EA047070B1 - COMPOSITIONS AND METHODS FOR TREATING CANCER - Google Patents

Description

Cross references to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/686,165, filed June 18, 2018; the contents of which are incorporated herein by reference in their entirety; including any drawings.

Link to sequence list

This application is submitted together with a sequence listing in electronic format. The sequence listing is provided as a file called CD39-9_ST25, created on May 27, 2019, and has a size of 72 kB. The sequence listing information in electronic format is incorporated herein by reference in its entirety.

Technical field

The invention relates to antigen-binding compounds (eg antibodies) that inhibit the enzymatic activity of soluble human CD39. The present invention also relates to cells producing such compounds; methods for preparing such compounds and antibodies, fragments, variants and derivatives thereof; pharmaceutical compositions containing them; methods of using the compounds for the diagnosis, treatment or prevention of diseases, such as cancer.

State of the art

Eight different ENTPD genes encode member proteins of the NTPDh3 family. Individual subtypes of NTPDases differ in cellular location and functional properties. Plasma membrane-bound nucleoside triphosphate diphosphohydrolases control cell surface nucleotide levels by hydrolyzing c and b nucleotide phosphates.

KTHOase 1 (ectonucleoside triphosphate diphosphohydrolase 1), also known as CD39/ENTPD1 or vascular CD39, functions in concert with another enzyme, CD73 (ecto-5'-nucleotidase), to hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds with adenosine receptors and inhibits T cell and (NK) cell responses, thereby suppressing the immune system. Generation of adenosine through the CD73/CD39 pathway is recognized as a major mechanism of the immunosuppressive function of regulatory T cells (Tregs). The number of CD39+ Tregs is increased in several human cancers, and the importance of CD39+ 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 signaling pathway. CD39 in cancer cells exhibits ATPase activity and, together with CD73, generates adenosine. CD73+CD39+ cancer cells inhibited the proliferation of CD4 and CD8 T cells and the generation of cytotoxic effector CD8 T cells (CTL) in a CD39- and adenosine-dependent manner. CD39 levels have been reported to be elevated in several solid tumors (colorectal cancer, head and neck cancer, pancreatic cancer) as well as in chronic lymphocytic leukemia. Antibodies that bind and inhibit CD39 in CD39-expressing cells are disclosed in WO 2009/095478. Antibody A1 (eBiosciences, Inc.) was used for staining and was not able to neutralize CD39 activity in cells. In Hayes et al. (2015) Am. J. Transl. Res. 7(6): 11811188 uses an anti-CD39 molecule that binds FcyR and has effector function but is said to also block it. The expression of CD39 on various cell types, including leukocytes and tumor cells, coupled with the use of antibodies that either do not effectively block CD39 or are not absolute blockers, creates a challenging environment for assessing underlying antibody activity. To date, the only reported active site inhibitor of CD39 remains small molecule non-hydrolysable ATP analogues, such as ARL67156, suggesting the need for direct active site inhibition. ARL67156, however, is not specific for CD39 and also inhibits other NTPDases such as mouse NTPDase1, NTPDase3, NPP1 or NTPDase8, and furthermore acts only as a weak competitive inhibitor (Levesque et al. (2007) Br. J. Pharmacol . 152:141-150).

CD39 has two transmembrane domains near the N- and C-termini, short cytoplasmic N- and C-terminal segments, and a large extracellular domain containing the active site. However, although CD39 is typically anchored to the membrane by two transmembrane domains at the two ends of the molecule, it has also recently been reported that a soluble catalytically active form of CD39 can be found in the bloodstream in humans and mice. (Yegutkin et al., (2012) FASEB J. 26(9): 3875-3883). Despite the various CD39 antibodies that have been described, no antibody has been reported that is capable of inhibiting the ATPase activity of soluble CD39 protein.

Brief description of the invention

The inventors have produced antibodies that inhibit the enzymatic (ATPase activity) activity of the soluble (extracellular domain) human CD39 protein. The antibodies additionally bind an epitope present on the human CD39 protein, which is expressed on the surface of cells, including tumor cells, and potentially inhibit the enzymatic (ATPase activity) activity of the cell membrane-associated CD39 enzyme (cell surface expressed CD39). These antibodies can be usefully used to achieve greater neutralization of CD39 activity in an individual through neutralization as membrane-bound,

- 1 047070 and soluble CD39 protein, including soluble CD39 released or secreted from tumor cells, thereby reducing immunosuppression, for example, in the treatment of cancer and/or infectious diseases. Although other anti-CD39 antibodies have previously been described that inhibit the enzymatic (ATPase activity) activity of the membrane-bound CD39 enzyme, such antibodies do not inhibit soluble CD39 protein, which is not associated with the cell membrane.

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

In one embodiment, an antigen binding domain of an anti-CD39 molecule or a protein that contains such an antigen binding domain is provided (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.), wherein said antigen binding domain or a protein containing such an antigen binding domain , contains a heavy chain variable region (VH) containing 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 of SEQ ID NO: 27- 34, and optionally further comprises one or more substitutions of an amino acid residue present in the human framework sequence with another residue (eg, a residue present in a non-human framework) at positions selected from the group consisting of 48, 67, 71 and 76 (numbering according to Kabat); and a light chain variable region (VL) comprising 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 of SEQ ID NO: 35-37 , characterized in that optionally phenylalanine is present at position 36 (numbering according to Kabat). In one embodiment, the VH further comprises one or more amino acid substitutions in the heavy chain CDR2 at positions 60 and/or 64 (Kabat numbering). Optionally, the residue present in the heavy chain at position 60 (numbering according to Kabat) is a serine residue. Optionally, the residue present in the heavy chain at position 64 (Kabat numbering) is a glutamine residue. In one embodiment, VL further comprises an amino acid substitution in the light chain CDR2 at position 24 of the light chain (Kabat numbering), further optionally the residue present in the light chain at position 24 being a lysine residue.

Optionally, the amino acid at position 48 of the heavy chain (Kabat numbering) is isoleucine. Optionally, the amino acid at position 67 of the heavy chain (numbering according to Kabat) is alanine. Optionally, the amino acid at position 71 of the heavy chain (numbering according to Kabat) is valine. Optionally, the amino acid at position 76 of the heavy chain (numbering according to Kabat) is arginine.

In one embodiment, VH contains an alanine residue at position 67 (numbering according to Kabat) and a valine residue at position 71.

In one embodiment, VH contains an isoleucine residue at position 48 (Kabat numbering), an alanine residue at position 67 (Kabat numbering), valine at position 71 (Kabat numbering), and an arginine residue at position 76 (Kabat numbering).

In one embodiment, VL contains phenylalanine at position 36 (Kabat numbering) (FR2). In one embodiment, VL contains a lysine at position 24 (numbering according to Kabat) (CDR1).

- 2 047070

In any embodiment, the antigen binding domain of an anti-CD39 molecule or a protein that contains such an antigen binding domain (e.g., a monoclonal antibody or antibody fragment, multispecific binding protein, bispecific antibody, etc.) can be characterized as containing a heavy chain variable region (VH ), containing an amino acid sequence 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) containing 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.

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

In any embodiment, VH may be characterized as containing a substitution at one, two, three, or all of positions 48, 67, 71, and 76 (numbering according to Kabat). In one embodiment, the residue at position 48 is isoleucine (eg, M48I substitution). In one embodiment, the residue at position 67 is an alanine (eg, substitution V67A). In one embodiment, the residue at position 71 is valine (eg, R71V substitution). In one embodiment, the residue at position 76 is an arginine (eg, S76R substitution). In any embodiment, VL may be characterized as containing a substitution at position 36 (numbering according to Kabat). In one embodiment, the residue at position 36 is a phenylalanine (eg, Y36F substitution).

In one embodiment, VH comprises human VH framework amino acid sequences, and VL comprises human VL framework amino acid sequences. In one embodiment, the VH segment of the human VH acceptor framework region is derived from a human IGHV1-3 gene segment, and optionally the J segment is derived from a human IGHJ1 gene segment. In one embodiment, the human VH framework region is derived from the human IGHV1-3*01 gene segment. In one embodiment, the human VL domain acceptor framework is derived from a human IGKV4-1 gene segment, and optionally the J segment is also derived from a human IGKJ4 gene segment.

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

(a) an antibody binding domain comprising a light chain variable region containing the amino acid sequence of SEQ ID NO: 36, and a heavy chain variable region containing an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 29, 30, 31, 32, 33 or 34; and (b) an antibody binding domain comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a heavy chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SeQ ID NO: 29, 30, 31 , 32, 33 or 34.

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 any of the amino acid sequences of SEQ ID NO: 23, 24, 25, or 26. In any embodiment, the antibody light chain comprises a human light chain constant domain, wherein the constant domain is optionally a human kappa domain.

In one embodiment, an anti-CD39 antibody is provided comprising a heavy chain comprising an amino acid sequence 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 a light chain containing an amino acid sequence 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.

In one embodiment, an anti-CD39 antibody containing a heavy chain is provided with

- 3 047070 containing an amino acid sequence 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 a light chain containing the amino acid sequence, 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.

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

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

In any embodiment, an antigen binding domain or protein containing such, optionally an antibody or antibody fragment, can be characterized to bind and inhibit or neutralize the ATPase activity of soluble CD39 protein (pCD39). In one embodiment, the pCD39 protein lacks two transmembrane domains (ie, transmembrane domains approximately N- and C-terminal) found in membrane-bound CD39. In one embodiment, pCD39 is a non-membrane associated pCD39 protein found in the bloodstream of, for example, a human subject. In one embodiment, pCD39 comprises or consists of the amino acid sequence of SEQ ID NO: 43, optionally further comprising a C-terminal tag or other amino acid sequence not derived from CD39; wherein optionally the amino acid sequence of SEQ ID NO: 43 is further devoid of N-terminal residues 1 to 37 of SEQ ID NO: 1. The pCD39 protein can be characterized as containing or consisting of the Thr38-Val478 fragment of CD39. The Thr38-Val478 protein with a C-terminal His tag is commercially available from R&D Systems, Inc. (product number 4397-EN). In one embodiment, the protein, antibody, or antibody fragment inhibits the ATPase activity of pCD39 when incubated with pCD39 in solution, e.g., in accordance with cell-free methods or assays as described herein (see, e.g., Examples, Methods) , for example in tumor cell supernatants. In one embodiment, the protein, antibody, or antibody fragment specifically binds human CD39 protein, in both soluble (extracellular protein domain) and membrane-bound forms.

Without being bound by any theory, some antibodies can neutralize membrane-bound CD39 by inhibiting the movement of the membrane-bound CD39 domain (memCD39) without similarly affecting the activity of soluble CD39 protein (pCD39). MemCD39 has been reported to occur as a homo-multimer while pCD39 is a monomer, and furthermore, the transmembrane domains in memCD39 undergo dynamic movements that underlie the functional relationship with the active site. Therefore, unlike pCD39, memCD39 may have a setting that allows antibody-mediated neutralization. One possibility is that functional neutralization requires the use of a divalent antibody that binds simultaneously to two memCD39 molecules (eg, as part of a memCD39 homomultimer).

The present antibodies that neutralize the activity of pCD39 (and memCD39) may, in addition to being used as divalent binding molecules, also be effective as monovalent binding molecules, regardless of whether they target memCD39 in addition to pCD39. Therefore, in one embodiment, an antigen binding protein is provided that monovalently binds to human CD39 protein (pCD39 and/or memCD39) and neutralizes its enzymatic (ATPase) activity. An antigen binding protein may optionally be defined as binding to a single CD39 protein and/or having a single antigen binding domain capable of binding to a CD39 protein. In one embodiment, an antibody fragment, optionally an F(ab) fragment, a single chain antibody, pcFv, is provided, a multispecific antibody that monovalently binds to human CD39 protein (pCD39 and/or memCD39) and neutralizes its enzymatic (ATPase) activity. In one embodiment, the CD39 neutralizing antigen binding protein that binds monovalently 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, a CD39 neutralizing antigen binding protein that binds monovalently to human CD39 protein comprises a first (or only) antigen binding domain that binds CD39 (pCD39 and/or memCD39) and a second antigen binding domain that binds a protein other than CD39 .

Preferably, in one embodiment, the antibody comprises a human Fc domain that is modified to have reduced or substantially no binding to a human FCy receptor, for example, one or more (or all) human CD16, CD32a, CD32b and CD64. In one aspect, the antibodies do not rely on ADCC, CDC, or toxin-mediated depletion of CD39-expressing cells for their CD39 inhibitory activity. Such antibodies can be used as absolute CD39 blockers with immunomodulatory activity.

- 4 047070

In an alternative embodiment, the binding molecule may be designed such that it retains and/or mediates effector function through its Fc domain. In one embodiment, the antibody contains a human Fc domain that binds to a human FCy receptor, for example, one or more (or all) of human CD16, CD32a, CD32b and CD64.

In another embodiment, the FC domain may be modified to reduce FcY receptor binding, not necessarily by maintaining binding to one or more human Fcy receptors, but reducing binding to one or more other human FcY receptors.

In one aspect, the antibodies specifically bind vascular CD39, eg, the antibody binds a polypeptide having the sequence of SEQ ID NO: 1, but does not bind a secreted isoform of the CD39 polypeptide, eg, a CD39-L2 and/or -L4 polypeptide. Optionally, the anti-CD39 antibody specifically binds vascular CD39, eg, the antibody binds a polypeptide having the sequence SEQ ID NO: 1, but does not bind a membrane-bound isoform of CD39, eg, a CD39L1 and/or-L3 polypeptide.

The antibodies disclosed herein can inhibit the enzymatic activity of the membrane-bound protein CD39 expressed on the surface of cells.

In one aspect, the antibodies are not dependent on CD39 down-modulation for their CD39 inhibitory activity.

Antibodies disclosed herein may, in addition to inhibiting soluble CD39, be capable of inhibiting the enzymatic activity of membrane-bound CD39 protein expressed on the surface of cells, with or without induction of CD39 internalization, and with or without CD16 (FcyIII receptor) binding and/or with or without significantly conferring ADCC and/or CDC on a CD39-expressing cell. Optionally, the antibodies retain the Fc domain and retain binding to human FcRn.

While antibodies that function by inducing ADCC and/or CDC may be effective even without complete neutralization/inhibition of CD39 ATPase activity, as long as sufficient antibodies are bound to the CD39-expressing cell to induce ADCC, neutralizing nondepleting antibodies may require stronger inhibition of ATPase enzymatic activity. In one embodiment, the nondepleting antibody will provide at least a 50, 60, 70, 80, or 90% reduction in the ATPase activity of soluble CD39 protein (e.g., as assessed by methods described herein), optionally additionally at a concentration compatible with administration of the antibody to a human . In one embodiment, the non-depleting antibody will provide at least a 70, 80, 90% reduction in the ATPase activity of a CD39-expressing cell (e.g., as assessed by a reduction in AMP production by a CD39' cell, such as a B cell, a Ramos cell, as measured by methods disclosed herein), optionally also at a concentration compatible with administration of the antibody to a human.

The antibody-bound epitope on CD39 is present on CD39 polypeptides expressed by a variety of cells, e.g. cancer cells, CD4 T cells, CD8 T cells, B cells, transfected cells, and binds with high affinity, as determined by flow cytometry.

The antibody may optionally be characterized by an EC ₅₀ value, determined by flow cytometry, of 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 the CD39 polypeptide on their surface. In one embodiment, said cells are cells that are engineered to express CD39 on their surface. In one embodiment, the cells are cells that endogenously express CD39 on their surface, e.g., T regulatory cells (TReg), B cells, cancer cells, lymphoma cells (eg, Ramos cells), leukemia cells, bladder cancer cells, gliomas, glioblastoma cells, ovarian cancer cells, melanoma cells, prostate cancer cells, thyroid cancer cells, esophageal cancer cells or breast cancer cells.

In one aspect, an anti-CD39 antibody is capable of: (a) inhibiting the enzymatic activity of a membrane-bound CD39 protein (eg, comprising the amino acid sequence of SEQ ID NO: 1) expressed on the surface of cells, and (b) inhibiting the enzymatic activity of a soluble CD39 protein. In one embodiment, the antibodies do not substantially bind (e.g., through their Fc domain) to human FCY receptors (e.g., CD16, CD32a, CD32b, CD64) and/or C1q and/or do not substantially confer ADCC and/or CDC to a CD39-expressing cell. Optionally, the antibodies retain the Fc domain and retain binding to human FcRn.

In one embodiment, the antibodies are administered in an amount effective to neutralize the enzymatic activity of pCD39 and/or memCD39 for a desired period of time, for example, 1 week, 2 weeks, a month, before the next sequential administration of the anti-CD39 antibody.

In one embodiment, the antibodies are administered at a dosage and/or frequency to provide a blood concentration of the antibody equal to at least _EC50 , _EC70 , or _EC100 to inhibit ATPases.

- 5 047070 new activity of the pCD39 protein, optionally the concentration is maintained for at least 1 week, 2 weeks, a month or until the next sequential administration of the anti-CD39 antibody.

In one aspect, the antibody binds a CD39 epitope containing an amino acid residue (eg, one, two, or three residues) selected from the group consisting of R138, M139, and E142 (relative to SEQ ID NO: 1).

In one aspect, an anti-CD39 antibody exhibits reduced binding (e.g., substantially complete loss of binding) to a CD39 polypeptide having a mutation at one, two, or three residues selected from the group consisting of: R138, M139, and E142 (relative to SEQ ID NO : 1) compared to wild type CD39 polypeptide (CD39 polypeptide SEQ ID NO: 1); optionally, the mutant CD39 polypeptide has the following mutations: R138A, M139A and E142K. In one optional aspect, the antibody has no loss of binding to any of the mutant CD39 polypeptides from Table. 1, except mutant 19.

In one embodiment, CD39 neutralizing antibodies may be capable, in purified form, of causing a reduction in the ATPase activity of the pCD39 protein in a cell-free assay (e.g., pCD39 from tumor cell culture supernatant), in some cases causing a reduction in pCD39 AMP generation by at least 70 , 80 or 90%; optionally causing an increase in ATP present (compared to the negative control), for example, in accordance with the assessment of the assays disclosed herein. For example, pCD39 inhibition can be assessed by quantifying luminescence units that are proportional to the amount of ATP present after incubation with anti-CD39 antibody. In one embodiment, CD39-neutralizing antibodies can be characterized using EC ₅₀ to inhibit the ATPase activity of pCD39 protein by no more than 1 μg/ml, optionally no more than 0.5 μg/ml, optionally no more than 0.1 μg/ml.

Optionally, inhibition of the ATPase activity of the pCD39 protein is determined by quantifying luminescence units using Cell Titer Glo™ (Promega), a cell-free assay characterized in that dose ranges of the test antibody are incubated with the soluble recombinant human CD39 protein described in the Examples for 1 h at 37°C, with 20 µM ATP added to the plates for an additional 30 minutes at 37°C before adding Cell Titer Glo™ (CTG) reagent, and emitted light quantified using an Enspire™ luminometer after incubation for 5 minutes in the dark (see, for example, Examples, Methods).

In one embodiment, the pCD39 protein is a pCD39 protein found in or derived from a human tumor cell culture supernatant, in some cases from a tumor cell line that expresses CD39 at high levels, optionally from Ramos tumor cells.

Optionally, CD39-neutralizing antibodies may further be characterized by the ability, in purified form, to cause a reduction in the ATPase activity of CD39 cells, optionally causing a reduction in AMP generation by the CD39-expressing cell by at least 70%, 80%, or 90%. In one embodiment, CD39-neutralizing antibodies may be characterized by an EC ₅₀ for inhibiting the ATPase activity (e.g., an EC 50 for inhibiting the generation of AMP by a CD39 expressing cell) of CD39 expressed by the cell of no more than 1 μg/ml, optionally no more than 0.5 μg/ml, optional - no more than 0.1 μg/ml.

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

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

In one aspect, an anti-CD39 neutralizing antibody binds an antigenic determinant present on both pCD39 and cell surface expressed CD39 (memCD39).

In one aspect, the anti-CD39 neutralizing antibody competes for binding to a CD39 epitope bound by mAb20, mAb21 (or their parent antibody I-394) (e.g., which competes for binding to an epitope of a CD39 polypeptide with an antibody having heavy and light chain CDRs or variable region of any of mAb20, mAb21 or I-394).

In one aspect of any of the embodiments disclosed herein, the antigen binding compound binds the same epitope, and/or competes for binding to the CD39 polypeptide with antibodies mAb20, mAb21 (or their parent antibody I-394) (e.g., that binds the same

- 6 047070 epitope, and/or competes for binding to the CD39 polypeptide with an antibody having CD Rs heavy and light chains or variable regions mAb20, mAb21 (or I-394)). In one embodiment, the antigen binding compound binds the same epitope and/or competes for binding to a CD39 polypeptide with an antibody having the VH and VL regions, respectively, of SEQ ID NO: 38 and 39.

In one embodiment, an anti-CD39 antibody binds an epitope containing one, two, or three amino acid residues selected from the group consisting of CD39 amino acid residues bound by mAb20, mAb21 (or I-394).

In any embodiment, the binding molecule (e.g., an antibody or antibody fragment) comprises a heavy chain variable domain ( _VH ) containing heavy chain CDR1, 2, and 3 (e.g., as described herein) for antibody I-394, and a light chain variable domain (V _L ) containing light chain CDRs 1, 2, and 3 (e.g., as described herein) for the corresponding I-394 antibody, or an amino acid sequence in which the CDRs (or set of heavy and/or light chain CDRs ) has at least 70, 80, 90, or 95% amino acid identity to such CDR (or such set of heavy and/or light chain CDRs) wherein VH and VL each contain framework domains of human origin (e.g., VH and VL are different from their respective VH and VL in SEQ ID NO: 6 and 7). Optionally, CDRs are determined by numbering schemes according to Kabat or IMGT.

In one aspect, the protein that contains an antibody or antibody fragment contains 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 the CD16A, CD16B, CD32A, CD32B and/or polypeptides. or human CD64, wherein the antibody comprises: (i) a heavy chain comprising CDRs 1, 2 and 3 of the heavy chain variable region of SEQ ID NO: 31, and (ii) a light chain comprising CDRs 1, 2 and 3 of the light chain variable region of SEQ ID NO: 31 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 contains an amino acid substitution in the heavy chain constant region to any one, two, three, four, five or more residues selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237 , 238, 243, 264, 268, 297, 298, 299, 309, 310, 318, 320, 322, 327, 330 and 331 (numbering according to EU Kabat). In one embodiment, the antibody has an amino acid substitution in the heavy chain constant region of any three, four, five or more residues selected from the group consisting of: 234, 235, 237, 322, 330 and 331.

In one embodiment, the antibodies are administered to an individual having cancer in an amount and frequency sufficient to neutralize the activity of pCD39 in the tumor microenvironment and/or in the bloodstream. In one embodiment, the antibodies are administered in an amount and frequency sufficient to reduce the generation and/or concentration of adenosine in the tumor microenvironment. In one embodiment, the antibodies are administered in an amount and frequency sufficient to reduce the generation and/or concentration of AMP in the tumor microenvironment. In one embodiment, the antibodies are administered in an amount and frequency sufficient to neutralize the activity of CD39 expressed by the tumor cells. In one embodiment, the antibodies are 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.

Antibodies will be useful for inhibiting CD39-mediated ATP hydrolysis, which, for example, will lead to a decrease in adenosine concentrations in the tumor microenvironment and/or in the bloodstream. Therefore, these antibodies will be useful for reversing the immunosuppressive effects of CD39 and or adenosine on T cells, B cells and other cells expressing adenosine receptors (A2A receptors), for example, in the treatment of cancer. In one embodiment, the anti-CD39 antibody neutralizes adenosine-mediated inhibition of proliferation, cytokine production, cytotoxicity, and/or NFkB activity in T cells.

In another aspect, a method of treating an individual is provided, comprising administering to the individual (eg, an individual having a disease, tumor, etc.) a therapeutically active amount of any of the anti-CD39 antigen-binding compounds described herein.

Antibodies will be useful for inhibiting the production, amount and/or concentration of adenosine in the tumor microenvironment and/or in the bloodstream, including, but not limited to, tumors characterized by detectable, significant, elevated or increased adenosine generation, ATP catabolism or CD39/ axis catabolic activity. CD73 (eg, compared to control value). In addition, with increasing concentrations, antibodies that neutralize soluble CD39 provide almost complete inhibition of the catabolic activity of the CD39/CD73 axis. In one embodiment, antibodies can be used to inhibit the production, amount and/or concentration of adenosine in the tumor microenvironment in tumors characterized by the presence of the CD73 protein (eg, tumors with soluble CD73 and/or CD73-expressing cells; CD73-positive tumors).

In one embodiment, antibodies disclosed herein that neutralize soluble CD39 protein may preferably be used in combination with CD73 blockade, e.g.

- 7 047 070 measures, the antibodies disclosed in this application can be administered to an individual having cancer in combination with an agent that inhibits CD73 activity.

In one aspect, a method of treating an individual is provided, comprising, consisting of, or consisting of: administering to an individual (e.g., an individual having a disease, tumor, etc.) a therapeutically active amount of an antigen-binding compound disclosed herein that inhibits a CD39 polypeptide . In one embodiment, an anti-CD39 antigen-binding compound (eg, an antibody) is administered to an individual in combination with a second therapeutic agent. In one embodiment, the second therapeutic agent is an agent (eg, an antibody) that neutralizes the 5'-ectonucleotidase activity of human CD73. In one embodiment, the second therapeutic agent is an agent (eg, 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 includes an agent or method of treatment (e.g., a chemotherapeutic agent, taxane, anthracycline, camptothecin, epothilones, mitomycin, combretastatin, vinca alkaloid, nitrogen mustard, maytansinoids, calicheamicin, duocarmycin, tubulisin, dolastatin or aristatin, enedine, amatoxin, pyrrolobenzodiazepine, ethylenimine, radioisotope, therapeutic protein or peptide, or toxin) that induces extracellular ATP release from tumor cells and/or induces tumor cell death.

In one embodiment, an anti-CD39 antigen-binding compound (eg, an antibody) is administered to an individual who has cancer and has a poor response or is predicted to respond to treatment with an agent that neutralizes human PD-1 inhibitory activity. In one embodiment, the antibody inhibits CD39 polypeptide in a cell-based assay. In one embodiment, the compound is a non-depleting antibody (an antibody that does not deplete the cells to which it binds, such as an Fc region-silenced antibody). Optionally, the compound binds to CD39 polypeptides in a divalent manner. Optionally, the antibody is a chimeric, humanized, or human antibody. Optionally, the antibody contains a heavy chain constant region of an IgG isotype (eg, IgG1) modified to eliminate binding to human Fcy receptors (eg, CD16A, CD16B, CD32A, CD32B, and/or CD64).

In one aspect, there is provided a method of reducing ATP hydrolysis by a CD39-expressing cell (e.g., a white blood cell and/or a tumor cell in an individual) or a method of neutralizing the enzymatic activity of a cellular CD39, comprising: contacting the CD39-expressing cell with an antigen-binding compound (e.g., an antibody or antibody fragment) according to the present invention, which inhibits CD39. In one embodiment, the step of contacting a CD39-expressing cell with an antigen binding compound disclosed herein comprises administering to the individual a therapeutically active amount of an antigen binding compound that inhibits CD39. In one embodiment, the individual has cancer.

In one aspect, there is provided a method of reducing adenosine present in the environment of a tumor (e.g., in an individual), the method comprising, consisting of, or consisting of: administering to the individual a therapeutically active amount of an antigen-binding compound (e.g., an antibody or antibody fragment) disclosed herein, which inhibits CD39 polypeptide. In one embodiment, the individual has cancer.

In one embodiment, the active amount of an antibody that inhibits a CD39 polypeptide is an amount 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 essentially EC100 to inhibit CD39-mediated catabolism of ATP to AMP in an individual. In one embodiment, the active amount of an antigen binding compound that inhibits a CD39 polypeptide is an amount effective to achieve an EC50, optionally an _EC70 , optionally a substantially _EC100, to inhibit CD39-mediated catabolism of ATP to AMP in extravascular tissue of an individual. In one embodiment, the active amount of the antigen binding compound that inhibits the CD39 polypeptide is an amount effective to achieve EC ₅₀ , optionally EC ₇₀ , optionally substantially EC ₁₀₀ , for inhibiting CD39-mediated catabolism of ATP to AMP in an individual. In one embodiment, the active amount of the antigen binding compound that inhibits the CD39 polypeptide is from 1 to 20 mg/kg body weight. In one embodiment, the active amount is administered to the individual weekly, every two weeks, monthly, or every two months.

Optionally, the individual is a person who has or is susceptible to cancer. Optionally, the individual is a human having cancer or is susceptible to cancer characterized by malignant cells that express CD39 and/or have (secretion or release of) or soluble CD39 protein. Optionally, the individual is a person who has or is susceptible to cancer and has detectable levels of circulating soluble extracellular CD39 protein or tumor-infiltrating leukocytes expressing CD39.

- 8 047070

Optionally, the antibodies have a binding affinity (K _D ) for human CD39 polypeptide of less than (better) 10 -9 M, preferably less than 10 ^-10 M or preferably less than 10 ^-11 M and/or binding of human CD39 to an EC ₅₀ of less than (better) 1 μg /ml, preferably where the antibody has an EC ₅₀ of not more than 0.5 μg/ml, optionally not more than 0.2 μg/ml, optionally not more than 0.1 μg/ml, for binding to cells (for example, tumor cells) expressing Human CD39 on the cell surface.

Optionally, the antibodies are chimeric, human or humanized antibodies.

Optionally, the antibodies have an EC ₅₀ for neutralizing CD39 enzymatic activity in CD39-expressing cells (eg, Ramos tumor cells) of less than (better) 1 μg/ml, optionally less than 0.5 μg/ml.

In one embodiment, the antibody is a monoclonal antibody or fragment thereof that retains the binding specificity and ability to neutralize the enzymatic activity of CD39. In one embodiment, the antibody is an IgG1 antibody. For example, the antibody may be an antibody comprising a human IgG1 isotype Fc domain modified to reduce binding between the Fc domain and an FCY receptor (eg, CD16). In one embodiment, the antibody or fragment thereof does not have an Fc domain or contains an Fc domain that does not induce antibody-mediated cellular cytotoxicity (ADCC) and/or CDC; optionally, the antibody or fragment thereof contains an Fc domain that does not bind to the FcyRIIIA (CD16) polypeptide. In one embodiment, an FC domain (e.g., human IgG1, IgG2, IgG3, or IgG4 isotype) contains an amino acid modification (e.g., a substitution) relative to a wild-type Fc domain, wherein the substitution reduces the ability of the Fc domain (or antibodies containing it ) bind to the FcY receptor (eg CD16) and/or fix complement. In one embodiment, the antibody or fragment thereof is not associated with a toxic fragment.

Also provided are nucleic acids encoding a humanized antibody or antibody fragment having any of the previous properties, a vector containing such a nucleic acid, a cell containing such a vector, and a method for producing a human anti-CD39 antibody, including culturing such a cell under conditions suitable for expression of the antibody against CD39, and optionally reconstituting or purifying the resulting antibody. The invention also relates to compositions, such as pharmaceutically acceptable compositions and kits containing such proteins, nucleic acids, vectors and/or cells and, typically, one or more additional ingredients, which may be active ingredients or inactive ingredients, which contribute to the composition, delivery, stability or other characteristics of the composition (eg, different media). The invention also relates to various new and useful methods for producing and using such antibodies, nucleic acids, vectors, cells, organisms and/or compositions, such as modulation of CD39-mediated biological activity, for example, in the treatment of related diseases, in particular cancer.

The present invention also provides a method of potentiating the activity of lymphocytes (e.g., T cells) in an individual in need thereof, or to restore the activity of lymphocytes (e.g., T cells), or a method of relieving adenosine-mediated inhibition of lymphocytes (e.g., T cells) which comprises administering to an individual an effective amount of any of said compositions. In one embodiment, the individual is a patient having cancer. For example, the patient may have a solid tumor, such as colorectal cancer, kidney cancer, ovarian cancer, lung cancer, breast cancer, or malignant melanoma. Alternatively, the patient may have a hematopoietic cancer, such as acute myeloid leukemia, chronic myeloid leukemia, multiple myeloma, or non-Hodgkin's lymphoma.

The present invention also provides a method of treating a disease in an individual, wherein said treatment comprises administering to the individual an anti-CD39 antibody that neutralizes the enzymatic activity of CD39 over 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 between two successive administrations in the blood (serum) or extravascular tissue (eg, surrounding the tumor), a concentration of anti-CD39 antibody that corresponds to at least EC ₅₀ (eg, EC ₅₀ between 0.01 and 0.5 µg/ml), optional EC ₇₀ or optional EC1 ₀₀ , to neutralize the enzymatic activity of CD39 (e.g. ECio between 0.05 and 1 µg/ml, between 0.1 and 1 µg/ ml). The antibody can, for example, be administered in an amount to achieve and/or maintain a concentration in the bloodstream or in extravascular tissue (eg, surrounding a tumor) of at least about 0.1, 0.5, 1 or 2 μg/ml) . For example, to achieve a concentration in extravascular tissue of 0.05 to 1 μg/ml or 0.1 to 1 μg/ml, the anti-CD39 antibody is administered in amounts effective to achieve a circulating concentration of the anti-CD39 antibody of 0.5 to 10 μg /ml or from 1 to 10 μg/ml. Optionally, the anti-CD39 antibody is administered at least twice and in amounts effective to maintain a concentration of the anti-CD39 antibody at least at the indicated concentration for at least 1, 2, 3, 4

- 9 047 070 weeks between two consecutive administrations of the anti-CD39 antibody and/or throughout the entire administration cycle.

The present invention also provides a method of treating a disease in an individual, wherein said treatment comprises administering to the individual an anti-CD39 antibody that neutralizes the enzymatic activity of CD39 over at least one cycle of administration, wherein the anti-CD39 antibody is administered at least once, optionally at at least twice, in an amount effective to achieve and/or maintain, between two successive administrations of the anti-CD39 antibody, a concentration of the anti-CD39 antibody in the blood or tissues of at least 1 μg/ml, optionally at least 10 μg/ml, optionally from 1 to 100 µg/ml. Optionally, the anti-CD39 antibody is administered at least twice and in amounts effective to maintain a continuous concentration of the anti-CD39 antibody in the blood or tissues of at least 1 μg/ml, optionally at least 10 μg/ml, optionally from 1 to 100 μg/ml , for at least 1, 2, 3, 4 weeks between two consecutive administrations of the anti-CD39 antibody and/or throughout the entire administration cycle.

Such aspects will be more fully described in the specification provided herein, and additional aspects, features and advantages will be apparent from the following description.

Brief description of drawings

In fig. 1 shows a representative screening result showing antibodies I-397, I-398 and I399 compared to the positive control antibody I-394.

In fig. Figure 2A shows that antibodies BY40, I-394, I-395 and I-396 inhibit cell membrane-bound CD39, with both I-394 and I-395 demonstrating greater potency at all concentrations, as well as greater maximum cell membrane inhibition. CD39 versus BY40. In fig. 2B shows that antibodies I-395 and I-396 both inhibit soluble CD39 compared to negative control (BY40) and positive control (I-394) antibodies.

In fig. Figure 3A shows the position of the mutated residues in mutants 5 (M5), 15 (M15) and 19 (M19) on the surface of the CD39 protein. In fig. Figure 3B shows the binding results of various antibodies to mutants 5, 15 and 19.

In fig. 4 shows the binding of antibody I-394 to cells expressing human CD39 by flow cytometry. I-394 binds cells expressing human CD39 (CHOhuCD39), cells expressing cynomolgus CD39 (CHO-cyCD39), and Ramos lymphoma cells, but not cells expressing murine CD39 (CHO-moCD39).

In fig. 5 shows that antibody I-394 is highly effective in blocking CD39 enzymatic activity in tumor cells (Ramos), in cells expressing human CD39 (CHO-huCD39), and in cells expressing cynomolgus CD39 (CHO-cyCD39), by quantifying luminescence units that are proportional to the amount of ATP present.

In fig. 6 shows that antibody I-394 is highly effective in blocking the enzymatic activity of soluble recombinant human CD39 protein as measured by luminescence units that are proportional to the amount of ATP present.

In fig. 7 shows that antibody I-394 binds to human CD39, but not to any of the human CD39-L1, -L2, -L3 or -L4 isoforms, as determined by ELISA.

In fig. Figure 8 shows an experimental procedure to evaluate the effect of ATP-mediated DC activation on CD4 T cell activation, ATP-mediated DC activation was washed and then incubated with allogeneic CD4 T cells (1 mDC/4 T cell ratio) for a mixed lymphocyte response (MLR). ) within 5 days. T cell activation and proliferation were analyzed by CD25 expression and Cell Trace Violet dilution by flow cytometry.

In fig. 9 shows HLA-DR expression on mDCs, and FIG. Figure 10 shows the expression of CD83 on mDCs. These figures show that the blocking anti-CD39 antibody I-394 and chemical inhibitors of CD39 lead to activation of mDC at each of 0.125, 0.25 or 0.5 mM. However, anti-CD39 BY40 or anti-CD73 antibodies could not promote ATP-induced dendritic cell (DC) activation, suggesting that the antibodies are not able to sufficiently block enzymatic activity to avoid ATP catabolism. The legend from top to bottom corresponds to the bars in the graph from left to right.

In fig. 11 shows the expression of CD25, indicating that mDCs activated in the presence of ATP were able to induce T cell activation and proliferation in the MLR assay; enhancement of ATP-mediated mDC activation by the CD39 blocking antibody I-394 resulted in higher T cell proliferation and activation. The legend from top to bottom corresponds to the bars in the graph from left to right.

In fig. 12A shows the dose range of anti-CD73 antibodies on CD4 T cell proliferation in the presence of added ATP at 3 different doses of anti-pCD39 antibodies: 0.01, 0.1 and 1 μg/ml. Antibodies against CD39, which are able to neutralize soluble human CD39, show strong

- 10 047070 enhancement of antibodies against CD73 in relation to the restoration of proliferation of CD4 T cells. In fig. 12B shows the dose range of anti-CD73 antibodies on CD8 T cell proliferation, in the presence of added ATP, anti-pCD39 antibodies show a strong enhancement of anti-CD73 antibodies on restoring CD8 T cell proliferation.

In fig. 13 shows antibodies titrated on Ramos lymphoma cells by flow cytometry. Antibodies H2L1, H2L1*, H4L1 and h4I1* antibodies (mAb8, 9, 20 and 21, respectively) showed the best binding.

In fig. 14 shows inhibition of ATPase activity in the Ramos and Mino tumor cell lines expressing membrane-bound CD39. Antibodies H4L1 and H4L1* (mAb20 and mAb21) were most potent in blocking CD39 enzymatic activity.

In fig. 15A shows that antibody I-394 (parent light and heavy chains) has a higher temperature of aggregation (TAgg) and improved stability compared to antibody BY40. In fig. 15B shows that humanized variants of I-394 antibodies with variable regions H2L1 (mAb 8), H2L1 * (mAb9), H4L1 (mAb20) and H4L1 * (mAb21) all have a high temperature of aggregation (TAgg) and good stability.

Detailed Description of the Invention

Definitions.

Where the word containing is used, it may optionally be replaced by actually consisting of or consisting of.

Human CD39, also known as vascular CD39, NTPase1, ENTPD1, ATPDase and vascular ATPdiphosphohydrolase, exhibits ATPase activity. CD39 hydrolyzes extracellular ATP and ADP to AMP, which is further converted to adenosine by another enzyme, 5'-nucleotidase. The amino acid sequence of the mature human vascular CD39 polypeptide chain is shown in Genbank under accession number P49961, the full disclosure of which is incorporated herein by reference and is as follows:

MEDTKESNVK TFCSKNILAI LGFSSIIAVI ALLAVGLTQN KALPENVKYG IVLDAGSSHT

SLYIYKWPAE KENDTGVVHQ VEECRVKGPG ISKFVQKVNE IGIYLTDCME RAREVIPRSQ

121 HQETPVYLGA TAGMRLLRME SEELADRVLD VVERSLSNYP FDFQGARIIT GQEEGAYGWI

181 TINYLLGKFS QKTRWFSIVP YETNNQETFG ALDLGGASTQ VTFVPQNQTI ESPDNALQFR

241 LYGKDYNVYT HSFLCYGKDQ ALWQKLAKDI QVASNEILRD PCFHPGYKKV VNVSDLYKTP

301 CTKRFEMTLP FQQFEIQGIG NYQQCHQSIL ELFNTSYCPY SQCAFNGIFL PPLQGDFGAF

361 SAFYFVMKFL NLTSEKVSQE KVTEMMKKFC AQPWEEIKTS YAGVKEKYLS EYCFSGTYIL

421 SLLLQGYHFT ADSWEHIHFI GKIQGSDAGW TLGYMLNLTN MIPAEQPLST PLSHSTYVFL

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

As used herein, neutralize or neutralize CD39 polypeptide (eg, neutralize CD39, neutralize CD39 activity, or neutralize CD39 enzymatic activity) refers to a process in which the ATP hydrolysis activity of CD39 (ATPase) is inhibited. This includes, in particular, inhibition of CD39-mediated generation of AMP and/or ADP, that is, inhibition of CD39-mediated catabolism of ATP to AMP and/or ADP. For membrane-bound CD39, it can be measured, for example, in a cell-based assay that measures the ability of a test compound to inhibit the conversion of ATP to AMP and/or ADP, directly or indirectly. For soluble CD39, this can be measured by incubating recombinant soluble CD39, as described herein, with a test compound and measuring the conversion of ATP to AMP and/or ADP, directly or indirectly. For example, the disappearance of ATP and/or the formation 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 preparation causes a reduction in the conversion of ATP to AMP by at least 60%, a reduction in the conversion of ATP to AMP by at least 70%, or a reduction in the conversion of ATP to AMP by at least 80 or 90%, with reference to, for example, to the assays described herein (eg, ATP disappearance and/or AMP formation).

Whenever a cancer treatment or the like is mentioned with reference to an anti-CD39 binding agent (eg, an antibody), it may include: (a) a method of treating cancer, said method comprising the step of administering (at least one treatment) an anti-CD39 binding agent. CD39 (preferably in a pharmaceutically acceptable carrier) to an individual mammal, especially a human, in need of such treatment, in a cancer-treating dose (therapeutically effective amount), preferably in a dose (amount) as described herein;

- 11 047070 (b) the use of an anti-CD39 binding agent for the treatment of cancer or an anti-CD39 binding agent for use in said treatment (especially in humans); (c) using an anti-CD39 binding agent for the production of a pharmaceutical preparation for the treatment of cancer, a method of using an anti-CD39 binding agent for the production 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 containing an effective dose an anti-CD39 binding agent suitable for the treatment of cancer; or (d) any combination of clauses a), b) and c) in accordance with the subject matter eligible for patenting in the country in which the application is filed.

As used herein, the term antigen-binding domain refers to a domain containing a three-dimensional structure capable of immunospecifically binding to an epitope. Thus, in one embodiment, said domain may comprise 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 comprise at least one complementarity determining region (CDR) of an antibody chain. In another embodiment, the binding domain may comprise a polypeptide domain from a non-immunoglobulin framework region.

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

The term binds specifically to means that the antibody can bind preferentially in a competitive binding assay to a binding partner, such as CD39, as assessed using either recombinant forms of the proteins, their epitopes, or native proteins present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are described in detail below and are well known in the art.

When an antibody is said to compete with a particular monoclonal antibody (eg, antibody I-394), this means that the antibody competes with the monoclonal antibody in a binding assay using either recombinant CD39 molecules or surface expressed CD39 molecules. For example, if a test antibody reduces the binding of a reference antibody to a CD39 polypeptide or a CD39-expressing cell in a binding assay, then the antibody is said to compete with the reference antibody, respectively.

The term affinity as used herein refers to the strength of binding of an antibody to an epitope. The affinity of an antibody is determined 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 Ka is defined as 1/Kd. Methods for determining the affinity of monoclonal antibodies can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92: 589-601 (1983), references to which are incorporated herein by reference in their entirety. One standard method well known in the art for determining mAb affinity is surface plasmon resonance (SPR) screening (eg, by analysis with a BIAcore™ SPR assay).

As used herein, a determinant refers to a site of interaction or binding with a polypeptide.

The term epitope refers to an antigenic determinant and is the region or region on an antigen to which an antibody binds. A protein epitope may contain amino acid residues

- 12 047070 amino acid residues directly involved in binding, as well as amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide, that is, 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 combine with, for example, an antibody or receptor. Epitopes can be linear or conformational/structural. The term linear epitope is defined as an epitope consisting of amino acid residues that are contiguous in linear amino acid sequence (primary structure). The term conformational or structural epitope is defined as an epitope consisting of amino acid residues that are not all contiguous and thus represent separated portions of a linear sequence of amino acids that are brought into close proximity to each other by folding of the molecule (secondary, tertiary and/or quaternary structures ). The conformational epitope depends on the 3-dimensional structure. Therefore, the term conformational is often used as a synonym for the term structural.

The term internalization, used interchangeably with intracellular internalization, refers to the molecular, biochemical and cellular events associated with the process of translocation of a molecule from the extracellular surface of a cell to the intracellular surface of a cell. The processes responsible for the intracellular internalization of molecules are well known and may include, among others, the internalization of extracellular molecules (such as hormones, antibodies, and small organic molecules); membrane-associated molecules (such as cell surface receptors); and complexes of membrane-bound molecules bound to extracellular molecules (eg, a ligand bound to a transmembrane receptor or an antibody bound to a membrane bound molecule). Thus, induction and/or enhancement of internalization include events in which intracellular internalization is initiated and/or the rate and/or extent of intracellular internalization is increased.

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

For purposes of this application, a humanized antibody refers to an antibody characterized in that a constant and variable framework region of one or more human immunoglobulins is fused to a binding region, such as a CDR, of an animal-derived immunoglobulin. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody.

The term hypervariable region as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region typically contains amino acid residues from the complementarity determining region or CDR (for example, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain 31-35 (H1), 5065 ( H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991) and/or such residues from the hypervariable loop (eg 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 1987;196: 901- 917), or a similar system for determining the essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in this region is performed in the manner described in Kabat et al., supra. Phrases such as Kabat numbering, Kabat variable domain residue numbering, and Kabat numbering herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using Kabat's numbering system, the actual linear amino acid sequence of the peptide may contain fewer or additional amino acids corresponding to a truncation or insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain may contain one amino acid insertion (residue 52a, Kabat numbering) after residue 52 of CDR H2 and inserted residues (e.g., residues 82a, 82b and 82c, etc., Kabat numbering) after residue 82 FR heavy chain. Kabat numbering of residues can be determined for a given antibody by alignment in regions of homology of the antibody sequence with a standard Kabat numbered sequence.

By framework or FR residues, as used herein, is meant the region of the variable domain of an antibody, excluding those regions defined as CDRs. Each antibody variable domain framework region can be further subdivided into contiguous regions separated by CDRs (FR1, FR2, FR3 and FR4).

The terms Fc domain, Fc portion, and Fc region refer to the C-terminal portion of an antibody heavy chain, for example, from about amino acid (AA) 230 to about AA 450 of the human γ (gamma) heavy chain or its analogue sequence in other types of heavy chains. antibody chains

- 13 047070 (for example, α, δ, ε and μ for human antibodies), or their natural allotype. Unless otherwise noted, Kabat's generally accepted amino acid numbering for immunoglobulins is used in this invention (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

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

The terms polypeptide, peptide and protein are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers characterized in that one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as natural amino acid polymers and non-natural amino acid polymers.

The term recombinant, when used with reference to, for example, a cell or nucleic acid, protein (e.g., antibody or antibody fragment), or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or changes in the native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

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

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

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

Obtaining antibodies.

An anti-CD39 antigen-binding domain, or a protein (e.g., an antibody or antibody fragment) that contains such a domain, binds and neutralizes a soluble human CD39 polypeptide, e.g., 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 CD39-mediated adenosine generation. In one embodiment, the antibody inhibits CD39-mediated catabolism of ATP to AMP. In one embodiment, the antibody inhibits adenosine-mediated inhibition of lymphocyte (eg, T-cell) activity. In one aspect, the antibody is selected from a full-length antibody, an antibody fragment, and a synthetic or semi-synthetic molecule derived from the antibody.

Antibodies that potentially inhibit the enzymatic (ATPase) activity of the soluble (and optionally membrane-bound) CD39 protein may, in one embodiment, immobilize or restrict the domain movement of the soluble (and optionally membrane-bound) CD39 protein in one of its conformations, thereby preventing hydrolysis of its substrate. Antibodies

- 14 047070 can achieve this by binding to both the C- and N-terminal domains of soluble (and optionally membrane-bound) CD39 simultaneously.

In one embodiment, an anti-CD39 antigen binding domain or antigen binding protein that comprises an antigen binding domain (e.g., an antibody or antibody fragment, a multispecific binding protein, a bispecific antibody, etc.) contains complementarity determining regions (CDRs) and framework regions (FRs). . Antigen binding domains can be designed or modified to provide desired and/or improved properties.

In one embodiment, the anti-CD39 antigen binding protein is capable of binding and inhibiting the activity of a human CD39 polypeptide, the antigen binding protein comprises VH and VL, each of which contains a framework region (e.g., a framework region having an amino acid sequence of human origin) and CDR1, CDR2 and CDR3. In one embodiment, the antigen binding protein restricts the movement of the CD39 domain upon binding to CD39. Optionally, the VH and/or VL framework regions (eg, FR1, FR2, FR3 and/or FR4) are of human origin.

In a particular embodiment, the binding molecules and domains can be derived from immunoglobulin variable domains, for example, as associated _VL and VH domains found on two polypeptide chains, or a single chain antigen binding domain such as a scFv, VH domain, _VL domain, dAb , V-NAR domain or VHH domain.

In one aspect, the CD39 binding agent is an antibody selected from a fully human antibody, a humanized antibody, and a chimeric antibody.

In one aspect, the agent is an antibody fragment containing a constant or Fc domain derived from a human IgG1 constant or Fc domain, for example modified, as described hereinafter.

In one aspect, the agent contains an antibody fragment selected from a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an F(ab)2 fragment, an F(ab')2 fragment, an Fv fragment, a heavy chain Ig (llama or camel Ig) , V _HH fragment, single domain FV and single chain antibody fragment. In one aspect, the agent comprises a synthetic or semi-synthetic antibody molecule derived from scFV, dsFV, minibody, diabody, tribody, kappabody, IgNAR; and a multispecific (eg, bispecific) antibody. The agent may further comprise an Fc domain.

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

In one aspect, the antibody is in a substantially isolated form.

Antibodies can be produced using various methods known in the art. In one embodiment, antibodies of the present invention are produced by selection from an antibody library (eg, a phage display library). In another embodiment, antibodies are produced by immunizing a non-human animal, preferably a mouse, with an immunogen comprising a CD39 polypeptide, preferably a soluble human CD39 extracellular domain polypeptide. The CD39 polypeptide may optionally be or contain a fragment or derivative of the full-length CD39 polypeptide, usually an immunogenic fragment, i.e. part of a polypeptide containing an epitope expressed on the surface of cells expressing the CD39 polypeptide. Such fragments typically contain at least about 7 contiguous amino acids of the mature polypeptide sequence, even more preferably at least about 10 contiguous amino acids. The fragments are typically actually derivatives of the extracellular domain of the receptor. In one embodiment, the immunogen comprises a wild-type human CD39 polypeptide in a lipid membrane, typically on the surface of a cell. In a particular embodiment, the immunogen contains intact cells, in particular intact human cells, optionally treated or lysed. In another embodiment, the polypeptide is a recombinant CD39 polypeptide.

The step of immunizing a non-human mammal with an antigen can be performed by any method well known in the art to stimulate antibody production in the mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is incorporated by reference herein). Isolation of antibody-producing hybridomas is well known and can be accomplished by any method well known in the art.

Antibodies can also be prepared by screening combinatorial libraries of immunoglobulins, as described, for example, in (Ward et al. Nature, 341 (1989) p. 544, the entire disclosure of which is incorporated by reference herein).

The identification of one or more antibodies that bind to CD39, particularly to substantially or substantially the same region on CD39 as the monoclonal antibody mAb20 or mAb21, can be readily determined using any of a variety of immunoassays that may Antibody competition was assessed. Many such assays are routinely practiced and well known in the art (see, for example, US Pat. No. 5,660,827, issued Aug. 26, 1997, which

- 15 047070 ry is specifically incorporated into this application by reference).

For example, if the test antibodies to be tested are from different parent animals or even have different Ig isotypes, a simple competition assay can be used in which control (eg mAb20 or mAb21) and test antibodies are mixed (or pre-adsorbed) and applied to a sample containing CD39 polypeptides, for example, as described in PCT Publication No. WO 2018/167267, the disclosure of which is incorporated herein by reference. Protocols based on Western blotting and the use of the BIACORE assay are suitable for use in such competition studies.

Determining whether an antibody binds within an epitope region can be performed by methods known to one of ordinary skill in the art. As one example of such mapping/characterization methods, the epitope region for an anti-CD39 antibody can be determined by epitope footprinting using chemical modification of exposed amines/carboxyls in the CD39 protein. One specific example of this sole imprinting method is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry), in which hydrogen-deuterium exchange of receptor amide and protein ligand protons, binding and reverse exchange occurs, with the main amide groups involved in protein binding, are protected from reverse exchange and therefore remain deuterated. Relevant regions can be identified at this stage using peptic proteolysis, rapid microbial high performance liquid chromatography separation and/or electrospray ionization mass spectrometry. See, for example, Ehring H, Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999) Engen, J.R. and Smith, D.L. (2001) Anal. Chem. 73, 256A-265A. Another example of a suitable method for identifying epitopes is nuclear magnetic resonance epitope mapping (NMR), which typically compares the position of signals in two-dimensional NMR spectra of a free antigen and an antigen complexed with an antigen-binding peptide, such as an antibody. The antigen is typically selectively isotopically labeled with 15N, so that only signals corresponding to the antigen and no signals from the antigen-binding peptide are visible in the NMR spectrum. Antigen signals emanating from the amino acids involved in the interaction with the antigen-binding peptide will usually shift position in the spectrum of the complex compared to the spectrum of the free antigen, and thus the amino acids involved in the binding can be identified. See, for example, Ernst Schering Res Found Workshop. 2004; (44): 149-67; Huang et al., Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 Jun; 9 (3): 516-24.

Epitope mapping/characterization can also be performed using mass spectrometry techniques. See, for example, Downard, J Mass Spectrom. 2000 Apr; 35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9): 1792-1801. Protease cleavage methods may also be useful in the context of epitope mapping and identification. Regions/sequences relevant to antigenic determinants can be determined by protease digestion, for example using trypsin at a ratio of approximately 1:50 to CD39 or overnight at pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification . Peptides protected from trypsin digestion by the anti-CD39 binding molecule can subsequently be identified by comparing samples digested with trypsin and samples incubated with the antibody and then digested with, for example, trypsin (thereby revealing a footprint for the binding molecule). Other enzymes such as chymotrypsin, pepsin, etc. may also or alternatively be used in similar epitope characterization methods. In addition, enzymatic digestion may provide a rapid means of analyzing whether a potential antigenic determinant sequence is located in a region of the CD39 polypeptide that is not surface exposed and therefore most likely not relevant to immunogenicity/antigenicity.

Site-directed mutagenesis is another method that can be used to identify the binding epitope. For example, in an alanine scan, each residue in a protein segment is replaced by an alanine residue and the consequences on binding affinity are measured. If a mutation results in a significant decrease in binding affinity, then it is most likely involved in binding.

Monoclonal antibodies specific for structural epitopes (i.e., antibodies that do not bind unfolded protein) can be used to test that alanine substitution does not affect overall protein folding. See, for example, Clackson and Wells, Science 1995; 267:383-386; and Wells, Proc Natl Acad Sci USA 1996; 93:1-6.

Electron microscopy can also be used to determine the foot print of an epitope. For example, in Wang et al., Nature 1992; 355:275-278 used the coordinated use of cryo-electron microscopy, three-dimensional image reconstruction and x-ray crystallography to determine the physical imprint of the Fab fragment on the surface of the capsid of native cowpea mosaic virus.

Other forms of label-free analysis for epitope assessment include surface plasmon resonance (SPR, BIACORE) and reflectometric interference spectroscopy (RifS). See, on

- 16 047070 example, Fagerstam et al., Journal Of Molecular Recognition 1990;3:208-14; Nice et al., J. Chroma-togr. 1993; 646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37:3308-3311; Kroger et al., Biosensors and Bioelectronics 2002; 17:937-944.

Typically, an anti-CD39 antibody provided herein has an affinity for a CD39 polypeptide (e.g., the monomeric CD39 polypeptide produced in the Examples herein) ranging from about 104 to about 10 ^{PM -1} (e.g., about 108 to approximately 10 ¹⁰ M ^-1 ). For example, anti-CD39 antibodies may have an average dissociation constant (KD) of less than 1x10 ^-9 M relative to CD39, determined, for example, by surface plasmon resonance (SPR) screening (eg, analysis using a BIAcore™ SPR analytical device). In a more specific exemplary aspect, the invention provides anti-CD39 antibodies that have a KD of from about 1x10 ^-8 M to about 1x10 ^-10 M or from about 1x10 ^-9 M to about 1x10 ^-11 M for CD39.

Antibodies may be characterized, for example, by an average KD of no more than about (i.e., affinity better than) 100, 60, 10, 5, or 1 nanomolar, preferably subnanomolar, or optionally no more than about 500, 200, 100, or 10 picomolar . KD can be determined, for example, by immobilizing recombinantly produced human CD39 proteins on the surface of the chip and then adding the test antibody to the solution. In one embodiment, the method further includes step (d), selecting antibodies from (b) that are capable of competing for CD39 binding with antibody I-394 or, for example, with any of mAbs 1-24.

DNA encoding monoclonal antibodies of the present invention, such as mAb20 or mAb21, can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding mouse antibody heavy and light chains). In one aspect, a nucleic acid encoding the heavy chain or light chain of an anti-CD39 antibody according to any embodiment herein is provided. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. As described elsewhere herein, such DNA sequences may be modified for any of a variety of purposes, for example, to humanize antibodies, produce fragments or derivatives, or to modify the antibody sequence, for example, at the antigen binding site to optimize the binding specificity of the antibody. In one embodiment, an isolated nucleic acid sequence encoding the light chain and/or heavy chain of an antibody (eg, mAb20 or mAb21) is provided, as well as a recombinant host cell containing (eg, in its genome) such nucleic acid. Recombinant expression in bacterial DNA encoding an antibody is well known in the art (see, for example, Skerra et al., Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun, Immunol. 130, p. 151 (1992).

Once antibodies are identified that are capable of binding pCD39 and/or memCD39 and/or have other desirable properties, they are typically also assessed using methods such as those described herein for their ability to bind other polypeptides, including unrelated polypeptides. Ideally, antibodies bind with significant affinity only to CD39 and do not bind at a significant level to unrelated polypeptides or other polypeptides of the NTPDa3 family, particularly CD39-L1, L2, L3 and L4 or ILPOase8. However, it should be understood that as long as the affinity for CD39 is significantly greater (eg, 10x, 100x, 500x, 1000x, 10,000x or more) than for other, unrelated polypeptides, then the antibodies are suitable for use in the present methods.

In one embodiment, anti-CD39 antibodies may be formulated such that they do not have significant specific binding to human Fcy receptors, e.g., one or more of CD16A, CD16B, CD32A, CD32B, and/or CD64). Such antibodies may contain constant regions of various heavy chains that are known to have little or no binding to Fcy receptors. Alternatively, antibody fragments that do not contain (or contain parts of) constant regions, such as F(ab')2 fragments, can be used to prevent Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including, for example, testing antibody binding to the Fc receptor in the BIACORE assay. In addition, any isotype of IgG antibody can generally be used wherein 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, for example, WO 03/101485, the disclosure of which is incorporated by reference herein). Assays, such as cellular assays to assess Fc receptor binding, are well known in the art and are described, for example, in WO 03/101485.

In one embodiment, the antibody may contain one or more specific mutations in

- 17 047070 Fc regions, which result in Fc region silenced antibodies that have minimal interaction with effector cells. Suppression of effector functions can be achieved through mutations in the Fc region of antibodies, they are described in the art: N297A mutation, LALA mutations (Strohl, W., 2009, Curr. Opin. Biotechnol. Vol. 20(6): 685-691) ; and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69), see also Heusser et al., WO 2012/065950, the disclosure of which is incorporated herein by reference. In one embodiment, the antibody contains one, two, three, or more amino acid substitutions in the hinge region. In one embodiment, the antibody is IgG1 or IgG2 and contains one, two or three substitutions at residues 233-236, optionally 233-238 (EC numbering). In one embodiment, the antibody is IgG4 and contains one, two or three substitutions at residues 327, 330 and/or 331 (EC numbering). Examples of antibodies with a silenced IgG1 Fc region are LALA mutants containing the L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent Fc mutation is a mutation at residue D265 or at D265 and P329, for example used in the IgG1 antibody as the DAPA mutation (D265A, P329A) (US Pat. No. 6,737,056). Another silent IgG1 antibody contains a mutation at residue N297 (eg, N297A, N297S mutation), which results in aglycosylated/non-glycosylated antibodies. Other silent mutations include: substitutions at residues L234 and G237 (L234A/G237A); replacements for residues S228, L235 and R409 (S228P/L235E/R409K,T,M,L); replacements for residues H268, V309, A330 and A331 (H268Q/V309L/A330/A331S); substitutions at residues C220, C226, C229 and P238 (C220S/C226S/C229S/P238S); replacements for residues C226, C229, E233, L234 and L235 (C226S/C229S/E233P/L234V/L235A; replacements for residues K322, L235 and L235 (K322A/L234A/L235A); replacements for residues L234, L235 and P331 (L234F / L235E/P331S); replacements for residues 234, 235 and 297; replacements for residues E318, K320 and K322 (L235E/E318A/K320A/K322A); replacements for residues 243 and 264; substitutions at residues 297 and 299; substitutions such that residues 233, 234, 235, 237 and 238 according to the EU numbering system contain a sequence selected from PAAAP, PAAAS and SAAAS (see WO2011/066501).

In one embodiment, the antibody may contain one or more specific mutations in the Fc region. For example, such an antibody may contain a human-derived IgG1 Fc domain containing a mutation at residue(s) 234, 235, 237, 330 and/or 331 according to Kabat. One example of such an Fc domain contains substitutions at residues L234, L235 and P331 according to Kabat (for example, L234A/L235E/P331S or (L234F/L235E/P331S). Another example of such an Fc domain contains substitutions at residues L234, L235, G237 and P331 according to Kabat (e.g. L234A/L235E/G237A/P331S). Another example of such an Fc domain contains substitutions at residues L234, L235, G237, A330 and P331 according to Kabat (e.g. L234A/L235E/G237A/A330S/P331S) In one embodiment, the antibody contains an Fc domain, optionally of the human IgG1 isotype, containing: an L234X1 substitution, a L235X ₂ substitution, and a P331X ₃ substitution, where X1 is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, and X3 is any amino acid residue other than proline, wherein X1 is optionally alanine or phenylalanine, or conservative substitutions thereof; and optionally, _X2 is glutamic acid or conservative substitutions thereof; and optionally, _X3 is serine or conservative substitutions thereof; In another embodiment, the antibody contains an Fc domain, optionally of the human IgG1 isotype, containing: an L234X ₁ substitution, a L235X ₂ substitution, a G237X4 substitution, and a P331X4 substitution, where X1 is any amino acid residue other than leucine, X ₂ is any amino acid residue except leucine, X ₃ is any amino acid residue other than glycine, and X ₄ is any amino acid residue other than proline; wherein X1 is optionally alanine or phenylalanine or conservative substitutions thereof; wherein X2 optionally represents glutamic acid or a conservative substitute thereof; optionally X3 is alanine or conservative substitutions thereof; optionally X4 is serine or conservative substitutions thereof. In another embodiment, the antibody contains an Fc domain, optionally of the human IgG1 isotype, containing: an L234X ₁ substitution, a L235X ₂ substitution, a G237X4 substitution, a G330X4 substitution, and a P331X ₅ substitution, wherein X1 is any amino acid residue other than leucine, X ₂ is any an amino acid residue other than leucine, X ₃ is any amino acid residue other than glycine, X ₄ is any amino acid residue other than alanine, X ₅ is any amino acid residue other than proline; wherein X1 is optionally alanine or phenylalanine or conservative substitutions thereof; wherein X ₂ 2 optionally represents glutamic acid or a conservative substitute thereof; optionally X3 is alanine or conservative substitutions thereof; optionally X44 is serine or conservative substitutions thereof; optionally X5 is serine or conservative substitutions thereof. In the short notation used herein, the format is as follows: wild-type residue: polypeptide position: mutant residue, where residue positions are designated according to Kabat's EC numbering.

In one embodiment, the antibody comprises a heavy chain constant region comprising an amino acid sequence set forth below, or an amino acid sequence at least 90, 95, or 99% identical thereto but retaining amino acid residues in half

- 18 047070 marriages 234, 235 and 331 according to Kabat (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPASIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23).

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPASIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24).

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPSSIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25).

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPASIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26).

Antibodies with a silent Fc region mediate no or low ADCC activity, which means that an antibody with a silent Fc region exhibits ADCC activity that is below 50% specific cell lysis. Preferably, the antibody has substantially no ADCC activity, for example, an antibody with a silenced Fc region exhibits ADCC activity that is below 5% or below 1%. Antibodies with a silenced Fc region can also result in the absence of FcyR-mediated cross-linking of CD39 on the surface of CD39-expressing cells.

In one embodiment, the antibody has a heavy chain constant region substitution at any one, two, three, four, five or more residues selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 243, 264, 268, 297, 298, 299, 309, 310, 318, 320, 322, 327, 330, 331 and 409 (residue numbering in the heavy chain constant region corresponds to EU numbering according to Kabat). In one embodiment, the antibody contains a substitution at residues 234, 235, and 322. In one

- 19 047070 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 of the human IgG1 subtype. Amino acid residues are designated according to the EU numbering according to Kabat.

In one embodiment, the antibody contains an Fc domain containing an amino acid substitution that increases binding to human FcRn polypeptides to increase the half-life of the antibody in vivo. Typical mutations are described in Strohl, W., 2009, Curr. Opin. Biotechnol. Vol. 20(6):685-691, the disclosure of which is incorporated herein by reference. Examples of substitutions used in human IgG1 isotype antibodies are substitutions at residues M252, S254 and T256 according to Kabat; replacements for the remains of T250 and M428; substitutions based on N434 residues; substitutions at residues H433 and N434; replacements for residues T307, E380 and N434; replacements for residues T307, E380 and N434; substitutions at residues M252, S254, T256, H433, n434 and 436; substitutions at residue I253; substitutions at residues P257, N434, D376 and N434.

In one embodiment, the antibody contains an Fc domain containing an amino acid substitution that confers reduced sensitivity to protease degradation. Matrix metalloproteinases (MMPs) are the most prominent family of proteinases associated with tumorigenesis. While cancer cells can express MMPs, the bulk of extracellular MMPs are provided by various types of stromal cells that infiltrate the tumor and produce a distinct repertoire of proteinases and proteinase inhibitors that are released into the extracellular space and specifically alter the environment around the tumor. MMPs present in the tumor microenvironment can cleave antibodies at the hinge region and thus can lead to inactivation of therapeutic antibodies that are designed to function at the tumor site. In one embodiment, the Fc domain containing the amino acid substitution has reduced sensitivity to cleavage by any one, two, three, or more (or all) proteases selected from the group consisting of: 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 reduced cleavage sensitivity antibody comprises an Fc domain containing an amino acid substitution at residues E233-L234 and/or L235. In one embodiment, the antibody contains an Fc domain containing an amino acid substitution at residues E233, L234, L235, and G236. In one embodiment, the antibody contains an Fc domain containing an amino acid substitution at one or more residues 233-238, for example, such that the sequence E233-L234-L235-G236 is replaced by the sequence P233-V234-A235 (G236 is removed). See, for example, WO 99/58572 and WO 2012087746, the disclosure of which is incorporated herein by reference.

The antigen-binding compound may, at any desired stage, be evaluated for its ability to inhibit the enzymatic activity of CD39, in particular to block the ATPase activity of pCD39 and reduce the production of ADP and AMP (and, together with CD73, adenosine) by the soluble CD39 protein and, optionally, further by the cell expressing CD39, and, in turn, restore the activity and/or attenuate adenosine-mediated inhibition of lymphocytes.

The inhibitory activity (eg, immunostimulatory potential) of an antibody can be assessed, for example, in an assay to detect the disappearance (hydrolysis) of ATP and/or the formation of AMP.

The ability of an antibody to inhibit soluble recombinant human CD39 protein can be tested by detecting ATP after incubating the test antibody with soluble CD39 protein. Briefly, ATP can be quantified using Cell Titer Glo™ (Promega) in an assay in which dose ranges of the test antibody are incubated with the soluble recombinant human CD39 protein described in Example 1 for 1 hour at 37°C. 20 μM ATP was added to the plates for an additional 30 minutes at 37°C before adding CTG reagent. The emitted light is quantified using an Enspire™ luminometer after a short incubation period of 5 min in the dark.

The ability of an antibody to inhibit cells expressing CD39 protein can be tested by detecting ATP after incubation of the test antibody with cells (eg, Ramos cells, cells transfected with CD39, etc.). See, for example, Examples, Methods. Cells can be incubated for 1 hour at 37°C with the test antibody. Cells were then incubated with 20 μM ATP for 1 additional hour at 37°C. The plates are centrifuged for 2 minutes at 400g and the cell supernatant is transferred to a fluorescent microplate (white wells). CTG was added to the supernatant and the emitted light was quantified after a 5-minute incubation in the dark using an Enspire™ luminometer. The effectiveness of anti-CD39 antibodies is determined by comparing the light emitted in the presence of the antibody with ATP alone (maximum light emission) and ATP plus cells (minimal light emission).

A decrease in ATP hydrolysis to AMP and/or an increase in ATP and/or a decrease in AMP generation in the presence of the antibody indicates that the antibody inhibits CD39. In one embodiment

- 20 047070 the antibody preparation is capable of causing at least a 60% reduction in the enzymatic activity of the CD39 polypeptide expressed by the cell, preferably the antibody causes at least a 70, 80 or 90% reduction in the enzymatic activity of the CD39 polypeptide in the cell, according to assessment by detecting ATP using Cell Titer Gio™ (Promega) after incubating cells expressing CD39 polypeptide (eg, Ramos cells) with a test antibody, for example, as in the example methods.

In one embodiment, the antibody preparation is capable of causing at least a 60% reduction in the enzymatic activity of the soluble recombinant CD39 polypeptide (e.g., in the absence of cells), preferably at least a 70, 80, or 90% reduction in the enzymatic activity of the soluble recombinant CD39 polypeptide, assessed by detecting ATP using Cell Titer Glo™ (Promega) after incubating the soluble recombinant CD39 polypeptide with a test antibody, for example, as in the example methods.

The activity of an antibody can also be measured in an indirect assay for its ability to modulate the activity of immune cells (eg, immune cells expressing adenosine receptors; cells expressing A2A receptors), 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, an antibody may be assessed in an indirect assay for its ability to modulate lymphocyte proliferation.

Epitopes on CD39.

In one aspect, the antibodies bind an antigenic determinant present on CD39 expressed on the surface of a cell.

In one aspect, the antibodies bind substantially the same epitope as the antibodies having the VH and VL of mAb1-24 (or I-394). In one embodiment, the antibodies bind to a CD39 epitope that at least partially overlaps with, or contains at least one residue, an epitope bound by mAb1-24 (or I394). Antibody-bound residues can be defined as those present on the surface of a CD39 polypeptide, for example, on a CD39 polypeptide expressed on the surface of a cell.

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

In some embodiments, a significant reduction in binding means that the binding affinity and/or ability between the anti-CD39 antibody and the mutant CD39 polypeptide is reduced by more than 40%, more than 50%, more than 55%, more than 60%, more greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% relative to the binding between the antibody and the wild-type CD39 polypeptide. In some embodiments, binding is reduced below detectable limits. 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 wild-type CD39 polypeptide.

In some embodiments, anti-CD39 antibodies are provided that exhibit significantly lower binding to a mutant CD39 polypeptide characterized in that the residue in the segment containing the amino acid residue bound by the mAb1-24 (or I-394) antibody is replaced with a different amino acid, compared to with binding to a wild-type CD39 polypeptide not containing such substitution(s) (eg, polypeptide SEQ ID NO: 1).

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

Representative antibody variable region sequences.

Examples of antibodies according to the present invention include antibodies containing the VH domain and the VL domain of any of the antibodies mAb1 to mAb24. The sequences of VH and VL antibodies against CD39 mAb1mAb24 are presented in table. A and B (example 13).

- 21 047070

One of the exemplary highly effective anti-CD39 VH and VL pairs of the present invention is an antibody mAb20, the amino acid sequence of which is as follows (SEQ ID NO: 31) and the amino acid sequence of which is as follows (SEQ ID NO: 31). 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 containing the amino acid sequence of SEQ ID NO: 39.

Another exemplary highly effective anti-CD39 VH and VL pair of the present invention is the antibody mAb21, the amino acid sequence of which is shown below (SEQ ID NO: 31) and the amino acid sequence of which is shown 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 containing the amino acid sequence of SEQ ID NO: 40.

In any aspect, an isolated antibody that binds a human CD39 polypeptide can be defined as comprising VH and VL framework regions (eg, FR1, FR2, FR3, and FR4) of human origin. In one aspect, the antibody contains: HCDR1 containing the amino acid sequence: DYNMH (SEQ ID NO: 8), or a sequence of at least 4 contiguous amino acids, and optionally one or more of such amino acids may be replaced by another amino acid; HCDR2, containing the amino acid sequence: YIVPLNGGSTFNQKFKG (SEQ ID NO: 9), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, and optionally one or more of such amino acids may be replaced by another amino acid , wherein asparagine is optionally substituted at position 61 according to Kabat, and lysine is optionally substituted at position 65 according to Kabat; HCDR3, containing the amino acid sequence: GGTRFAY (SEQ ID NO: 10), or a sequence thereof of at least 4, 5 or 6 contiguous amino acids, and optionally one or more of such amino acids can be replaced by another amino acid; LCDR1 containing the amino acid sequence: RASESVDNFGVSFMY (SEQ ID NO: 11) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, and optionally one or more of such amino acids may be replaced by another amino acid, wherein the arginine at position 24 according to Kabat is optionally substituted; an LCDR2 region containing the amino acid sequence: GASNQGS (SEQ ID NO: 12) or a sequence of at least 4, 5 or 6 contiguous amino acids, and optionally one or more of these amino acids may be replaced by another amino acid; and/or an LCDR3 region containing the amino acid sequence: QQTKEVPYT (SEQ ID NO: 13) or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids, where optionally one or more of such amino acids may be deleted or replaced by another amino acid. The provisions in the CDR may correspond to the numbering according to Kabat.

In one embodiment, HCDR2 comprises the amino acid sequence of formula I: YIVPLNGGSTF-Xaaj - Q - K - F - Xaa ₂ - G (SEQ ID NO: 14), or a subsequence thereof, where Xaaj can be any amino acid residue, and optionally Xaa ₁ represents is asparagine or serine; wherein Xaa ₂ can be any amino acid residue, and additionally Xaa ₂ 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).

In one embodiment, LCDR1 contains the amino acid sequence of formula II: Xaa ₃ -ASESVDNFGVSFMY (SEQ ID NO: 17), where Xaa3 can be any amino acid residue, and Xaa3 is optionally lysine or arginine. In one embodiment, LCDR1 comprises the amino acid sequence: KASESVDNFGVSFMY (SEQ ID NO: 18).

In one embodiment, the antibody comprises a heavy chain framework region from the human IGHV1-3 subgroup (optionally together with IGHJ1), optionally IGHV1-3 being IGHV1-3*01. In one embodiment, the humanized antibody comprises a light chain framework region from a subset of the human IGKV4-1 gene (optionally together with IGKJ4).

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

(a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 8;

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

(c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 10;

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

(e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 12;

(f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 13; and (g) human heavy and light chain framework sequences.

The antibody may additionally contain one, two, three, four, five or more amino acids

- 22 047070 substitutions in the framework region of the human heavy and/or light chain, for example, to increase the affinity, stability or other properties of the antibody. Optionally, the substitution introduces a residue present at a specific position in a non-human mammal (eg, mouse or rat).

In any of the VH sequence variants reported herein, the amino acid at position 67 of the heavy chain (Kabat numbering) may be alanine.

In any of the VH sequence variants reported herein, the amino acid at position 71 of the heavy chain (Kabat numbering) is valine.

In any of the VH sequence variants reported herein, the amino acid at position 76 of the heavy chain (Kabat numbering) is arginine.

In some embodiments of the VH sequences herein, the amino acid at position 48 of the heavy chain (Kabat numbering) may be isoleucine. In some embodiments of the VH sequences herein, the amino acid at position 48 of the heavy chain (Kabat numbering) may be methionine.

In one embodiment, VH contains an alanine residue at position 67 (numbering according to Kabat) and a valine at position 71.

In one embodiment, VH contains an isoleucine residue at position 48 (Kabat numbering), an alanine residue at position 67 (Kabat numbering), a valine at position 71 (Kabat numbering), and an arginine at position 76 (Kabat numbering).

In any of the VL sequence variants reported herein, VL contains a phenylalanine at position 36 (Kabat numbering) (FR2). In one embodiment, VL contains a lysine at position 24 (Cabat numbering) (CDR1).

Positions in the VH and VL domains are described herein using the numbering system according to Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD ).

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 greater identity) with a heavy chain having the amino acid sequence of SEQ ID NO: 38.

In one aspect, an 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 greater identity) with a light chain having the amino acid sequence of SEQ ID NO: 39 or 40.

In any aspect, said heavy chains, light chains, variable region, FR and/or CDR sequences may contain one or more sequence modifications, such as a substitution (1, 2, 3, 4, 5, 6, 7, 8 or more consecutive modifications) . In one embodiment, the replacement is a conservative modification.

A further object of the present invention also includes functionally conservative variants of the antibodies disclosed in this application. Functionally conserved variants are those 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, substitution of an amino acid with an amino acid having similar properties (such as, for example, polarity, hydrogen bond potential, acidity, basicity, hydrophobicity, aromaticity and the like). Amino acids other than those indicated as conserved may differ in a protein such that the percentage of protein or amino acid sequence similarity between any two proteins of similar function may vary and may range from, for example, 70 to 99%, as determined in accordance with the scheme alignment, such as Cluster Mode, where similarity is based on the MEGALIGN algorithm. A functionally conserved variant also includes a polypeptide that has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.

In any embodiment, the antibody or antibody fragment may optionally be defined as antibodies other than antibody I-394 (eg, having the amino acid sequences VH and VL of SEQ ID NO: 6 and 7, respectively). In any embodiment, the antibody or antibody fragment may optionally be defined as antibodies other than antibodies I-395, I-396, I-397, I398, or I-399 (e.g., having the amino acid sequences VH and VL disclosed in the PCT patent application No. PCT/EP2018/056661, filed March 16, 2018, the disclosure of which is incorporated herein by reference). In any embodiment, the antibody or antibody fragment may optionally be defined as an antibody other than the By40, Ba54g, or BY12 antibody disclosed in U.S. Patent Publication No. US 2016/0137747A1 (e.g., having the VH and VL amino acid sequences of By40, Ba54g, or BY12; an antibody different from the BY40 antibody produced by a hybridoma cell line producing such an antibody.

- 23 047070

Antibody fragments and derivatives (which are encompassed by the term antibody or antibodies as used herein unless otherwise indicated or clearly contrary to context) can be prepared by methods known in the art. The fragments include part of an intact antibody, typically an antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2 and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one contiguous sequence of contiguous amino acid residues (referred to herein as a single-chain antibody fragment or single-chain polypeptide), including, without limitation, (1) single-chain Fv molecules (2) single-chain polypeptides containing only one light chain variable domain, or a fragment thereof, that contains three light chain variable domain CDRs, without the associated heavy chain portion, and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof, containing three variable region CDRs heavy chain, without the associated light chain portion; and multispecific (eg, bispecific) antibodies formed from antibody fragments. This includes, but is not limited to, a nanobody, domain antibody, single domain antibody, or dAb.

The anti-CD39 antibody may be included in a pharmaceutical formulation containing concentrations of from 1 mg/ml to 500 mg/ml, which composition has a pH of from 2.0 to 10.0. The composition may further contain a buffer system, preservative(s), tonic agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment, the pharmaceutical composition is an aqueous composition, that is, a composition containing water. This composition is usually a solution or suspension. In another embodiment, the pharmaceutical composition is an aqueous solution. The term aqueous composition is defined as a composition containing at least 50% wt./wt. water. Likewise, the term aqueous solution is defined as a solution containing at least 50% w/w. water, and the term aqueous suspension is defined as a suspension containing at least 50% wt./wt. water.

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

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

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

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

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

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

It is possible that other ingredients may be present in the peptide pharmaceutical composition. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, excipients, tonicity regulators, chelating agents, metal ions, oily carriers, proteins (eg, human serum albumin, gelatin or proteins) and zwitterions (eg, amino acids such as betaine, taurine , arginine, glycine, lysine and histidine). Such additional ingredients should, of course, not adversely affect the overall stability of the pharmaceutical composition.

Pharmaceutical compositions containing the antibody can be administered to a patient in need of such treatment at multiple sites, for example, at topical sites, such as on the skin and mucous membranes, at sites that bypass absorption, such as injection into an artery, into a vein , in the heart, and at sites that involve absorption, such as injection into the skin, subcutaneously, into a muscle, or into the abdominal cavity. The administration of pharmaceutical compositions can be carried out by several routes of administration, for example subcutaneous, intramuscular, intraperitoneal, intravenous, lingual

- 24 047070 nim, sublingual, buccal, oral, oral, into the stomach and intestines, nasal, pulmonary, for example, through bronchioles and alveoli or a combination thereof, epidermal, cutaneous, transdermal, vaginal, rectal, ocular, for example, through the conjunctiva, urethral and parenteral patients requiring such treatment.

Suitable antibody formulations can also be determined by studying experience with other therapeutic monoclonal antibodies already developed. Some monoclonal antibodies have been shown to be effective in clinical situations, such as Rituxan (Rituximab), Herceptin (Trastuzumab), Xolair (omalizumab), Bexar (Tositumomab), Campat (Alemtuzumab), Zevalin, Oncolym and similar formulations can be used with such antibodies . For example, the monoclonal antibody can be supplied at a concentration of 10 mg/ml in either 100 mg (10 ml) or 500 mg (50 ml) in single-dose vials prepared for intravenous 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 provided in a formulation containing approximately 20 mM Na Citrate, approximately 150 mM NaCl, at a pH of approximately 6.0.

Diagnosis and treatment of the disease.

Also provided are methods of treating an individual, in particular a human individual, using an anti-CD39 agent according to the present invention. In one embodiment, the invention 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 or is at risk of developing cancer or an infectious disease (eg, viral infection, bacterial infection). In one embodiment, the individual has detectable soluble (extracellular) CD39 protein in the bloodstream and/or in a tissue sample (eg, a tumor or tumor-adjacent tissue sample).

For example, in one aspect, there is provided a method of restoring or potentiating lymphocyte activity in an individual in need thereof, comprising the step of administering to said individual a neutralizing anti-CD39 antibody or antibody fragment according to the present invention. In one embodiment, the method is directed to increasing the activity of lymphocytes (e.g., T cells) in an individual having a disease in which increased lymphocyte activity is beneficial or which is caused or characterized by immunosuppression, immunosuppressive cells, or, for example, adenosine generated by CD4 T cells , CD8 T cells, B cells). Such methods will be particularly useful, for example, for treating an individual with a solid tumor in which it is suspected that the tumor microenvironment (and CD39-mediated adenosine production therein) may contribute to a lack of recognition by the immune system (immune escape). The tumor environment (tumor tissue or tumor adjacent tissue) may, for example, be characterized by the presence of CD39-expressing immune cells, eg CD4 T cells, CD8 T cells, B cells.

More specifically, the methods and compositions are used to treat various types of cancer and other proliferative diseases, as well as infectious diseases. Because these methods work by reducing adenosine, which inhibits anti-target cellular (eg, antitumor) activity of lymphocytes, and perhaps additionally by increasing ATP, which can increase the antitumor activity of lymphocytes, they will be useful in a very wide range of cancers and infections. diseases. In one embodiment, anti-CD39 compositions are useful for treating cancer in individuals who do not respond well (or are not responsive) to treatment with an agent that neutralizes the inhibitory activity of human PD-1, for example, that inhibits the interaction between PD-1 and PD-L1. Representative examples of cancers that can be treated include, in particular, solid tumors, where adenosine in the tumor microenvironment can play a strong role in suppressing the antitumor immune response. In one embodiment, the human patient treated with the anti-CD39 antibody has liver cancer, bone cancer, pancreatic cancer, skin cancer, head or 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, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvic carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell cancer, environmentally induced cancer, including those caused by asbestos, hematological malignancies, including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma/primary mediastinal B-large cell lymphoma, non-Hodgkin's lymphomas, acute leukemia, lymphoma, chronic myelo

- 25 047070 leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, B-lymphoblastic leukemia from progenitor cells, lymphoma from mantle cells, acute lymphoblastic leukemia, fungoid granuloma, anaplastic large cell lymphoma , T-cell lymphoma, and acute lymphoblastic leukemia of T-lymphocyte precursor cells, as well as any combination of these cancers. The present invention is also useful in the treatment of metastatic cancer. Patients may be tested or screened for one or more of the clinical features described above before, during, or after treatment.

In one embodiment, an anti-CD39 antibody or antibody fragment is used in the treatment of cancer characterized by detectable and/or elevated levels of soluble (extracellular) CD39 protein, for example, in the bloodstream and/or in tissues, such as in a tumor or tumor-adjacent tissue.

In one embodiment, an anti-CD39 antibody or antibody fragment is used in the treatment of cancer characterized by malignant cells expressing CD39.

In one embodiment, the anti-CD39 antibody or antibody fragment is administered in an amount effective to achieve and/or maintain in the individual (e.g., for 1, 2, 3, 4 weeks and/or until subsequent administration of the antigen-binding compound) a blood concentration of at least EC50, optionally EC70, optionally substantially _EC100 , to neutralize the enzymatic activity of CD39, optionally pCD39, optionally memCD39. In one embodiment, the active amount of an anti-CD39 antibody is an amount effective to achieve an EC50, optionally _EC70 , optionally substantially _EC100 , to neutralize the enzymatic activity of CD39, optionally pCD39, optionally memCD39 in extravascular tissue of an individual. In one embodiment, the active amount of an anti-CD39 antibody is an amount effective to achieve (or maintain) an individual's EC50, optionally EC70, optionally substantially EC100, to inhibit the neutralizing enzymatic activity of CD39, optionally pCD39, optionally memCD39.

Optionally, in one embodiment, unlike some antibodies that are directed to deplete CD39-expressing tumor cells via ADCC (which, for example, may provide full effectiveness at concentrations equal to or substantially lower than those that saturate the receptor), an anti CD39 does not exhibit significant Fcy receptor-mediated activity and is administered in an amount effective to neutralize the enzymatic activity, optionally in addition to CD39, without significantly reducing the modulation of CD39 expression, for a desired period of time, e.g., 1, 2 weeks, months, before the next sequential administration of an antibody against CD39.

In one embodiment, the anti-CD39 antibody or antibody fragment is administered in an amount effective to achieve and/or maintain (e.g., for 1, 2, 3, 4 weeks and/or until subsequent administration of the anti-CD39 antibody) in the subject's blood a concentration of at least EC50, optionally _EC70 , optionally substantially _EC100 , to inhibit CD39-mediated catabolism of ATP to AMP (e.g., by assessing neutralization of the ATPase activity of pCD39; by assessing neutralization of the ATPase activity of soluble (extracellular) CD39 protein, see examples, ways).

In one embodiment, a method is provided for treating or preventing cancer in an individual, comprising administering to the individual having the disease an anti-CD39 antibody or antibody fragment in an amount that achieves or maintains for a specified period of time a concentration in the bloodstream, optionally in an extravascular tissue of interest. (eg, tumor or tumor environment) that is higher than the concentration required for 50, 70%, or complete (eg, 90%) saturation of the receptors by CD39-expressing cells in the bloodstream (eg, as assessed by PBMC). Optionally, the concentration achieved is at least 20, 50 or 100% higher than the concentration required to saturate the specified receptor.

In one embodiment, a method of treating or preventing cancer in an individual is provided, comprising administering to the individual an anti-CD39 antibody or antibody fragment in an amount that achieves or maintains a concentration in the circulation for a specified period of time, optionally in an extravascular tissue of interest (eg, a tumor or tumor environment) that is higher than the EC ₅₀ , optionally EC ₇₀ or optionally EC ₁₀₀ , for binding to CD39-expressing cells (e.g., as assessed by flow cytometry by titrating an anti-CD39 antibody on CD39-expressing cells, e.g. Ramos cells) , as in the examples, Methods). Optionally, the concentration achieved is at least 20, 50 or 100% higher than the EC ₅₀ , optionally EC ₇₀ or optionally EC ₁₀₀ for binding to CD39-expressing cells.

EC ₅₀ , EC ₇₀ or EC ₁₀₀ can be assessed, for example, in a cell-based assay to neutralize CD39 enzymatic activity, as shown in the Examples herein, for example neutralizing ATPase activity in B cells by quantifying hydrolysis

- 26 047070

ATP to AMP (or ATP to descending adenosine), see examples, methods. The EC50 for neutralizing CD39 enzymatic activity refers to the effective concentration of an anti-CD39 antibody that produces 50% of its maximum response or effect in neutralizing enzymatic activity. EU ₇₀ for neutralizing CD39 enzymatic activity refers to the effective concentration of the anti-CD39 antibody that produces 70% of its maximum response or effect. EC ₁₀₀ for neutralizing CD39 enzymatic activity refers to the effective concentration of the anti-CD39 antibody that produces its substantially maximum response or effect in relation to such neutralizing enzymatic activity.

In some embodiments, particularly for the treatment of solid tumors, the concentration achieved is intended to result in a tissue concentration (outside the vasculature, e.g., in the tumor or tumor surroundings) that corresponds to at least EC ₅₀ or EC ₇₀ to neutralize enzymatic activity, optionally about or at least about EC ₁₀₀ .

In one embodiment, the amount of anti-CD39 antibody is from 1 to 20 mg/kg body weight. In one embodiment, the amount is administered to the individual weekly, every two weeks, monthly, or every two months.

In one embodiment, a method of treating a human patient with cancer is provided, comprising administering to the individual an effective amount of an anti-CD39 antibody of the present invention over at least one administration cycle (optionally at least 2, 3, 4 or more administration cycles), wherein the cycle represents is a period of eight weeks or less, with one, two, three or four doses of anti-CD39 antibody at a dose of 1-20 mg/kg body weight administered for each of at least one cycle. In one embodiment, the anti-CD39 antibody is administered via intravenous infusion.

Suitable treatment protocols for treating humans include, for example, administering to a patient an amount described herein of an anti-CD39 antibody, the method comprising at least one administration cycle characterized in that at least one dose of an anti-CD39 antibody is administered. Optionally, at least 2, 3, 4, 5, 6, 7 or 8 doses of anti-CD39 antibody are administered. In one embodiment, the administration cycle is from 2 to 8 weeks.

In one embodiment, a method is provided for treating or preventing a disease (e.g., cancer, solid tumor, hematologic tumor) in an individual, comprising administering to the individual having the disease (e.g., cancer, solid tumor, hematologic tumor) an anti-CD39 antibody that neutralizes the enzymatic activity CD39 for at least one administration cycle, wherein the administration cycle includes at least the first and second (and optionally 3-, 4-, 5-, 6-, 7- and/or 8th or more) administering an anti-CD39 antibody, wherein the anti-CD39 antibody is administered in an amount effective to achieve or maintain, between two successive administrations, a blood (serum) concentration of the anti-CD39 antibody of at least 0.1 μg/mL, optionally at least 0.2 μg/mL ml, optionally at least 1 μg/ml or optionally at least 2 μg/ml (for example, for the treatment of a hematological tumor), or optionally at least about 1, 2, 10 or 20 μg/ml, for example between 1-100 µg/ml, 1-50 µg/ml, 1-20 µg/ml or 1-10 µg/ml (eg, for the treatment of a solid tumor, for the treatment of a hematological tumor). In one embodiment, a predetermined continuous blood concentration is maintained without the blood concentration falling significantly below the predetermined concentration for a predetermined period of time (e.g., between two antibody administrations, number of weeks, 1, 2, 3, 4 weeks), i.e. e. although the blood concentration may vary over a given period of time, the target maintained blood concentration represents the trough or trough concentration. In one embodiment, a therapeutically active amount of an anti-CD39 antibody is an amount of such antibody capable of providing (at least) an EC50 concentration, optionally an _EC70 concentration, optionally an _EC100 concentration in blood and/or tissue to neutralize CD39 enzymatic activity over a period at least about 1 week, about 2 weeks, or about one month after administration of the antibody.

Before or during a course of treatment with an anti-CD39 antibody according to the present invention, the presence or levels of 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 a patient is suitable for treatment (eg, to predict whether a patient will respond to treatment). Increased presence or levels of soluble (extracellular) CD39, CD39 expressing cells, levels of adenosine, ATP, ADP and/or AMP may indicate that the individual is suitable for treatment (e.g., likely to benefit) with an anti-CD39 antibody of the present invention (including, but not limited to, an antibody that inhibits substrate-bound CD39).

Before or during a course of treatment with an anti-CD39 antibody according to the present invention, levels of adenosine, ADP and/or AMP may be further assessed within and/or adjacent to a patient's tumor to assess whether the patient will benefit from treatment with an anti-CD39 antibody. Reduced levels

- 27 047070 adenosine, ATP, ADP and/or AMP after administration (or antibody dosing) compared to levels before treatment (or antibody dosing) may indicate that the individual benefits from treatment with an anti-CD39 antibody disclosure (including but not limited to it, an antibody that inhibits substrate-bound CD39). Optionally, if the patient benefits from treatment with an anti-CD39 antibody, the methods may further include administering an additional dose of the anti-CD39 antibody to the patient (eg, continuing treatment).

In one embodiment, assessing the levels of adenosine, ADP, and/or AMP in a tissue sample within and/or adjacent to a patient's tumor comprises obtaining from the patient a biological human tissue sample selected from the group consisting of tissue from a patient with cancer, e.g., cancer tissue, tissue proximal or peripheral to cancer, adjacent cancer tissue, adjacent non-tumor tissue, or normal adjacent tissue, and detecting levels of adenosine, ATP, ADP, and/or AMP within the tissue. Levels from a patient can be compared to a reference level, for example, that of a healthy individual.

In one embodiment, the invention provides a method of treating or preventing cancer in an individual in need thereof, the method comprising:

a) detection of soluble (extracellular) CD39 protein and/or CD39-expressing cells in the bloodstream or surrounding the tumor, optionally within the tumor and/or in adjacent tissue, and

B) upon determination that soluble (extracellular) CD39 protein and/or CD39-expressing cells are present in the bloodstream or tumor environment, optionally at a level that is elevated relative to a reference level (e.g., the level observed in healthy tissue; optionally a level corresponding to a healthy individual or an individual not receiving significant benefit from an anti-CD39 antibody), the individual is administered an anti-CD39 antibody. CD39 expressing cells may contain tumor cells or leukocytes, eg circulating or tumor infiltrating cells, eg CD4 T cells, CD8 T cells, TReg cells, B cells.

In one embodiment, the invention provides a method of treating or preventing cancer in an individual in need thereof, the method comprising:

a) assessing whether the individual has detectable soluble (extracellular) CD39, optionally located in the bloodstream, optionally within the tumor and/or adjacent tissue, and

b) upon detection of soluble (extracellular) CD39, optionally at a level that is elevated relative to a reference level (eg, corresponding to a healthy individual or an individual not significantly benefiting from the anti-CD39 antibody of the present invention), administering to the individual the anti-CD39 antibody of the present invention invention.

Optionally, in any of the methods of implementation, detection of soluble CD39 protein and/or CD39-expressing cells (or adenosine, ATP, ADP and/or AMP) in the tumor environment involves obtaining from an individual a biological sample that contains cancerous tissue and/or tissue proximal or at the periphery of the cancer (eg, adjacent cancer tissue, adjacent non-tumor tissue, or normal adjacent tissue), and detection of pCD39 protein levels of 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.

An individual with cancer can be treated with an anti-CD39 antibody without a prior detection step to assess the presence of pCD39 and/or CD39 expression on circulating cells or cells in the tumor microenvironment (eg, tumor cells, CD4 T cells, CD8 T cells, TReg, B cells). Optionally, the method of treatment may include the step of detecting a CD39 nucleic acid or polypeptide in a biological sample from blood or tumor from an individual (eg, cancerous tissue, tissue proximal or peripheral to cancer, cancerous adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue). Determining that a biological specimen contains cells expressing CD39 (eg, prominently expressing; expressing CD39 at a high level; high staining intensity with anti-CD39 antibody compared to reference, such as healthy tissue) indicates that the patient has cancer that may benefit greatly from treatment with a CD39 inhibitory agent. In one embodiment, the method includes determining the expression level of a CD39 nucleic acid or polypeptide in a biological sample and comparing the level to a reference level corresponding to a healthy individual (eg, healthy tissue). Determination that a biological sample contains pCD39 protein and/or cells expressing CD39 nucleic acid or polypeptide at a level that is elevated above the reference level indicates that the patient has a cancer that can be successfully treated with an anti-CD39 antibody according to the present invention. In one embodiment, detecting a CD39 polypeptide in a biological sample includes detecting soluble extracellular CD39 protein. In one embodiment, detecting a CD39 polypeptide in a biological sample includes detecting a CD39 polypeptide expressed on the surface of a cancer cell, CD4 T cell, CD8 T cell, TReg cell, B cell. In one embodiment, determining that the biological sample contains cells that significantly express CD39 nucleic acid or polypeptide indicates that the patients have cancer that can be successfully treated

- 28 047070 using an anti-CD39 antibody according to the present invention. Pronounced, when referring to a CD39 polypeptide, means that the CD39 polypeptide is expressed in a significant number of cells taken from a given patient. Although the definition of pronounced is not bound by exact percentage, in some examples a receptor that is said to be prominent will be present in at least 10, 20, 30, 40, 50°, 60, 70, 80%, or more tumor cells taken from the patient.

Determining whether an individual has a cancer characterized by cells that express the CD39 polypeptide may, for example, involve obtaining a biological sample (eg, by performing a biopsy) from the individual that contains cells from the environment of the cancer (eg, the tumor or tissue adjacent to the cancer). tumor), contacting said cells with an antibody that binds a CD39 polypeptide, and determining whether the cells express CD39 on their surface. Optionally, determining whether an individual has cells expressing CD39 involves performing an immunohistochemical analysis.

In one embodiment, the anti-CD39 antibodies described herein can be used primarily to treat cancer that is CD73-positive cancer. CD73 expression has been reported in a number of tumor cells, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, and breast cancer, among others. CD73 expression has also been associated with a prometastatic phenotype in melanoma and breast cancer.

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

A patient with cancer may receive anti-CD39 antibody treatment with or without a prior detection step to assess CD73 expression on cells in the tumor microenvironment (eg, tumor cells, CD4 T cells, CD8 T cells, B cells). Optionally, the treatment methods may include the step of detecting a CD73 nucleic acid or polypeptide in a tumor biological sample from an individual (eg, cancer tissue, tissue proximal or peripheral to the cancer, cancerous adjacent tissue, adjacent non-tumor tissue, or normal adjacent tissue). Determining that a biological specimen contains cells expressing CD73 (e.g., prominently expressing; expressing CD73 at high levels; high intensity of anti-CD73 staining compared to reference, e.g., healthy tissue) indicates that the patient has cancer. which may benefit greatly from treatment with a pCD39 inhibitory agent (optionally also in combination with a CD73 inhibitory agent). 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, for example, a healthy individual (eg, healthy tissue). Determining that a biological sample contains cells expressing CD73 nucleic acid or polypeptide at a level that is elevated relative to control levels indicates that the patient has cancer that can be treated with an anti-CD39 antibody. Optionally, detection of a CD73 polypeptide in a biological sample includes detection of a CD73 polypeptide expressed on the surface of a cancer cell, CD4 T cell, CD8 T cell, B cell. In one embodiment, determining that a biological sample contains cells expressing a CD73 nucleic acid or polypeptide indicates that the patients have cancer that may particularly benefit from treatment with an anti-CD39 antibody. The CD73 polypeptide may, for example, be expressed in a significant number of cells taken from a given patient, for example, CD73 may be detected on at least 10, 20, 30, 40, 50, 60, 70, 80%, or more of tumor cells taken from patient.

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

In one embodiment, the invention provides a method of treating or preventing cancer in an individual in need thereof, the method comprising:

a) detection of CD73-expressing cells in the surroundings of the tumor, optionally within the tumor and/or in adjacent tissue, and

- 29 047070

b) when determining the presence of CD73-expressing cells in the tumor environment, optionally at a level that is elevated compared to a reference level (for example, the level of observed healthy tissue), administering to the individual an antibody according to the present invention that binds and inhibits the activity of soluble human CD39 protein. Optionally, detecting CD73-expressing cells in the tumor environment involves obtaining from an individual a biological sample that contains cancer tissue and/or tissue proximal or peripheral to the cancer (eg, adjacent cancer tissue, adjacent non-tumor tissue, or normal adjacent tissue), and detecting levels of CD73-expressing cells. cells (for example, through immunohistochemical analysis). CD73-expressing cells may include, for example, tumor cells, CD4 T cells, CD8 T cells, B cells.

The anti-CD39 antibody compositions of the present invention can be used as monotherapy or combination treatment with one or more other therapeutic agents, including agents typically used for the particular therapeutic purpose for which the antibody is administered. The additional therapeutic agent is typically administered in amounts and treatment regimens that would typically be used for that agent in monotherapy for the particular disease or condition being treated.

In one embodiment, the anti-CD39 antibody compositions of the present invention may be used in combination treatment with a chemotherapeutic agent capable of inducing extracellular ATP release from tumor cells.

In one embodiment, neutralizing antibodies against CD39 do not bind to human CD16, but potentiate the activity of CD16-expressing effector cells (eg, NK or effector T cells). Accordingly, in one embodiment, the second or additional second therapeutic agent is an antibody or other Fc domain-containing protein capable of inducing ADCC to a cell to which it is associated, for example, through CD16 expressed by an NK cell. Typically, such a second agent antibody or other protein will comprise a domain that binds to an antigen of interest, such as an antigen present on a tumor cell (tumor antigen), and an Fc domain or portion thereof, and will exhibit binding to the antigen through antigen-binding domain and with FcY receptors (for example, CD16) through the Fc domain. In one embodiment, its ADCC activity will be mediated, at least in part, by CD16. In one embodiment, the additional therapeutic agent is an antibody having a native or modified human Fc domain, such as the Fc domain of 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 nonspecific cytotoxic cells expressing Fc receptors (FcRs) recognize bound antibody on a target cell and subsequently induce lysis of target cells. Nonspecific 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 exhibits ADCC as measured by assay(s) known to those skilled in the art. Such activity is typically characterized by 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 exhibit ADCC may be, for example, due to its subclass (such as IgG1 or IgG3), mutations introduced into the Fc region, or due to modifications of carbohydrate patterns in the Fc region of the antibody. Examples of ADCC-inducing antibodies include rituximab (for the treatment of lymphomas, CLL, trastuzumab (for the treatment of breast cancer), alemtuzumab (for the treatment of chronic lymphocytic leukemia), and cetuximab (for the treatment of colorectal cancer, squamous cell carcinoma of the head and neck). Examples of antibodies with enhanced ADCC include, but are not limited to: GA-101 (a hypofucosylated anti-CD20 molecule), margetuximab (an Fc-enhanced anti-HER2 molecule), mepolizumab, MEDI-551 (an Fc-engineered anti-CD19 molecule), obinutuzumab (an Fc-enhanced based on glycosilin engineering/hypofucosuylated anti-CD20 molecule), ocaratuzumab (Fc-engineered anti-CD20 molecule), XmAb®5574/MOR208 (Fc-engineered anti-CD19 molecule).

In one embodiment, anti-CD39 neutralizing antibodies enhance the effectiveness of agents that neutralize human PD-1 inhibitory activity, such as those that inhibit the interaction between PD-1 and PD-L1, particularly in individuals who respond poorly to (or are not responsive to) treatment with the agent. which neutralizes the inhibitory activity of human PD-1. Accordingly, in one embodiment, the second or additional second therapeutic agent is an antibody or other agent that neutralizes human PD-1 inhibitory activity.

Programmed cell death protein 1 (PD-1) (also called programmed cell death protein 1) is a member of the CD28 family of receptors with inhibitory properties. The complete sequence of human PD-1 can be found in GenBank under accession number U64863.

- 30 047070

Inhibiting or neutralizing the inhibitory activity of PD-1 may involve the use of a polypeptide agent (eg, antibody, Fc domain fusion polypeptide, immunoadhesin, etc.) that prevents PD-L1-induced PD-1 signaling. There are currently at least six agents that block the PD-1/PD-L1 pathway that are marketed or undergoing clinical evaluation. One agent is BMS-936558 (Nivolumab/ONO-4538, Bristol - Myers Squibb; formerly MDX-1106). Nivolumab (trade name Opdivo®), is a fully FDA-approved anti-PD-L1 human IgG4 mAb that inhibits PDL1 ligand binding to both PD-1 and CD80, and is described as antibody 5C4 in WO 2006/121168, the disclosure of which is included herein by reference. For melanoma patients, the most significant OR was observed at 3 mg/kg, while for other cancer types it was 10 mg/kg. Nivolumab is usually administered at a dose of 10 mg/kg every 3 weeks until the cancer progresses. The terms reduce human PD-1 inhibitory activity, neutralize PD-1, or neutralize human PD-1 inhibitory activity refer to a process characterized in that PD-1 is inhibited in its signal transducing ability as a result of the interaction of PD-1 with one or more of its binding partners such as PD-L1 or PD-L2. An anti-PD-1 inhibitory agent reduces, blocks, inhibits, abolishes or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. Such an agent may thus reduce the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes to enhance T cell effector functions such as proliferation, cytokine production and/or cytotoxicity.

MK-3475 (human IgG4 anti-PD-L1 mAb from Merck), also called lambrolizumab or pembrolizumab (trade name Keytruda®), has been approved by the FDA for the treatment of melanoma and is being tested in other cancers. Pembrolizumab was tested at a dose of 2 or 10 mg/kg every 2 or 3 weeks until disease progression. MK-3475, also known as Merck 3745 or SCH900475, is also described in WO2009/114335.

MPDL3280A/RG7446 (atezolizumab, brand name Tecentriq™, anti-PD-L1 molecule from Roche/Genentech) is an anti-human PD-L1 mAb that contains an engineered Fc domain designed to optimize efficacy and safety by minimizing FcyR binding and as a consequence of antibody-dependent cellular cytotoxicity (ADCC). Doses of <1, 10, 15, and 25 mg/kg MPDL3280A were administered every 3 weeks for 1 year. In the phase 3 study, MPDL3280A is administered at a dose of 1,200 mg via intravenous infusion every three weeks in NSCLC.

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

Pidlizumab (CT-011; CureTech) (humanized anti-PD1 IgG1 mAb from CureTech/Teva), pidlizumab (CT-011; CureTech) (see, for example, WO 2009/101611) is another example; The drug was tested in thirty patients with relapsed FL sensitive to rituximab who were administered 3 mg/kg IV ST-011 every 4 weeks for 4 infusions in combination with rituximab at a dose of 375 mg/m ² weekly for 4 weeks, starting from 2 weeks after the first infusion of ST-011.

Other known anti-PD-1 antibodies and other PD-1 inhibitors include AMP-224 (a B7-DC/IgG1 fusion protein licensed from GSK), AMP-514 described in WO 2012/145493, MEDI-4736 antibody (durvalumab, trade name Imfinzi™, anti-PD-L1 molecule developed by AstraZeneca/Medimmune), described in WO 2011/066389 and US 2013/034559, antibody YW243.55.S70 (anti-PD-L1 molecule), described in WO 2010/077634, MDX- 1105, also known as BMS-936559, is an anti-PD-L1 antibody developed by Bristol-Myers Squibb described in WO 2007/005874, and antibodies and inhibitors described in WO 2006/121168, WO 2009/014708, WO 2009/ 114335 and WO 2013/019906, the disclosures of which are incorporated herein by reference. Other examples of anti-PD-L1 antibodies are disclosed in WO 2015/085847 (Shanghai Hengrui Pharmaceutical Co. Ltd.), for example, antibodies having CDR1, 2 and 3 of the light chain variable domain SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 8, respectively, and antibodies with CDR1, 2 and 3 of the heavy chain variable domain of SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, respectively, and references to SEQ ID NO are numbering in accordance with WO 2015/085847, 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 may also be used.

In some embodiments, the PD-1 neutralizing agent is an anti-PD-L1 mAb that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 neutralizing agent is an anti-PD-L1 mAb that inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 neutralizing agent 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).

- 31 047070

In methods of treatment, the anti-CD39 antibody and the second therapeutic agent may be administered separately, together or sequentially, or in a cocktail. In some embodiments, the antigen binding compound is administered prior to administration of the second therapeutic agent. For example, the anti-CD39 antibody may be administered approximately 0-30 days prior to administration of the second therapeutic agent. In some embodiments, the anti-CD39 antibody is administered over about 30 minutes to about 2 weeks, about 30 minutes to about 1 week, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to 1 day, or about 1 to 5 days before administration of the second therapeutic agent. In some embodiments, the anti-CD39 antibody is administered concomitantly with the administration of the second therapeutic agent. In some embodiments, the anti-CD39 antibody is administered after administration of the second therapeutic agent. For example, the anti-CD39 antibody may be administered approximately 0-30 days after administration of the second therapeutic agent. In some embodiments, the anti-CD39 antibody is administered over about 30 minutes to about 2 weeks, about 30 minutes to about 1 week, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to 1 day, or about 1 to 5 days after administration of the second therapeutic agent.

Examples. Ways.

Generation of CD39 mutants.

CD39 mutants were obtained by PCR. Amplified sequences were analyzed on an agarose gel and purified using the Macherey Nagel PCR Clean-Up Gel Extraction kit (reference 740609). Purified PCR products obtained for each mutant were then ligated into an expression vector using the ClonTech InFusion system. Vectors containing mutant sequences were prepared as Miniprep and sequenced. After sequencing, vectors containing mutant sequences were prepared as Midiprep using the Promega PureYield™ Plasmid Midiprep System. HEK293T cells were grown in DMEM (Invitrogen), transfected with vectors using Invitrogen 2000 Lipofectamine and incubated at 37°C in a CO ₂ incubator for 48 hours before testing for transgene expression. The mutants were transfected into Hek-293Y cells as shown in the table below. Target amino acid mutations in table. 1 below are shown using SEQ ID NO: 1.

Table 1

Mutan t Substitutions 1 Q444 G445 V77G H79Q TO D 2A V81S E82A R111A V115A 2B E110A R113T E114A 3 Q120 Q122 R118A S119A TO N E123A 4 D150A E153S R154A S157K N158A L278F 5 Q96A N99A E143A R147E 6 K188R Replacing residues 190 to 207 with KTPGGS 7 A273S N275A I277S R279A 8 S294A K298G KZOZA E306A T308K Q312A 9 K288E K289A V290A E315R 10A Q354A D356S E435A H436Q 10V H428A T430A A431D D432A eleven Insert N371K L372K E375A K376G 377V V377S 12 K388N Q392K P393S E396A 13 А402Р G403A K405A E406A 15 K87A E100A D107A 16 Q323A Q324A Q327A E331K 17 N334A S336A Y337G N346A 18 Q228A I230S D234A Q238A 19 R138A M139A E142K

Cloning, production and purification of soluble huCD39

- 32 047070

Molecular biology.

The huCD39 protein was cloned from human PBMC cDNA using the following primers:

TACGACTCACAAGCTTGCCGCCACCATGGAAGATACAAAGGAGTC (SEQ ID NO: 41) (forward) and CCGCCCCGACTCTAGATCACTTGTCATCTCTTTGTAATCGACATAGGTGGAGGAGAGAG (SEQ ID NO: 42) (reverse). The purified PCR product was then cloned into an expression vector using an infusion cloning system. An M2 tag (FLAG tag as underlined in SEQ ID NO: 44) was added to the C-terminal portion of the protein for the purification step; It should be understood that the CD39 extracellular domain protein (eg, SEQ ID NO: 44) may, in any embodiment, optionally be provided without the M2 tag.

Expression and purification of huCD39 proteins.

After validation of the cloned sequence, CHO cells were nucleofected and the production pool was then subcloned to obtain a cell clone producing the huCD39 protein. The supernatant from the huCD39 clone grown in roller bottles was collected and purified using an M2 chromatography column and eluted using M2 peptide. The purified proteins were then loaded onto an S200 size chromatography column. The purified protein corresponding to the monomer was prepared in TBS buffer pH 7.5. The amino acid sequence of the extracellular domain of the recombinant CD39-M2 protein without the M2 tag was as follows:

MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGSSHTSLYIY

KWPAEKENDTGWHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERAREVIPRSQHQETPV YLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGK FSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQTIESPDNALQFRLYGKDYNVY GKDQALWQKLAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLP FQQFEIQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFGAFSAFYFVMKFLNL TSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADS KIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHSTYV (SEQ ID NO: 43).

The final amino acid sequence of the extracellular domain of the M2-tagged recombinant CD39-M2 protein was as follows:

MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGSSHTSLYIY

KWPAEKENDTGWHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERAREVIPRSQHQETPV YLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGK FSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQTIESPDNALQFRLYGKDYNVY GKDQALWQKLAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLP FQQFEIQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFGAFSAFYFVMKFLNL TSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADS KIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHSTYVDYKDDDDK (SEQ ID NO: 44).

Inhibition of enzymatic activity of soluble CD39.

Antibody inhibition of the enzymatic activity of the produced soluble CD39 protein was assessed using Cell Titer Glo™ (Promega, reference G7571), which assesses ATP hydrolysis using a reagent that generates a luminescent signal proportional to the amount of ATP present. In this way, inhibition of soluble CD39-mediated ATP hydrolysis can be assessed. Briefly, dose ranges of anti-CD39 antibodies from 100 μg/ml to 6 x 10' ³ μg/ml were incubated with 400 ng/ml soluble recombinant human CD39 protein having the amino acid sequence described in the methods section (SEQ ID NO: 44) for 1 h at 37°C. 20 μM ATP was added to the plates for an additional 30 minutes at 37°C before adding CTG reagent (Ceil Titer Gio). Emitted light was quantified using an Enspire™ luminometer after a short incubation period of 5 min in the dark. The effectiveness of anti-CD39 antibodies was determined by comparing light emission in the presence of the antibody with ATP alone (maximum light emission) and ATP plus soluble CD39 protein (minimal light emission).

Inhibition of enzymatic activity of cellular CD39.

Inhibition of CD39 enzymatic activity in CD39-expressing cells by antibodies was assessed using Cell Titer Gio™ (Promega, reference G7571), which allows the assessment of ATP hydrolysis using a reagent that generates a luminescent signal proportional to the amount of ATP present. Therefore, the assay was designed to assess the inhibition of ATP hydrolyzed by CD39 in cell culture supernatant. Briefly, 5x10 ⁴ human cells

- 33 047070 Ramos lymphoma, 5x10 ³ CHO cells expressing human CD39, macaque CD39 and murine CD39 were incubated for 1 hour at 37°C with 30 μg/ml to 5x10 ^-4 μg/ml anti-CD39 antibodies. Cells were then incubated with 20 μM ATP for 1 additional hour at 37°C. The plates were centrifuged for 2 min at 400 g and 50 μl of cell supernatant was transferred to a fluorescent microplate (white wells). 50 μl of CellTiter-Glo™ (CTG) reagent was added to the supernatant and the emitted light was quantified after a 5-minute incubation in the dark using an Enspire™ luminometer. The effectiveness of anti-CD39 antibodies was determined by comparing light emission in the presence of the antibody with ATP alone (maximum light emission) and ATP plus cells (minimal light emission).

Antibody generation: immunization and screening in mice.

To generate antibodies to human CD39, Balb/c mice were immunized with the extracellular domain of the recombinant human CD39-M protein described above. Mice received one primary immunization with an emulsion of 50 μg of CD39 protein and complete Freund's adjuvant intraperitoneally, a 2nd immunization with an emulsion of 50 μg of CD39 protein and incomplete Freund's adjuvant intraperitoneally, and finally an increased dose of 10 μg of CD39 protein intravenously. Spleen immune cells were decanted 3 days after immortalized X63.Ag8.653 B cells and cultured in the presence of irradiated spleen cells. Hybridomas were seeded in semi-solid methylcellulose-containing medium and the grown clones were selected using a Clonepix™ 2 apparatus (Molecular Devices Corp).

Example 1: Epitope mapping of known CD39 neutralizing mAbs.

To gain insight into how antibodies are capable of inhibiting the enzymatic (ATPase) activity of cellular CD39, we examined epitopes bound by antibodies that have been reported to inhibit CD39 ATPase activity in cell-based assays: BY40 disclosed in PCT Publication No. WO 2009/095478 .

To define epitopes of anti-CD39 antibodies, we developed CD39 mutants defined by amino acid substitutions exposed on the molecular surface above the surface of CD39. The mutants were transfected into Hek-293E cells as shown in Table. 1, using SEQ ID NO: 1.

Dose ranges of I-394 (10 - 2.5 - 0.625 - 0.1563 - 0.0391 - 0.0098 - 0.0024 - 0.0006 μg/ml) were tested on 20 generated mutants by flow cytometry. BY40 antibodies had complete loss of binding to cells expressing CD39 mutant 5, with no loss of binding to any other mutant. 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 Fig. 3A.

Example 2. Known neutralizing monoclonal antibodies to CD39 are not able to inhibit the ATPase activity of recombinant soluble CD39 protein.

Two antibodies that have been reported to inhibit the ATPase activity of CD39 in cell-based assays (BY40 and BY12) were evaluated to determine whether they were able to inhibit the ATPase activity of recombinant soluble CD39 protein. Antibody inhibition of the enzymatic activity of soluble CD39 protein produced as described above was assessed using Cell Titer Glo™ (Promega, reference G7571). Antibody inhibition of the enzymatic activity of the cellular CD39 protein was assessed as described above.

As expected, BY40 inhibited the ATPase activity of CD39 protein in cells. However, BY40 was unable to inhibit the enzymatic activity of soluble CD39 protein. In fig. 2B shows a comparison of BY40 with the new antibodies identified herein.

Example 3: Screening of new mAbs to block the activity of pCD39.

A series of immunizations was carried out to search for antibodies that neutralize the ATPase activity of pCD39. To generate anti-human CD39 antibodies, animals were immunized with the recombinant extracellular domain of recombinant human CD39-M2 described above. In total, the series of immunizations involved different protocols and different animals, including different strains of mice, rats and rabbits.

In the original immunization protocols, primary screening involved testing the supernatant (SN) of growing clones by flow cytometry using wild-type and CHO cell lines expressing huCD39. Cells were stained with 0.1 and 0.005 μM CFSE, respectively. For flow cytometry screening, all cells were mixed equally, and the presence of reactive antibodies in the supernatants was detected using an APC-labeled polyclonal goat anti-mouse antibody (pAb). For huCD39 binding antibodies, supernatants were then screened for inhibition of soluble CD39 enzymatic activity using the screening assay developed and described above (Methods).

The results showed that, while numerous specific CD39-binding antibodies can be obtained, none of the antibodies from any of these immunizations showed any inhibition of the enzymatic activity of soluble CD39. One possibility is that the dominant epitopes on CD39 do not contain any epitopes suitably located on or near this CD39 catalytic site. Due to the paucity of available antibodies that inhibit cellular CD39 and the known difficulty of inhibiting catalytic sites of enzymes with antibodies, the lack of antibodies that neutralize pCD39 may indicate the inability to obtain antibodies that inhibit the soluble (extracellular domain) of CD39. Other possibilities relate to nonfunctional screening assays and/or misfolded or functional soluble CD39 protein, especially since the lack of any antibody that can inhibit soluble CD39 has hampered the validation of pCD39 blockade assays.

In the absence of antibodies capable of inhibiting soluble CD39, further immunization was performed using a screening protocol designed to generate antibodies that bind the active site of CD39 identified by the BY40 antibody epitope. Briefly, the primary screen involved testing the supernatant (SN) of growing clones by flow cytometry using wild-type CHO and huCD39-expressing CHO cell lines as in previous immunizations, followed by screening for loss of binding of I lek-293'T mutant-expressing cells CD39 5, compared to wild type CD39, as shown in Table. 1. Mutant 5 has substitutions in residues Q96, N99, E143 and R147. However, again the results showed that although many specific CD39-binding antibodies could be obtained that showed loss of binding to mutant 5, none of the antibodies from any of the initial immunizations showed any inhibition of the enzymatic activity of soluble CD39.

Example 4 Identification of Antibody I-394.

We sought to identify anti-CD39 antibodies that do not bind 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 should not have any ability to block the ATPase activity of CD39, may be useful for pharmacological studies of antibodies that inhibit cellular CD39 that binds to the BY40 binding site, for example, to detect and quantify free CD39 proteins on cells in the presence of BY40 or Ly40-like antibodies that inhibit cellular CD39.

Based on the immunization results of Example 3, in which hybridomas were screened for loss of binding to the CD39 mutant 5, a hybridoma was selected that was not among those that showed loss of binding to the CD39 mutant 5. This hybridoma (I-394) was part of a larger pool due to inconclusive data indicating a possible partial decrease in binding to mutant 5, but did not lose binding to mutant 5 and was therefore not initially retained.

In the context of ongoing screening of supernatants from further immunizations for inhibition of soluble CD39 enzymatic activity, the cloned and produced antibody I-394 was included as a control. Surprisingly, although antibody I-394 was not among the clones retained in the epitope-directed screen, this antibody showed potent inhibition of the enzymatic activity of soluble CD39 in the assay described above (Methods).

I-394 was produced with constant regions of the human IgG1 isotype, with a modified Fc domain having mutations L234A/L235E/G237A/A330S/P331S (numbering according to EU Kabat), which led to the lack of binding to FCY receptors CD16A, CD16B, CD32A, CD32B and Human CD64, in brief, the VH and Vk sequences of antibody I-394 (VH and Vk variable regions shown in SEQ ID NO: 6 and 7, respectively) were cloned into expression vectors containing huIgG1 constant domains containing the indicated mutations and a huCk constant domain, respectively. The two resulting vectors were co-transfected into CHO cells. The created pool of cells was used to produce antibodies in CHO medium. The antibody was then purified with Protein A. The amino acid sequences of the corresponding heavy and light chain variable domains of I-394 are shown below (Kabat CDRs are underlined).

I-394 heavy chain variable domain sequence

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGRTLEWIGYIVPLNGGSTF

NQKFKGRATLTVNTSSRTAYMELRSLTSEDSAAYYCARGGTRFAYWGQGTLVTVSA (SEQ

ID NO: 6).

I-394 light chain variable domain sequence

DIVLTQSPASLAVSLGQRATISCRASESVDNFGVSFMYWFQQKPGQPPNLLIYGASNQGSG

VPARFRGSGSGTDFSLNIHPMEADDTAMYFCQQTKEVPYTFGGGTKLEIK (SEQ ID NO:

7)·

Antibody I-394 was then tested for loss of binding to CD39 mutants, as determined by amino acid substitutions exposed on the molecular surface above the surface of CD39. The mutants were transfected into Hek-293T cells as shown in Table. 1 using SEQ ID NO: 1. Dosage ranges of I-394 antibodies were tested on 20 mutants by flow cytometry. As shown in FIG. 3B, I-394 showed complete loss of binding to cells expressing mutant 19 of CD39. Mutant 19 contains substitutions at residues R138, M139, and E142. So the main

- 35 047070 epitope I-394 contains one or more (or all) of residues R138, M139 and E142.

Unlike the predecessor antibody BY40, which loses binding to mutant 5 and has the ability to inhibit cellular CD39 but not soluble CD39, antibody I-394 loses binding to the neighboring mutant 19, with greatly reduced binding to mutant 5 (but with some residual binding to mutant 5). Interestingly, residues from mutant 19 are in close proximity or adjacent to residues from mutant 5, so I-394 may represent a shift in epitope compared to BY40. Thus, antibody I-394 represents a valuable new epitope for anti-CD39 antibodies that allows inhibition of the ATPase activity of soluble CD39 protein. It also provided a specific positive control that allows validation and testing of screening assays to detect further antibodies that neutralize the ATPase activity of soluble CD39 protein.

Example 5. Non-epitope-directed screening of monoclonal antibodies that neutralize pCD39.

Based on the results of Example 4 indicating the possibility of antibody-mediated inhibition of soluble CD39, fusions from different immunizations using different protocols from Example 3 were revised to search for antibodies that neutralize the ATPase activity of pCD39.

Different screening approaches for ATPase inhibition were then evaluated. In one experiment, antibody I-394 was used to spike supernatants from immunization hybridomas of Example 3, which were found to be negative in their ability to inhibit the ATPase activity of soluble CD39. This addition of I-394 to the supernatant did not restore the ability of the negative supernatants to inhibit CD39 ATPase activity. Antibody I-394 was then purified from the negative supernatant using protein A-coated beads, and the inventors observed that purified I-394 was again able to inhibit ATPase activity.

In view of the above results, new immunization and screening protocols were developed in which growing clones from new and past immunizations were screened by flow cytometry using huCD39-expressing and wild-type CHO cell lines, without assessing inhibition of soluble CD39 or cellular CD39 ATPase activity. and without epitope screening bias. While data on loss of binding to mutant 5 or 19 were available for some hybridomas, such data were not used for clone selection and were retained only for the purpose of preserving hybridomas for cloning in the event of negative ATPase blocking assay results. CD39 binding hybridomas were selected and cloned and then purified using protein A according to the following protocol.

Add 10 μl of protein A beads to 300 μl of hybridoma supernatant.

Add NaCl to a final concentration of 1.5 M.

Rotate the tubes for 3-4 hours at a temperature of 4°C.

Centrifuge for 1 min at 1500 rpm.

Remove the supernatant and perform three washes with 1 ml TBS.

Remove all TBS after the third wash.

Add 50 μl of citrate 0.1 M pH 3, homogenize and incubate at room temperature for 5 minutes.

Centrifuge the beads for 1 minute at 1500 rpm.

Collect 50 µl of eluate and quickly add 450 µl of TBS and store at 4°C.

The resulting antibodies were then comparatively analyzed for their ability to inhibit the ATPase activity of CD39 to the same extent as I-394. Assays used to inhibit the enzymatic activity of soluble and cellular CD39 have been 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). In fig. 1 shows a representative screening result showing antibodies I-397, I-398 and I-399 compared to the positive control antibody I394. Similarly, antibodies I-395 and I-396 from various immunizations inhibited the enzymatic activity of soluble CD39 protein. In fig. 2A and 2B show results for antibodies I-395 and I-396, for which more antibodies were available for additional experiments for both soluble and cellular neutralization of CD39. In fig. 2A shows that antibodies I-395 and I-396 both inhibit cell membrane-associated CD39 compared to antibodies BY40 and I-394, with both antibodies I-394 and I-395 demonstrating greater potency and maximum inhibition of cellular CD39 compared to BY40. In fig. 2B shows that antibodies I-395 and I-396 both inhibit soluble CD39 compared to BY40 and I-394 antibodies. While BY40 does not inhibit soluble CD39 at any concentration, I-394, I-395 and I-396 all inhibit soluble CD39, with I-394 showing the greatest potency, followed by I-395 and then I396 with lower potency .

These results raise the possibility that factor(s) in hybridoma supernatants rapidly hydrolyze ATP in both cell culture and soluble CD39 assays such that a signal for ATP is not detected in antibody screens using traditional methods. Dissolve

- 36 047070 This factor may be CD39 or some other enzyme, for example produced by a fusion partner.

Antibodies were then cloned with modification to obtain constant regions with the human IgG1 Fc domain having mutations L234A/L235E/G237A/A330S/P331S (EU Kabat numbering), resulting in a lack of binding to the Fcy receptors CD16A, CD16B, CD32A, CD32B and human CD64 in the same manner as shown herein for I-394. The resulting antibodies can then be subjected to titration and then more detailed activity assessment as shown in Example 7-9 (Titration, ATPase Activity Inhibition) to evaluate EC ₅₀ and IC ₅₀ determinations to rank antibodies by potential.

Example 6: Epitope mapping of pCD39 neutralizing mAbs.

As shown in Example 4, I-394 showed complete loss of binding to cells expressing CD39 mutant 19, but did not lose binding to mutant 5. To determine the epitopes of the other anti-CD39 antibodies from Example 5, they were tested for loss of binding to a panel of CD39 mutants , as described in example 1 and table. 1. Mutants were transfected into Hek-293T cells as shown in Table. 1, using SEQ ID NO: 1. Test antibody dose ranges (10 - 2.5 - 0.625 - 0.1563 0.0391 - 0.0098 - 0.0024 - 0.0006 μg/ml) were tested on 20 generated mutants by flow cytometry.

The results showed that the antibodies selected in Example 5 for their ability to inhibit soluble CD39 represented several different epitopes. Among the antibodies that showed inhibition of soluble extracellular CD39 in Example 5, antibody I-395 is an example of an antibody that showed loss of binding to mutant 5, having substitutions at residues Q96, N99, E143 and R147, as well as loss of binding to mutant 19. having substitutions in residues R138, M139 and E142. Mutant 19 contains substitutions at residues R138, M139, and E142. Thus, the core epitope on CD39 I-395 contains one, two, three, or four residues Q96, N99, E143, and R147, as well as one, two, or three residues R138, M139, and E142.

Antibody I-398, on the other hand, is an example of an antibody that showed loss of binding to mutant 19, having substitutions at residues R138, M139, and E142, but had no reduction or loss of binding to mutant 5, having substitutions at residues Q96, N99 , E143 and R147.

Other antibodies that showed inhibition of soluble extracellular CD39 in example 5 had very different epitopes and did not show loss of binding to either mutant 5 or 19, suggesting that soluble CD39 may also be inhibited by binding to other sites on pCD39. For some antibodies, loss of binding to one of the 20 mutants in Table. 1 allowed the binding site to be localized to CD39, while for the others the binding site remained uncertain because they did not lose binding to any of the 20 mutants. Among the antibodies demonstrating inhibition of the ATPase activity of soluble CD39 in Example 5, antibody I-396 showed loss of binding to mutant 15, having the K87A, E100A and D107A substitutions, without loss of binding to any of the other 20 mutants. Thus, the core epitope on CD39 of this antibody contains one or more (or all) of residues K87, E100 and D107. Antibody I-399 showed loss of binding to mutant 11, which has substitutions N371K, L372K, E375A, K376G, V377A, and a valine insertion between K376 and V377 (listed in Table 1 as insertion 377V), without loss of binding to any of the other 20 mutants. Thus, the core epitope on CD39 of this antibody contains one or more (or all) of residues N371, L372, E375, K376 and V377. In fig. 3A shows the position of residues mutated in mutants 5 (M5), 15 (M15) and 19 (M19) on the surface of the CD39 protein. In fig. ZV shows the results of binding to mutants of 5, 15 and 19 different antibodies.

Thus, our results indicate that antibodies that inhibit soluble CD39 can be generated against a variety of epitopes. Epitopes include epitopes defined by one or more residues of mutant 19 that are located adjacent to the binding site of BY40 or BY40-like antibodies that inhibit only cellular CD39 but not soluble CD39 (which lose binding to mutant 5), epitopes that are defined by one or more residues of mutant 19, but also some of mutant 5, indicating perhaps a smaller shift compared to BY40 or BY40-like antibodies, epitopes defined by one or more residues of mutant 19 rather than residues of mutant 5, and other epitopes, such as those detected by one or more residues of mutant 11 or one or more residues of mutant 15, or further by other antibodies that have no reduced binding to any of mutants 5, 15 or 19, for which the localization of epitopes has not yet been determined.

Example 7. Titration of antibodies on CD39-expressing cells by flow cytometry.

Antibody I-394 was tested in duplicate for binding to CHO cells expressing human CD39, CHO cells expressing macaque CD39, CHO cells expressing murine CD39, and human Ramos lymphoma cells (ATCC™, reference CRL-1596 ). Cells were incubated with various concentrations of unlabeled anti-antibody.

- 37 047070

CD39 from 30 µg/ml to 5x10 ^-4 µg/ml for 30 minutes at a temperature of 4°C. After washing, cells were incubated with labeled secondary goat anti-mouse antibody H+L for 30 min at 4°C.

The results are shown in Fig. 4. Antibody I-394 bound to cells expressing human CD39 (CHO-huCD39), cells expressing macaque CD39 (CHO-cyCD39) and Ramos lymphoma cells, but not to cells expressing murine CD39 (CHO-moCD39). I-394 bound to Ramos cells with EC50 values of 0.16 μg/ml and 0.19 μg/ml in the respective first and second sets of experiments. Several other anti-CD39 antibodies showed comparable EC ₅₀ values for binding to Ramos cells.

Example 8 Determination of IC50 for inhibition of cellular ATPase activity.

Antibody I-394 inhibition of CD39 ATPase activity in CD39-expressing cells was assessed using an assay used to inhibit cellular CD39 enzymatic activity as described above (Methods).

The results are presented in Fig. 5. I-394 was highly effective in blocking CD39 enzymatic activity in tumor cells (Ramos) with greater efficacy than all other antibodies tested. I-394 also blocked CD39 enzymatic activity in cells expressing human CD39 (CHO-huCD39) and in cells expressing macaque CD39 (CHO-cyCD39). Cells expressing mouse CD39 (CHO-moCD39) are shown as a negative control. The calculated IC ₅₀ (50% inhibition of CD39 enzymatic activity expressed by 50,000 Ramos cells) is 0.05 μg/ml. The maximum inhibition was 81.6%. Isotype control had no effect.

Example 9 Determination of IC50 for inhibition of ATPase activity of recombinant soluble CD39 protein.

Antibody I-394 inhibition of the ATPase activity of soluble CD39 protein was assessed using assays used to inhibit the enzymatic activity of soluble CD39 protein as described above (Methods). The results are shown in Fig. 6. I-394 inhibited the enzymatic activity of soluble CD39 protein. BY40 antibody, in comparison, did not inhibit the enzymatic activity of soluble CD39 protein. The calculated IC ₅₀ is 0.003 μg/ml. The maximum inhibition was 74.9%.

Example 10. Titration of ELISA on isoforms CD39-L1, L2, L3, L4.

Antibody I-394 was tested for binding to recombinant human CD39 isoforms (Rec-huCD39 isoforms) having the amino acid sequences shown below, coated in a 96-well plate in PBS 1X at 500 ng/ml or 1 μg/ml at 4°C during the night. Wells were washed in TBS-Tween 20 and further saturated for 2 h at room temperature in TBS blocking buffer. A range of primary antibody concentrations were incubated in TBS blocking buffer for 2 h at room temperature. Wells were washed in TBS Tween 20. Secondary antibody (GAM-HRP or GAH-HRP in TBS blocking buffer) was incubated for 1 h at room temperature and detected using TMB. Optical density was measured on Enspire™ at OD=450.

Amino acid sequence of cloned huCD39 (vascular isoform): human CD39-L1, also known as NTPDase2 or ENTPD2

- 38 047070

MAGKVRSLLP PLLLAAAGLA GLLLLCVPTR DVREPPALKY GIVLDAGSSH TSMFIYKWPA

DKENDTGIVG QHSSCDVPGG GISSYADNPS GASQSLVGCL EQALQDVPKE RHAGTPLYLG

121 ATAGMRLLNL TNPEASTSVL MAVTHTLTQY PFDFRGARIL SGQEEGVFGW VTANYLLENF

181 IKYGWVGRWF RPRKGTLGAM DLGGASTQIT FETTSPAEDR ASEVQLHLYG QHYRVYTHSF

241 LCYGRDQVLQ RLLASALQTH GFHPCWPRGF STQVLLGDVY QSPCTMAQRP QNFNSSARVS

301 LSGSSDPHLC RDLVSGLFSF SSCPFSRCSF NGVFQPPVAG NFVAFSAFFY TVDFLRTSMG

361 LPVATLQQLE AAAVNVCNQT WAQQLLSRGY GFDERAFGGV IFQKKAADTA VGWALGYMLN

421 LTNLIPADPP GLRKGTDFSS WWLLLLFAS ALLALVLLL RQVHSAKLPS Tl (SEQ ID NO: 2).

Human CD39-L2, also known as NTPDasa 6 or ENTPD6:

MKKGIRYETS RKTSYIFQQP QHGPWQTRMR KISNHGSLRV AKVAYPLGLC VGVFIYVAYI

KWHRATATQA FFSITRAAPG ARWGQQAHSP LGTAADGHEV FYGIMFDAGS TGTRVHVFQF

121 TRPPRETPTL THETFKALKP GLSAYADDVE KSAQGIRELL DVAKQDIPFD FWKATPLVLK

181 ATAGLRLLPG EKAQKLLQKV KEVFKASPFL VGDDCVSIMN GTDEGVSAWI TINFLTGSLK

241 TPGGSSVGML DLGGGGSTQIA FLPRVEGTLQ ASPPGYLTAL RMFNRTYKLY SYSYLGLGLM

301 SARLAILGGV EGQPAKDGKE LVSPCLSPSF KGEWEHAEVT YRVSGQKAAA SLHELCAARV

361 SEVLQNRVHR TEEVKHVDFY AFSYYYDLAA GVGLIDAEKG GSLVVGDFEI AAKYVCRTLE

421 TQPQSSPFSC MDLTYVSLLL QEFGFPRSKV LKLTRKIDNV ETSWALGAIF HYIDSLNRQK

481 SPAS (SEQ ID NO: 3).

Human CD39-L3, also known as NTPDasa 3 or ENTPD3:

MFTVLTRQPC EQAGLKALYR TPTIIALVVL LVSIVVLVSI TVIQIHKQEV LPPGLKYGIV

LDAGSSRTTV YVYQWPAEKE NNTGWSQTF KCSVKGSGIS SYGNNPQDVP RAFEECMQKV

121 KGQVPSHLHG STPIHLGATA GMRLLRLQNE TAANEVLESI QSYFKSQPFD FRGAQIISGQ

181 EEGVYGWITA NYLMGNFLEK NLWHMWVHPH GVETTGALDL GGASTQISFV AGEKMDLNTS

241 DIMQVSLYGY VYTLYTHSFQ CYGRNEAEKK FLAMLLQNSP TKNHLTNPCY PRDYSISFTM

301 GHVFDSLCTV DQRPESYNPN DVITFEGTGD PSLCKEKVAS IFDFKACHDQ ETCSFDGVYQ

361 PKIKGPFVAF AGFYYTASAL NLSGSFSLDT FNSSTWNFCS QNWSQLPLLL PKFDEVYARS

421 YCFSANYIYH LFVNGYKFTE ETWPQIHFEK EVGNSSIAWS LGYMLSLTNQ IPAESPLIRL

481 PIEPPVFVGT LAFFTAAALL CLAFLAYLCS ATRRKRHSEH AFDHAVDSD (SEQ ID NO: 4).

Human CD39-L4, also known as NTPDasa 5 or ENTPD5:

MATSWGTVFF MLWSCVCSA VSHRNQQTWF EGIFLSSMCP INVSASTLYG IMFDAGSTGT

RIHVYTFVQK MPGQLPILEG EVFDSVKPGL SAFVDQPKQG AETVQGLLEV AKDSIPRSHW

121 KKTPVVLKAT AGLRLLPEHK AKALLFEVKE IFRKSPFLVP KGSVSIMDGS DEGILAWVTV

181 NFLTGQLHGH RQETVGTLDL GGASTQITFL PQFEKTLEQT PRGYLTSFEM FNSTYKLYTH

241 SYLGFGLKAA RLATLGALET EGTDGHTFRS ACLPRWLEAE WIFGGVKYQY GGNQEGEVGF

301 EPCYAEVLRV VRGKLHQPEE VQRGSFYAFS YYYDRAVDTD MIDYEKGGIL KVEDFERKAR

361 EVCDNLENFT SGSPFLCMDL SYITALLKDG FGFADSTVLQ LTKKVNNIET GWALGATFHL

421 LQSLGISH (SEQ ID NO: 5).

I-394 bound to CD39 but not to any of the CD39-L1, -L2, -L3, or -L4 isoforms. Antibodies con

- 39 047070 troll isotype (IC) did not bind to any CD39 or CD39-L molecule. The results are shown in Fig. 7.

Example 11. Activation of dendritic cells.

While ATP has proinflammatory activity, CD39-mediated ATP catabolism is thought to be capable of impairing dendritic cell (DC) activation, in turn altering the broader adaptive immune response against tumor antigen. To evaluate whether CD39 blockade with anti-CD39 antibodies can overcome the CD39-mediated alteration of dendritic cell (DC) activation in the presence of ATP, we incubated monocyte-derived DCs (mDCs) with anti-CD39 antibodies in the presence of ATP.

Briefly, human monocytes were purified from healthy human blood and differentiated into mDCs in the presence of GM-CSF and IL-4 for 6 days. MoDCs were then activated in the presence of ATP (Sigma, 0.25 - 1 mM) for 24 hours, and DC activation was assessed by analyzing the expression of CD80, CD83 and HLA-DR by flow cytometry. Optionally, mDCs are preincubated for 1 hour in the presence of CD39 inhibitor: ARL6716 (Tocris, 250 μM), CD73 inhibitor: APCP (Tocris 50 μM), CD39 blocking antibody I-394 or BY40 (for BY40, see WO 2009 /095478), or blocking antibodies against CD73. LPS (Invivogen, 10 ng/ml) was used as a positive control. To assess the net effect of ATP-mediated DC activation on CD4 T cell activation, ATP-activated DCs were washed and then incubated with allogeneic CD4 T cells (1 mDC/4 T cell ratio) for a mixed lymphocyte reaction (MLR) for 5 days. T cell activation and proliferation were analyzed by CD25 expression and Cell Trace Violet dilution by flow cytometry (Fig. 8).

The results are presented in Fig. 9, 10 and 11. In the presence of a negative control (medium), moDC activation was observed in the presence of 1 mM ATP, but ATP at 0.125 mM, 0.25 mM or 0.5 mM did not allow moDC activation. Addition of chemical CD39 inhibitors, which are believed to completely block CD39 enzymatic activity through binding to the active site, resulted in moDC activation at each of 0.125, 0.25, or 0.5 mM. However, anti-CD39 antibodies, such as BY40 or anti-CD73 antibodies, were not able to promote ATP-induced dendritic cell (DC) activation, suggesting that the antibodies are not able to block enzymatic activity sufficiently to avoid ATP catabolism. Surprisingly, the anti-CD39 blocking antibody I-394 (shown in the figures at a concentration of 10 μg/ml), which almost completely blocks the ATPase activity of CD39 and therefore can allow ATP accumulation, allows mDC activation, assessed by the expression of HLA-DR or CD83 on each of 0.125, 0.25 or 0.5 mM (Figs. 9 and 10). Interestingly, mDCs activated in the presence of ATP were able to induce better T cell activation and proliferation in the MLR assay. In addition, enhancement of ATP-mediated activation of mDCs by the anti-CD39 blocking antibody I-394 resulted in higher T cell proliferation and activation (Fig. 11).

Assessing the ability of CD39 inhibitors to activate DCs in the presence of ATP provides a way to identify and evaluate anti-CD39 antibodies capable of achieving high levels of CD39 inhibition. In addition, the possibility of using anti-CD39 antibodies to attenuate the immunosuppressive effect of CD39 on DCs may provide an enhanced adaptive immune response to antigens, especially on tumor cells. In addition, such anti-CD39 antibodies may be of particular interest when used to enhance the immunogenic effect of chemotherapeutic agents. Numerous chemotherapeutic agents that cause tumor cell necrosis are capable of inducing ATP;

combination use of such agents with anti-CD39 antibodies may be particularly useful in enhancing the antitumor response.

Example 12 Antibodies that inhibit the ATPase activity of recombinant soluble CD39 protein strongly potentiate CD73 blockade in the presence of ATP.

T cell proliferation assay.

Peripheral blood from healthy donors was obtained from EPS, and mononuclear cells were isolated on a Ficoll™ gradient. Lymphocytes were further enriched on a 52% Percoll™ gradient by collecting cell pellets and stained with Cell Trace (Thermofisher) according to the TDS provided by the manufacturer. 5x104-1x105 stained cells were distributed into 96 round bottom plates, incubated for 1 hour at 37°C with anti-huCD73 antibodies (6E1 antibody described in WO 2016/131950) and/or anti-huCD39 mAb (described herein as I-394) and activated for 3–5 days by adding anti-CD3/CD28-coated beads (bead:cell = 1:4; Life Technologies). Inhibition of T cell proliferation was achieved by adding ATP (200 μM). T cell proliferation and the ability of Abs to block the immunosuppressive effect of AMP were assessed by flow cytometry by quantifying dye dilution in the proliferating subset of T cells.

Percentage of proliferating T cells compared to anti-CD73 Ab concentration per line

- 40 047070 graphs using GraphPad Prism™ software.

Results.

Antibodies were tested for their ability to restore proliferation of CD4 or CD8 T cells in the presence of added ATP, designed to represent conditions that might be found in the tumor environment. Each of the anti-CD73 and anti-CD39 molecules was tested over a dose range at 3 different doses of anti-CD73 or anti-CD39 antibody. Anti-CD39 antibody I-394 significantly enhanced the effect of anti-CD73 antibodies in restoring proliferation of CD4 or CD8 T cells, such that even low concentrations of anti-CD73 antibodies (eg, below 0.01 μg/ml, below 0.001 μg/ml, and even below 0.001 μg/ml) ml) strongly enhanced the proliferation of CD4 or CD8 T cells when used in combination with anti-CD39 antibodies. Additionally, when tested only over a dose range, the anti-CD73 antibody I-394 anti-CD39 resulted in a marked increase in CD4 or CD8 T cell proliferation at concentrations of 0.1 and 1 μg/mL. In fig. 12A shows the dose range of the anti-CD73 antibody 6E1 on CD4 T cell proliferation at 3 different doses of the anti-CD39 antibody I-394, either 0.01, 0.1 and 1 μg/ml. Antibodies against CD39, capable of neutralizing soluble and/or monomeric human CD39, demonstrate a strong potentiation of the effect of anti-CD73 antibodies in restoring CD4 T-cell proliferation. The effect was particularly strong at concentrations where anti-CD73 antibodies were suboptimally active, consistent with the concentration ranges that can be observed in tumor tissues during a course of anti-CD73 antibody treatment. At a concentration of 0.01 μg/ml, anti-CD39 provided an approximately 1-log increase in the activity of anti-CD73 antibodies, and at a concentration of 0.1 μg/ml, anti-CD39 provided an approximately 4-log increase in the activity of anti-CD73 antibodies. Thus, anti-CD39 antibodies may be useful for enhancing the activity of anti-CD73 antibodies, especially in tumor tissue, such as tumors containing CD73-expressing cells. In addition, while the anti-CD73 antibodies tested (which are capable of neutralizing soluble CD73 protein) were highly capable of restoring CD4 T cell proliferation, other antibodies had lower activity (eg, as assessed in the enzyme inhibition assay, in the T cell proliferation assay or other suitable assay) and may further benefit from combination with anti-pCD39 antibodies. In fig. 12B shows the dose range of anti-CD73 antibodies for CD8 T cell proliferation. Again, anti-CD39 antibodies show a strong synergy and/or additive effect with anti-CD73 antibodies in restoring CD8 T cell proliferation. The effect was particularly strong at concentrations where anti-CD73 antibodies were suboptimally active, consistent with the concentration ranges that can be observed in tumor tissues during a course of anti-CD73 antibody treatment.

Example 13 Generation of Strong Humanized Variants of Antibody I-394.

The parent antibody I-394, having the amino acid sequences VH and VL SEQ ID NO: 6 and 7, respectively, was modified by introducing into the VH heavy chain framework regions (FR1, FR2, FR3) from the human IGHV1-3 subgroup together with IGHJ1 * 01 ( FR4), and the introduction of light chain framework regions (FR1, FR2, FR3) from the human IGKV4-1 gene subgroup together with IGKJ4 * 01 (FR4) into the VL.

Three-dimensional models based on different segments of the human VH and VL genes were superimposed, and all amino acid differences were carefully studied one by one. In silico molecular design was challenged using 3D models of both the original chimeric (HPLP) and humanized (HOLO) antibodies. 3D models of Fab fragments were built using the Discovery Studio antibody modeling protocol (DS version 4.5).

Heavy and light chain sequences used to model a chimeric version of Fab I-394 with human IgG1 constant regions containing an Fc domain containing the N297S substitution (without #97-linked glycosylation) or the L234A/L235E/G237A/A330S/P331S substitution (retaining the # 97-linked glycosylation) were as follows.

I-394-LP (original Fab light chain)

DIVMTQSPASLAVSLGQRATISCRASESVDNFGVSFMYWFQQKPGQPPNLLIYGAS NQGSGVPARFRGSGSGTDFSLNIHPMEADDTAMYFCQQTKEVPYTFGGGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19).

I-394-HP (original Fab heavy chain):

EVKLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSPGRTLEWIGYIVPLN

GGSTFNQKFKGRATLTVNTSSRTAYMELRSLTSEDSAAYYCARGGTRFAYWGQGTLVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK (SEQ ID NO: 20).

The template structures of the heavy and light chains were identified, and references 4M7K, 1I7Z and 3D85 from the Protein Data Bank (PDB) were used for the VH/VL interface, LC and HC modeling, respectively

- 41 047070 no. The PDB used is the RCSB PDB from the Research Collaboration in Structural Bioinformatics, managed by RCSB members Rutgers and UCSD/SDSC, see Www.rcsb.org and N.M. Berman, et al. (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242. Reference PDB entry 4M7K: Teplyakov, A., et al. (2014) Proteins 82: 1563–1582. Reference PDB entry 1I7Z: Larsen, N. A., et al., (2001) J. Mol. Biol.311: 9-15. Reference PDB entry 3D85: Beyer, V. M., et al. (2008) J. Mol. Biol. 382:942-955.

The heavy and light chain sequences used to model the humanized version of Fab I-394 were as follows.

Light chain I-394 Fab L0

DIVMTQSPDSLAVSLGERATINCRASESVDNFGVSFMYWYQQKPGQPPKLLIYGAS

NQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTKEVPYTFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21).

Heavy chain I-394 Fab H0

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYIVPL NGGSTFNQKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARGGTRFAYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDK (SEQ ID NO: 22).

For the humanized version of Fab I-394, PDB references 4NWT, 4I77, and 4JPI were used to model the VH/VL, LC, and HC interface, respectively. Reference PDB entry 4NWT: Bowers, P. M., et al., (2014) J. Biol. Chem.289: 33557-33567. Reference PDB entry 4I77: Ultsch, M., et al., (2013) J. Mol.Biol. 425: 1330-1339. Reference PDB entry 4JPI: Jardine, J., et al., (2013) Science 340: 711-716.

For intermediate selection of humanized light and heavy chain variants, HPLP and HOLO 3D models were superimposed, and all amino acid differences were carefully examined one by one. Intra- and extra-strand connections between residues were also assessed to ensure that no important low-energy connection was disrupted by introducing a back mutation into a given chain. Additionally, for the humanized light and heavy chain variants, amino acids affected by the discrepancy between the Kabat and IMGT CDR numbering schemes were specifically examined in an overlay of the HPLP and HOLO 3D models; the findings prompted the design of VH variants (designated * as H2*, H3* and H4*) that retained the parent residue (tyrosine) present at residue 50 in Kabat's VH (the Kabat residue but not the CDR2 IGMT), but it was not conserved parent residues at positions 60 and 64 (both CDR2 residues according to Kabat). In a similar manner, the VL L1* variant was obtained, which did not retain the original residue at position 24 (residue CDR1 according to Kabat).

Amino acid modifications were introduced into the original sequences. The VH and VL sequences of the anti-CD39 antibody are presented in Table 1 below. A. Compared to the original VH HO from SEQ ID NO: 27, H1 contains the replacement R72V (FR3); H2 contains replacement V68A (FR3) and R72V (FR3); H2 * contains replacement V68A (FR3) and R72V (FR3), as well as replacement N61S (CDR2); H3 contains replacement M48I (FR2), V68A (FR3) and R72V (FR3); H3 contains replacements M48I (FR2), V68A (FR3) and R72V (FR3), as well as replacements N61S and K65Q in CDR2; H4 contains replacement M48I (FR2), V68A (FR3), R72V (FR3) and S77R FR3); and H4 * contains substitutions M48I (fR2), V68A (FR3), R72V (FR3) and S77R (FR3), as well as substitutions N61S and K65Q in CDR2. Compared to the parent chain L0 VL SEQ ID NO: 35, L1 contains a Y40F substitution (FR2), and L1* contains a Y40F substitution (FR2) and an R24K substitution (CDR1).

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Table A

Domain V SEQ ID Amino acid sequence VH “BUT” 27 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLE WMGYIVPLNGGSTFNQKFKGRVTITRDTSASTAYMELSSLRSEDTAVY YCARGGTRFAYWGQGTLVTVSS VH “NG” 28 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLE WMGYIVPLNGGSTFNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVY YCARGGTRFAYWGQGTLVTVS VH Ή2” 29 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLE WMGYIVPLNGGSTFNQKFKGRATITVDTSASTAYMELSSLRSEDTAVY YCARGGTRFAYWGQGTLVTVSS VH Ή3” thirty QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLE WIGYIVPLNGGSTFNQKFKGRATITVDTSASTAYMELSSLRSEDTAVYY CARGGTRFAYWGQGTLVTVSS VH Ή4” 31 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLE WIGYIVPLNGGSTFNQKFKGRATITVDTSARTAYMELSSLRSEDTAVYY CARGGTRFAYWGQGTLVTVSS VH Ή2*” 32 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLE WMGYIVPLNGGSTFSQKFKGRATITVDTSASTAYMELSSLRSEDTAVY YCARGGTRFAYWGQGTLVTVSS VH Ή3*” 33 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLE WIGYIVPLNGGSTFSQKFQGRATITVDTSASTAYMELSSLRSEDTAVYY CARGGTRFAYWGQGTLVTVSS VH Ή4*” 34 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLE WIGYIVPLNGGSTFSQKFQGRATITVDTSARTAYMELSSLRSEDTAVYY CARGGTRFAYWGQGTLVTVSS VL “L0” 35 DIVMTQSPDSLAVSLGERATINCRASESVDNFGVSFMYWYQQKPGQP PKLLIYGASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQT KEVPYTFGGGTKVEIK VL “1_G 36 DIVMTQSPDSLAVSLGERATINCRASESVDNFGVSFMYWFQQKPGQP PKLLIYGASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQT KEVPYTFGGGTKVEIK VL “L1*” 37 DIVMTQSPDSLAVSLGERATINCKASESVDNFGVSFMYWFQQKPGQPP KLLIYGASNQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTK EVPYTFGGGTKVEIK

Antibodies having the variable regions VH and VL were produced as recombinant chimeric antibodies with the silent Fc region of human IgG1 with substitutions in the heavy chain L234A/L235E/G237A/A330S/P331S (numbering according to EU Kabat), which leads to loss of binding to Fcy receptors human CD16A, CD16B, CD32A, CD32B and CD64.

Briefly, the VH and Vk sequences shown below were cloned into vectors containing the huIgG1 constant domains (containing the L234A/L235E/G237A/A330S/P331S substitutions) and the huCk constant domain, respectively. The two resulting vectors were cotransfected into CHO cells in a combinatorial manner to generate combinations of VH and VL. The created pool of cells was used for the production of antibodies in CHO medium. The antibody was then purified using protein A.

In addition to the original CDR-grafted humanized antibody (mAb1), 23 additional humanized antibody variants were constructed that contained different amino acid substitutions compared to the original CDR-grafted version. All antibody variants were successfully produced in CHO cells as human IgG1 antibodies. VH and VL of the obtained antibodies mmAb1-mAb24 are shown in table. IN.

- 43 047070

Table B

gnAb standard VH VL GPAAY HOLO But (SEQ ID NO: 27) L0 (SEQ ID NO: 35) mAL2 H0L1 BUT (SEQ ID NO: 27) L1 (SEQ ID NO: 36) TLS H0L1* BUT L1* (SEQ ID NO: 27) (SEQ ID NO: 37) mAL4 H1L0 H1 (SEQ ID NO: 28) L0 (SEQ ID NO: 35) mAL5 H1L1 H1 (SEQ ID NO: 28) L1 (SEQ ID NO: 36) tABb H1L1* H1 (SEQ ID NO: 28) L1* (SEQ ID NO: 37) tAL7 H2L0 H2 (SEQ ID NO: 29) L0 (SEQ ID NO: 35) tAL8 H2L1 H2 (SEQ ID NO: 29) L1 (SEQ ID NO: 36) tAL9 H2L1* H2 (SEQ ID NO: 29) L1* (SEQ ID NO: 37) tAL10 H2*L0 H2* (SEQ ID NO: 32) L0 (SEQ ID NO: 35) tAL11 H2*L1 H2* (SEQ ID NO: 32) L1 (SEQ ID NO: 36) tAL12 H2*L1* H2* (SEQ ID NO: 32) L1* (SEQ ID NO: 37) tAL13 H3L0 H3 (SEQ ID NO: 30) L0 (SEQ ID NO: 35) tAL14 H3L1 H3 (SEQ ID NO: 30) L1 (SEQ ID NO: 36) tAL15 H3L1* H3 (SEQ ID NO: 30) L1* (SEQ ID NO: 37) tAL16 H3*L0 H3* (SEQ ID NO: 33) L0 (SEQ ID NO: 35) tAL17 H3*L1 H3* (SEQ ID NO: 33) L1 (SEQ ID NO: 36) tAL18 H3*L1* H3* (SEQ ID NO: 33) L1* (SEQ ID NO: 37) tAL19 H4L0 H4 (SEQ ID NO: 31) L0 (SEQ ID NO: 35) tAL20 H4L1 H4 (SEQ ID NO: 31) L1 (SEQ ID NO: 36)

- 44 047070

tAL21 H4L1* H4 (SEQ ID NO: 31) Li* (SEQ ID NO: 37) tAL22 H4*L0 H4* (SEQ ID NO: 34) L0 (SEQ ID NO: 35) tAL23 N4*1_1 H4* (SEQ ID NO: 34) L1 (SEQ ID NO: 36) tAL24 H4*1_1* H4* (SEQ ID NO: 34) Li* (SEQ ID NO: 37)

Antibodies mAb1-21 were assessed for binding to CD39 by flow cytometry as described in Example 7 using human CD39-expressing CHO cells and macaque CD39-expressing CHO cells (macaca fascicularis). All mAbs showed comparable binding to the parent antibody I-394 in human and macaque CHO CD39 cell lines.

Example 14. Activity of humanized variants of antibody I-394.

MAbs1-24 were further evaluated for binding and inhibition of CD39 using various assays, including binding to CD39 present on Ramos tumor cell lines that were found to express particularly high levels of CD39, inhibition of the enzymatic activity of recombinant produced pCD39, and inhibition enzymatic activity of the pCD39 protein isolated in cell culture supernatants from CHO cells expressing human CD39 and from Ramos tumor cells.

Antibodies were titrated on Ramos lymphoma cells by flow cytometry according to the methods described in Example 7. The results showed that all L0 variants bound less strongly on the Ramos cell line compared to the parent antibody I-394, while antibodies H2L1, H2L1*, H4L1 and H4L1* (mAbs8, 9, 20 and 21, respectively) showed the best binding and were all similar to the parent antibody I-394. See fig. 13.

Antibodies were tested for the ability to inhibit the ATPase activity of soluble CD39 protein using assays used to inhibit the enzymatic activity of soluble CD39 protein as described above (Methods). All antibodies showed good activity, with H3L1, H3L1*, H4L1 and H4L1* (mAbs14, 15, 20 and 21, respectively) being comparable to the parent antibody I-394, while the other antibodies had slightly lower activity. The antibodies were also tested for the ability to inhibit the ATPase activity of soluble CD39 protein released in cell culture supernatant from CHO cells expressing human CD39 using assays used to inhibit the enzymatic activity of soluble CD39 as described above (Methods). All antibodies showed good activity, with H4L1 and H4L1* (mAb20 and mAb21) being comparable to the parent antibody I-394, while the other antibodies had slightly lower activity.

Antibodies were tested to evaluate their effectiveness in reducing T cell suppression in the assay described in Example 12. When testing the 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 mDC/4 T cells) for a mixed lymphocyte reaction (MLR) for 5 days. T cell activation and proliferation were analyzed by CD25 expression and Cell Trace Violet dilution by flow cytometry. The results showed that antibodies with H2, H3 or H4 heavy chains combined with L1 light chains were as effective as the parent antibody I-394, while antibodies with L0 light chains were less effective.

Antibodies were tested for their ability to inhibit ATPase activity in cell lines expressing membrane-bound CD39. Antibody inhibition of CD39 ATPase activity in CD39-expressing cells was assessed using an assay used to inhibit cellular CD39 enzymatic activity as described above (Methods). The antibodies were first evaluated in CHO cells expressing human CD39; under these conditions, no significant differences were observed between the mAb1-24 variants and the parent antibody I-394 on transfected CHO cell lines. However, when the antibodies were evaluated on the Ramos and Mino tumor cell lines, the H4L1 and H4L1* antibodies (mAb20 and mAb21) were more potent in blocking CD39 enzymatic activity compared to all other antibodies. In fig. Figure 14 shows the results on Mino cells. Differences observed between transfectant and tumor cell assays may arise from the particularly high expression of CD39 found in such tumor cell lines compared to CHO cells, allowing for differences in potency between antibodies to be identified. Antibodies having a heavy chain H4* and light chains L1 or L1* were also tested on Ramos tumor cells; in this case, the H4* antibodies mAb23 and mAb24 had slightly lower efficacy than mAb20 and mAb21. Thus, the most potent inhibitors of tumor cell-derived pCD39 (e.g., in tumor cells expressing high levels of CD39) were

- 45 047070 H4 heavy chain antibodies, followed by H2 heavy chain antibodies, followed by H3 heavy chain antibodies, in each case with L1 light chain. One possible explanation is that there is a deleterious back mutation (BM) in the H3 variant that makes them less potent than the H2 variants, but which is in turn counterbalanced by a substitution in the H4 heavy chain variants that restores activity to baseline levels I-394. Thus, the most effective antibodies among the humanized variants were the H4L1 antibody (having VH SEQ ID NO: 31 and VL SEQ ID NO: 36) and the H4L1* antibody (having VH SEQ ID NO: 31 and VL SEQ ID NO: 37). MAb20 has the corresponding heavy and light chain CDRs SEQ ID NO: 8-13, with the heavy chain framework regions (FR1, FR2, FR3) from the human IGHV1-3 gene together with the IGHJ1*01 gene (FR4), and the following substitutions (numbered according to Kabatu): M48I (FR2), V68A (FR3), R72V (FR3) and S77R (FR3); and light chain framework regions (FR1, FR2, FR3) from the human IGKV4-1 gene subgroup together with IGKJ4*01 (FR4) and the Y40F substitution (FR2). MAb21 additionally contains a substitution in the light chain CDR1 at residue 24 according to Kabat (R24K substitution ).

The complete heavy chain of the H4L1 antibody (mAb20) with substitutions L234A/L235E/G237A/A330S/P331S is shown below:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWIGYIVPLNGGST

FNQKFKGRATITVDTSARTAYMELSSLRSEDTAVYYCARGGTRFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLF PPKPKDTLMISRTPEV TCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQ LSLSPGK (SEQ ID NO: 38).

The complete light chain of the H4L1 antibody (mAb20) is shown below:

DIVMTQSPDSLAVSLGERATINCRASESVDNFGVSFMYWFQQKPGQPPKLLIYGASNQGS

GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTKEVPYTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 39).

The complete light chain of the H4L1* antibody (mAb21) is shown below:

DIVMTQSPDSLAVSLGERATINCKASESVDNFGVSFMYWFQQKPGQPPKLLIYGASNQGS

GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTKEVPYTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40).

Example 15: Stability of Humanized Variants of Antibody I-394.

Prior art mAb1-24 and anti-CD39 BY40, all produced as human IgG1 isotypes with L234A/L235E/G237A/A330S/P331S substitutions, were tested for stability in the following reference formulation at a concentration of approximately 7 mg/mL: pH 6.0 .; histidine buffer (10 mM); sucrose (200 mM); NaCl (50 mM); Polysorbate 80 (PS80) (0.2 g/l). The stability of the compositions was monitored under two storage conditions (at +5±3°C and at +40±3°C. For each study, 3 time points were carried out: T0, T15D (15 days) and T1M (1 month). For format comparison freeze-thaw (F/T) and thermal shift stability analysis (TSSA) were performed. To perform F/T cycles, samples were frozen for at least 2 hours at -20°C and thawed for at least 1 hour at room temperature, F/T cycle. was repeated three times and samples were tested 24 hours after the last freeze/thaw cycle. The following were tested at each time point:

Particulate matter (MFI).

Visual Inspection (appearance).

Impurities (SE-HPLC).

Turbidity (400 Nm).

Protein concentration (280 Nm) (performed using Nanodrop, Thermo Fisher Scientific Inc.).

The resulting antibodies H2L1 (mAb 8), H2L1* (mAb9), H4L1 (mAb20) and H4L1* (mAb21) showed good physicochemical stability. The aggregation temperature (T _Agg ) is shown in FIG. 15A for parent antibody I-394 compared to antibody BY40 and FIG. 15B for antibodies having combinations of H2L1, H2L1*, H4L1 or H4L1* chains. Antibodies I-394 and H2L1, H2L1*, H4L1 and H4L1* each showed a T _Agg approaching 70°C. Compared to BY40, which has a T _Agg closer to 60°C, antibodies H2L1, H2L1*, H4L1 and H4L1* showed a significant advantage in stability. One possible reason for the relatively low internal stability of the BY40 antibody is the numerous aromatic amino acid residues on the surface of the mAb, located in the CDRs,

- 46 047070 especially in the CDR3 of the heavy chain, which give the BY40 antibody a relatively high predicted hydrophobicity.

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

When the singular is used, the plural of the noun is also implied unless specifically stated otherwise. Unless otherwise stated, all precise values presented herein are representative of the corresponding approximate values (e.g., all precise approximate values presented for a particular factor or measurement may be considered to also provide the corresponding approximate measurement, modified approximately where appropriate ).

The description of any aspect or embodiment in this application using terms such as comprising, having, including or containing with reference to an element or elements is intended to provide support for a similar aspect or embodiment in this application which consists of, consists in fact of or essentially contains that particular element or elements unless otherwise indicated or clearly inconsistent with the context (e.g., a composition described herein as containing a particular element should be understood to also describe a composition consisting of that element unless otherwise indicated or is clearly consistent with the context).

The use of any and all examples or exemplary statements (eg, such as) set forth in this application is intended only to better illuminate the invention and does not limit the scope of the invention unless otherwise stated. Nothing in the specification should be construed as indicating any element not claimed as essential to the practice of the invention.

Claims (15)

1. An antibody or antibody fragment that binds a human CD39 polypeptide and that is capable of inhibiting the ATPase activity of a soluble extracellular domain of a human CD39 polypeptide, wherein said antibody or antibody fragment comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 31, and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 36 or 37. 2. An antibody or antibody fragment according to claim 1, wherein said antibody or antibody fragment comprises a heavy chain containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 38, and a light chain containing the amino acid sequence , at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 39 or 40. 3. An antibody or antibody fragment according to claim 1 or 2, characterized in that said antibody or antibody fragment contains the amino acid sequences of the heavy chain framework regions FR1, FR2 and FR3 from the human IGHV1-3 gene; and the amino acid sequences of the light chain framework regions FR1, FR2 and FR3 from the human IGKV4-1 gene. 4. An antibody or antibody fragment, wherein said antibody or antibody fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain containing the amino acid sequence of SEQ ID NO: 39. 5. An antibody or antibody fragment, wherein said antibody or antibody fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain containing the amino acid sequence of SEQ ID NO: 40. 6. An antibody or antibody fragment according to any of the previous paragraphs, characterized in that said antibody is capable of inhibiting the ATPase activity of the pCD39 protein secreted into the cell culture supernatant by CD39-expressing human tumor cells. 7. An antibody or antibody fragment as claimed in any one of the preceding claims, wherein said antibody inhibits the ATPase activity of the CD39 protein in the presence of exogenously added ATP, wherein optionally the exogenously added ATP is provided at a concentration of 20 μM. 8. An antibody or antibody fragment according to any of the preceding claims, characterized in that said antibody is capable of binding human CD39 protein on the surface of a cell and is capable of inhibiting the ATPase activity of said CD39 protein on the surface of a cell. 9. An antibody or antibody fragment according to any of the preceding claims, characterized in that said antibody is capable of causing an increase in the expression of a cell surface activation marker in monocyte-derived dendritic cells when such mDCs are incubated in vitro with said - 47 047070 antibody and ATP, wherein exogenously added ATP is provided at a concentration of 0.125, 0.25 or 0.5 mM. 10. The antibody or antibody fragment of any one of the preceding claims, wherein said antibody is an antibody having a human Fc domain that is modified to reduce binding between said Fc domain and a human Fcy receptor. 11. An antibody or antibody fragment as claimed in any one of the preceding claims, wherein said antibody comprises an Fc domain, wherein the Fc domain optionally contains an amino acid modification relative to the wild type Fc domain that reduces or abolishes binding to one or more or with all human Fcy receptors CD16A, CD16B, CD32A, CD32B and/or CD64. 12. A pharmaceutical composition containing an antibody according to any of the previous paragraphs and a pharmaceutically acceptable carrier. 13. Nucleic acid encoding the heavy and light chain of an antibody or antibody fragment according to any one of paragraphs. 1-11. 14. A recombinant host cell producing an antibody or antibody fragment according to any one of claims 1 to 11. 15. Use of an antibody or antibody fragment according to any one of claims 1 to 11 or a composition according to claim 12 in the treatment of cancer.