] Exemplary Antibody Variable Region Sequences

[0180] Examples of antibodies of the present disclosure include antibodies comprising the VH domain and the VL domain of any one of antibodies mAb 1 to mAb 24. The VH and VL sequences of anti-CD39 antibodies mAb 1 to mAb 24 are provided in Tables A and B (Example 13).

[0181] An exemplary highly effective anti-CD39 VH and VL pair according to the present disclosure is the VH and VL pair of antibody mAb 20, the amino acid sequence of its heavy chain variable region is listed below (SEQ ID NO: 31), and the amino acid sequence of its light chain variable region is listed below (SEQ ID NO: 36). Such an antibody may, for example, have 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.

[0182] Another exemplary highly effective anti-CD39 VH and VL pair according to the present disclosure is the VH and VL pair of antibody mAb 21, the amino acid sequence of its heavy chain variable region is listed below (SEQ ID NO: 31), and the amino acid sequence of its light chain variable region is listed below (SEQ ID NO: 37). Such an antibody may, for example, have 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: 40.

[0183] In any aspect, an isolated antibody that binds to a human CD39 polypeptide can be specified as comprising VH and VL frameworks (eg, FR1, FR2, FR3, and FR4) of human origin. In one aspect, the antibody comprises: HCDR1 comprising the amino acid sequence: DYNMH (SEQ ID NO: 8), or a sequence of at least 4 consecutive amino acids thereof, optionally wherein one or more of these amino acids can be substituted with a different amino acid; HCDR2 comprising the amino acid sequence: YIVPLNGGSTFNQKFKG (SEQ ID NO: 9), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, optionally wherein one or more of these amino acids can be substituted with a different amino acid, optionally wherein the asparagine at Kabat position 61 is substituted, optionally wherein the lysine at Kabat position 65 is substituted; HCDR3 comprising the amino acid sequence: GGTRFAY (SEQ ID NO: 10), or a sequence of at least 4, 5 or 6 consecutive amino acids thereof, optionally wherein one or more of these amino acids can be substituted with a different amino acid; LCDR1 comprising the amino acid sequence: RASESVDNFGVSFMY (SEQ ID NO: 11). NO: 11), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, optionally wherein one or more of these amino acids can be substituted with a different amino acid, optionally wherein the arginine at Kabat position 24 is substituted; a LCDR2 region comprising the amino acid sequence: GASNQGS (SEQ ID NO: 12), or a sequence of at least 4, 5 or 6 consecutive amino acids thereof, optionally wherein one or more of these amino acids can be substituted with a different amino acid; and / or a LCDR3 region comprising the amino acid sequence: QQTKEVPYT (SEQ ID NO: 13), or a sequence of at least 4, 5, 6, 7 or 8 consecutive amino acids thereof, optionally wherein one or more of these amino acids can be deleted or substituted with a different amino acid. The CDR positions can be numbered according to Kabat.

[0184] In one embodiment, HCDR2 comprises the amino acid sequence of Formula I:

[0185] YIVPLNGGSTF-Xaa1-QKF-Xaa2-G (SEQ ID NO: 14), or a subsequence thereof, wherein Xaa1 can be any amino acid residue, optionally wherein Xaa1 is asparagine or serine; wherein Xaa2 can be any amino acid residue, optionally wherein Xaa2 is lysine or glutamine. In one embodiment, HCDR2 comprises the amino acid sequence: YIVPLNGGSTFSQKFKG (SEQ ID NO: 15). In one embodiment, HCDR2 comprises the amino acid sequence: YIVPLNGGSTFSQKFQG (SEQ ID NO: 16).

[0186] In one embodiment, LCDR1 comprises the amino acid sequence of Formula II:

[0187] Xaa3-ASESVDNFGVSFMY (SEQ ID NO: 17), wherein Xaa3 can be any amino acid residue, optionally wherein Xaa3 is lysine or arginine. In one embodiment, LCDR1 comprises the amino acid sequence: KASESVDNFGVSFMY (SEQ ID NO: 18).

[0188] In one embodiment, the antibody comprises a heavy chain framework from human subgroup IGHV1-3 (optionally together with IGHJ1), optionally IGHV1-3 is IGHV1-3*01. In one embodiment, the humanized antibody comprises a light chain framework from human subgroup IGKV4-1 (optionally together with IGKJ4).

[0189] In one aspect, the present invention provides an antigen binding domain or antibody that binds to a human CD39 polypeptide, the antigen binding domain or antibody comprising:

[0190] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8;

[0191] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 9, 14, 15 or 16;

[0192] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10;

[0193] (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11, 17 or 18;

[0194] (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 12;

[0195] (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13; and

[0196] (g) Human heavy and light chain framework sequences.

[0197] The antibody may further include one, two, three, four, five or more amino acid substitutions across human heavy and / or light chain frameworks, such as to enhance affinity, stability or other properties of the antibody. Optionally, substitutions introduce residues present at specific positions of non-human mammals (e.g., mice or rats).

[0198] In any of the embodiments of the VH sequences herein, the amino acid at position 67 of the Kabat heavy chain can be alanine.

[0199] In any of the embodiments of the VH sequences herein, the amino acid at position 71 of the Kabat heavy chain is valine.

[0200] In any of the embodiments of the VH sequences herein, the amino acid at position 76 of the Kabat heavy chain can be arginine.

[0201] In some embodiments of the VH sequences herein, the amino acid at position 48 of the Kabat heavy chain can be isoleucine. In other embodiments of the VH sequences herein, the amino acid at position 48 of the Kabat heavy chain can be methionine.

[0202] In one embodiment, VH comprises an alanine residue at position 67 and a valine at position 71 of Kabat.

[0203] In one embodiment, VH includes an isoleucine residue at Kabat position 48, an alanine residue at Kabat position 67, a valine at Kabat position 71, and an arginine at Kabat position 76.

[0204] In any of the embodiments of the VL sequences herein, the VL includes a phenylalanine (FR2) at Kabat position 36. In one embodiment, the VL includes a lysine (CDR1) at Kabat position 24.

[0205] Positions within the VH and VL domains herein are described using the Kabat numbering system (Kabat et al. (1991) Protein Sequences of Immunological Significance, 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, MD).

[0206] In one aspect, an anti-CD39 antibody comprises a heavy chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98%, 99% or more identity) to a heavy chain having the amino acid sequence of SEQ ID NO:38.

[0207] In one aspect, the anti-CD39 antibody comprises a light chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98%, 99% or more identity) to a light chain having an amino acid sequence of SEQ ID NO: 39 or 40.

[0208] In any aspect, the specified heavy chain, light chain, variable region, FR and / or CDR sequence can include one or more sequence modifications, such as substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more sequence modifications). In one embodiment, the substitution is a conservative modification.

[0209] The present invention also encompasses functional conservative variants of antibodies disclosed herein. "Functional conservative variants" are variants in which a given amino acid residue in a protein or enzyme has been changed without changing the overall conformation and function of the polypeptide, including but not limited to replacing an amino acid with an amino acid having similar properties (e.g., polarity, hydrogen bonding potential, acidity, alkalinity, hydrophobicity, aromaticity, etc.). Amino acids other than those indicated as conservative amino acids may be different in a protein, so that the percentage of protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, 70% to 99% as determined by a clustering method, according to an alignment scheme, wherein the similarity is based on a MEGALIGN algorithm. "Functional conservative variants" also include polypeptides having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, still preferably at least 90% and even more preferably at least 95%, as determined by a BLAST or FASTA algorithm, and having the same or substantially similar properties or functions as the native or parent protein to which it is compared.

[0210] In any embodiment, the antibody or antibody fragment may optionally be designated as an antibody other than antibody 1-394 (e.g., having VH and VL amino acid sequences shown in SEQ ID NOs: 6 and 7, respectively). In any embodiment, the antibody or antibody fragment may optionally be designated as an antibody other than antibody 1-395, 1-396, 1-397, 1-398 or 1-399 (e.g., having VH and VL amino acid sequences disclosed in PCT patent application No. PCT / EP2018 / 056661 filed on March 16, 2018, the disclosure of which is incorporated herein by reference). In any embodiment, the antibody or antibody fragment can optionally be designated as an antibody other than antibody By40, Ba54g or BY12 disclosed in U.S. Patent Publication No. US 2016 / 0137747A1 (e.g., an antibody other than antibody BY40 produced by a hybridoma cell line producing such an antibody, having the VH and VL amino acid sequences of By40, Ba54g or BY12).

[0211] Fragments and derivatives of antibodies (which are covered by the terms "antibody" or "antibodies" as used in this application, unless otherwise specified or clearly contradictory in context) can be produced by techniques known in the art. "Fragments" include a portion of an intact antibody, typically the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2 and Fv fragments; diabodies; any antibody fragment having a polypeptide with a primary structure consisting of an uninterrupted sequence of continuous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including but not limited to (1) single-chain Fv molecules (2) single-chain polypeptides containing only one light chain variable domain, or containing three CDRs of a light chain variable domain, without its associated heavy chain portion and (3) single-chain polypeptides containing only one heavy chain variable region, or containing three CDRs of a heavy chain variable region, without its associated light chain portion; and multispecific (e.g., bispecific) antibodies formed by antibody fragments. In particular, nanobodies, domain antibodies, single domain antibodies or "dAbs" are included.

[0212] Anti-CD39 antibodies can be incorporated into a pharmaceutical formulation comprising a concentration of 1 mg / ml to 500 mg / ml, wherein the pH of the formulation is 2.0 to 10.0. The formulation may further include a buffer system, one or more preservatives, one or more tonicity agents, one or more chelating agents, stabilizers, and surfactants. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation comprising water. Such formulations are typically solutions or suspensions. In further embodiments, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution comprising at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w water.

[0213] In another embodiment, the pharmaceutical formulation is a lyophilized formulation and the physician or patient adds solvents and / or diluents prior to use.

[0214] In another embodiment, the pharmaceutical formulation is a dry formulation (eg, freeze-dried or spray-dried) and is ready for use without any prior dissolution.

[0215] In another aspect, the pharmaceutical formulation comprises an aqueous solution of such an antibody and a buffer, wherein the antibody is present at a concentration of 1 mg / ml or greater, and wherein the pH of the formulation is from about 2.0 to about 10.0.

[0216] In another embodiment, the pH of the formulation is in a range selected from the list consisting of about 2.0 to about 10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, and about 5.5 to about 7.5.

[0217] In further embodiments, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate and tris(hydroxymethyl)-aminomethane, bicine, trimethylglycine, malic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment.

[0218] In other embodiments, the formulation further comprises a pharmaceutically acceptable preservative. In other embodiments, the formulation further comprises an isotonic agent. In other embodiments, the formulation further comprises a chelating agent. In other embodiments, the formulation further comprises a stabilizer. In other embodiments, the formulation further comprises a surfactant. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0219] Other ingredients may be present in the peptide drug formulation. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, fillers, tension modifiers, chelating agents, metal ions, oily vehicles, proteins (e.g., human serum albumin, gelatin or protein) and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine and histidine). Of course, such additional ingredients should not adversely affect the overall stability of the drug formulation.

[0220] Pharmaceutical compositions containing antibodies can be administered to patients in need of such treatment at several sites, such as local sites, such as skin and mucosal sites, sites of bypass absorption, such as in arteries, veins, heart, and sites involved in absorption, such as skin, subcutaneous, muscle, or abdomen. Administration of the pharmaceutical composition can be by several routes of administration, such as subcutaneous, intramuscular, intraperitoneal, intravenous, lingual, sublingual, buccal, oral, gastric and intestinal, nasal, pulmonary, such as through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, such as through the conjunctiva, urethra, and parenteral to patients in need of such treatment.

[0221] Suitable antibody formulations can also be determined by examining the experience of other developed therapeutic monoclonal antibodies. Several monoclonal antibodies have been shown to be effective in clinical situations, such as Rituximab, Herceptin (Trastuzumab), Sorel (Omalizumab), Bexar (Tositumomab), Campas (Alemtuzumab), Zevalin, Oncolym and similar formulations can be used with antibodies. For example, monoclonal antibodies can be supplied at a concentration of 10 mg / mL in 100 mg (10 mL) or 500 mg (50 mL) disposable vials, formulated for IV administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80 and sterile water for injection. The pH is adjusted to 6.5. In another embodiment, the antibody is supplied with a formulation including about 20 mM sodium citrate, about 150 mM NaCl, and the pH is about 6.0.

[0222] Diagnosis and treatment of diseases

[0223] Also provided is a method for treating an individual, especially a human individual, with an anti-CD39 agent of the present disclosure. In one embodiment, the present disclosure provides the use of an antibody or antibody fragment as described herein in the preparation of a pharmaceutical composition for administration to a human patient. Typically, the individual suffers from cancer or an infectious disease (e.g., viral infection, bacterial infection) or is at risk thereof. In one embodiment, the individual has detectable soluble (extracellular) CD39 protein in the circulation and / or in a tissue sample (e.g., a tissue sample of a tumor or adjacent tumor).

[0224] For example, on the one hand, there is provided a method for restoring or enhancing the activity of lymphocytes in an individual in need, the method comprising administering to the individual a step of neutralizing anti-CD39 antibodies or antibody fragments disclosed herein. In one embodiment, the method relates to increasing the activity of lymphocytes (e.g., T cells) in an individual with a disease, in which the increased lymphocyte activity is beneficial, or the disease is caused or characterized by immunosuppression, immunosuppressive cells, or adenosine generated, for example, by CD4 T cells, CD8 T cells, or B cells. The method is particularly useful, for example, for treating individuals with solid tumors, in which it is suspected that the tumor microenvironment (and CD39-mediated adenosine production therein) may lead to a lack of recognition by the immune system (immune escape). The tumor environment (tumor tissue or tissue adjacent to the tumor) can be characterized, for example, by the presence of CD39-expressing immune cells, such as CD4 T cells, CD8 T cells, or B cells.

[0225] More specifically, methods and compositions are used to treat a variety of cancers and other proliferative diseases and infectious diseases. Because these methods work by reducing adenosine that inhibits the anti-target cell (e.g., anti-tumor) activity of lymphocytes and may additionally increase ATP that can increase the anti-tumor activity of lymphocytes, the methods are applicable to a very wide range of cancers and infectious diseases. In one embodiment, anti-CD39 compositions can be used to treat cancer in individuals who are poorly responsive (or insensitive) to treatment with agents that neutralize the inhibitory activity of human PD-1, such as inhibiting the interaction between PD-1 and PD-L1. Representative examples of cancers that can be treated specifically include solid tumors, in which adenosine in the tumor microenvironment may play a powerful role in suppressing anti-tumor immune responses. In one embodiment, the human patient treated with an anti-CD39 antibody has liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, including head and neck squamous cell carcinoma (HNSCC), breast cancer, lung cancer, non-small cell lung cancer (NSCLC), castration-resistant prostate cancer (CRPC), melanoma, uterine cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin's lymphoma (non-Hodgkin's Cancers of the esophagus, small intestine, endocrine system, thyroid, parathyroid, adrenal, soft tissue sarcomas, urethra, penis, solid tumors of childhood, lymphocytic lymphoma, bladder, kidney or ureter, renal pelvis, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers, including those induced by asbestos, hematological malignancies, including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma, The present invention relates to a method for treating a patient with a primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma and precursor T-lymphoblastic lymphoma, and any combination of the above-mentioned cancers. The present disclosure is also applicable to the treatment of metastatic cancers. One or more of the above-mentioned clinical attributes of the patient can be tested or selected before, during or after treatment.

[0226] In one embodiment, an anti-CD39 antibody or antibody fragment is used to treat a cancer characterized by detectable and / or elevated levels of soluble (extracellular) CD39 protein, e.g., in the circulation and / or in a tissue, e.g., in a tumor or tissue adjacent to a tumor.

[0227] In one embodiment, the anti-CD39 antibody or antibody fragment is used to treat a cancer characterized by malignant cells expressing CD39.

[0228] In one embodiment, the present invention is effective to achieve and / or maintain a blood concentration of at least EC in a subject (e.g., for 1 week, 2 weeks, 3 weeks, 4 weeks, and / or until subsequent administration of an antigen binding compound). 50 , optionally EC 70 , optionally substantially EC 100 The anti-CD39 antibody or antibody fragment is administered in an amount to neutralize the enzymatic activity of CD39, optionally sCD39, optionally memCD39. In one embodiment, the active amount of the anti-CD39 antibody is effective to achieve EC 50 , optionally EC 70 , optionally substantially EC 100 An amount of an anti-CD39 antibody is effective to achieve (or maintain) EC in an individual. 50 , optionally EC 70 , optionally substantially EC 100 An amount for inhibiting and neutralizing the enzymatic activity of CD39, optionally sCD39, optionally memCD39.

[0229] Optionally, in one embodiment, the anti-CD39 antibody does not exhibit significant Fcγ receptor-mediated activity compared to some antibodies involved in depletion of CD39-expressing tumor cells by ADCC (e.g., the antibody may provide full efficacy at concentrations equal to or substantially below those that provide receptor saturation), and is administered in an amount effective to neutralize, and optionally further the enzymatic activity of CD39, without substantially causing downregulation of CD39 expression, for a desired period of time, e.g., 1 week, 2 weeks, a month, until the next consecutive administration of the anti-CD39 antibody.

[0230] In one embodiment, the present invention is effective to achieve and / or maintain (e.g., for 1 week, 2 weeks, 3 weeks, 4 weeks, and / or until subsequent administration of an anti-CD39 antibody) a blood concentration of at least EC 50 , optionally EC 70 , optionally substantially EC 100An anti-CD39 antibody or antibody fragment is administered in an amount to inhibit CD39-mediated catabolism of ATP to AMP (e.g., by assessing neutralization of ATPase activity of sCD39; by assessing neutralization of ATPase activity of soluble (extracellular) CD39 protein, see Examples, Methods).

[0231] In one embodiment, a method for treating or preventing cancer in an individual is provided, the method comprising: administering an anti-CD39 antibody or antibody fragment to an individual with the disease in an amount that achieves or maintains a concentration in the circulation, optionally in an extravascular tissue of interest (e.g., a tumor or tumor environment) above the concentration required for 50%, 70% or complete (e.g., 90%) receptor saturation of CD39 expressing cells in the circulation (e.g., as assessed in PBMCs) for a specified period of time. Optionally, the concentration achieved is at least 20%, 50% or 100% higher than the concentration required for the specified receptor saturation.

[0232] In one embodiment, a method for treating or preventing cancer in an individual is provided, the method comprising: achieving or maintaining a concentration in the circulation, optionally in an extravascular tissue of interest (e.g., a tumor or tumor environment) above the EC 50 , optionally EC 70 or optionally EC 100 An anti-CD39 antibody or antibody fragment is administered to an individual in an amount for binding to CD39-expressing cells (e.g., by titrating the anti-CD39 antibody on CD39-expressing cells, e.g., Ramos cells as in the example methods, as assessed by flow cytometry). Optionally, the concentration achieved is less than EC 50 , optionally EC 70 or optionally EC 100 At least 20%, 50% or 100% higher for binding to CD39 expressing cells.

[0233] EC 50 ,EC 70 or EC 100 This can be assessed, for example, in a cellular assay for neutralizing the enzymatic activity of CD39 as shown in the Examples herein, for example by quantifying the hydrolysis of ATP to AMP (or ATP to downstream adenosine) to neutralize the enzymatic activity of ATP in B cells, see Examples, Methods. With respect to neutralization of the enzymatic activity of CD39, "EC 50 ” refers to the effective concentration of anti-CD39 antibody that produces 50% of its maximum response or efficacy with respect to neutralization of the enzymatic activity. With respect to neutralization of the enzymatic activity of CD39, “EC 70 ” refers to the effective concentration of an anti-CD39 antibody that produces 70% of its maximal response or efficacy. With respect to neutralization of the enzymatic activity of CD39, “EC 100” refers to the effective concentration of anti-CD39 antibody that produces a substantially maximal response or efficacy with respect to such neutralization of enzyme activity.

[0234] In some embodiments, particularly for the treatment of solid tumors, the concentration achieved is designed to result in a concentration in the tissue (outside the vasculature, e.g., in a tumor or tumor environment) that is at least as high as the EC for neutralizing enzyme activity. 50 or EC 70 , optionally about, or at least about EC 100 Corresponding.

[0235] In one embodiment, the amount of anti-CD39 antibody is between 1 mg / kg and 20 mg / kg body weight. In one embodiment, the amount is administered to the individual once a week, once every two weeks, once a month, or once every two months.

[0236] In one embodiment, a method of treating a human subject suffering from cancer is provided, comprising: administering to the subject an effective amount of an anti-CD39 antibody of the present disclosure for at least one administration cycle (optionally at least 2, 3, 4 or more administration cycles), wherein the cycle is a period of eight weeks or less, wherein for each of at least one cycle, one, two, three or four doses of the anti-CD39 antibody dose are administered at a dose of 1-20 mg / kg body weight. In one embodiment, the anti-CD39 antibody is administered by intravenous infusion.

[0237] Suitable treatment regimens for treating humans include, for example, administering to a patient an amount of an anti-CD39 antibody as disclosed herein, wherein the method comprises administering at least one dose of the anti-CD39 antibody in at least one administration cycle. Optionally, at least 2, 3, 4, 5, 6, 7, or 8 doses of the anti-CD39 antibody are administered. In one embodiment, the administration cycle is between 2 and 8 weeks.

[0238] In one embodiment, a method for treating or preventing a disease (e.g., cancer, solid tumor, hematological tumor) in an individual is provided, the method comprising administering to an individual suffering from a disease (e.g., cancer, solid tumor, hematological tumor) an anti-CD39 antibody that neutralizes the enzymatic activity of CD39 for at least one administration cycle, the administration cycle comprising at least a first and a second (and optionally a third, fourth, fifth, sixth, seventh and / or eighth or further) administration of the anti-CD39 antibody, wherein the anti-CD39 antibody is administered at an amount effective to achieve or maintain a level of CD39 between two consecutive administrations. The blood (serum) concentration of the anti-CD39 antibody is 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 (e.g., for treating blood tumors), or optionally at least about 1 μg / ml, 2 μg / ml, 10 μg / ml or 20 μg / ml, such as 1-100 μg / ml, 1-50 μg / ml, 1-20 μg / ml or 1-10 μg / ml (e.g., for treating solid tumors, for treating blood tumors). In one embodiment, a specified continuous blood concentration is maintained, wherein the blood concentration does not substantially drop below the specified blood concentration over the duration of a specified time period (e.g., between two administrations of the antibody, several weeks, 1 week, 2 weeks, 3 weeks, 4 weeks), i.e., although the blood concentration may vary during a specified time period, the maintained specified blood concentration represents a minimum or "trough" concentration. In one embodiment, a therapeutically active amount of an anti-CD39 antibody is one that provides (at least) an EC for neutralizing the enzymatic activity of CD39 in the blood and / or tissues. 50 Concentration, optionally EC 70 Concentration, optionally EC 100 The concentration is for a period of at least about 1 week, about 2 weeks, or about one month, followed by administration of the antibody in such amount.

[0239] Prior to or during a course of treatment with an anti-CD39 antibody of the present disclosure, the presence or level or soluble (extracellular) CD39 protein, CD39-expressing cells, adenosine, ATP, ADP, and / or AMP levels can be assessed within and / or adjacent to the patient's tumor to assess whether the patient is suitable for treatment (e.g., to predict whether the patient is likely to respond to treatment). Increased presence or level or soluble (extracellular) CD39, CD39-expressing cells, adenosine, ATP, ADP, and / or AMP levels can indicate that the individual is suitable for (e.g., may benefit from) treatment with an anti-CD39 antibody of the present disclosure (including, but not limited to, antibodies that inhibit substrate-bound CD39).

[0240] Before or during the course of treatment with an anti-CD39 antibody of the present disclosure, adenosine, ADP and / or AMP levels may also be optionally assessed within and / or adjacent to the patient's tumor to assess whether the patient is benefiting from treatment with an anti-CD39 antibody. A decrease in the level of adenosine, ATP, ADP and / or AMP after administration (or administration of an antibody) compared to the level before treatment (or administration of an antibody) may indicate that the individual is benefiting from treatment with an anti-CD39 antibody of the present disclosure (including, but not limited to, an antibody that inhibits substrate binding to CD39). Optionally, if the patient is benefiting from treatment with an anti-CD39 antibody, the method may further include administering an additional dose of an anti-CD39 antibody to the patient (e.g., continuing treatment).

[0241] In one embodiment, the adenosine, ADP and / or AMP levels in and / or adjacent to the patient's tumor are assessed, the tissue sample comprises a biological sample of a human tissue selected from the group consisting of tissues from a cancer patient: for example, cancer tissue, tissue close to or in the periphery of the cancer, tissue adjacent to the cancer, adjacent non-tumor tissue or normal adjacent tissue, and adenosine, ATP, ADP and / or AMP levels in the tissue are detected. The levels from the patient can be compared to reference levels (e.g., corresponding to healthy individuals).

[0242] In one embodiment, the present disclosure provides a method for treating or preventing cancer in an individual in need thereof, the method comprising:

[0243] a) detecting soluble (extracellular) CD39 protein and / or CD39 expressing cells in the circulation or in the tumor environment, optionally within the tumor and / or adjacent tissue, and

[0244] b) upon determining that soluble (extracellular) CD39 protein and / or CD39-expressing cells are included in the circulation or tumor environment, optionally administering an anti-CD39 antibody to the individual at a level increased compared to a reference level (e.g., a level observed in healthy tissue; optionally a level corresponding to a healthy individual or an individual who has not obtained a substantial benefit from the anti-CD39 antibody). CD39-expressing cells may include tumor cells or leukocytes, such as circulating or tumor-infiltrating cells, such as CD4 T cells, CD8 T cells, TReg cells, B cells.

[0245] In one embodiment, the present disclosure provides a method for treating or preventing cancer in an individual in need thereof, the method comprising:

[0246] a) assessing whether the individual has detectable soluble (extracellular) CD39, optionally in the circulation, optionally within the tumor and / or in adjacent tissues, and

[0247] b) upon detection of soluble (extracellular) CD39, optionally administering to the individual an anti-CD39 antibody of the disclosure at a level increased compared to a reference level (e.g., corresponding to a healthy individual or an individual who does not derive substantial benefit from an anti-CD39 antibody of the disclosure).

[0248] Optionally, in any method, detecting soluble CD39 protein and / or CD39-expressing cells (or adenosine, ATP, ADP and / or AMP) within the tumor environment comprises obtaining a biological sample comprising cancer tissue and / or tissue proximal to or peripheral to the cancer (e.g., tissue adjacent to the cancer, adjacent non-tumor tissue, or normal adjacent tissue) from an individual, and detecting the level of sCD39 protein, CD39-expressing cells (or adenosine, ATP, ADP and / or AMP). CD39-expressing cells may include, for example, tumor cells, CD4 T cells, CD8 T cells, TReg cells, B cells.

[0249] Individuals with cancer can be treated with anti-CD39 antibodies without a prior detection step to assess the presence of sCD39 and / or the expression of CD39 on circulating cells or cells in the tumor microenvironment (e.g., on tumor cells, CD4 T cells, CD8 T cells, TReg cells, B cells). Optionally, the method of treatment may include a step of detecting CD39 nucleic acids or polypeptides in a biological sample from blood or from an individual's tumor (e.g., in cancer tissue, tissue close to or at the periphery of cancer, tissue adjacent to cancer, adjacent non-tumor tissue, or normal adjacent tissue). Determining that a biological sample includes cells expressing (e.g., prominent expression; expressing CD39 at high levels and high intensity with an anti-CD39 antibody compared to a reference (e.g., healthy tissue)) CD39 indicates that the patient has a cancer that may benefit greatly from treatment with an agent that inhibits CD39. In one embodiment, the method includes determining the expression level of CD39 nucleic acids or polypeptides in a biological sample, and comparing the level with a reference level corresponding to a healthy individual (e.g., healthy tissue). Determining that a biological sample includes sCD39 protein and / or cells expressing CD39 nucleic acid or polypeptide at an increased level compared to a reference level indicates that the patient suffers from a cancer that can be advantageously treated with an anti-CD39 antibody of the present disclosure. In one embodiment, detecting a CD39 polypeptide in a biological sample includes detecting a soluble extracellular CD39 protein. In one embodiment, detecting a CD39 polypeptide in a biological sample includes detecting CD39 polypeptides expressed on the surface of malignant cells, CD4 T cells, CD8 T cells, TReg cells, and B cells. In one embodiment, determining that a biological sample includes cells that prominently express CD39 nucleic acid or polypeptides indicates that the patient suffers from a cancer that can be advantageously treated with an anti-CD39 antibody of the present disclosure. When referring to a CD39 polypeptide, "prominent expression" refers to the expression of a CD39 polypeptide in a large number of cells taken from a given patient. Although the definition of the term "prominent expression" is not subject to an exact percentage value, in some instances, a receptor referred to as "prominent expression" will be present on at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of tumor cells taken from a patient.

[0250] Determining whether an individual has a cancer characterized by cells expressing a CD39 polypeptide can, for example, include obtaining a biological sample (e.g., by performing a biopsy) from an individual that includes cells from a cancer environment (e.g., a tumor or tumor-adjacent tissue), contacting the cells with an antibody that binds to a CD39 polypeptide, and detecting whether the cells express CD39 on their surface. Optionally, determining whether an individual has cells that express CD39 includes performing an immunohistochemical assay.

[0251] In one embodiment, the anti-CD39 antibodies described herein can be advantageously used to treat CD73-positive cancers. CD73 expression has been reported in a range of tumor cells, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, and breast cancer. CD73 expression is also associated with a pro-metastatic phenotype in melanoma and breast cancer.

[0252] Thus, a method for treating or preventing cancer or an infectious disease in an individual with a CD73-positive cancer is provided, the method comprising administering to the individual an anti-CD39 antibody or antibody fragment of the present disclosure. In one embodiment, the present disclosure provides a method for treating or preventing a CD73-positive cancer in an individual, the method comprising administering to the individual an antibody of the present disclosure that binds to and inhibits the activity of a soluble human CD39 protein. In one embodiment, a CD73-positive cancer is a cancer known to be generally characterized by the presence of CD73-expressing cells in a tumor or tumor environment.

[0253] Patients with cancer can be treated with anti-CD39 antibodies without a prior detection step to assess the expression of CD73 on cells in the tumor microenvironment (e.g., on tumor cells, CD4 T cells, CD8 T cells, B cells). Optionally, the method of treatment can include a step of detecting CD73 nucleic acid or polypeptide in a biological sample from a tumor of an individual (e.g., in cancer tissue, tissue proximal to or peripheral to the cancer, tissue adjacent to the cancer, adjacent non-tumor tissue, or normal adjacent tissue). Determining that the biological sample includes cells expressing CD73 (e.g., prominent expression; expressing CD73 at high levels, high intensity staining with an anti-CD73 antibody compared to a reference (e.g., healthy tissue)) indicates that the patient has a cancer that may benefit greatly from treatment with an agent that inhibits sCD39 (optionally further combined with an agent that inhibits CD73). In one embodiment, the method includes determining the expression level of a CD73 nucleic acid or polypeptide in a biological sample, and comparing the level to a reference level corresponding to a healthy individual (e.g., healthy tissue). Determining that a biological sample includes cells expressing a CD73 nucleic acid or polypeptide at an increased level compared to a reference level indicates that the patient has a cancer that can be treated with an anti-CD39 antibody. Optionally, detecting a CD73 polypeptide in a biological sample includes detecting a CD73 polypeptide expressed on the surface of a malignant cell, a CD4 T cell, a CD8 T cell, or a B cell. In one embodiment, determining that a biological sample includes cells expressing a CD73 nucleic acid or polypeptide indicates that the patient has a cancer that can obtain a particular benefit from treatment with an anti-CD39 antibody. The CD73 polypeptide can be expressed, for example, in a large number of cells taken from a given patient, for example, CD73 can be detected on at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the tumor cells taken from the patient.

[0254] Determining whether an individual has a cancer characterized by cells expressing a CD73 polypeptide can, for example, comprise obtaining a biological sample (e.g., by performing a biopsy) from an individual that includes cells from a cancer environment (e.g., a tumor or tissue adjacent to a tumor), contacting the cells with an antibody that binds a CD73 polypeptide, and detecting whether the cells express CD73 on their surface. Optionally, determining or detecting whether an individual has cells that express CD73 comprises performing an immunohistochemical assay.

[0255] In one embodiment, the present disclosure provides a method for treating or preventing cancer in an individual in need thereof, the method comprising:

[0256] a) detecting CD73 expressing cells in a tumor environment, optionally within the tumor and / or adjacent tissue, and

[0257] b) upon determining that the tumor environment includes CD73-expressing cells, optionally administering to the individual an antibody of the present disclosure that binds to and inhibits the activity of soluble human CD39 protein at a level increased compared to a reference level (e.g., the level observed for healthy tissue). Optionally, detecting CD73-expressing cells within the tumor environment comprises obtaining a biological sample from the individual that includes cancer tissue and / or tissue proximal to or in the periphery of the cancer (e.g., tissue adjacent to the cancer, adjacent non-tumor tissue, or normal adjacent tissue), and detecting the level of CD73-expressing cells (e.g., by performing an immunohistochemical assay). CD73-expressing cells may include, for example, tumor cells, CD4 T cells, CD8 T cells, B cells.

[0258] The anti-CD39 antibody compositions of the present disclosure can be used in monotherapy or combination therapy with one or more other therapeutic agents, including agents typically used for the specific therapeutic purpose of administering the antibody. The additional therapeutic agent will generally be administered in a monotherapy for the specific disease or condition being treated in an amount and treatment regimen typically used for the agent.

[0259] In one embodiment, the anti-CD39 antibody compositions of the present disclosure can be used in combination therapy with chemotherapeutic agents that are capable of releasing ATP from the extracellular space of tumor cells.

[0260] In one embodiment, the anti-CD39 neutralizing antibody lacks binding to human CD16, but enhances the activity of CD16-expressing effector cells (e.g., NK cells or effector T cells). Therefore, in one embodiment, the second or additional second therapeutic agent is an antibody or other protein containing Fc domains that can, for example, induce ADCC toward cells bound thereto by CD16 expressed by NK cells. Typically, such second agent antibodies or other proteins will include a domain bound to an antigen of interest, such as an antigen (tumor antigen) present on a tumor cell, and an Fc domain or a portion thereof, and will exhibit binding to the antigen through the antigen binding domain and binding to Fcγ receptors (e.g., CD16) through the Fc domain. In one embodiment, its ADCC activity will be mediated at least in part by CD16. In one embodiment, another therapeutic agent is an antibody with a natural or modified human Fc domain, such as an Fc domain from a human IgG1 or IgG3 antibody. The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well known in the art, and refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils. The term "ADCC-inducing antibody" refers to an antibody that demonstrates ADCC as measured by one or more assays known to those skilled in the art. Such activity is typically characterized by the binding of the Fc region to various FcRs. Without being limited to any particular mechanism, those skilled in the art will recognize that the ability of an antibody to demonstrate ADCC can be, for example, by virtue of its subclass (such as IgG1 or IgG3), by mutations introduced into the Fc region, or by virtue of modifications to the carbohydrate pattern in the Fc region of the antibody. Examples of antibodies that induce ADCC include rituximab (for the treatment of lymphoma, CLL), trastuzumab (for the treatment of breast cancer), alemtuzumab (for the treatment of chronic lymphocytic leukemia) and cetuximab (for the treatment of colorectal cancer, head and neck squamous cell carcinoma). Examples of ADCC enhancing antibodies include, but are not limited to: GA-101 (hypofucosylated anti-CD20), margetuximab (Fc enhanced anti-HER2), mepolizumab, MEDI-551 (Fc engineered anti-CD19), obinutuzumab (glycoengineered / hypofucosylated anti-CD20), ocaratuzumab (Fc engineered anti-CD20), 5574 / MOR208 (Fc engineered anti-CD19).

[0261] In one embodiment, the anti-CD39 neutralizing antibody enhances the efficacy of an agent that neutralizes the inhibitory activity of human PD-1, such as inhibiting the interaction between PD-1 and PD-L1, particularly in individuals who are poorly responsive (or insensitive) to treatment with an agent that neutralizes the inhibitory activity of human PD-1. Therefore, in one embodiment, the second or additional second therapeutic agent is an antibody or other agent that neutralizes the inhibitory activity of human PD-1.

[0262] Programmed death 1 (PD-1) (also known as "programmed cell death 1") is an inhibitory member of the CD28 receptor family. The complete human PD-1 sequence can be found under GenBank accession number U64863. Inhibition or neutralization of the inhibitory activity of PD-1 can involve the use of polypeptide agents (e.g., antibodies, polypeptides fused to an Fc domain, immunoadhesins, etc.) that block PD-1 signaling induced by PD-L1. There are currently at least six agents that block the PD-1 / PD-L1 pathway, which are sold on the market or in clinical evaluation. One agent is BMS-936558 (nivolumab / ONO-4538, Bristol-Myers Squibb; formerly MDX-1106). Nivolumab (trade name ) is an FDA-approved fully human IgG4 anti-PD-L1 mAb that inhibits the binding of the PD-L1 ligand to both PD-1 and CD80, and is described as antibody 5C4 in WO 2006 / 121168, the disclosure of which is incorporated herein by reference. For melanoma patients, the most significant OR was observed at a dose of 3 mg / kg, while for other cancer types, it can be observed at 10 mg / kg. Nivolumab is usually administered at 10 mg / kg every 3 weeks until cancer progresses. The terms "reducing the inhibitory activity of human PD-1", "neutralizing PD-1" or "neutralizing the inhibitory activity of human PD-1" refer to the process in which the signal transduction ability of PD-1 is inhibited due to the interaction of PD-1 with one or more of its binding partners, such as PD-L1 or PD-L2. Agents that neutralize the inhibitory activity of PD-1 reduce, block, inhibit, eliminate or interfere with signal transduction caused by the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and PD-L2. Therefore, such agents can reduce negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes, thereby enhancing T cell effector functions such as proliferation, cytokine production and / or cytotoxicity.

[0263] MK-3475 (human IgG4 anti-PD1 mAb from Merck), also known as lambrolizumab or pembrolizumab (trade name ) has been approved by the FDA for the treatment of melanoma and is being tested in other cancers. Pembrolizumab is tested at 2 mg / kg or 10 mg / kg every 2 or 3 weeks until disease progression. MK-3475, also known as Merck 3745 or SCH-900475, is also described in WO2009 / 114335.

[0264] MPDL3280A / RG7446 (Atezolizumab from Roche / Genentech, trade name Tecentriq TM MPDL3280A (MPDL3280A, anti-PD-L1) is a human anti-PD-L1 mAb containing an engineered Fc domain designed to optimize efficacy and safety by minimizing FcγR binding and the resulting antibody-dependent cellular cytotoxicity (ADCC). Doses of ≤1 mg / kg, 10 mg / kg, 15 mg / kg, and 25 mg / kg MPDL3280A were administered every 3 weeks for up to 1 year. In a Phase 3 trial, 1200 mg of MPDL3280A was administered by intravenous infusion every three weeks in NSCLC.

[0265] AMP-224 (Amplimmune and GSK) is an immunoadhesin that includes the extracellular domain of PD-L2 fused to an Fc domain. Other examples of agents that neutralize PD-1 may include antibodies that bind to PD-L2 (anti-PD-L2 antibodies) and block the interaction between PD-1 and PD-L2.

[0266] Pidlizumab (CT-011; CureTech) (a humanized IgG1 anti-PD1 mAb from CureTech / Teva), Pidlizumab (CT-011; Pidlizumab) (see, e.g., WO2009 / 101611) is another example; the agent was tested in thirty patients with rituximab-sensitive relapsed FL, treated with 3 mg / kg intravenous CT-011 every 4 weeks in combination with rituximab dosed weekly at 375 mg / m2 for 4 infusions for 4 weeks, starting 2 weeks after the first infusion of CT-011.

[0267] Further known PD-1 antibodies and other PD-1 inhibitors include AMP-224 (B7-DC / IgG1 fusion protein licensed from GSK), AMP-514 described in WO 2012 / 145493, and the antibody MEDI-4736 described in WO 2011 / 066389 and US 2013 / 034559 (durvalumab, trade name Imfinzi TM, anti-PD-L1 developed by AstraZeneca / Medimmune), antibody YW243.55.S70 (anti-PD-L1) described in WO2010 / 077634, MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody developed by Bristol-Myers Squibb described in WO2007 / 005874, and antibodies and inhibitors described in WO2006 / 121168, WO2009 / 014708, WO2009 / 114335 and WO2013 / 019906, the disclosures of which are hereby incorporated by reference. Additional examples of anti-PD1 antibodies are disclosed in WO2015 / 085847 (Shanghai Hengrui Pharmaceutical Co. Ltd.), such as antibodies having light chain variable domain CDR1, 2 and 3 of SEQ ID NO: 6, SEQ ID NO: 7 and / or SEQ ID NO: 8, respectively, and antibodies having heavy chain variable domain CDR1, 2 and 3 of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, respectively, wherein SEQ ID NO references numbering according to WO2015 / 085847, the disclosure of which is incorporated herein by reference. Antibodies that compete with any of these antibodies for binding to PD-1 or PD-L1 can also be used.

[0268] In some embodiments, the PD-1 neutralizer is an anti-PD-L1 mAb that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 neutralizer is an anti-PD1 mAb that inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 neutralizer is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)).

[0269] In the method of treatment, anti-CD39 antibody and the second therapeutic agent can be applied separately, together or sequentially, or applied in the form of a mixture. In some embodiments, the antigen binding compound is applied before the second therapeutic agent is applied. For example, CD39 antibody can be applied about 0 to 30 days before the second therapeutic agent is applied. In some embodiments, anti-CD39 antibody is applied 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 the second therapeutic agent is applied. In some embodiments, anti-CD39 antibody is applied simultaneously with the application of the second therapeutic agent. In some embodiments, anti-CD39 antibody is applied after the second therapeutic agent is applied. For example, anti-CD39 antibody can be applied about 0 to 30 days after the second therapeutic agent is applied. In some embodiments, the anti-CD39 antibody is administered about 30 minutes to about 2 weeks, about 30 minutes to about 1 week, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to 1 day, or about 1 to 5 days after administration of the second therapeutic agent.

[0270] Examples

[0271] method

[0272] Generation of CD39 mutants

[0273] CD39 mutants were generated by PCR. The amplified sequences were run on an agarose gel and purified using the Macherey Nagel PCR Clean-Up Gel Extraction Kit (reference 740609). The purified PCR products generated for each mutant were then ligated into expression vectors using the ClonTech InFusion system. The vectors containing the mutant sequences were prepared as Minipreps and sequenced. After sequencing, the vectors were purified using the Promega PureYield TM Plasmid Midiprep system, the vector containing the mutant sequence is prepared as Midiprep. Before testing transgenic expression, HEK293T cells were grown in DMEM medium (Invitrogen), transfected with the vector using Invitrogen's Lipofectamine 2000 and incubated for 48 hours at 37°C in a CO2 incubator. Mutants were transfected in Hek-293T cells as shown in the table below. The target amino acid mutations in Table 1 below are shown using the numbering of SEQ ID NO:1.

[0274] Table 1

[0275]

[0276] Cloning, production and purification of soluble huCD39

[0277] Molecular Biology

[0278] The huCD39 protein was cloned from human PBMC cDNA using the following primers: TACGACTCACAAGCTTGCCGCCACCATGGAAGATACAAAGGAGTC (SEQ ID NO: 41) (forward), and CCGCCCCGACTCTAGATCACTTGTCATCGTCATCTTTGTAATCGACATAGGTGGAGTGGGAGAG (SEQ ID NO: 42) (reverse). The purified PCR product was then cloned into an expression vector using the InFusion cloning system. An M2 tag (the FLAG tag underlined in SEQ ID NO: 44) was added at the C-terminal portion of the protein for the purification step; it should be understood that in any embodiment, the CD39 extracellular domain protein (e.g., of SEQ ID NO: 44) can optionally be specified as lacking an M2 tag.

[0279] Expression and purification of huCD39 protein

[0280] After verifying the sequence of the clone, CHO cells were nucleofected and the pool was then subcloned to obtain cell clones producing huCD39 protein. The supernatant from the huCD39 clone grown in the drum was harvested and purified using an M2 column and eluted using the M2 peptide. The purified protein was then loaded onto an S200 size exclusion column. The purified protein corresponding to the monomer was prepared in TBS PH7.5 buffer. The amino acid sequence of the CD39-M2 extracellular domain recombinant protein without the M2 tag is as follows:

[0281] MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGSSHTSLYIYKWPAEKENDTGVVHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERAREVIPRS QHQETPVYLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGKFSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQTIESPDNALQF RLYGKDYNVYTHSFLCYGKDQALWQKLAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLPFQQFEIQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFG AFSAFYFVMKFLNLTSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADSWEHIHFIGKIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHSTYV

[0282] (SEQ ID NO:43).

[0283] The final amino acid sequence of the CD39-M2 extracellular domain recombinant protein with M2 tag is as follows:

[0284] MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGSSHTSLYIYKWPAEKENDTGVVHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERAREVIPRS QHQETPVYLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGKFSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQTIESPDNALQF RLYGKDYNVYTHSFLCYGKDQALWQKLAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLPFQQFEIQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFG AFSAFYFVMKFLNLTSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADSWEHIHFIGKIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHSTYV DYKDDDDK

[0285] (SEQ ID NO:44).

[0286] Inhibits the enzymatic activity of soluble CD39

[0287] Using Cell Titer Glo TM (Promega, Ref. G7571), which allows for the assessment of ATP hydrolysis by using an agent that generates a luminescent signal proportional to the amount of ATP present, to assess the inhibition of the enzymatic activity of the produced soluble CD39 protein. In this way, the inhibition of soluble CD39-mediated ATP hydrolysis can be assessed. Briefly, 100 μg / ml to 6×10 -3 A dose range of anti-CD39 antibodies at μg / ml was incubated with 400ng / ml of soluble recombinant human CD39 protein having the amino acid sequence described in the Methods section (SEQ ID NO: 44) at 37°C for 1 hour. 20μM ATP was added to the plate for another 30 minutes at 37°C before adding CTG (Cell TiterGlo) reagent. After a brief incubation of 5 minutes in the dark, the plates were incubated with Enspire TMThe emitted light is quantified by a luminometer. Anti-CD39 antibody efficacy is determined by comparing the emitted light in the presence of the antibody to ATP alone (maximum light emission) and ATP together with soluble CD39 protein (minimum light emission).

[0288] Inhibits the enzymatic activity of CD39

[0289] Using Cell Titer Glo TM (Promega, Ref. G7571), which allows the evaluation of antibody inhibition of CD39 enzymatic activity in CD39-expressing cells by assessing ATP hydrolysis using a reagent that generates a luminescent signal proportional to the amount of ATP present. Therefore, the assay was designed to allow the evaluation of inhibition of ATP hydrolysis by CD39 in cell culture supernatants. Briefly, 5×10 4 Ramos human lymphoma cells, 5×10 3 Human CD39-expressing, cynomolgus monkey CD39-expressing and mouse CD39-expressing CHO cells were incubated with 30 μg / ml to 5×10 -4 The cells were incubated with 20 μM ATP at 37°C for 1 hour. The plates were centrifuged at 400 g for 2 minutes and 50 μl of cell supernatant was transferred to a luminescent microplate (white wells). 50 μl CellTiter-Glo TM Reagent (CTG) was added to the supernatant and after incubation in the dark for 5 min, Enspire TM The luminometer quantifies the emitted light. Anti-CD39 antibody efficacy is determined by comparing the emitted light in the presence of the antibody to ATP alone (maximum light emission) and ATP with cells (minimum light emission).

[0290] Antibody Generation: Immunization and Screening in Mice

[0291] In order to obtain anti-human CD39 antibodies, Balb / c mice were immunized with the above-mentioned recombinant human CD39-M2 extracellular domain recombinant protein. The mice received a primary immunization of an emulsion of 50 μg CD39 protein and complete Freund Adjuvant intraperitoneally, received a second immunization with an emulsion of 50 μg CD39 protein and incomplete Freund Adjuvant intraperitoneally, and finally boosted immunization intravenously with 10 μg CD39 protein. After boosting, immune spleen cells were fused with X63.Ag8.653 immortalized B cells 3 days later and cultured in the presence of irradiated spleen cells. Hybridomas were plated in a medium containing semisolid methylcellulose and clonepix was used. TMGrowing clones were picked using the ELISA kit (Molecular Devices Corp.).

[0292] Example 1: Epitope mapping of known neutralizing CD39 mAbs

[0293] To gain further insight into antibodies capable of inhibiting the enzymatic (ATPase) activity of cellular CD39, the epitope bound by an antibody that reportedly inhibits the ATPase activity of CD39 in a cellular assay was investigated: BY40 disclosed in PCT Publication No. WO2009 / 095478.

[0294] To define the epitope of the anti-CD39 antibody, CD39 mutants defined by substitution of surface-exposed amino acids above the surface of CD39 were designed. The mutants were transfected in Hek-293T cells as shown in Table 1 using the numbering of SEQ ID NO: 1.

[0295] A dose range of I-394 (10–2.5–0.625–0.1563–0.0391–0.0098–0.0024–0.0006 μg / ml) was tested on the 20 generated mutants by flow cytometry. The BY40 antibody all completely lost binding to cells expressing mutant 5 of CD39, without losing binding to any of the other mutants. Mutant 5 contains amino acid substitutions at residues Q96, N99, E143, and R147. The position of mutant 5 on the surface of CD39 is shown in Figure 2. Figure 3A shown.

[0296] Example 2: Known neutralizing CD39 mAbs cannot inhibit the ATPase activity of recombinant soluble CD39 protein

[0297] Two antibodies that have been reported to inhibit the ATPase activity of CD39 in a cell-based assay (BY40 and BY12) were evaluated to determine if they could inhibit the ATPase activity of recombinant soluble CD39 protein. TM (Promega, reference G7571), and the antibodies were evaluated for inhibition of the enzymatic activity of soluble CD39 protein produced as described above. The antibodies were evaluated for inhibition of the enzymatic activity of cellular CD39 protein as described above.

[0298] As expected, BY40 inhibited the ATPase activity of CD39 protein in cells. However, BY40 failed to inhibit the enzyme activity of soluble CD39 protein. Figure 2B A comparison of BY40 with the novel antibodies identified herein is shown.

[0299] Example 3: Screening of new mAbs to block sCD39 activity

[0300] A series of immunizations were performed to find antibodies that neutralize the ATPase activity of sCD39. To obtain anti-human CD39 antibodies, animals were immunized with the above-mentioned recombinant human CD39-M2 extracellular domain recombinant protein. In summary, the series of immunizations included different protocols and in different animals, including different mouse strains, rats and rabbits.

[0301] In the initial immunization protocol, the preliminary screening involved testing the supernatants (SN) of growing clones by flow cytometry using wild-type CHO and CHO-expressing huCD39 cell lines. The cells were stained with 0.1 μM and 0.005 μM CFSE, respectively. For flow cytometry screening, all cells were mixed evenly and the presence of reactive antibodies in the supernatant was revealed with APC-labeled goat anti-mouse polyclonal antibody (pAb). For bound huCD39 antibodies, the supernatant was then screened for inhibition of the enzymatic activity of soluble CD39 using the screening assay developed and described above (Methods).

[0302] The results show that, although many specific CD39 binding antibodies can be obtained, antibodies from any of these immunizations do not show any inhibition of the enzymatic activity of soluble CD39. One possibility is that the dominant epitope on CD39 does not contain any epitope that is properly positioned at or near the catalytic site of the CD39. In view of the known difficulties of a few antibodies that can be used to inhibit cellular CD39 and the catalytic site of the enzyme using antibodies, the absence of antibodies that neutralize sCD39 may indicate that it is impossible to obtain antibodies that inhibit soluble (extracellular domain) CD39. Other possibilities relate to non-functional screening assays and / or improperly folded or functional soluble CD39 proteins, particularly because the lack of any antibodies that can inhibit soluble CD39 has hindered the verification of sCD39 blocking assays.

[0303] In view of the absence of antibodies capable of inhibiting soluble CD39, further immunization was performed with a screening protocol designed to facilitate the generation of antibodies that bind to the active site of CD39, as identified by the epitope of antibody BY40. In brief, the initial screening involved testing the supernatant (SN) of the growing clones by flow cytometry using wild-type CHO and CHO-expressing huCD39 cell lines, as in the aforementioned immunization, followed by screening for the loss of binding Hek-293T cells expressing CD39 mutant 5 compared to wild-type CD39, as shown in Table 1. Mutant 5 has substitutions at residues Q96, N99, E143, and R147. However, the results again show that although many specific CD39 binding antibodies can be obtained, which show loss of binding to mutant 5, antibodies from any initial immunization do not show any inhibition of the enzymatic activity of soluble CD39.

[0304] Example 4: Labeling of I-394 Antibody

[0305] An attempt was made to identify anti-CD39 antibodies that do not bind to the Q96, N99, E143 and R147 region (defined by mutant 5) in order to have antibodies that do not compete with BY40-like antibodies. Such antibodies, which do not necessarily have any ability to block the ATPase activity of CD39, can be used in pharmacological studies of antibodies that inhibit cellular CD39 binding to the BY40 binding site, such as detecting and quantifying free CD39 protein on cells in the presence of BY40 or BY40-like antibodies that inhibit cellular CD39.

[0306] Starting from the immunization results of Example 3, in which hybridomas were screened for loss of binding to CD39 mutant 5, a hybridoma was selected that was not among those that showed loss of binding to CD39 mutant 5. This hybridoma (I-394) was part of the broader pool due to inconclusive data indicating that binding to mutant 5 may be partially reduced, but not lost, and was therefore not initially retained.

[0307] In the context of ongoing screening of supernatants from additional immunizations for inhibition of the enzymatic activity of soluble CD39, the already cloned and generated antibody I-394 was included as a control. Surprisingly, although antibody I-394 was not retained in the clones in the epitope-directed screening, this antibody showed strong inhibition of the enzymatic activity of soluble CD39 in the above assay.

[0308] I-394 with a modified Fc domain was generated with a human constant region of the IgG1 isotype, wherein the modified Fc domain has mutations L234A / L235E / G237A / A330S / P331S (Kabat EU numbering) that result in a lack of binding to human Fcγ receptors CD16A, CD16B, CD32A, CD32B, and CD64. Briefly, the VH and Vk sequences of the I-394 antibody (VH and Vk variable regions shown in SEQ ID NOs: 6 and 7, respectively) were cloned into expression vectors containing huIgG1 constant domains and huCk constant domains with the aforementioned mutations, respectively. The two vectors obtained were co-transfected into a CHO cell line. The established cell pool was used to produce antibodies in CHO culture medium. Protein A was then used to purify the antibodies. The amino acid sequences of the corresponding heavy chain variable domains and light chain variable domains of I-394 are shown below (Kabat CDRs are underlined).

[0309] I-394 heavy chain variable domain sequence:

[0310] EVQLQQSGPELVKPGASVKMSCKASGYTFT DYNMHWVKQSHGRTLEWIG YIVPLNGGSTFNQKFKG RATLTVNTSSRTAYMELRSLTSEDSAAYYCAR GGTRFAY WGQGTLVTVSA (SEQ ID NO: 6).

[0311] I-394 light chain variable domain sequence:

[0312] DIVLTQSPASLAVSLGQRATISC RASESVDNFGVSFMY WFQQKPGQPPNLLIY GASNQGS GVPARFRGSGSGTDFSLNIHPMEADDTAMYFC QQTKEVPYT FGGGTKLEIK (SEQ ID NO: 7).

[0313] Antibody I-394 was then tested for loss of binding to CD39 mutants defined by substitution of amino acids exposed on the surface of the molecule above the CD39 surface. Using the numbering of SEQ ID NO: 1, the mutants were transfected in Hek-293T cells as shown in Table 1. A dose range of antibody I-394 was tested on the 20 mutants by flow cytometry. Figure 3B As shown, I-394 exhibits a complete loss of binding to cells expressing CD39 mutant 19. Mutant 19 comprises substitutions at residues R138, M139, and E142. Thus, the core epitope of I-394 comprises one or more (or all) residues R138, M139, and E142.

[0314] Unlike antibody BY40, which previously lost binding to mutant 5 and had the ability to inhibit cellular CD39 but not soluble CD39, antibody I-394 lost binding to neighboring mutant 19, greatly reduced binding to mutant 5 (but had some residue binding to mutant 5). Interestingly, the residues of mutant 19 are very close or adjacent to those of residue 5, so that I-394 can represent a shift in the epitope compared to BY40. Therefore, antibody I-394 presents a valuable new epitope for anti-CD39 antibodies, which allows inhibition of the ATPase activity of soluble CD39 proteins. It also provides a specific positive control, which allows validation and testing of screening assays to detect additional antibodies that neutralize the ATPase activity of soluble CD39 proteins.

[0315] Example 5: Non-epitope-directed screening for sCD39 neutralizing mAbs

[0316] Based on the results of Example 4 indicating that antibody-mediated inhibition of soluble CD39 was possible, the fusion of different immunizations from different protocols of Example 3 was re-used to search for antibodies that neutralize the ATPase activity of sCD39.

[0317] Different methods for screening ATPase inhibition were then evaluated. In one experiment, the supernatant from the immunized hybridoma of Example 3 was stimulated with the I-394 antibody and found to be negative for its ability to inhibit the ATPase activity of soluble CD39. Addition of this I-394 to the supernatant did not restore the ability of the negative supernatant to inhibit the ATPase activity of CD39. The antibody I-394 was then purified from the negative supernatant using protein A-coated beads, and it was observed that the purified I-394 was again able to inhibit the recovery of ATPase activity.

[0318] In view of the above results, a new immunization and screening protocol was developed in which growing clones from new and past immunizations were screened by flow cytometry using wild-type CHO and CHO-expressing huCD39 cell lines without assessing inhibition of soluble CD39 or cellular CD39 ATPase activity and without epitope bias for screening. Although data on loss of binding to mutants 5 or 19 were available for some hybridomas, such data were not used for clonal selection but were retained only in the event of a negative result in the ATPase blocking assay to rescue hybridomas for cloning purposes. Hybridomas that bind CD39 were selected and cloned and then purified using protein A according to the following protocol:

[0319] - Add 10 μl of Protein A beads to 300 μl of hybridoma supernatant

[0320] - Add NaCl to a final concentration of 1.5 M

[0321] - Rotate the tubes at 4°C for 3-4 hours

[0322] - Centrifuge at 1500 rpm for 1 minute

[0323] - Eliminate supernatant and wash three times with 1 ml TBS

[0324] -After the third wash, remove all TBS

[0325] - Add 50 μl citrate 0.1 M pH 3, homogenize and incubate for 5 minutes at room temperature

[0326] - Centrifuge the beads at 1500 rpm for 1 minute

[0327] - Harvest 50 μl of eluate and quickly add 450 μl TBS and store at 4°C

[0328] The antibodies obtained were then screened for their ability to inhibit the ATPase activity of CD39 in a comparative assay to a similar extent as I-394. The assays for inhibiting the enzymatic activity of soluble and cellular CD39 are as described above (Methods). Surprisingly, among the exemplary antibodies produced in this manner, several showed inhibition of soluble CD39 (as well as inhibition of cellular CD39). Figure 1 Representative screening results are shown, showing antibodies I-397, I-398 and I-399 compared to the positive control I-394 antibody. Similarly, antibodies I-395 and I-396 from different immunizations inhibited the enzymatic activity of soluble CD39 protein. Figure 2A and 2B Results are shown for antibodies 1-395 and 1-396, where larger amounts of antibody were available for additional experiments of both soluble and cellular CD39 neutralization. Figure 2A It was shown that antibodies I-395 and I-396 both inhibited cell membrane-bound CD39 compared to BY40 and I-394 antibodies, with both I-394 and I-395 showing higher potency and maximal inhibition of cellular CD39 compared to BY40. Figure 2B It is shown that antibodies I-395 and I-396 both inhibit soluble CD39 compared to BY40 and I-394 antibodies. While BY40 did not inhibit soluble CD39 at any concentration, I-394, I-395, and I-396 all inhibited soluble CD39, with I-394 showing the greatest potency, followed by I-395, and then I-396 with lower potency.

[0329] The results obtained raise the possibility that one or more factors in the hybridoma supernatant rapidly hydrolyze ATP in both cell culture and soluble CD39 assays, such that no ATP signal is detected in screening antibodies using conventional methods. The soluble factor can be CD39 or some other enzyme, such as produced by the fusion partner.

[0330] The antibody is then cloned and modified to have a human constant region of an IgG1 Fc domain with mutations L234A / L235E / G237A / A330S / P331S (Kabat EU numbering), which results in a lack of binding to human Fcγ receptors CD16A, CD16B, CD32A, CD32B and CD64, in the same manner as described herein for I-394. The resulting antibody can then be subjected to titration and then a more detailed activity assessment, as described in Examples 7-9 (titration, inhibition of ATPase activity), to assess EC 50 and IC 50 Determined to rank antibodies according to potency.

[0331] Example 6: Epitope mapping of sCD39 neutralizing mAbs

[0332] As shown in Example 4, 1-394 showed complete loss of binding to cells expressing mutant 19 of CD39, but not to mutant 5. To define the epitopes of the additional anti-CD39 antibodies of Example 5, the epitopes were tested for loss of binding to a panel of CD39 mutants as described in Example 1 and Table 1. As shown in Table 1, the mutants were transfected in Hek-293T cells using the numbering of SEQ ID NO: 1. A dose range of the test antibodies (10–2.5–0.625–0.1563–0.0391–0.0098–0.0024–0.0006 μg / ml) was tested on 20 generated mutants by flow cytometry.

[0333] The results show that the antibodies selected in Example 5 that can inhibit soluble CD39 represent several different epitopes. Among the antibodies that inhibit soluble extracellular CD39 shown in Example 5, antibody I-395 is an example of an antibody that shows loss of binding to mutant 5 with substitutions at residues Q96, N99, E143 and R147, and loss of binding to mutant 19 with substitutions at residues R138, M139 and E142. Mutant 19 contains substitutions at residues R138, M139 and E142. Therefore, the core epitope on CD39 of I-395 includes one, two, three or four residues of residues Q96, N99, E143 and R147 and one, two or three residues of residues R138, M139 and E142.

[0334] On the other hand, antibody 1-398 is an example of an antibody that shows loss of binding to mutant 19 having substitutions at residues R138, M139, and E142, but does not have reduced or lost binding to mutant 5 having substitutions at residues Q96, N99, E143, and R147.

[0335] Other antibodies that showed inhibition of soluble extracellular CD39 in Example 5 had very different epitopes and did not show loss of binding to any of mutants 5 or 19, indicating that soluble CD39 may also be inhibited by binding to other sites on sCD39. For some antibodies, loss of binding to one of the 20 mutants of Table 1 allowed localization of the binding site on CD39, while for other antibodies, the binding site remains to be determined because they do not lose binding to any of the 20 mutants. Among the antibodies that showed inhibition of the ATPase activity of soluble CD39 in Example 5, antibody 1-396 showed loss of binding to mutant 15 with substitutions K87A, E100A, and D107A, without loss of binding to any of the other 20 mutants. Therefore, the core epitope on CD39 for this antibody includes one or more (or all) of residues K87, E100, and D107. Antibody I-399 showed loss of binding to mutant 11 with substitutions N371K, L372K, E375A, K376G, V377A, and an inserted valine between K376 and V377 (referred to as "insertion 377V" in Table 1), but not to any of the other 20 mutants. Thus, the core epitope on CD39 for this antibody includes one or more (or all) of residues N371, L372, E375, K376, and V377. Figure 3A The positions of the residues mutated in mutants 5 (M5), 15 (M15), and 19 (M19) on the surface of the CD39 protein are shown. Figure 3B Results for binding to mutants 5, 15 and 19 are shown for the different antibodies.

[0336] The results thus show that antibodies that inhibit soluble CD39 can be obtained against different epitopes. The epitopes include an epitope defined by one or more residues of mutant 19, which is located near the binding site of BY40 or BY40-like antibodies, which only inhibit cellular CD39 but not soluble CD39 (which loses binding to mutant 5); an epitope defined by one or more residues of mutant 19, but also partially by mutant 5, indicating that there may be a smaller shift compared to BY40 or BY40-like antibodies; an epitope defined by one or more residues of mutant 19, but not by residues of mutant 5; and other epitopes, such as those defined by one or more residues of mutant 11 or one or more residues of mutant 15, or further by other antibodies that do not have any reduction in binding to any of mutants 5, 15 or 19, and the location of the epitope remains to be determined.

[0337] Example 7: Antibody titration on CD39 expressing cells by flow cytometry

[0338] In duplicate experiments, antibody I-394 was tested against CHO cells expressing human CD39, CHO cells expressing cynomolgus monkey (macaca fascicularis) CD39, CHO cells expressing mouse CD39, and human Ramos lymphoma cells (ATCC TM , refer to CRL-1596). Cells were treated with 30 μg / ml to 5×10- 4 The cells were incubated with various concentrations of unlabeled anti-CD39 antibody at 1 μg / ml for 30 minutes at 4°C. After washing, the cells were incubated with goat anti-mouse H+L labeled secondary antibody at 4°C for 30 minutes.

[0339] The results are shown in Figure 4 Antibody I-394 bound to cells expressing human CD39 (CHO-huCD39), cells expressing cynomolgus CD39 (CHO-cyCD39), and to Ramos lymphoma cells, but not to cells expressing murine CD39 (CHO-moCD39). I-394 bound to EC in the corresponding first and second sets of experiments. 50 Several other anti-CD39 antibodies showed EC values ​​comparable to Ramos cell binding. 50 value.

[0340] Example 8: IC50 determination of inhibition of cellular ATPase activity

[0341] As described above (Methods), the inhibition of the ATPase activity of CD39 in CD39 expressing cells by antibody 1-394 was assessed using an assay for inhibition of the enzymatic activity of cellular CD39.

[0342] The results are shown in Figure 5 I-394 was highly effective in blocking CD39 enzymatic activity in tumor (Ramus) cells, with higher potency than all other antibodies tested. I-394 also blocked CD39 enzymatic activity in cells expressing human CD39 (CHO-huCD39) and cells expressing cynomolgus monkey CD39 (CHO-cyCD39). Cells expressing mouse CD39 (CHO-moCD39) are shown as negative controls. The calculated IC 50 (50% inhibition of the enzymatic activity of CD39 expressed by 50,000 Ramos cells) was 0.05 μg / ml. The maximum inhibition was 81.6%. The isotype control was ineffective.

[0343] Example 9: IC50 determination of inhibition of ATPase activity of recombinant soluble CD39 protein

[0344] As described above (Methods), the inhibition of the ATPase activity of soluble CD39 protein by antibody I-394 was evaluated using an assay for inhibition of the enzymatic activity of soluble CD39. The results are shown in Figure 6 I-394 inhibited the enzymatic activity of soluble CD39 protein. In contrast, antibody BY40 did not inhibit the enzymatic activity of soluble CD39 protein. The calculated IC 50 The maximum inhibition value was 74.9%.

[0345] Example 10: ELISA titration on CD39-L1, L2, L3, L4 isoforms

[0346] Antibody I-394 was tested for binding to recombinant human CD39 isoforms (recombinant huCD39 isoforms) having the amino acid sequence shown below, which were coated in 96-well plates at 500 ng / ml or 1 μg / ml in PBS 1X overnight at 4°C. The wells were washed in TBS Tween 20 and further saturated in TBS blocking buffer for 2 hours at room temperature. Dose range concentrations of the primary antibody were incubated in TBS blocking buffer for 2 hours at room temperature. The wells were washed in TBS Tween 20. The secondary antibody (GAM-HRP or GAH-HRP in TBS blocking buffer) was incubated for 1 hour at room temperature and revealed with TMB. The plates were then stained with Enspire ELISA kit (see Figure 5). TM The optical density was measured at OD=450.

[0347] Amino acid sequence of cloned huCD39 (vascular isotype):

[0348] Human CD39-L1, also known as NTPDase2 or ENTPD2:

[0349] 1MAGKVRSLLP PLLLAAAGLA GLLLLCVPTR DVREPPALKY GIVLDAGSSH TSMFIYKWPA

[0350] 61DKENDTGIVG QHSSCDVPGG GISSYADNPS GASQSLVGCL EQALQDVPKE RHAGTPLYLG

[0351] 121ATAGMRLLNL TNPEASTSVL MAVTHTLTQY PFDFRGARIL SGQEEGVFGW VTANYLLENF

[0352] 181IKYGWVGRWF RPRKGTLGAM DLGGASTQIT FETTSPAEDR ASEVQLHLYG QHYRVYTHSF

[0353] 241LCYGRDQVLQ RLLASALQTH GFHPCWPRGF STQVLLGDVY QSPCTMAQRP QNFNSSARVS

[0354] 301LSGSSDPHLC RDLVSGLFSF SSCPFSRCSF NGVFQPPVAG NFVAFSAFFY TVDFLRTSMG

[0355] 361LPVATLQQLE AAAVNVCNQT WAQQLLSRGY GFDERAFGGV IFQKKAADTA VGWALGYMLN

[0356] 421LTNLIPADPP GLRKGTDFSS WVVLLLLFAS ALLAALVLLL RQVHSAKLPS TI(SEQ IDNO:2)。

[0357] Human CD39-L2, also known as NTPDase6 or ENTPD6:

[0358] 1MKKGIRYETS RKTSYIFQQP QHGPWQTRMR KISNHGSLRV AKVAYPLGLC VGVFIYVAYI

[0359] 61KWHRATATQA FFSITRAAPG ARWGQQAHSP LGTAADGHEV FYGIMFDAGS TGTRVHVFQF

[0360] 121TRPPRETPTL THETFKALKP GLSAYADDVE KSAQGIRELL DVAKQDIPFD FWKATPLVLK

[0361] 181ATAGLRLLPG EKAQKLLQKV KEVFKASPFL VGDDCVSIMN GTDEGVSAWI TINFLTGSLK

[0362] 241TPGGSSVGML DLGGGSTQIA FLPRVEGTLQ ASPPGYLTAL RMFNRTYKLY SYSYLGLGLM

[0363] 301SARLAILGGV EGQPAKDGKE LVSPCLSPSF KGEWEHAEVT YRVSGQKAAA SLHELCAARV

[0364] 361SEVLQNRVHR TEEVKHVDFY AFSYYYDLAA GVGLIDAEKG GSLVVGDFEI AAKYVCRTLE

[0365] 421TQPQSSPFSC MDLTYVSLLL QEFGFPRSKV LKLTRKIDNV ETSWALGAIF HYIDSLNRQK

[0366] 481SPAS

[0367] (SEQ ID NO:3)。

[0368] Human CD39-L3, also known as NTPDase3 or ENTPD3:

[0369] 1MFTVLTRQPC EQAGLKALYR TPTIIALVVL LVSIVVLVSI TVIQIHKQEV LPPGLKYGIV

[0370] 61LDAGSSRTTV YVYQWPAEKE NNTGVVSQTF KCSVKGSGIS SYGNNPQDVP RAFEECMQKV

[0371] 121KGQVPSHLHG STPIHLGATAGMRLLRLQNE TAANEVLESI QSYFKSQPFD FRGAQIISGQ

[0372] 181EEGVYGWITA NYLMGNFLEK NLWHMWVHPH GVETTGALDL GGASTQISFV AGEKMDLNTS

[0373] 241DIMQVSLYGY VYTLYTHSFQ CYGRNEAEKK FLAMLLQNSP TKNHLTNPCY PRDYSISFTM

[0374] 301GHVFDSLCTV DQRPESYNPN DVITFEGTGD PSLCKEKVAS IFDFKACHDQ ETCSFDGVYQ

[0375] 361PKIKGPFVAF AGFYYTASAL NLSGSFSLDT FNSSTWNFCS QNWSQLPLLL PKFDEVYARS

[0376] 421YCFSANYIYH LFVNGYKFTE ETWPQIHFEK EVGNSSIAWS LGYMLSLTNQ IPAESPLIRL

[0377] 481PIEPPVFVGT LAFFTAAALL CLAFLAYLCS ATRRKRHSEH AFDHAVDSD

[0378] (SEQ ID NO:4)。

[0379] Human CD39-L4, also known as NTPDase5 or ENTPD5:

[0380] 1MATSWGTVFF MLVVSCVCSA VSHRNQQTWF EGIFLSSMCP INVSASTLYG IMFDAGSTGT

[0381] 61RIHVYTFVQK MPGQLPILEG EVFDSVKPGL SAFVDQPKQG AETVQGLLEV AKDSIPRSHW

[0382] 121KKTPVVLKAT AGLRLLPEHK AKALLFEVKE IFRKSPFLVP KGSVSIMDGS DEGILAWVTV

[0383] 181NFLTGQLHGH RQETVGTLDL GGASTQITFL PQFEKTLEQT PRGYLTSFEM FNSTYKLYTH

[0384] 241SYLGFGLKAA RLATLGALET EGTDGHTFRS ACLPRWLEAE WIFGGVKYQY GGNQEGEVGF

[0385] 301EPCYAEVLRV VRGKLHQPEE VQRGSFYAFS YYYDRAVDTD MIDYEKGGIL KVEDFERKAR

[0386] 361EVCDNLENFT SGSPFLCMDL SYITALLKDG FGFADSTVLQ LTKKVNNIET GWALGATFHL

[0387] 421LQSLGISH

[0388] (SEQ ID NO:5).

[0389] I-394 binds to CD39, but not to any isotype CD39-L1, -L2, -L3 or -L4. The isotype control antibody (IC) does not bind to any CD39 or CD39-L molecules. The results are shown in Figure 7 middle.

[0390] Example 11: Activation of dendritic cells

[0391] Despite the proinflammatory activity of ATP, CD39-mediated ATP catabolism is believed to impair dendritic cell (DC) activation, thereby altering the broader adaptive immune response to tumor antigens. To assess whether CD39 blockade using anti-CD39 antibodies can overcome CD39-mediated alterations in DC activation in the presence of ATP, monocyte-derived DCs (moDCs) were incubated with anti-CD39 antibodies in the presence of ATP.

[0392] In brief, human monocytes were purified from healthy human blood and differentiated into MoDCs in the presence of GM-CSF and IL-4 over a period of 6 days. MoDCs were then activated in the presence of ATP (Sigma, 0.25-1 mM) over a period of 24 hours, and DC activation was assessed by flow cytometry by analyzing CD80, CD83, and HLA-DR expression. In some cases, MoDCs were pre-incubated for 1 hour in the presence of CD39 inhibitors: ARL6716 (Tocris, 250 μM), CD73 inhibitors: APCP (Tocris, 50 μM), anti-CD39 blocking antibodies I-394 or BY40 (for BY40, see WO2009 / 095478) or anti-CD73 blocking antibodies. LPS (Invivogen, 10 ng / ml) was used as a positive control. To evaluate the resulting effect of ATP-mediated DC activation on CD4 T cell activation, ATP-activated DCs were washed and then incubated with allogeneic CD4 T cells (ratio 1 MoDC / 4 T cells) for 5 days for mixed lymphocyte reactions (MLR). T cell activation and proliferation were analyzed by CD25 expression and Cell Trace Violet dilution by flow cytometry ( Figure 8 ).

[0393] The results are shown in Fig. 9 , 10and 11. In the presence of the negative control (culture medium), moDC activation was observed in the presence of 1mM ATP, however, 0.125mM, 0.25mM or 0.5mM ATP did not allow moDC activation. The addition of chemical inhibitors of CD39 is believed to completely block CD39 enzymatic activity by binding to the active site, resulting in moDC activation at each of 0.125mM, 0.25mM or 0.5mM. However, anti-CD39 antibodies, such as BY40 or anti-CD73 antibodies, were unable to support ATP-induced dendritic cell (DC) activation, suggesting that the antibodies were unable to adequately block enzymatic activity to avoid ATP catabolism. Surprisingly, the anti-CD39 blocking antibody I-394 (shown in the figure at a concentration of 10μg / ml), which essentially completely blocks the ATPase activity of CD39 and can therefore allow the accumulation of ATP, allows moDC activation, as assessed by HLA-DR or CD83 expression at each of 0.125mM, 0.25mM or 0.5mM ( Fig. 9 and 10 Interestingly, MoDC activated in the presence of ATP were able to induce better T cell activation and proliferation in the MLR assay. Furthermore, enhancement of ATP-mediated MoDC activation by the anti-CD39 blocking antibody I-394 resulted in higher T cell proliferation and activation ( Fig.11 ).

[0394] The ability of CD39 inhibitors to activate DC in the presence of ATP provides a method for identifying and evaluating anti-CD39 antibodies that can achieve a high degree of inhibition of CD39. In addition, the possibility of using anti-CD39 antibodies to reduce the immunosuppressive effects of CD39 on DC can provide an enhancement of the adaptive immune response to antigens, particularly to tumor cells. In addition, such anti-CD39 antibodies may be particularly interesting when used to enhance the immunogenic effects of chemotherapeutic agents. Many chemotherapeutic agents that cause tumor cell necrosis are able to induce ATP; the combined use of such agents and anti-CD39 antibodies can be particularly useful for enhancing anti-tumor responses.

[0395] Example 12: Antibodies that inhibit the ATPase activity of recombinant soluble CD39 protein strongly enhance CD73 blockade in the presence of ATP.

[0396] T cell proliferation assay

[0397] Peripheral blood from healthy donors was obtained from EFS and cultured on Ficoll TM Mononuclear cells were isolated by gradient. Cell pellets were collected and washed with 52% Percoll TMLymphocytes were further enriched on the gradient and stained with Cell Trace dye (Thermofisher) following the TDS provided by the manufacturer. 5x104 to 1x105 of stained cells were distributed in 96 round bottom plates, incubated with anti-huCD73 antibodies (antibody 6E1 described in WO2016 / 131950) and / or anti-huCD39Ab (I-394 described herein) for 1 hour at 37°C, and activated for 3 to 5 days by adding anti-CD3 / anti-CD28 coated beads (beads: cells = 1:4; Life Technologies). Inhibition of T cell proliferation was achieved by adding ATP (200 μM). T cell proliferation and the ability of Ab to block the immunosuppressive effect of AMP were evaluated by flow cytometry by quantifying the dye dilution in the proliferating T cell subsets.

[0398] Using GraphPad Prism TM The software plotted the percentage of proliferating T cells relative to the anti-CD73 Ab concentration in a graph.

[0399] result

[0400] The ability of the test antibodies to restore CD4 or CD8 T cell proliferation in the presence of added ATP is intended to represent conditions such as may be found in a tumor environment. Each of anti-CD73 and CD39 was tested within a dose range of 3 different doses of the other of the anti-CD73 or anti-CD39 antibodies. The anti-CD39 antibody I-394 strongly enhances the effect of the anti-CD73 antibody in restoring CD4 or CD8 T cell proliferation, such that even low concentrations of anti-CD73 antibodies (e.g., less than 0.01 μg / ml, less than 0.001 μg / ml, and even less than 0.001 μg / ml) strongly enhance CD4 or CD8 T cell proliferation when used in combination with anti-CD39 antibodies. In addition, when tested within a separate dose range without anti-CD73, the anti-CD39 antibody I-394 resulted in a significant enhancement of CD4 or CD8 T cell proliferation at concentrations of 0.1 μg / ml and 1 μg / ml. Fig. 12AThe dose range of anti-CD73 antibody 6E1 on CD4 T cell proliferation is shown at 3 different doses (0.01 μg / ml, 0.1 μg / ml and 1 μg / ml) of anti-CD39 antibody I-394. Anti-CD39 antibodies capable of neutralizing soluble and / or monomeric human CD39 show a strong enhancement of the effect of anti-CD73 antibodies in restoring CD4 T cell proliferation. This effect is particularly strong at concentrations where the anti-CD73 antibodies are suboptimally active, corresponding to the concentration range that can be observed in tumor tissue during the course of treatment with anti-CD73 antibodies. At a concentration of 0.01 μg / ml, anti-CD39 antibodies provide approximately a 1-log increase in the potency of anti-CD73 antibodies, and at a concentration of 0.1 μg / ml, anti-CD39 antibodies provide approximately a 4-log increase in the potency of anti-CD73 antibodies. Therefore, anti-CD39 antibodies can be used to enhance the activity of anti-CD73 antibodies, particularly in tumor tissues, such as in tumors with CD73-expressing cells. Furthermore, although the tested anti-CD73 antibodies (which are able to neutralize soluble CD73 protein) have a high capacity to restore CD4 T cell proliferation, other antibodies have lower potency (e.g., as assessed in enzyme inhibition assays, in T cell proliferation assays or other suitable assays) and may benefit even more from combination with anti-sCD39 antibodies. Fig. 12B A dose range of anti-CD73 antibodies on CD8 T cell proliferation is shown. Again, anti-CD39 antibodies showed strong synergistic and / or additive effects with anti-CD73 antibodies in restoring CD8 T cell proliferation. This effect was particularly strong at concentrations where the anti-CD73 antibodies were suboptimally active, corresponding to the concentration range that can be observed in tumor tissue during the course of treatment with anti-CD73 antibodies.

[0401] Example 13: Generation of Effective Humanized Variants of Antibody I-394

[0402] The parent antibody I-394 having the VH and VL amino acid sequences of SEQ ID NOs: 6 and 7 was modified by introducing the heavy chain framework (FR1, FR2, FR3) from human subgroup IGHV1-3 together with IGHJ1*01 (FR4) into VH and the light chain framework (FR1, FR2, FR3) from human subgroup IGKV4-1 together with IGKJ4*01 (FR4) into VL, respectively.

[0403] The three-dimensional models based on different human VH and VL gene segments were superimposed, and all amino acid differences were checked one by one. The 3D models of both parent chimeric (HPLP) and humanized (H0L0) antibodies were used to stimulate computer molecular design. The 3D model of the Fab fragment was constructed using the model antibody program (DS version 4.5) of Discovery Studio.

[0404] The heavy and light chain sequences used to model a chimeric Fab version of I-394 with a human IgG1 constant region comprising an Fc domain including the N297S substitution (lacking N297-linked glycosylation) or the L234A / L235E / G237A / A330S / P331S substitutions (retaining N297-linked glycosylation) are as follows:

[0405] I-394-LP (parental Fab light chain):

[0406] DIVMTQSPASLAVSLGQRATISCRASESVDNFGVSFMYWFQQKPGQPPNLLIYGASNQGSGVPARFRGSGSGTDFSLNIHPMEADDTAMYFCQQTKEVPYTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:19).

[0407] I-394-HP (parental Fab heavy chain):

[0408] EVKLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSPGRTLEWIGYIVPLNGGSTFNQKFKGRATLTVNTSSRTAYMELRSLTSEDSAAYYCARGGTRFAYWGQGTLV TVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK(SEQ ID NO:20).

[0409] Heavy and light chain template structures were identified, and protein database (PDB) references 4M7K, 1I7Z, and 3D85 were used for VH / VL interface, LC, and HC modeling, respectively. The PDB database used was the RCSB PDB from the Structural Bioinformatics Research Collaboratory managed by RCSB members Rutgers University and UCSD / SDSC, see www.rcsb.org, and HM Bergman et al., (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242. PDB entry 4M7K reference: Teplyakov, A. et al. (2014) Proteins 82: 1563-1582. PDB entry 1I7Z reference: Larsen, NA et al., (2001) J. Mol. Biol. 311: 9-15. Citation for PDB entry 3D85: Beyer, BM et al. (2008) J. Molecular Biology 382: 942-955.

[0410] The heavy and light chain sequences used to model the humanized Fab version of 1-394 are as follows.

[0411] I-394Fab L0 Light Chain:

[0412] DIVMTQSPDSLAVSLGERATINCRASESVDNFGVSFMYWYQQKPGQPPKLLIYGASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTKEVPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:21).

[0413] I-394Fab H0 Heavy Chain:

[0414] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYIVPLNGGSTFNQKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARGGTRFAYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK(SEQ ID NO:22).

[0415] For the humanized Fab version of I-394, PDB references 4NWT, 4I77, and 4JPI were used for VH / VL interface, LC, and HC modeling, respectively. Citation of PDB entry 4NWT: Bowers, PM, et al. (2014) J. Biol. Chem. 289:33557-33567. Citation of PDB entry 4I77: Ultsch, M., et al. (2013) J. Molecular Biology 425:1330-1339. Citation of PDB entry 4JPI: Jardine, J., et al. (2013) Science 340:711-716.

[0416] For the intermediate selection of light chain and heavy chain humanized variants, HPLP and H0L0 3D models are superimposed, and all amino acid differences are checked one by one. The intrachain connection and the extrachain connection between the residues are also evaluated, so as not to destroy any important low-energy bond by introducing back mutations (back-mutation) in a given chain. In addition, for light chain and heavy chain humanized variants, the amino acid affected by the difference between Kabat and IMGT CDR numbering schemes is specifically examined in detail in the HPLP and H0L0 3D model superpositions; It is found that the design of VH variants (designated as H2*, H3* and H4* by *) is promoted, and the variant retains the parental residue (tyrosine) present at Kabat residue 50 in VH (Kabat but not IGMT CDR2 residues) but does not retain the parental residues (two Kabat CDR2 residues) at positions 60 and 64. Similarly, a VL variant L1* that does not retain the parental residue (Kabat CDR1 residue) at position 24 is produced.

[0417] The amino acid modifications were introduced into the parent sequence. The anti-CD39 antibody VH and VL sequences are provided in Table A below. Compared to the parent H0VH of SEQ ID NO: 27, H1 contains R72V substitution (FR3); H2 contains V68A (FR3) and R72V (FR3) substitutions; H2* contains V68A (FR3) and R72V (FR3) substitutions and N61S (CDR2) substitution; H3 contains M48I (FR2), V68A (FR3) and R72V (FR3) substitutions; H3 contains M48I (FR2), V68 A (FR3) and R72V (FR3) substitutions, and N61S and K65Q substitutions in CDR2; H4 contains M48I (FR2), V68A (FR3), R72V (FR3) and S77R (FR3) substitutions; and H4* contains M48I (FR2), V68A (FR3), R72V (FR3) and S77R (FR3) substitutions, and N61S and K65Q substitutions in CDR2. Compared to the parent L0 chain VL of SEQ ID NO: 35, L1 contains Y40F substitution (FR2) and L1* contains Y40F substitution (FR2) and R24K substitution (CDR1).

[0418] Table A

[0419]

[0420] Antibodies with VH and VL variable regions were generated as Fc-silent recombinant chimeric human IgG1 antibodies with heavy chain substitutions L234A / L235E / G237A / A330S / P331S (Kabat EU numbering) mutations resulting in loss of binding to human Fcγ receptors CD16A, CD16B, CD32A, CD32B and CD64.

[0421] Briefly, the VH and Vk sequences shown below were cloned into vectors containing huIgG1 constant domains (with L234A / L235E / G237A / A330S / P331S substitutions) and huCk constant domains, respectively. The two vectors obtained were co-transfected into a CHO cell line in a combined manner to produce a combination of VH and VL. The established cell pool was used to produce antibodies in CHO culture medium. The antibodies were then purified using protein A.

[0422] In addition to the humanized antibody (mAb 1) grafted with parental CDR, 23 other humanized variants of antibodies were constructed, which contained different amino acid substitutions compared to the version grafted with parental CDR. All antibody variants were successfully produced in CHO cells as human IgG1 antibodies. The VH and VL of the resulting antibodies mAb 1 to mAb 24 are shown in Table B.

[0423] Table B

[0424]

[0425]

[0426] Binding of antibodies mAb 1-21 to CD39 was assessed by flow cytometry using CHO cells expressing human CD39 and CHO cells expressing cynomolgus monkey ( macaca fascicularis ) CD39 as described in Example 7. All mAbs showed comparable binding to the parental 1-394 antibody on both human and cynomolgus monkey CD39 CHO cell lines.

[0427] Example 14: Activity of humanized variants of antibody I-394

[0428] Binding and inhibition of CD39 by mAb 1-24 were further evaluated by a variety of assays, including binding to CD39 as present on the Ramos tumor cell line, which was found to express particularly high levels of CD39, inhibition of the enzymatic activity of recombinantly produced sCD39, and inhibition of the enzymatic activity of sCD39 protein shed in cell culture supernatants from CHO cells expressing human CD39 and from Ramos tumor cells.

[0429] The antibodies were titrated by flow cytometry on Ramos lymphoma cells according to the method described in Example 7. The results showed that all L0 variants bound less strongly on the Ramos cell line than the parental I-394 antibody, while the H2L1, H2L1*, H4L1 and H4L1* antibodies (mAbs 8, 9, 20 and 21, respectively) showed the best binding and were all similar to the parental I-394 antibody. Fig.13 .

[0430] Using the assay (method) for inhibiting the enzymatic activity of soluble CD39 as described above, the ability of the antibodies to inhibit the ATPase activity of soluble CD39 protein was tested. All antibodies showed good activity, with H3L1, H3L1*, H4L1 and H4L1* (mAb 14, 15, 20 and 21, respectively) all comparable to the parental I-394 antibody, while the efficacy of the other antibodies was slightly lower. The ability of the antibodies to inhibit the ATPase activity of soluble CD39 protein released from the cell culture supernatant of CHO cells expressing human CD39 was also tested using the assay (method) for inhibiting the enzymatic activity of soluble CD39 as described above. All antibodies showed good activity, with both H4L1 and H4L1* (mAb 20 and mAb 21) being comparable to the parental I-394 antibody, while the efficacy of the other antibodies was slightly lower.

[0431] In the assay described in Example 12, the antibodies were tested to assess their efficacy in reducing T cell suppression. The effect of ATP-mediated DC activation on CD4 T cell activation was tested, ATP-activated DCs were washed, and then incubated for 5 days with allogeneic CD4 T cells (ratio 1 MoDC / 4 T cells) for mixed lymphocyte reaction (MLR). T cell activation and proliferation were analyzed by CD25 expression and by Cell Trace Violet dilution by flow cytometry. The results showed that antibodies with heavy H2, H3 or H4 chains in combination with L1 light chains were all as effective as the parental 1-394 antibody, while antibodies with L0 light chains were less efficient.

[0432] The test antibody inhibits the effectiveness of ATPase activity in cell lines expressing membrane-bound CD39. Using the assay (method) for inhibiting the enzymatic activity of cellular CD39 as described above, the inhibition of the ATPase activity of CD39 in CD39-expressing cells by the antibody was evaluated. The antibody was first evaluated on CHO cells expressing human CD39; in this case, no significant difference between the variant mAb 1-24 and the parent I-394 antibody was observed on the CHO transfected cell line. However, when the antibody was evaluated on the tumor cell lines Ramos and Minnow, H4L1 and H4L1* antibodies (mAb 20 and mAb 21) were more effective in blocking CD39 enzymatic activity compared to all other antibodies. Fig.14Results in mino cells are shown. The differences observed between the assays in transfectants and tumor cells may result from the particularly high CD39 expression found in these tumor cell lines compared to CHO cells, thereby allowing the disclosure of differences in efficacy between antibodies. Antibodies with H4* heavy chains and L1 or L1* light chains were also tested on Ramos tumor cells; in this case, the efficacy of H4* antibodies mAb 23 and mAb 24 was slightly lower than mAb 20 and mAb 21. In summary, the most effective inhibitor of tumor cell-derived sCD39 (e.g., in tumor cells expressing high levels of CD39) is an antibody with an H4 heavy chain, followed by an antibody with an H2 heavy chain, followed by an antibody with an H3 heavy chain, with an L1 light chain in each case. A possible explanation is that there is a deleterious back mutation (BM) in the H3 variant, making the variant less potent than the H2 variant, but this is then balanced by substitutions in the H4 heavy chain variant, which restore activity to the level of parent I-394. Thus, the most potent antibodies overall among the humanized variants were the H4L1 antibody (having a VH of SEQ ID NO: 31 and a VL of SEQ ID NO: 36) and the H4L1* antibody (having a VH of SEQ ID NO: 31 and a VL of SEQ ID NO: 37). mAb 20 has the corresponding heavy and light chain CDRs shown in SEQ ID NOs: 8-13, wherein the heavy chain framework (FR1, FR2, FR3) is from the human IGHV1-3 gene together with the IGHJ1*01 gene (FR4), and the following substitutions (Kabat numbering): M48I (FR2), V68A (FR3), R72V (FR3), and S77R (FR3); and the light chain framework (FR1, FR2, FR3) is from human subgroup IGKV4-1 together with IGKJ4*01 (FR4) and a Y40F substitution (FR2). mAb 21 additionally carries a substitution in the light chain CDR1 at Kabat residue 24 (R24K substitution).

[0433] The full heavy chain of the H4L1 antibody (mAb 20) with L234A / L235E / G237A / A330S / P331S substitutions is shown below:

[0434] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWIGYIVPLNGGSTFNQKFKGRATITVDTSARTAYMELSSLRSEDTAVYYCARGGTRFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:38).

[0435] The full light chain of the H4L1 antibody (mAb 20) is shown below:

[0436] DIVMTQSPDSLAVSLGERATINCRASESVDNFGVSFMYWFQQKPGQPPKLLIYGASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTKEVPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:39).

[0437] The full light chain of the H4L1* antibody (mAb 21) is shown below:

[0438] DIVMTQSPDSLAVSLGERATINCKASESVDNFGVSFMYWFQQKPGQPPKLLIYGASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTKEVPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:40).

[0439] Example 15: Stability of humanized variants of antibody I-394

[0440] The stability of the test antibodies mAb 1-24 and the prior art anti-CD39 antibody BY40 (all produced as human IgG1 isotype with L234A / L235E / G237A / A330S / P331S substitutions) at a concentration of approximately 7 mg / mL in the following reference formulations: pH 6.0; histidine buffer (10 mM); sucrose (200 mM); NaCl (50 mM); polysorbate 80 (PS80) (0.2 g / L). The stability of the formulations was monitored under two storage conditions (at +5°C ± 3°C and at +40 ± 3°C). For each study, 3 time points were performed: T0, T15D (15 days) and T1M (1 month). Freeze-thaw (F / T) and thermal shift stability assays (TSSA) were performed for format comparison. For F / T cycling, samples were frozen at -20°C for at least 2 hours and thawed at room temperature for at least 1 hour, the F / T cycling was repeated three times and the samples were tested 24 hours after the last freeze / thaw cycle. At each time point, the following tests were performed:

[0441] ●Particulate matter (MFI)

[0442] ●Visual inspection (appearance)

[0443] ●Impurities (SE-HPLC)

[0444] ●Turbidity (400nm)

[0445] • Protein concentration (280 nm) (performed with Nanodrop, Thermo Fisher Scientific Inc.).

[0446] The obtained antibodies H2L1 (mAb 8), H2L1* (mAb 9), H4L1 (mAb 20) and H4L1* (mAb 21) all showed good physicochemical stability. Compared with antibody BY40, the aggregation temperature (T Agg ) is shown in Fig.15A and the aggregation temperatures of antibodies with H2L1, H2L1*, H4L1 or H4L1* chain combinations are shown in Fig. 15B Each of I-394 and H2L1, H2L1*, H4L1, and H4L1* antibodies showed a T close to 70°C. Agg . With T Agg Compared with antibody BY40, which is closer to 60°C, H2L1, H2L1*, H4L1 and H4L1* antibodies show a significant stability advantage. One possible reason for the relatively low intrinsic stability of antibody BY40 is the many aromatic amino acid residues located at the surface of the mAb in the CDRs, especially in the heavy chain CDR3, which endow the BY40 antibody with a relatively high predicted hydrophobicity.

[0447] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of whether incorporation of a specific document referenced elsewhere herein is provided separately.

[0448] The use of the terms "a," "an," and "the" and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0449] Unless otherwise indicated, all exact values ​​provided herein represent corresponding approximate values ​​(eg, all exact exemplary values ​​provided for a particular factor or measurement can be considered to also provide the corresponding approximate measurement modified by "about" where appropriate).

[0450] The description of any aspect or embodiment herein using terms such as "comprising," "having," "including," or "containing" is intended to provide support for similar aspects or embodiments herein "consisting of," "consisting essentially of," or "consisting essentially of the one or more specific elements," unless otherwise stated or clearly contradicted by context (for example, a composition described herein comprising a particular element should be understood to also describe a composition consisting of the element, unless otherwise stated or clearly contradicted by context).

[0451] Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. Any language in this specification should not be construed as indicating any unclaimed element as necessary to practice the present invention. Sequence Listing <110> INNATE PHARMA <120> Compositions and methods for treating cancer <130> CD39-9 <150> US 62 / 686,165 <151> 2018-06-18 <160> 44 <170> PatentIn version 3.5 <210> 1 <211> 510 <212> PRT <213> Homo sapiens <400> 1 Met Glu Asp Thr Lys Glu Ser Asn Val Lys Thr Phe Cys Ser Lys Asn 1 5 10 15 Ile Leu Ala Ile Leu Gly Phe Ser Ser Ile Ile Ala Val Ile Ala Leu 20 25 30 Leu Ala Val Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys 35 40 45 Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile 50 55 60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln 65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln 85 90 95 Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185 190 Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr 195 200 205 Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val 210 215 220 Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg 225 230 235 240 Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr 245 250 255 Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val 260 265 270 Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys 275 280 285 Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290 295 300 Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly 305 310 315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro 340 345 350 Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430 Ser Trp Glu His Ile His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435 440 445 Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala 450 455 460 Glu Gln Pro Leu Ser Thr Pro Leu Ser His Ser Thr Tyr Val Phe Leu 465 470 475 480 Met Val Leu Phe Ser Leu Val Leu Phe Thr Val Ala Ile Ile Gly Leu 485 490 495 Leu Ile Phe His Lys Pro Ser Tyr Phe Trp Lys Asp Met Val 500 505 510 <210> 2 <211> 472 <212> PRT <213> Homo sapiens <400> 2 Met Ala Gly Lys Val Arg Ser Leu Leu Pro Pro Leu Leu Leu Ala Ala 1 5 10 15 Ala Gly Leu Ala Gly Leu Leu Leu Leu Cys Val Pro Thr Arg Asp Val 20 25 30 Arg Glu Pro Pro Ala Leu Lys Tyr Gly Ile Val Leu Asp Ala Gly Ser 35 40 45 Ser His Thr Ser Met Phe Ile Tyr Lys Trp Pro Ala Asp Lys Glu Asn 50 55 60 Asp Thr Gly Ile Val Gly Gln His Ser Ser Cys Asp Val Pro Gly Gly 65 70 75 80 Gly Ile Ser Ser Tyr Ala Asp Asn Pro Ser Gly Ala Ser Gln Ser Leu 85 90 95 Val Gly Cys Leu Glu Gln Ala Leu Gln Asp Val Pro Lys Glu Arg His 100 105 110 Ala Gly Thr Pro Leu Tyr Leu Gly Ala Thr Ala Gly Met Arg Leu Leu 115 120 125 Asn Leu Thr Asn Pro Glu Ala Ser Thr Ser Val Leu Met Ala Val Thr 130 135 140 His Thr Leu Thr Gln Tyr Pro Phe Asp Phe Arg Gly Ala Arg Ile Leu 145 150 155 160 Ser Gly Gln Glu Glu Gly Val Phe Gly Trp Val Thr Ala Asn Tyr Leu 165 170 175 Leu Glu Asn Phe Ile Lys Tyr Gly Trp Val Gly Arg Trp Phe Arg Pro 180 185 190 Arg Lys Gly Thr Leu Gly Ala Met Asp Leu Gly Gly Ala Ser Thr Gln 195 200 205 Ile Thr Phe Glu Thr Thr Ser Pro Ala Glu Asp Arg Ala Ser Glu Val 210 215 220 Gln Leu His Leu Tyr Gly Gln His Tyr Arg Val Tyr Thr His Ser Phe 225 230 235 240 Leu Cys Tyr Gly Arg Asp Gln Val Leu Gln Arg Leu Leu Ala Ser Ala 245 250 255 Leu Gln Thr His Gly Phe His Pro Cys Trp Pro Arg Gly Phe Ser Thr 260 265 270 Gln Val Leu Leu Gly Asp Val Tyr Gln Ser Pro Cys Thr Met Ala Gln 275 280 285 Arg Pro Gln Asn Phe Asn Ser Ser Ala Arg Val Ser Leu Ser Gly Ser 290 295 300 Ser Asp Pro His Leu Cys Arg Asp Leu Val Ser Gly Leu Phe Ser Phe 305 310 315 320 Ser Ser Cys Pro Phe Ser Arg Cys Ser Phe Asn Gly Val Phe Gln Pro 325 330 335 Pro Val Ala Gly Asn Phe Val Ala Phe Ser Ala Phe Phe Tyr Thr Val 340 345 350 Asp Phe Leu Arg Thr Ser Met Gly Leu Pro Val Ala Thr Leu Gln Gln 355 360 365 Leu Glu Ala Ala Ala Val Asn Val Cys Asn Gln Thr Trp Ala Gln Gln 370 375 380 Leu Leu Ser Arg Gly Tyr Gly Phe Asp Glu Arg Ala Phe Gly Gly Val 385 390 395 400 Ile Phe Gln Lys Lys Ala Ala Asp Thr Ala Val Gly Trp Ala Leu Gly 405 410 415 Tyr Met Leu Asn Leu Thr Asn Leu Ile Pro Ala Asp Pro Pro Gly Leu 420 425 430 Arg Lys Gly Thr Asp Phe Ser Ser Trp Val Val Leu Leu Leu Leu Phe 435 440 445 Ala Ser Ala Leu Leu Ala Ala Leu Val Leu Leu Leu Arg Gln Val His 450 455 460 Ser Ala Lys Leu Pro Ser Thr Ile 465 470 <210> 3 <211> 484 <212> PRT <213> Homo sapiens <400> 3 Met Lys Lys Gly Ile Arg Tyr Glu Thr Ser Arg Lys Thr Ser Tyr Ile 1 5 10 15 Phe Gln Gln Pro Gln His Gly Pro Trp Gln Thr Arg Met Arg Lys Ile 20 25 30 Ser Asn His Gly Ser Leu Arg Val Ala Lys Val Ala Tyr Pro Leu Gly 35 40 45 Leu Cys Val Gly Val Phe Ile Tyr Val Ala Tyr Ile Lys Trp His Arg 50 55 60 Ala Thr Ala Thr Gln Ala Phe Phe Ser Ile Thr Arg Ala Ala Pro Gly 65 70 75 80 Ala Arg Trp Gly Gln Gln Ala His Ser Pro Leu Gly Thr Ala Ala Asp 85 90 95 Gly His Glu Val Phe Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly 100 105 110 Thr Arg Val His Val Phe Gln Phe Thr Arg Pro Pro Arg Glu Thr Pro 115 120 125 Thr Leu Thr His Glu Thr Phe Lys Ala Leu Lys Pro Gly Leu Ser Ala 130 135 140 Tyr Ala Asp Asp Val Glu Lys Ser Ala Gln Gly Ile Arg Glu Leu Leu 145 150 155 160 Asp Val Ala Lys Gln Asp Ile Pro Phe Asp Phe Trp Lys Ala Thr Pro 165 170 175 Leu Val Leu Lys Ala Thr Ala Gly Leu Arg Leu Leu Pro Gly Glu Lys 180 185 190 Ala Gln Lys Leu Leu Gln Lys Val Lys Glu Val Phe Lys Ala Ser Pro 195 200 205 Phe Leu Val Gly Asp Asp Cys Val Ser Ile Met Asn Gly Thr Asp Glu 210 215 220 Gly Val Ser Ala Trp Ile Thr Ile Asn Phe Leu Thr Gly Ser Leu Lys 225 230 235 240 Thr Pro Gly Gly Ser Ser Val Gly Met Leu Asp Leu Gly Gly Gly Ser 245 250 255 Thr Gln Ile Ala Phe Leu Pro Arg Val Glu Gly Thr Leu Gln Ala Ser 260 265 270 Pro Pro Gly Tyr Leu Thr Ala Leu Arg Met Phe Asn Arg Thr Tyr Lys 275 280 285 Leu Tyr Ser Tyr Ser Tyr Leu Gly Leu Gly Leu Met Ser Ala Arg Leu 290 295 300 Ala Ile Leu Gly Gly Val Glu Gly Gln Pro Ala Lys Asp Gly Lys Glu 305 310 315 320 Leu Val Ser Pro Cys Leu Ser Pro Ser Phe Lys Gly Glu Trp Glu His 325 330 335 Ala Glu Val Thr Tyr Arg Val Ser Gly Gln Lys Ala Ala Ala Ser Leu 340 345 350 His Glu Leu Cys Ala Ala Arg Val Ser Glu Val Leu Gln Asn Arg Val 355 360 365 His Arg Thr Glu Glu Val Lys His Val Asp Phe Tyr Ala Phe Ser Tyr 370 375 380 Tyr Tyr Asp Leu Ala Ala Gly Val Gly Leu Ile Asp Ala Glu Lys Gly 385 390 395 400 Gly Ser Leu Val Val Gly Asp Phe Glu Ile Ala Ala Lys Tyr Val Cys 405 410 415 Arg Thr Leu Glu Thr Gln Pro Gln Ser Ser Pro Phe Ser Cys Met Asp 420 425 430 Leu Thr Tyr Val Ser Leu Leu Leu Gln Glu Phe Gly Phe Pro Arg Ser 435 440 445 Lys Val Leu Lys Leu Thr Arg Lys Ile Asp Asn Val Glu Thr Ser Trp 450 455 460 Ala Leu Gly Ala Ile Phe His Tyr Ile Asp Ser Leu Asn Arg Gln Lys 465 470 475 480 Ser Pro Ala Ser <210> 4 <211> 529 <212> PRT <213> Homo sapiens <400> 4 Met Phe Thr Val Leu Thr Arg Gln Pro Cys Glu Gln Ala Gly Leu Lys 1 5 10 15 Ala Leu Tyr Arg Thr Pro Thr Ile Ile Ala Leu Val Val Leu Leu Val 20 25 30 Ser Ile Val Val Leu Val Ser Ile Thr Val Ile Gln Ile His Lys Gln 35 40 45 Glu Val Leu Pro Pro Gly Leu Lys Tyr Gly Ile Val Leu Asp Ala Gly 50 55 60 Ser Ser Arg Thr Thr Val Tyr Val Tyr Gln Trp Pro Ala Glu Lys Glu 65 70 75 80 Asn Asn Thr Gly Val Val Ser Gln Thr Phe Lys Cys Ser Val Lys Gly 85 90 95 Ser Gly Ile Ser Ser Tyr Gly Asn Asn Pro Gln Asp Val Pro Arg Ala 100 105 110 Phe Glu Glu Cys Met Gln Lys Val Lys Gly Gln Val Pro Ser His Leu 115 120 125 His Gly Ser Thr Pro Ile His Leu Gly Ala Thr Ala Gly Met Arg Leu 130 135 140 Leu Arg Leu Gln Asn Glu Thr Ala Ala Asn Glu Val Leu Glu Ser Ile 145 150 155 160 Gln Ser Tyr Phe Lys Ser Gln Pro Phe Asp Phe Arg Gly Ala Gln Ile 165 170 175 Ile Ser Gly Gln Glu Glu Gly Val Tyr Gly Trp Ile Thr Ala Asn Tyr 180 185 190 Leu Met Gly Asn Phe Leu Glu Lys Asn Leu Trp His Met Trp Val His 195 200 205 Pro His Gly Val Glu Thr Thr Gly Ala Leu Asp Leu Gly Gly Ala Ser 210 215 220 Thr Gln Ile Ser Phe Val Ala Gly Glu Lys Met Asp Leu Asn Thr Ser 225 230 235 240 Asp Ile Met Gln Val Ser Leu Tyr Gly Tyr Val Tyr Thr Leu Tyr Thr 245 250 255 His Ser Phe Gln Cys Tyr Gly Arg Asn Glu Ala Glu Lys Lys Phe Leu 260 265 270 Ala Met Leu Leu Gln Asn Ser Pro Thr Lys Asn His Leu Thr Asn Pro 275 280 285 Cys Tyr Pro Arg Asp Tyr Ser Ile Ser Phe Thr Met Gly His Val Phe 290 295 300 Asp Ser Leu Cys Thr Val Asp Gln Arg Pro Glu Ser Tyr Asn Pro Asn 305 310 315 320 Asp Val Ile Thr Phe Glu Gly Thr Gly Asp Pro Ser Leu Cys Lys Glu 325 330 335 Lys Val Ala Ser Ile Phe Asp Phe Lys Ala Cys His Asp Gln Glu Thr 340 345 350 Cys Ser Phe Asp Gly Val Tyr Gln Pro Lys Ile Lys Gly Pro Phe Val 355 360 365 Ala Phe Ala Gly Phe Tyr Tyr Thr Ala Ser Ala Leu Asn Leu Ser Gly 370 375 380 Ser Phe Ser Leu Asp Thr Phe Asn Ser Ser Thr Trp Asn Phe Cys Ser 385 390 395 400 Gln Asn Trp Ser Gln Leu Pro Leu Leu Leu Pro Lys Phe Asp Glu Val 405 410 415 Tyr Ala Arg Ser Tyr Cys Phe Ser Ala Asn Tyr Ile Tyr His Leu Phe 420 425 430 Val Asn Gly Tyr Lys Phe Thr Glu Glu Thr Trp Pro Gln Ile His Phe 435 440 445 Glu Lys Glu Val Gly Asn Ser Ser Ile Ala Trp Ser Leu Gly Tyr Met 450 455 460 Leu Ser Leu Thr Asn Gln Ile Pro Ala Glu Ser Pro Leu Ile Arg Leu 465 470 475 480 Pro Ile Glu Pro Pro Val Phe Val Gly Thr Leu Ala Phe Phe Thr Ala 485 490 495 Ala Ala Leu Leu Cys Leu Ala Phe Leu Ala Tyr Leu Cys Ser Ala Thr 500 505 510 Arg Arg Lys Arg His Ser Glu His Ala Phe Asp His Ala Val Asp Ser 515 520 525 Asp <210> 5 <211> 428 <212> PRT <213> Homo sapiens <400> 5 Met Ala Thr Ser Trp Gly Thr Val Phe Phe Met Leu Val Val Ser Cys 1 5 10 15 Val Cys Ser Ala Val Ser His Arg Asn Gln Gln Thr Trp Phe Glu Gly 20 25 30 Ile Phe Leu Ser Ser Met Cys Pro Ile Asn Val Ser Ala Ser Thr Leu 35 40 45 Tyr Gly Ile Met Phe Asp Ala Gly Ser Thr Gly Thr Arg Ile His Val 50 55 60 Tyr Thr Phe Val Gln Lys Met Pro Gly Gln Leu Pro Ile Leu Glu Gly 65 70 75 80 Glu Val Phe Asp Ser Val Lys Pro Gly Leu Ser Ala Phe Val Asp Gln 85 90 95 Pro Lys Gln Gly Ala Glu Thr Val Gln Gly Leu Leu Glu Val Ala Lys 100 105 110 Asp Ser Ile Pro Arg Ser His Trp Lys Lys Thr Pro Val Val Leu Lys 115 120 125 Ala Thr Ala Gly Leu Arg Leu Leu Pro Glu His Lys Ala Lys Ala Leu 130 135 140 Leu Phe Glu Val Lys Glu Ile Phe Arg Lys Ser Pro Phe Leu Val Pro 145 150 155 160 Lys Gly Ser Val Ser Ile Met Asp Gly Ser Asp Glu Gly Ile Leu Ala 165 170 175 Trp Val Thr Val Asn Phe Leu Thr Gly Gln Leu His Gly His Arg Gln 180 185 190 Glu Thr Val Gly Thr Leu Asp Leu Gly Gly Ala Ser Thr Gln Ile Thr 195 200 205 Phe Leu Pro Gln Phe Glu Lys Thr Leu Glu Gln Thr Pro Arg Gly Tyr 210 215 220 Leu Thr Ser Phe Glu Met Phe Asn Ser Thr Tyr Lys Leu Tyr Thr His 225 230 235 240 Ser Tyr Leu Gly Phe Gly Leu Lys Ala Ala Arg Leu Ala Thr Leu Gly 245 250 255 Ala Leu Glu Thr Glu Gly Thr Asp Gly His Thr Phe Arg Ser Ala Cys 260 265 270 Leu Pro Arg Trp Leu Glu Ala Glu Trp Ile Phe Gly Gly Val Lys Tyr 275 280 285 Gln Tyr Gly Gly Asn Gln Glu Gly Glu Val Gly Phe Glu Pro Cys Tyr 290 295 300 Ala Glu Val Leu Arg Val Val Arg Gly Lys Leu His Gln Pro Glu Glu 305 310 315 320 Val Gln Arg Gly Ser Phe Tyr Ala Phe Ser Tyr Tyr Tyr Asp Arg Ala 325 330 335 Val Asp Thr Asp Met Ile Asp Tyr Glu Lys Gly Gly Ile Leu Lys Val 340 345 350 Glu Asp Phe Glu Arg Lys Ala Arg Glu Val Cys Asp Asn Leu Glu Asn 355 360 365 Phe Thr Ser Gly Ser Pro Phe Leu Cys Met Asp Leu Ser Tyr Ile Thr 370 375 380 Ala Leu Leu Lys Asp Gly Phe Gly Phe Ala Asp Ser Thr Val Leu Gln 385 390 395 400 Leu Thr Lys Lys Val Asn Asn Ile Glu Thr Gly Trp Ala Leu Gly Ala 405 410 415 Thr Phe His Leu Leu Gln Ser Leu Gly Ile Ser His 420 425 <210> 6 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic <400> 6 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Lys Gln Ser His Gly Arg Thr Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asn Thr Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> 7 <211> 111 <212> PRT <213> artificial <220> <223> synthetic <400> 7 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Val Ser Phe Met Tyr Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Asn Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60 Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Met Glu Ala Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 8 <211> 5 <212> PRT <213> House mouse (Mus musculus) <400> 8 Asp Tyr Asn Met His 1 5 <210> 9 <211> 17 <212> PRT <213> House mouse <400> 9 Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 10 <211> 7 <212> PRT <213> House mouse <400> 10 Gly Gly Thr Arg Phe Ala Tyr 1 5 <210> 11 <211> 15 <212> PRT <213> House mouse <400> 11 Arg Ala Ser Glu Ser Val Asp Asn Phe Gly Val Ser Phe Met Tyr 1 5 10 15 <210> 12 <211> 7 <212> PRT <213> House mouse <400> 12 Gly Ala Ser Asn Gln Gly Ser 1 5 <210> 13 <211> 9 <212> PRT <213> House mouse <400> 13 Gln Gln Thr Lys Glu Val Pro Tyr Thr 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> X <222> (12)..(12) <223> X can be any amino acid residue <220> <221> X <222> (16) <223> X can be any amino acid residue <400> 14 Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Xaa Gln Lys Phe Xaa 1 5 10 15 Gly <210> 15 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic <400> 15 Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Ser Gln Lys Phe Lys 1 5 10 15 Gly <210> 16 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic <400> 16 Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Ser Gln Lys Phe Gln 1 5 10 15 Gly <210> 17 <211> 15 <212> PRT <213> Artificial <220> <223> Synthetic <220> <221> X <222> (1) <223> X can be any amino acid residue <400> 17 Xaa Ala Ser Glu Ser Val Asp Asn Phe Gly Val Ser Phe Met Tyr 1 5 10 15 <210> 18 <211> 15 <212> PRT <213> Artificial <220> <223> Synthetic <400> 18 Lys Ala Ser Glu Ser Val Asp Asn Phe Gly Val Ser Phe Met Tyr 1 5 10 15 <210> 19 <211> 218 <212> PRT <213> Artificial <220> <223> Synthetic <400> 19 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Val Ser Phe Met Tyr Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Asn Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60 Arg Phe Arg Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Met Glu Ala Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 20 <211> 221 <212> PRT <213> artificial <220> <223> synthetic <400> 20 Glu Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Met His Trp Val Lys Gln Ser Pro Gly Arg Thr Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Val Asn Thr Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Ala Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 <210> 21 <211> 218 <212> PRT <213> Artificial <220> <223> synthetic <400> 21 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Val Ser Phe Met Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 22 <211> 221 <212> PRT <213> Artificial <220> <223> synthetic <400> 22 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 <210> 23 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 24 <211> 330 <212> PRT <213> artificial <220> <223> synthetic <400> 24 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> 25 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 25 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> 26 <211> 330 <212> PRT <213> Artificial <220> <223> Synthetic <400> 26 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> 27 <211> 116 <212> PRT <213> Artificial <220> <223> synthetic <400> 27 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 28 <211> 115 <212> PRT <213> Artificial <220> <223> Synthetic <400> 28 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Val 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser 115 <210> 29 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic <400> 29 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Val 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 30 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic <400> 30 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Val 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 31 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic <400> 31 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Val Asp Thr Ser Ala Arg 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 32 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic <400> 32 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Ser Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Val 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 33 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic <400> 33 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Ser Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Val 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 34 <211> 116 <212> PRT <213> Artificial <220> <223> Synthetic <400> 34 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Ser Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Ile Thr Val Asp Thr Ser Ala Arg 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 35 <211> 111 <212> PRT <213> Artificial <220> <223> synthetic <400> 35 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Val Ser Phe Met Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 36 <211> 111 <212> PRT <213> Artificial <220> <223> synthetic <400> 36 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Val Ser Phe Met Tyr Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 37 <211> 111 <212> PRT <213> Artificial <220> <223> synthetic <400> 37 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Val Ser Phe Met Tyr Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 38 <211> 446 <212> PRT <213> Artificial <220> <223> Synthetic <400> 38 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 Asp Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Val Pro Leu Asn Gly Gly Ser Thr Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Val Asp Thr Ser Ala Arg 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 Gly Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Ala Glu Gly Ala Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 39 <211> 218 <212> PRT <213> Artificial <220> <223> Synthetic <400> 39 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Val Ser Phe Met Tyr Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 40 <211> 218 <212> PRT <213> Artificial <220> <223> Synthetic <400> 40 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Val Ser Phe Met Tyr Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Gln Gly Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Thr Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 41 <211> 45 <212> DNA <213> Homo sapiens <400> 41 tacgactcac aagcttgccg ccaccatgga agatacaaag gagtc 45 <210> 42 <211> 64 <212> DNA <213> Homo sapiens <400> 42 ccgccccgac tctagatcac ttgtcatcgt catctttgta atcgacatag gtggagtggg 60 agag 64 <210> 43 <211> 478 <212> PRT <213> Homo sapiens <400> 43 Met Glu Asp Thr Lys Glu Ser Asn Val Lys Thr Phe Cys Ser Lys Asn 1 5 10 15 Ile Leu Ala Ile Leu Gly Phe Ser Ser Ile Ile Ala Val Ile Ala Leu 20 25 30 Leu Ala Val Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys 35 40 45 Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile 50 55 60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln 65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln 85 90 95 Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185 190 Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr 195 200 205 Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val 210 215 220 Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg 225 230 235 240 Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr 245 250 255 Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val 260 265 270 Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys 275 280 285 Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290 295 300 Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly 305 310 315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro 340 345 350 Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430 Ser Trp Glu His Ile His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435 440 445 Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala 450 455 460 Glu Gln Pro Leu Ser Thr Pro Leu Ser His Ser Thr Tyr Val 465 470 475 <210> 44 <211> 486 <212> PRT <213> Artificial <220> <223> Synthetic <400> 44 Met Glu Asp Thr Lys Glu Ser Asn Val Lys Thr Phe Cys Ser Lys Asn 1 5 10 15 Ile Leu Ala Ile Leu Gly Phe Ser Ser Ile Ile Ala Val Ile Ala Leu 20 25 30 Leu Ala Val Gly Leu Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys 35 40 45 Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile 50 55 60 Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp Thr Gly Val Val His Gln 65 70 75 80 Val Glu Glu Cys Arg Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln 85 90 95 Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala 100 105 110 Arg Glu Val Ile Pro Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu 115 120 125 Gly Ala Thr Ala Gly Met Arg Leu Leu Arg Met Glu Ser Glu Glu Leu 130 135 140 Ala Asp Arg Val Leu Asp Val Val Glu Arg Ser Leu Ser Asn Tyr Pro 145 150 155 160 Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr Gly Gln Glu Glu Gly Ala 165 170 175 Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys 180 185 190 Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr 195 200 205 Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val 210 215 220 Pro Gln Asn Gln Thr Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg 225 230 235 240 Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr 245 250 255 Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val 260 265 270 Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys 275 280 285 Lys Val Val Asn Val Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg 290 295 300 Phe Glu Met Thr Leu Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly 305 310 315 320 Asn Tyr Gln Gln Cys His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser 325 330 335 Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro 340 345 350 Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys 355 360 365 Phe Leu Asn Leu Thr Ser Glu Lys Val Ser Gln Glu Lys Val Thr Glu 370 375 380 Met Met Lys Lys Phe Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser 385 390 395 400 Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly 405 410 415 Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp 420 425 430 Ser Trp Glu His Ile His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala 435 440 445 Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala 450 455 460 Glu Gln Pro Leu Ser Thr Pro Leu Ser His Ser Thr Tyr Val Asp Tyr 465 470 475 480 Lys Asp Asp Asp Asp Lys 485

Claims

1. An antibody or antibody fragment that binds to a human CD39 polypeptide and is capable of inhibiting the ATPase activity of a soluble extracellular domain human CD39 polypeptide, wherein the antibody or antibody fragment comprises a heavy chain variable region (VH) of SEQ ID NO: 31 and a light chain variable region (VL) of SEQ ID NO: 36 or 37.

2. The antibody or antibody fragment according to claim 1, wherein the antibody or antibody fragment comprises the heavy chain variable region of SEQ ID NO: 31 and the light chain variable region of SEQ ID NO:

36.

3. The antibody or antibody fragment according to claim 1, wherein the antibody or antibody fragment comprises the heavy chain variable region of SEQ ID NO: 31 and the light chain variable region of SEQ ID NO:

37.

4. The antibody or antibody fragment according to claim 1, wherein the antibody is capable of inhibiting the ATPase activity of sCD39 protein shed in the cell culture supernatant of human CD39-expressing tumor cells. 5 . The antibody or antibody fragment of claim 1 , wherein the antibody inhibits the ATPase activity of CD39 protein in the presence of exogenously added ATP, wherein the exogenously added ATP is provided at a concentration of 20 μM. 6 . The antibody or antibody fragment according to claim 1 , wherein the antibody is capable of binding to human CD39 protein at the surface of a cell and is capable of inhibiting the ATPase activity of the CD39 protein at the surface of a cell.

7. The antibody or antibody fragment of claim 1, wherein the antibody is capable of increasing the expression of cell surface activation markers in monocyte-derived dendritic cells when such moDCs are incubated with the antibody and ATP in vitro, wherein the exogenously added ATP is provided at 0.125 mM, 0.25 mM or 0.5 mM.

8. The antibody or antibody fragment of claim 1, wherein the antibody is an antibody having a human Fc domain that is modified to reduce binding between the Fc domain and a human Fcγ receptor.

9. The antibody or antibody fragment of claim 1, wherein the antibody comprises an Fc domain whose binding to one or more or all of the human Fcγ receptors CD16A, CD16B, CD32A, CD32B and / or CD64 is reduced or abolished.

10. An antibody or antibody fragment, wherein the antibody or antibody fragment comprises the heavy chain described in SEQ ID NO:38 and the light chain described in SEQ ID NO:

39.

11. An antibody or antibody fragment, wherein the antibody or antibody fragment comprises the heavy chain described in SEQ ID NO: 38 and the light chain described in SEQ ID NO:

40.

12. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. A nucleic acid encoding the heavy chain and / or light chain of the antibody or antibody fragment according to any one of claims 1 to 11.

14. A recombinant host cell which produces the antibody or antibody fragment according to any one of claims 1 to 11.

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