EA045378B1 - INCREASED EFFECT OF ADENOSINE TRIPHOSPHATE (ATP) RELEASE - Google Patents

Description

Cross reference to related applications

The application for this invention claims priority to US Provisional Application Nos. 62/586,224 filed November 15, 2017, 62/686,149 filed June 18, 2018, and 62/733,175 filed September 2018; all of which are incorporated herein by reference in their entirety, including any drawings.

Link to sequence list

The application for this invention is filed together with a sequence listing in electronic format. The sequence listing is presented as a file called CD39-8_ST25, created on November 14, 2018, which is 64 KB in size. The information provided electronically in the sequence listing is incorporated herein by reference in its entirety.

Technical field

The present invention relates to the use of CD39 neutralizing agents for the treatment of cancer.

State of the art

NTPDase 1 (ectonucleoside triphosphate diphosphohydrolase 1), also known as CD39/ENTPD1 or vascular CD39, functions in conjunction with another enzyme CD73 (ecto-5'-nucleotidase) to hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to form adenosine, which binds to adenosine receptors and inhibits the responses of T cells and natural killer cells (NK cells), thereby suppressing the immune system. Generation of adenosine in the CD73/CD39 pathway is recognized as a major mechanism of regulatory immunosuppressive function of T cells (regulatory T cells, Tregs). CD39 has two transmembrane domains near the N- and C-terminal regions, short cytoplasmic N- and C-terminal regions, and a large extracellular domain containing the active site. However, although CD39 is typically membrane-anchored by two transmembrane domains at the two ends of the molecule, it has also recently become known that a soluble, catalytically active form of CD39 can be found in circulation in humans and mice (Yegutkin et al., (2012), FASEB J. 26( 9):3875-3883).

Radiation therapy and some chemotherapeutic agents have been shown to induce a specific immune response leading to immunogenic death of cancer cells (Martins et al. 2009, Cell Cycle, 8(22):3723-3728). The antitumor immune response induced by these therapies depends on the ability of dendritic cells (DCs) to present antigen from dying cancer cells and to prime tumor-specific cytotoxic T lymphocytes (CTLs). To provide a CTL response, DCs must absorb antigens from stressed or dying cells, acquire the ability to process antigens at the stage of their maturation, and present antigenic peptides associated with MHC molecules (major histocompatibility complex) in the context of co-stimulatory signals and cytokines that stimulate differentiation/ activation of specific CTLs.

However, there remains a need to improve the effectiveness of current treatments designed to kill cancer cells, including radiation therapy and chemotherapy agents.

Brief description of the invention

The present invention originates, inter alia, from the discovery that antibodies that neutralize the ATPase activity of the CD39 protein in the presence of significant concentrations of ATP are able to reverse the immunosuppressive effect of CD39 on dendritic cells (DCs) in the presence of exogenously added ATP. In addition, antibodies are capable of inducing or increasing the proliferation of T cells co-cultured with DCs. The ability to reduce CD39-mediated inhibition of DC activation provides advantages when using antibodies in combination with agents or treatments that induce extracellular ATP release from tumor cells, particularly agents or treatments that induce immunogenic death of cancer cells, e.g. that cause the death of tumor cells (inter alia, chemotherapeutic agents, radiation therapy). Although ATP release has the potential to be immunogenic and promote DC activation, ATP may also be subject to catabolism by CD39, which in turn suppresses the immunogenic effect of extracellular ATP. In addition, during chemotherapy, increased ATP levels are associated with increased expression of CD39 on the surface of DCs. In an ATP-enriched tumor microenvironment, infiltrating DCs can promote ATP degradation by modulating CD39 expression, which in turn reduces the rate of immunogenic tumor cell death induced by chemotherapy. Thus, when used in combination with anti-CD39 antibodies, it is possible to enhance the immunogenic effect of agents or treatments that cause death (eg, apoptosis or necrosis) of tumor cells.

Accordingly, one aspect of the present invention provides improved methods of enhancing an antitumor immune response through the use of antibodies that bind to CD39 and neutralize CD39 in the presence of ATP, in combination with an agent or therapy that causes tumor cell death, such as an agent or therapy that is capable of causing extracellular release of ATP from tumor cells, agent or therapy that causes immunogenic death

- 1,045,378 cancer cells. In one aspect, the present invention provides improved methods for enhancing an antitumor immune response by using antibodies that are capable of binding to CD39 and neutralizing CD39 in the presence of ATP, in combination with an agent that causes tumor cell death, such as an agent that causes apoptosis and/or extracellular release of ATP from tumor cells. In one embodiment of the present invention, an agent or therapy (or agent) that is capable of causing extracellular ATP release from tumor cells includes an anthracycline, oxaliplatin, cisplatin, x-ray, PARP inhibitor, taxane, anthracycline, DNA- noxious agent, camptothecin, epothilone, mitomycin, combretastatin, vinca alkaloid, nitrogen mustard, maytansinoid, calicheamicin, duocarmycin, tubulisin, dolastatin, auristatin, enediine, amatoxin, pyrrolebenzodiazepine, ethyleneimine, radioactive isotope, therapeutic protein or peptide toxin, or antibody that binds antigen expressed by the tumor cell and which mediates antibody-dependent cellular cytotoxicity (ADCC). In one embodiment of the present invention, an antibody that is capable of binding to CD39 and neutralizing CD39 is capable of neutralizing the activity of both soluble CD39 extracellular domain protein (sCD39) and membrane-bound CD39 protein (memCD39). As demonstrated herein, antibodies that are capable of reversing the immunosuppressive effect of CD39 on dendritic cells (DCs) in the presence of exogenously added ATP are also characterized by the ability to neutralize the activity of both soluble CD39 extracellular domain protein (sCD39) and membrane-bound CD39 protein (memCD39). ). Notably, the BY40 antibody, which fails to reverse the immunosuppressive effect of CD39 on dendritic cells (DCs) in the presence of exogenously added ATP, also fails to neutralize the ATPase activity of soluble CD39 extracellular domain protein (sCD39) and has a lower maximum inhibitory effect on membrane ATPase activity. -associated protein CD39 (memCD39).

Without wishing to be bound by theory, the present inventors believe that antibodies that neutralize membrane-bound CD39 on the cell surface act by inhibiting the movement of membrane-bound CD39 (memCD39) domains, but do not have such an effect on soluble protein activity CD39 (sCD39). It is reported that memCD39 occurs as homomultimers (e.g., tetramers and/or other multimers other than monomeric forms), whereas SCD39 is a monomer, and that the transmembrane domains in memCD39 move dynamically, which underlies their functional association with the active site ( Schulte am Esch et al. 1999, Biochem 38(8):2248-58). Antibodies that block only memCD39 can recognize CD39 outside the enzyme's active site and prevent multimerization without blocking the monomeric form of CD39. Blocking multimerization can reduce enzyme activity, and multimerization of CD39 has been reported to significantly increase ATPase activity. Conversely, antibodies that also block SCD39 may interfere with the CD39 substrate and inhibit the monomeric form of the enzyme. Such antibodies may also prevent memCD39 multimerization, thereby providing a second mechanism for inhibiting CD39 enzymatic activity. In the presence of ATP (eg, as in the tumor environment), partial inhibition of CD39 by preventing multimerization without blocking sCD39 may result in the presence of residual AMP in sufficient quantities to prevent a detectable additive effect on DC activation. Therefore, antibodies that bind to and inhibit the ATPase activity of monomeric and/or soluble CD39 (e.g., monomeric SCD39) may be advantageously used to achieve greater neutralization of CD39 activity in an individual by neutralizing both membrane-bound and soluble CD39 protein (extracellular domain protein in solution).

In one aspect of the present invention, there is provided an agent that binds CD39 and inhibits the enzymatic (ATPase activity) activity of the human CD39 protein for use in the treatment of cancer, wherein the agent that binds CD39 is administered in combination with an agent that causes extracellular release of ATP from tumor cells , optionally an agent that causes tumor cell death, optionally inducing apoptosis and/or necrosis. In another aspect of the present invention, there is provided an agent that causes the extracellular release of ATP from tumor cells, optionally an agent that causes the death of tumor cells, optionally inducing apoptosis and/or necrosis, for use in the treatment of cancer, wherein the agent that causes the extracellular release of ATP from tumor cells cells are administered in combination with an agent that binds to CD39 and inhibits the enzymatic (ATPase activity) activity of the human CD39 protein.

In one embodiment, the present invention provides a method of treating or preventing cancer in an individual, the method comprising administering to the individual (a) an agent that binds to the CD39 protein and inhibits the ATPase activity of the CD39 protein, and (b) an agent that is capable of causing extracellular ATP release from tumor cells.

In one embodiment, the present invention provides a method of enhancing the antitumor effect of an antibody that is capable of binding to CD39 and inhibiting the ATPase activity of CD39 in the presence of exogenously added ATP, the method comprising administering an agent or therapy that causes extracellular release of ATP from tumor cells and/or

- 2 045378 death of tumor cells.

In one embodiment, the present invention provides a method of treating cancer in an individual who has a weak immune response or has been diagnosed with a weak immune response to treatment with an agent or a treatment that causes extracellular release of ATP from tumor cells and/or death of tumor cells (in the absence of combination anti-inflammatory treatment). -CD39 antibody), the method comprising administering to an individual an antibody that is capable of binding to CD39 and inhibiting the ATPase activity of CD39 in the presence of exogenously added ATP.

In one embodiment of the present invention, an agent that is capable of causing the extracellular release of ATP from tumor cells directly causes apoptosis of the tumor cells. In one embodiment of the present invention, an agent that is capable of causing the extracellular release of ATP from tumor cells directly causes necrosis of the tumor cells. In one embodiment of the present invention, the agent includes a cytotoxic agent that directly causes the death of tumor cells, optionally a chemotherapeutic agent used in the treatment of cancer. In one embodiment of the present invention, the agent comprises a depleting antibody. In one embodiment of the present invention, the agent comprises an immunoconjugate comprising an antibody that specifically binds to a protein expressed by a tumor cell and a cytotoxic agent. In one embodiment of the present invention, the agent includes an antibody that specifically binds to a protein expressed by a tumor cell and that is not conjugated to a cytotoxic agent (eg, a naked antibody). Optionally, the antibody is capable of directly inducing apoptosis of tumor cells.

In one embodiment of the present invention, an agent that binds to CD39 and inhibits the ATPase activity of the human CD39 protein is capable of neutralizing the ATPase activity of CD39 in the presence of exogenously added ATP.

In one embodiment of the present invention, an agent that binds to CD39 and inhibits the ATPase activity of a human CD39 protein is capable of neutralizing the ATPase activity of a soluble human CD39 extracellular domain protein. Optionally, the agent is capable of neutralizing the ATPase activity of soluble human CD39 extracellular domain protein in the presence of exogenously added ATP, where optionally ATP is added at a concentration of 20 μM. Assays may be, for example, as shown in the examples herein, for example, the anti-CD39 antibody is incubated in soluble recombinant human CD39 protein plates for 1 hour at 37° C. before adding CTG reagent (CellTiter Glo ) 20 μM ATP was added to the plates for an additional 30 minutes at 37°C, and the amount of light emitted was quantified using an Enspire™ luminometer after a short incubation period of 5 minutes in the dark.

Optionally, the antibody is capable of causing a reduction in the ATPase activity of human CD39 extracellular domain protein in solution by more than 50%, optionally by more than 60, 70, 75 or 80%.

In one embodiment of the present invention, an agent that binds to CD39 will provide a reduction in the ATPase activity of soluble human CD39 protein by at least 50, 60, 70, 75, 80, or 90% (e.g., when assessed by the methods described herein), optionally further in a concentration compatible with administration of the antibody to a human.

In one embodiment of the present invention, an agent that binds to CD39 and inhibits the ATPase activity of the human CD39 protein is capable of increasing cell surface activation marker expression in monocyte-derived dendritic cells (moDCs) when such moDCs are incubated in vitro with antibody and ATP, wherein the concentration of exogenously added ATP is optionally 0.125, 0.25 or 0.5 mM.

In one embodiment of the present invention, an agent that binds to CD39 and inhibits the ATPase activity of the human CD39 protein is capable of binding to and neutralizing the ATPase activity of the human CD39 protein on the surface of a cell. In one embodiment of the present invention, the agent is capable of enhancing the activation of dendritic cells in the presence of ATP. In one embodiment of the present invention, the agent is capable of increasing cell surface expression of an activation marker in monocyte-derived dendritic cells when such moDCs are incubated in vitro with antibody and ATP. Optionally, ATP is ATP exogenously added at a concentration of 0.125, 0.25 or 0.5 mM. Optionally, the increase in cell surface activation marker expression is assessed by incubating the moDC in the presence of ATP for 24 hours and analyzing the cell surface expression of CD80, CD83 and/or HLA-DR on the moDC surface by flow cytometry. Optionally, the increase in cell surface marker expression is at least 40, 50, 75, or 80% compared to a negative control (eg, medium).

In one aspect of any embodiment of the present invention, the agent that inhibits or neutralizes the ATPase activity of the CD39 protein is or contains an antibody or antibody fragment that binds the CD39 protein (eg, a monospecific antibody, a bispecific antibody, or a multispecific antibody).

Combination use of agents will be useful in promoting an adaptive immune response against a tumor by increasing the amount of available ATP in the tumor microenvironment. Next

- 3 045378 Therefore, these antibodies will be useful in reversing the immunosuppressive effect of CD39 on dendritic and/or T cell activity. In one embodiment of the present invention, the described methods are used to enhance or enhance antitumor immunity, reduce immunosuppression, enhance the adaptive antitumor immune response, or activate and/or enhance the activity of DCs, tumor-infiltrating T cells, and/or tumor-specific T cells in an individual.

In one aspect of any embodiment of the present invention, the sCD39 protein can be characterized as lacking two transmembrane domains (ie, transmembrane domains near the N- and C-terminal regions) found in membrane-bound CD39. In one embodiment of the present invention, sCD39 is a non-membrane-bound sCD39 protein found in the bloodstream, for example, in humans. In one embodiment of the present invention, sCD39 contains or consists of the amino acid sequence of SEQ ID NO: 2 (optionally further comprising a C-terminal tag or other amino acid sequence derived from non-CD39), such as the sCD39 protein obtained in the examples provided in this description. In one embodiment of the present invention, the protein, antibody, or antibody fragment inhibits or neutralizes the ATPase activity of SCD39 when incubated with sCD39 in solution, for example, in accordance with the methods disclosed herein. In one embodiment of the present invention, the protein, antibody, or antibody fragment specifically binds to human CD39 protein in both soluble (extracellular domain protein) and membrane-bound forms.

In one aspect of any embodiment of the present invention, the individual may be defined as a human.

In one embodiment of the present invention, the anti-CD39 antibody is administered in a therapeutically effective amount.

In one embodiment of the present invention, an anti-CD39 antibody is administered to an individual having cancer in an amount and frequency sufficient to neutralize the activity of CD39 (sCD39 and/or memCD39) at the periphery and/or within the tumor microenvironment. In one embodiment of the present invention, the antibody is administered in an amount and frequency sufficient to reduce ATP catabolism in the tumor microenvironment. Optionally, the antibody is administered in an amount and at a frequency sufficient to provide sustained inhibition of CD39 (sCD39 and/or memCD39) activity at the periphery and/or within the tumor microenvironment and/or continuous reduction of ATP catabolism in the tumor microenvironment during the period of time between two successive administrations an agent that can cause extracellular release of ATP from tumor cells.

In one embodiment of the present invention, an agent capable of causing extracellular release of ATP from tumor cells is administered in a therapeutically effective amount. In one embodiment of the present invention, an agent capable of inducing the extracellular release of ATP from tumor cells is administered to an individual having cancer in an amount and frequency sufficient to cause tumor cell death, apoptosis and/or necrosis. In one embodiment of the present invention, an agent capable of inducing extracellular ATP release from tumor cells is administered to an individual having cancer in an amount and frequency sufficient to induce extracellular ATP release in the tumor microenvironment.

In one embodiment of the present invention, an anti-CD39 antibody and an agent capable of causing extracellular ATP release from tumor cells are each administered for at least one application cycle, wherein the application cycle includes at least the first and second (and optionally 3- , 4, 5, 6, 7 and/or 8 or more) times of administration of an anti-CD39 antibody and an agent capable of causing extracellular release of ATP from tumor cells.

In one embodiment of the present invention, the cancer is leukemia, glioma, or glioblastoma, or cancer of the bladder, breast, colon, esophagus, kidney, liver, lung, ovary, uterus, prostate, pancreas, stomach, cervix, thyroid, head and neck (squamous cell carcinoma of the head and neck), and skin (eg melanoma). In one embodiment of the present invention, the cancer is a progressive and/or refractory solid tumor. In one embodiment of the present invention, the cancer is a progressive and/or refractory solid tumor. In one limiting embodiment of the present invention, the cancer (e.g., advanced refractory solid tumor) is selected from the group consisting of non-small cell lung cancer (NSCLC), kidney cancer, pancreatic or esophageal adenocarcinoma, breast cancer, renal cell carcinoma (RCC), melanoma, colorectal cancer and ovarian cancer (and optionally one other type of cancer described in this document).

In some optional aspects of the present invention, an anti-CD39 agent can be used in the treatment of cancer in an individual having a cancer or tumor characterized by immunosuppression, optionally absent or insufficient immune infiltrate in the tumors, optionally absent or insufficient antitumor immunity.

In certain optional aspects of the present invention, the methods of treatment described in

- 4 045378 herein can be used to treat cancer in an individual with a poor prognosis of the disease, in particular, a poor prognosis of response to treatment with an anticancer agent, for example an agent capable of causing extracellular release of ATP from tumor cells, a cytotoxic agent, a chemotherapeutic agent or an agent that inhibits the enzymatic activity of CD39. An individual with a poor prognosis for a disease, for example, is at higher risk of progression of that disease based on one or more prognostic factors. In one embodiment of the present invention, the prognostic factor(s) include the presence or absence of mutations in one or more genes. In one embodiment of the present invention, the prognostic factor(s) include the expression level(s) of one or more genes or proteins, or exemplary inhibitory or activating receptors on immune effector cells. In one embodiment of the present invention, the prognostic factor(s) include the presence (e.g., number) of cells in the bloodstream or tumor environment expressing CD39, and/or levels of CD39 expression on the surface of cells in the bloodstream or tumor environment ; in one embodiment of the present invention, the cells are tumor cells; in one embodiment of the present invention, the cells are white blood cells, eg, B cells, regulatory T cells (Treg); in one embodiment of the present invention, the cells are dendritic cells. Increased expression of CD39 and/or increased number of CD39-expressing cells may indicate that an individual has a poor prognosis for response to treatment with a CD39 neutralizing antibody.

In any aspect of the present invention, the individual may be an individual who does not respond to treatment, or who has had a partial or incomplete response to treatment with an agent capable of causing extracellular ATP release from tumor cells, or who has had disease relapse or disease progression after treatment with the agent , capable of inducing extracellular release of ATP from tumor cells.

In one aspect of the present invention, an anti-CD39 agent is provided for use in the treatment of cancer in an individual who is not responding to treatment, or who has had a partial or incomplete response to treatment with an agent capable of causing extracellular release of ATP from tumor cells, or whose disease has relapsed or progressed after treatment with an agent capable of inducing extracellular ATP release from tumor cells. In one embodiment of the present invention, an anti-CD39 agent is used in combination with a therapy (eg, an agent) capable of inducing extracellular ATP release from tumor cells. Optionally, the anti-CD39 agent is capable of binding to and inhibiting the ATPase activity of CD39 in the presence of ATP and/or is capable of binding to soluble human CD39 extracellular domain protein and inhibiting the ATPase activity of soluble human CD39 extracellular domain protein.

In one embodiment of the present invention, the anti-CD39 agent competes for binding to an epitope or determinant on CD39 with antibody I-394, I-395, I-396, I-397, I-398, or I-399. In one embodiment of the present invention, the anti-CD39 agent competes for binding to CD39 with an antibody having the heavy and light chains of SEQ ID NOs: 37 and 38, respectively. The agent may be, for example, a human or humanized anti-CD39 antibody. In one embodiment of the present invention, the anti-CD39 antibody is an antibody comprising the heavy chain CDR regions of the heavy chain SEQ ID NO: 37 and the light chain CDR regions of the light chain SEQ ID NO: 37, respectively. In one embodiment of the present invention, the anti-CD39 antibody comprises a heavy chain comprising an amino acid sequence that is at least 60, 70, 75, 80, 85 or 90% identical to the amino acid sequence of the heavy chain of SEQ ID NO: 37, as well as a light chain, containing an amino acid sequence that is at least 60, 70, 75, 80, 85 or 90% identical to the light chain amino acid sequence of SEQ ID NO: 38, respectively.

In some optional aspects of the present invention, individuals may be identified as suitable for treatment with a CD39-neutralizing agent and an agent capable of inducing extracellular ATP release from tumor cells by assessing whether the patient is a poor responder (poor prognosis for response) to therapy with the anticancer agent ( for example, a composition containing a cytotoxic compound, a chemotherapeutic agent, an antibody depleting agent). A patient with a poor response can be treated with a combination of a CD39-neutralizing agent and an agent capable of inducing extracellular ATP release from tumor cells.

In some optional aspects of the present invention, patients can be identified as suitable for treatment with a CD39-neutralizing agent by assessing the presence of extracellular ATP in a tumor sample (e.g., tumor tissue and/or tumor-adjacent tissue), optionally where a predetermined concentration of ATP indicates that It is optional that an individual is suitable for treatment with an anti-CD39 agent, wherein the extracellular ATP concentration is at least 0.01, 0.02, 0.05, 0.125, 0.25 or 0.5 mM.

In other embodiments of the present invention, the treatment methods described herein can be used in combination with any other suitable treatment methods. In one

- 5 045378 embodiment of the present invention, the methods of treatment described herein further comprise administering to the individual an agent, optionally an antibody, that neutralizes the inhibitory activity of human PD-1. In one embodiment of the present invention, the methods of treatment described herein further comprise administering to the individual an agent, optionally an antibody, that neutralizes the 5'-ectonucleotidase activity of the human CD73 protein.

In other embodiments, the present invention provides pharmaceutical compositions and kits, as well as methods for using them. In one embodiment, the present invention provides a pharmaceutical composition comprising a compound that neutralizes the ATPase activity of human CD39 polypeptide and an agent capable of causing extracellular ATP release from tumor cells. In one embodiment, the present invention provides a kit comprising a compound that neutralizes the inhibitory activity of human CD39 polypeptide and an agent capable of causing extracellular ATP release from tumor cells.

In other embodiments, the present invention provides methods for predicting or assessing the effectiveness/suitability of an anticancer agent for combination use with an antibody that is capable of binding to soluble human CD39 extracellular domain protein and inhibiting the ATPase activity of soluble human CD39 extracellular domain protein, the methods comprising determining or evaluating (e.g., in vitro), whether the anticancer agent causes extracellular release of ATP from cells (e.g., tumor cells), where a determination that the anticancer agent causes extracellular release of ATP from cells (e.g., tumor cells) indicates that the agent can used in the treatment of cancer in combination with the specified antibody, which is capable of binding to soluble human CD39 extracellular domain protein and inhibiting the ATPase activity of soluble human CD39 extracellular domain protein. Determining or assessing whether an anticancer agent induces extracellular ATP release from tumor cells may involve, for example, contacting cells (eg, tumor cells) with the agent in vitro and assessing extracellular ATP release.

These aspects are more fully described in the description of the invention according to this application, and additional aspects, features and advantages will become clear from the description of the invention proposed herein.

Brief description of drawings

In fig. 1 shows a representative screen showing antibodies I-397, I-398 and I-399 compared to antibody I-394, which is a positive control.

In fig. 2A shows that antibodies BY40, I-394, I-395 and I-396 inhibit cell membrane-bound CD39, with I-394 and I-395 showing greater activity at all concentrations, as well as greater maximum inhibition of cellular CD39 compared to BY40.

In fig. 2B shows that antibodies I-395 and I-396 inhibit soluble CD39 compared to negative control (BY40) and positive control (I-394) antibodies.

In fig. Figure 3A shows the position of the residues mutated in mutant forms 5 (M5), 15 (M15) and 19 (M19) on the surface of the CD39 protein.

In fig. Figure 2B 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 (CHO-huCD39), 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 has a high ability to block the enzymatic activity of CD39 in tumor cells (Ramos lymphoma), in cells expressing human CD39 (CHO-huCD39), and in cells expressing cynomolgus CD39 (CHO-cyCD39), according to evaluation of determining the number of luminescence units proportional to the amount of ATP present.

In fig. 6 shows that antibody I-394 has a high ability to block the enzymatic activity of soluble recombinant human CD39 protein, as assessed by determining the number of luminescence units proportional to the amount of ATP present.

In fig. 7 shows that antibody I-394 binds to human CD39, but not to one of the human CD39 isoforms-L1, -L2, -L3 or -L4, as assessed by enzyme-linked immunosorbent assay (ELISA).

In fig. Figure 8 shows the experimental procedure to evaluate 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 (at a ratio of 1 moDC/4 T cells) to perform a mixed lymphocyte reaction (MLR) for 5 days. T cell activation and proliferation were analyzed through CD25 expression and the CellTrace™ Violet flow cytometry kit.

In fig. Figure 9 shows HLA-DR expression on moDCs.

In fig. 10 shows the expression of CD83 on moDCs.

Fig. 9 and 10 show that the anti-CD39 blocking antibody I-394 and chemical inhibitors

- 6 045378

CD39 leads to moDC activation at concentrations of 0.125, 0.25, or 0.5 mM. However, aHmu-CD39 antibody

BY40 or anti-CD73 antibodies were unable to stimulate ATP-induced dendritic cell (DC) activation, suggesting that the antibodies were unable to block enzymatic activity sufficiently to avoid ATP catabolism. The top-to-bottom notation corresponds to the bars in the graphs from left to right.

In fig. 11 shows the expression of CD25, indicating that moDC activated in the presence of ATP are able to induce T cell activation and proliferation in the RSL assay; enhancement of ATP-mediated activation of moDCs by the anti-CD39 blocking antibody I-394 resulted in increased T cell proliferation and activation. The top-to-bottom notation corresponds to the bars in the graphs from left to right.

In fig. Figure 12 shows tumor growth and survival in mice treated with control PBS on day 5 after tumor cell engraftment (one group) or treated with oxaliplatin chemotherapy (two groups). In parallel, one group of mice treated with oxaliplatin was treated with anti-CD39 antibody twice weekly, with anti-CD39 antibody therapy starting just one day before oxaliplatin therapy (Day 4). This ensured that oxaliplatin would trigger ATP release in a tumor environment where CD39 was already present and completely inhibited, and thus provided optimal prevention of ATP degradation by intratumoral CD39.

In fig. 13 shows tumor growth and survival in mice treated on day 5 after tumor cell engraftment with either control PBS (one group), anti-CD39 antibody, oxaliplatin, or a combination of oxaliplatin and anti-CD39 antibody. Oxaliplatin injection was repeated one week after the first oxaliplatin injection and then again one day after antibody I-394 therapy to ensure optimal inhibition of ATP degradation.

Detailed description

Definitions.

The singular forms used in this specification (in the English version of the application, expressed by the articles a or an) can mean one or more. The singular forms used in the claims, when combined with the word containing/including/consisting of, can mean one or more than one. As used herein, the term other/another may mean at least a second or more.

Where containing/including is used, the term may optionally be replaced by consisting essentially of or consisting of.

Human CD39, also known as NTPdase1, ENTPD1, ATPDase and vascular ATP diphosphohydrolase, exhibits ATPase activity. CD39 is a membrane-bound protein that hydrolyzes extracellular ATP and ADP to AMP, which is then converted to adenosine by another enzyme, 5'-nucleotidase. The amino acid sequence of the mature human CD39 polypeptide chain is available in the Genbank genetic database under accession number P49961. Its complete description is incorporated herein by reference and is as follows:

MEDTKESNVK TFCSKNILAI LGFSSIIAVI ALLAVGLTQN KALPENVKYG IVLDAGSSHT

SLYIYKWPAE KENDTGVVHQ VEECRVKGPG ISKFVQKVNE IGIYLTDCME RAREVIPRSQ HQETPVYLGA TAGMRLLRME SEELADRVLD VVERSLSNYP FDFQGARIIT GQEEGAYGWI TINYLLGKFS QKTRWFSIVP YETNNQETFG ALDLGGASTQ VTFVPQNQTI ESPDNALQFR LYG KDYNVYT HSFLCYGKDQ ALWQKLAKDI QVASNEILRD PCFHPGYKKV VNVSDLYKTP CTKRFEMTLP FQQFEIQGIG NYQQCHQSIL ELFNTSYCPY SQCAFNGIFL PPLQGDFGAF SAFYFVMKFL NLTSEKVSQE KVTEMMKKFC AQPWEEIKTS YAGVKEKYLS EY CFSGTYIL SLLLQGYHFT ADSWEHIHFI GKIQGSDAGW TLGYMLNLTN MIPAEQPLST PLSHSTYVFL MVLFSLVLFT VAIIGLLIFH KPSYFWKDMV (SEQ ID NO: 1).

In the context of the present invention, inhibit, inhibit, neutralize or neutralize when referring to a CD39 polypeptide (eg, neutralize CD39, neutralize the activity of CD39, or neutralize the enzymatic activity of CD39, etc.) refers to the process where the ATP hydrolysis (ATPase) activity of CD39 is inhibited . This includes, in particular, inhibition of CD39-mediated formation of AMP and/or ADP, i.e. inhibition of CD39-mediated catabolism of ATP to AMP and/or ADP. This can be determined, for example, by a cell-based assay, which can measure the ability of a test compound to inhibit the conversion of ATP to AMP and/or ADP, directly or indirectly. For example, as described herein, the disappearance of ATP and/or the formation of AMP can be assessed. In one embodiment of the present invention, the antibody preparation causes at least a 60% reduction in the conversion of ATP to AMP, at least a 70% reduction in the conversion of ATP to AMP, or at least an 80% or 90% reduction in the conversion of ATP to AMP. AMP, referring, for example, to the assays described herein (eg, ATP disappearance and/or AMP formation).

EC ₅₀ for agent and specific activity (e.g. cell binding, inhibition

- 7 045378 tion of enzymatic activity, activation or inhibition of an immune cell) means the effective concentration of the agent at 50% of its maximum response or effect with respect to such activity. EC ₁₀₀ with respect to an agent and a particular activity means the effective concentration of the agent that provides substantially its maximum response with respect to that activity.

As used herein, the term antibody refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies belong to one of five main classes: IgA (immunoglobulin A), IgD (immunoglobulin D), IgE (immunoglobulin E), IgG (immunoglobulin G) and IgM (immunoglobulin M). Some of them are further classified into subclasses or isotypes such as IgG1, IgG2, IgG3, IgG4, etc. The typical structural unit of an immunoglobulin (antibody) contains a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, with 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 these light and heavy chains, respectively. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. IgG is the typical class of antibodies used in the present invention because they are the most common antibodies physiologically and because they are the easiest to produce in the laboratory. The antibody is optionally a monoclonal antibody. Antibodies include, but are not limited to, humanized, chimeric, human or otherwise suitable for human use. Antibodies also include any fragment or derivative of any of the antibodies described herein.

The term specifically binds means that the antibody can bind, preferably in a competitive binding assay, to a binding partner, for example, CD39, as assessed using either recombinant forms of proteins, epitopes, or native proteins present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are well known in the art. For example, binding can be detected using radioactive labels, physical methods such as mass spectrometry, or using direct or indirect fluorescent labels detected by, for example, cytofluorometric analysis (eg, FACS _can ). Binding at an amount greater than that observed with the control nonspecific agent indicates that the agent binds to the target.

When an antibody is said to compete with a particular monoclonal antibody, it means that the antibody competes with the monoclonal antibody in a binding assay using either recombinant molecules (eg, CD39) or surface expressed molecules (eg, CD39). For example, if, in a binding assay, the test antibody reduces the binding of the antibody having the heavy chain of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4 to a CD39 polypeptide or a cell expressing CD39, then the antibody is said to compete with such an antibody.

The term affinity as used herein means the strength of binding of an antibody to an epitope. The affinity of an antibody is determined by the dissociation constant K _d 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 molar concentration of unbound antigen. The affinity constant K _a is defined as 1/Kd. Information on methods for determining the affinity of monoclonal antibodies can be found in the following works: Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Coligan et al., Eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY (1992, 1993) and Muller, Meth. Enzymol. 92:589-601 (1983). These works are incorporated herein by reference in their entirety. One standard method for determining the affinity of monoclonal antibodies, well known in the art, is screening using surface plasmon resonance (SPR) (eg, SPR analysis and the BIAcore™ analytical device).

As used herein, a determinant refers to an interaction or binding region on a polypeptide.

The term epitope refers to an antigenic determinant and is the region or region on the surface of an antigen to which an antibody binds. A protein epitope may contain amino acid residues directly involved in binding, as well as amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide, i.e. amino acid residues in the antibody recognition region. It is the simplest form or smallest structural region on the surface of a complex antigen molecule that can combine, for example, with an antibody or receptor. Epitopes can be linear or conformational/structural.

- 8 045378

The term linear epitope means an epitope consisting of amino acid residues that are adjacent to a linear sequence of amino acids (primary structure).

The term conformational or structural epitope means an epitope consisting of amino acid residues that are not all contiguous and thus represent distinct parts of a linear sequence of amino acids that are brought closer together when the molecule is bent (secondary, tertiary and/or quaternary structures). The conformational epitope depends on the three-dimensional structure. Therefore, the term conformational is often used interchangeably with the term structural.

The term deplete or deplete, with respect to tumor cells, means a process, method or compound that results in or causes destruction, elimination, lysis, thereby adversely affecting the number of such tumor cells present in a sample or subject.

The term internalization, used interchangeably with the term intracellular internalization, refers to the molecular, biochemical and cellular events associated with the process of movement/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, but are not limited to, the internalization of extracellular molecules (such as hormones, antibodies, and small organic molecules); membrane-bound molecules (such as cell surface receptors); and complexes of membrane-associated molecules bound to extracellular molecules (eg, a ligand bound to a transmembrane receptor or an antibody bound to a membrane-associated molecule). Thus, the term inducing and/or enhancing internalization includes events that initiate intracellular internalization and/or increase the rate and/or extent of intracellular internalization.

The term agent is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract from biological materials. The term therapeutic agent means a biologically active agent.

For purposes of the present invention, a humanized or human antibody means an antibody in which the constant and variable framework regions of one or more human immunoglobulins are fused to a binding region, for example 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 and to prevent an immune response to the non-human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been bred specifically to produce specific human antibodies in response to immunization with an antigen (see, for example, Green et al. (1994), Nature Genet, 7:13; Lonberg et al. (1994), Nature, 368:856; Taylor et al. (1994), Int. Immun. 6:579, all of which are incorporated herein by reference). A fully human antibody can also be produced by genetic or chromosomal transfection methods known in the art, as well as by phage display technology (see, for example, McCafferty et al. (1990), Nature, 348:552-553). Human antibodies can also be generated by in vitro activated B cells (see, for example, US Pat. Nos. 5,567,610 and 5,229,275, which are incorporated by reference in their entirety).

A chimeric antibody is an antibody molecule in which (a) the constant region or part thereof is altered or replaced such that the antigen binding site (variable region) is associated with a constant region of a different or altered class, effector function and/or species, or an entirely different one a molecule that imparts new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or part thereof is altered or replaced by a variable region having a different or altered antigenic specificity.

The term hypervariable region as used herein refers to the amino acid residues of an antibody that are responsible for binding to an antigen. The hypervariable region typically contains amino acid residues from the complementarity determining region (complementarity determining region) or CDR (for example, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991) and/or residues from the hypervariable loop (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917) or a similar system to determine the essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in this region is performed in the manner described above in the work of Kabat et al. Phrases such as Kabat position, variable domain residue numbering as in Kabat and Kabat herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of the peptide may contain fewer amino acids or additional amino acids according to the location of insertion into the framework region or CDR region of the variable domain or abbreviation thereof. For example, a heavy chain variable domain may include one amino acid insertion (Kabat residue 52a) after residue 52 of the H2 CDR, as well as inserted residues (e.g., Kabat residues 82a, 82b, 82c, etc.) after residue 82 of the heavy chain framework region. chains. The Kabat residue nomenclature for a particular antibody can be determined by aligning the antibody sequence with a standard numbered Kabat sequence at regions of homology.

By framework or FR residues, as used herein, we mean the variable domain region of an antibody, excluding those regions defined as CDRs. Each antibody variable domain framework region can be further subdivided into adjacent 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 human γ (gamma) heavy chain or its analogous sequence in other types of antibody heavy chains ( for example, α, δ, ε and μ for human antibodies), or its natural allotype. Unless otherwise noted, Kabat's generally accepted amino acid numbering for immunoglobulins is used herein (see Kabat et al. (1991), Sequences of Protein of Immunological Interest, ^5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

The terms isolated, purified or biologically pure refer to a material that is substantially or substantially free of the components that normally accompany it in its natural state. Purity and uniformity are usually determined by analytical chemistry methods such as polyacrylamide gel electrophoresis or high performance liquid chromatography (HPLC). 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 in which one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

The term recombinant, when used with reference to, for example, a cell or nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by alteration of a native nucleic acid or protein , or that the cell 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 to a polypeptide or epitope means an antibody that binds to the specified determinant with specificity and/or affinity.

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

Identity determination methods are designed to determine the best match between test sequences. Methods for determining identity are described in publicly available computer programs. Methods for computer determination of 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 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.). You can also use the well-known Smith-Waterman algorithm to determine identity.

- 10 045378

CD39 inhibitory agents.

The agent that binds to CD39 and inhibits CD39 for use in the present invention may be an antigen binding domain or a protein containing such a domain, optionally an antibody or antibody fragment that binds to the CD39 protein and inhibits or neutralizes the ATPase activity of the CD39 protein, e.g. soluble CD39 protein (sCD39), monomeric CD39 protein, membrane-bound CD39 protein (memCD39), for example, expressed on the cell surface.

In one embodiment of the present invention, the sCD39 protein is a CD39 protein without the two transmembrane domains (ie, the transmembrane domains near the N- and C-terminal regions) found in membrane-bound CD39. In one embodiment of the present invention, sCD39 is a non-membrane-bound SCD39 protein circulating in the bloodstream of, for example, a human. In one embodiment of the present invention, sCD39 contains or consists of the amino acid sequence of SEQ ID NO: 2 (which optionally further contains a C-terminal tag or other amino acid sequence not derived from CD39). In one embodiment of the present invention, the protein, antibody, or antibody fragment inhibits the ATPase activity of SCD39 when incubated with SCD39 in solution, for example, in accordance with the methods disclosed herein. In one embodiment of the present invention, the protein, antibody, or antibody fragment specifically binds to human CD39 protein in both soluble (extracellular domain protein) and membrane-bound forms.

In one embodiment of the present invention, the anti-CD39 antibody does not increase or cause intracellular internalization, or more generally down-modulation, of cell surface expressed CD39, and/or the inhibitory activity of such CD39 antibody is independent of CD39.

In one aspect of the present invention, an anti-CD39 antibody is capable of (a) inhibiting the enzymatic activity of a membrane-bound CD39 protein (e.g., 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 ( for example, a CD39 protein containing the amino acid sequence of SEQ ID NO: 2, a CD39 protein without its transmembrane domains).

In one embodiment of the present invention, the anti-CD39 antibody does not substantially bind (eg, through its Fc domain) to human Fcy (eg, CD16, CD32a, CD32b, CD64) and/or C1q receptors and/or does not substantially target ADCC and /or CC against a cell expressing CD39. Optionally, the antibody retains the Fc domain (eg, human IgG isotype) and continues to bind to human FcRn.

In one embodiment of the present invention, CD39 neutralizing antibodies may be characterized by the ability to cause a reduction in the ATPase activity of an SCD39 polypeptide and/or a monomeric CD39 polypeptide, without necessarily causing a reduction in the formation of AMP from a soluble monomeric human CD39 protein, for example, a CD39 protein consisting of the amino acid sequence of SEQ ID NO: 2 by at least 50, 60, 70, 80 or 90%.

In one embodiment of the present invention, CD39 neutralizing antibodies may be characterized by the ability to cause a reduction in the ATPase activity of CD39 cells, optionally causing a reduction in AMP production in a cell expressing CD39 by at least 50, 60, 70, 80 or 90%. In one embodiment of the present invention, CD39 neutralizing antibodies may have an EC ₅₀ for inhibiting ATPase activity (e.g., an EC ₅₀ for inhibiting the generation of AMP by a cell expressing CD39) of CD39 expressed by the cell of no more than 1 μg/ml, optionally no more than 0. 5 μg/ml, optionally not more than 0.2 μg/ml.

In one embodiment of the present invention, CD39 neutralizing antibodies may have the ability to cause an increase in cell surface expression of an activation marker in human monocyte-derived dendritic cells (moDCs) when such moDCs are incubated in vitro with the antibody and ATP, where the ATP is optionally exogenously added ATP and wherein ATP is optionally added at a concentration of 0.125, 0.25 or 0.5 mM.

The antigen binding compound can be prepared as described later herein. At the same time, at any stage, one can evaluate its ability to inhibit the enzymatic activity of CD39, in particular to block the ATPase activity of SCD39 and reduce the production of ADP and AMP (and together with CD73, adenosine) by the soluble CD39 protein and, optionally, by the cell expressing CD39, as well as, in in turn, restore ATP-mediated activation of dendritic cell activity and/or T-cell proliferation.

The inhibitory activity (eg, immune enhancing potential) of an antibody can also be assessed, for example, by assay detecting 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 determining the presence of ATP after incubation of the test antibody with soluble CD39 protein (e.g., CD39 protein having the amino acid sequence SEQ ID NO: 2,

- 11 045378 as in example 1, optionally further including a purification tag or other functional or non-functional amino acid sequence not derived from CD39). See Examples section. Briefly, ATP can be quantified using the CellTiter Glo™ kit (Promega) in an assay where a dose range of the antibody of interest is incubated with the soluble recombinant human CD39 protein described in the Examples section for 1 hour at 37°C. Before adding the CTG reagent, 20 μM ATP was added to the plates for an additional 30 min incubation at 37°C. The emitted light was 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 determining the presence of ATP after incubation of the test antibody with cells (eg, Ramos cells, cells transfected with CD39, etc.). See Examples section. Cells can be incubated for 1 hour at 37°C with the antibodies being tested. The cells are then further incubated with the addition of 20 μM ATP for 1 h at 37°C. The plates are centrifuged for 2 minutes at 400 g, after which the cell supernatant is transferred to a microplate (in white wells) for luminescent analysis. CTG is added to the supernatant and the emitted light is 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 emission in the presence of the antibody with ATP (maximum light emission) and the antibody with ATP and cells (minimal light emission).

A decrease in ATP hydrolysis to AMP and/or an increase in ATP and/or a decrease in AMP production in the presence of the antibody indicates that the antibody inhibits CD39. In one embodiment of the present invention, 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 as assessed by the method detecting the presence of ATP using CellTiter Glo™ (Promega) after incubating cells expressing CD39 polypeptide (eg, Ramos cells) with the test antibody, for example, as in Example 1.

In one embodiment of the present invention, the antibody preparation is capable of causing at least a 60% reduction in the enzymatic activity of a soluble recombinant CD39 polypeptide, preferably at least a 70, 80, or 90% reduction in the enzymatic activity of a soluble recombinant CD39 polypeptide, as assessed by the presence detection method ATP using CellTiter Glo™ (Promega) after incubation of soluble recombinant CD39 polypeptide with test antibody, for example, as in Example 1.

One example provides a method for producing or identifying in vitro an anti-CD39 antibody or antigen binding domain that can be used in the present invention (for example, in combination with an agent that causes the release of ATP from tumor cells) and which includes the following steps:

(a) providing a plurality of antibodies that bind to human CD39 polypeptide;

(b) contacting each of the antibodies with human monocyte-derived dendritic cells (moDC) in the presence of ATP, optionally the ATP being exogenously added ATP; and (c) selecting that antibody from step (b) that results in increased expression of the cell surface activation marker in the moDC.

Optionally, in any embodiment of the present invention, the anti-CD39 neutralizing antibody binds to an antigenic determinant present on both SCD39 and cell surface expressed CD39 (memCD39).

Optionally, in any embodiment of the present invention, the anti-CD39 neutralizing antibody competes for binding to an epitope on CD39 bound to antibody I-394 (e.g., which competes for binding to an epitope on a CD39 polypeptide with an antibody having CDRs in the heavy and light chains or variable regions identical to any of I-394).

Optionally, in any embodiment of the present invention, the anti-CD39 neutralizing antibody binds to the same epitope and/or competes for binding to the CD39 polypeptide with the monoclonal antibody I-394 (for example, which competes for binding to the CD39 polypeptide with an antibody having a CDR in the heavy and light chains or variable regions identical to I-394). In one embodiment of the present invention, a neutralizing anti-CD39 antibody binds to the same epitope and/or competes for binding to a CD39 polypeptide with an antibody having the VH and _VL regions of SEQ ID NOs: 3 and 4, respectively.

Optionally, in any embodiment of the present invention, the anti-CD39 antibody binds to an epitope comprising one, two, or three amino acid residues selected from the group consisting of amino acid residues on CD39 linked to I-394.

Optionally, in any embodiment of the present invention, the binding molecule (eg, an anti-CD39 antibody) contains a heavy chain variable domain ( _VH ) containing CDR1, 2 and

- 12 045378 light chain, as described herein, and a light chain variable domain (VL) containing CDR1, 2 and 3 of the heavy chain, as described herein, or an amino acid sequence in which the CDR region (or series of CDRs heavy and/or light chain) is at least 60, 70, 80, 90 or 95% identical to the listed CDRs (or the listed series of heavy and/or light chain CDRs). In one aspect of any embodiment of the present invention, the antibody may comprise a heavy chain including three CDRs of the heavy chain variable region (VH) of antibody I-394, and a light chain including three CDRs of the variable light chain (VL) region of antibody I-394.

Optionally, in any embodiment of the present invention, the anti-CD39 antibody contains an Fc domain that is modified (compared to a wild-type Fc domain of the same isotype) to reduce the association between the Fc domain and human CD16A, CD16B, CD32A, CD32B and/or CD64 polypeptides wherein, optionally, the antibody comprises (i) a heavy chain comprising CDRs 1, 2 and 3 of the heavy chain variable region of SEQ ID NO: 3, and (ii) a light chain comprising CDRs 1, 2 and 3 of the light chain variable region of SEQ ID NO: 3, and (ii) a light chain comprising CDRs 1, 2 and 3 of the light chain variable region of SEQ ID NO: 4. In one aspect of the present invention, the Fc domain is modified (compared to a wild-type Fc domain of the same isotype) to reduce the association between the Fc domain and a human C1q polypeptide. In one embodiment of the present invention, the antibody contains an amino acid substitute in the heavy chain constant region at any one, two, three, four, five or more residues selected from the group consisting of 220, 226, 229, 233, 234, 235, 236, 237 , 238, 243, 264, 268, 297, 298, 299, 309, 310, 318, 320, 322, 327, 330 and 331 (Kabat numbering). In one embodiment of the present invention, the antibody has an amino acid substituent in the heavy chain constant region at any three, four, five, or more residues selected from the group consisting of 234, 235, 237, 322, 330, and 331. In one embodiment of the present invention the antibody contains an Fc domain containing the amino acid sequence shown below.

In one embodiment of the present invention, the antibody comprises a heavy chain constant region or Fc domain 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 Nos. 234, 235 and 331 according to Kabat's numbering (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPASIEK TISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44)

In one embodiment of the present invention, the antibody comprises a heavy chain constant region or Fc domain 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 No. 234, 235 and 331 according to Kabat's numbering (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPASIEK TISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45)

In one embodiment of the present invention, the antibody comprises a heavy chain constant region or Fc domain 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 No. 234, 235 , 237, 330 and 331 according to Kabat's numbering

- 13 045378 (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPSSIEK TISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 46)

In one embodiment of the present invention, the antibody comprises a heavy chain constant region or Fc domain 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 No. 234, 235 , 237 and 331 according to Kabat's numbering (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPASIEK TISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47)

In one aspect of the present invention, the anti-CD39 antibody binds to the same epitope as the I-394, I-395, I-396, I-397, I-398, or I-399 antibody. In one embodiment of the present invention, the antibodies bind to a CD39 epitope that at least partially overlaps with or includes at least one residue in the epitope associated with the antibody I-394, I-395, I-396, I-397, I -398 or I-399. Antibody-associated residues may be indicated as being present on the surface of a CD39 polypeptide, for example, in 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 shown in the table) means that there is a decrease in binding affinity (e.g., as measured by known methods such as FACS analysis of cells expressing a particular mutant, or analysis of binding to mutant polypeptides using Biacore systems) and/or a decrease in the overall binding capacity of the anti-CD39 antibody (as evidenced, for example, by a decrease in B _max on a graph of anti-CD39 antibody concentration versus polypeptide concentration). The significant decrease in binding indicates that the mutated residue is directly involved in the binding process of the anti-CD39 antibody or is in close proximity to the binding protein when the anti-CD39 antibody is already bound to CD39.

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

In some embodiments of the present invention, anti-CD39 antibodies exhibit significantly lower binding strength to a mutant CD39 polypeptide in which a residue in a segment

- 14 045378 comprising an amino acid residue associated with antibodies I-394, I-395, I-396, I-397, I-398 or I-399, replaced by another amino acid.

Some embodiments of the present invention provide anti-CD39 antibodies (e.g., other than I-394) that bind to an epitope on CD39 associated with antibody I-394, I-395,

I-396, I-397, I-398 or I-399.

In one aspect of the present invention, the antibody binds to an epitope on CD39 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 of the present invention, an anti-CD39 antibody exhibits a reduced degree of binding (eg, 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 to SEQ ID NO: 1), compared to wild-type CD39 polypeptide (CD39 polypeptide from SEQ ID NO: 1); optionally, the mutant CD39 polypeptide has the following mutations: R138A, M139A and E142K. In one optional aspect of the present invention, the antibody has no loss of binding to any of the mutant CD39 polypeptides in the table except mutant 19. In another optional aspect of the present invention, the anti-CD39 antibody exhibits a reduced degree of binding (optionally reduced, but not substantially complete loss of binding ; or, optionally, substantially complete loss of binding) with a CD39 polypeptide having a mutation at one, two, three or four residues selected from the group consisting of Q96, N99, E143 and R147 (relative to SEQ ID NO: 1) compared to wild type CD39 polypeptide (CD39 polypeptide of SEQ ID NO: 1); optionally, the mutant CD39 polypeptide has the following mutations: Q96A, N99A, E143A and R147E.

In one aspect of the present invention, the antibody binds to an epitope on CD39 containing an amino acid residue (e.g., one, two, three, or four residues) selected from the group consisting of Q96, N99, E143, and R147 (relative to SEQ ID NO: 1 ). In one aspect of the present invention, the antibody has a reduced degree of binding (eg, substantially complete loss of binding) to a mutant CD39 polypeptide containing a mutation at 1, 2, 3, or 4 residues selected from the group consisting of Q96, N99, E143, and R147 (relative to SEQ ID NO: 1), in each case relative to the extent of binding of the antibody to a wild-type CD39 polypeptide containing the amino acid sequence of SEQ ID NO: 1.

In one aspect of the present invention, the antibody binds to an epitope on CD39 comprising (a) an amino acid residue (e.g., one, two, or three residues) selected from the group consisting of R138, M139, and E142 (relative to SEQ ID NO: 1) , and (b) an amino acid residue (eg, one, two, three or four residues) selected from the group consisting of Q96, N99, E143 and R147.

In one aspect of the present invention, the anti-CD39 antibody exhibits a reduced degree (eg, substantially complete loss) of binding to either (a) a CD39 polypeptide having a mutation at one, two, three, or four residues selected from the group consisting of Q96, N99 , E143 and R147 (with respect to SEQ ID NO: 1), and with (b) a CD39 polypeptide having a mutation at one, two or three residues selected from the group consisting of R138, M139 and E142 (with respect to SEQ ID NO: 1), in each case compared to the wild-type CD39 polypeptide (CD39 polypeptide of SEQ ID NO: 1). Optionally, the mutant CD39 polypeptide of (a) has the following mutations: Q96A, N99A, E143A and R147E. Optionally, the mutant CD39 polypeptide of (b) has the following mutations: R138A, M139A and E142K. Optionally, the antibody has no loss of binding to any of the mutant CD39 polypeptides in the table except mutants 5 and 19.

In one aspect of the present invention, the antibody binds to an epitope on CD39 containing an amino acid residue (eg, one, two, three or four residues) selected from the group consisting of K87, E100 and D107 (relative to SEQ ID NO: 1).

In one aspect of the present invention, an anti-CD39 antibody exhibits a reduced degree of binding (eg, substantially complete loss of binding) to a CD39 polypeptide having a mutation at one, two, three, or four residues selected from the group consisting of K87, E100, and D107 ( relative to SEQ ID NO: 1), versus wild-type CD39 polypeptide (CD39 polypeptide of SEQ ID NO: 1); optionally, the mutant CD39 polypeptide has the following mutations: K87A, E100A and D107A. Optionally, the antibody has no loss of binding to any of the mutant CD39 polypeptides in the table except mutant 15.

In one aspect of the present invention, the antibody binds to an epitope on CD39 containing an amino acid residue (e.g., one, two, three, or four residues) selected from the group consisting of N371, L372, E375, K376, and V377 (relative to SEQ ID NO : 1).

In one aspect of the present invention, the anti-CD39 antibody exhibits a reduced degree (eg, substantially complete loss) of binding to a CD39 polypeptide having a mutation at one, two, three, four or five residues selected from the group consisting of N371, L372, E375 , K376 and V377 (relative to SEQ ID NO: 1), compared to the wild type CD39 polypeptide (CD39 polypeptide of SEQ ID NO: 1); optionally, the mutant CD39 polypeptide has the following mutations: N371K, L372K, E375A, K376G and V377S and a valine insertion between residues 376 and 377. Optionally,

- 15 045378 antibody has no loss of binding to any of the mutant CD39 polypeptides from the table, except mutant 11.

The Ahtu-CD39 antibody may, for example, comprise HCDR1 containing the amino acid sequence DYNMH (SEQ ID NO: 5) or a sequence of at least 4 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; HCDR2 comprising the amino acid sequence YIVPLNGGSTFNQKFKG (SEQ ID NO: 6) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid ; HCDR3 comprising the amino acid sequence GGTRFAY (SEQ ID NO: 7) or a sequence of at least 4, 5 or 6 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; LCDR1 containing the amino acid sequence RASESVDNFGVSFMY (SEQ ID NO: 8) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid ; an LCDR2 region containing the amino acid sequence GASNQGS (SEQ ID NO: 9) or a sequence of at least 4, 5 or 6 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; and/or the LCDR3 I-394 region containing the amino acid sequence QQTKEVPYT (SEQ ID NO: 10) or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids, where optionally one or more of these amino acids may be removed or replaced by another amino acid. The positions of the CDR areas can be in accordance with the Kabat numbering.

An example of an anti-CD39 antibody with a _VH and _VL pair that inhibits the enzymatic activity of human sCD39 protein is antibody I-394, the amino acid sequence of the heavy chain variable region of which is given below (SEQ ID NO: 3), and the amino acid sequence of the light chain variable region the circuits of which are also given below (SEQ ID NO: 4). The CDR regions in SEQ ID NO: 3 and 4 are underlined according to Kabat's numbering. Optionally, V _H and V _L contain (eg, modified to include) human acceptor framework regions. In one embodiment of the present invention, the anti-CD39 antibody comprises a V _H CDR1, CDR2, and/or CDR3 (e.g., Kabat numbering) heavy chain variable region having the amino acid sequence SEQ ID NO: 3. In one embodiment of the present invention, the anti-CD39 the antibody contains a V _L CDR1, CDR2 and/or CDR3 (e.g., according to Kabat numbering) of a light chain variable region having the amino acid sequence of SEQ ID NO: 4.

1-394 _VH :

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGRTLEWIGYIVPLNGGSTF NQKFKGRATLTVNTSSRTAYMELRSLTSEDSAAYYCARGGTRFAYWGQGTLVTVSA (SEQ ID NO: 3).

1-394 V _L :

DIVLTQSPASLAVSLGQRATISCRASESVDNFGVSFMYWFQQKPGQPPNLLIYGASNQGSG VPARFRGSGSGTDFSLNIHPMEADDTAMYFCQQTKEVPYTFGGGTKLEIK (SEQ ID NO: 4).

Another example of an anti-CD39 antibody with a V _H and V _L pair in accordance with the present invention is the antibody I-395, the amino acid sequence of the variable region of the heavy chain of which is indicated below (SEQ ID NO: 11), and the amino acid sequence of the variable region of the light chain of which is also listed below (SEQ ID NO: 12). The CDR regions in SEQ ID NO: 11 and 12 are underlined in accordance with Kabat's numbering. Optionally, V _H and V _L contain (eg, modified to include) human acceptor framework regions. In one embodiment of the present invention, the anti-CD39 antibody comprises a V _H CDR1, CDR2, and/or CDR3 (e.g., Kabat numbering) of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 11. In one embodiment of the present invention, the anti-CD39 the antibody contains a V _L CDR1, CDR2 and/or CDR3 (e.g., according to Kabat numbering) of a light chain variable region having the amino acid sequence of SEQ ID NO: 12.

1-395 _VH :

EVQLQQSGPELVKPGASVRMSCKASGYTFTDYNMHWVKKNHGKGLEWIGYINPNNGGTT YNQKFKGKATLTVNTSSKTAYMELRSLTSEDSAVYYCTRGGTRFASWGQGTLVTVSA (SEQ ID NO: 11).

I-395 V _L :

NIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMYWFQQKPGQPPKLLIYAASTQGSG VPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPFTFGSGTKLEIK (SEQ ID NO: 12).

The Ahtu-CD39 antibody may, for example, comprise HCDR1 from I-395 containing the amino acid sequence DYNMH (SEQ ID NO: 13) or a sequence of at least 4 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another

- 16 045378 amino acids; HCDR2 from I-395, containing the amino acid sequence YINPNNGGTTYNQKFKG (SEQ ID NO: 14) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; HCDR3 from I-395 containing the amino acid sequence GGTRFAS (SEQ ID NO: 15) or a sequence of at least 4, 5, 6 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; LCDRI region I-395 containing the amino acid sequence RASESVDNYGISFMY (SEQ ID NO: 16) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids can be replaced by another amino acid; LCDR2 region I-395 containing the amino acid sequence AASTQGS (SEQ ID NO: 17) or a sequence of at least 4, 5 or 6 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; and/or an I-395 LCDR3 region comprising the amino acid sequence QQSKEVPFT (SEQ ID NO: 18) or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids, where optionally one or more of these amino acids may be removed or replaced with another amino acid. The positions of the CDR areas can be in accordance with the Kabat numbering.

Another example of an anti-CD39 antibody with a V _H and V _L pair in accordance with the present invention is antibody I-396, the amino acid sequence of the variable heavy chain region of which is indicated below (SEQ ID NO: 19), and the amino acid sequence of the variable light chain region of which is also listed below (SEQ ID NO: 20). The CDR regions in SEQ ID NOs: 19 and 20 are underlined in accordance with Kabat's numbering. Optionally, V _H and V _L contain (eg, modified to include) human acceptor framework regions. In one embodiment of the present invention, the anti-CD39 antibody comprises a V _H CDR1, CDR2, and/or CDR3 (e.g., Kabat numbering) of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19. In one embodiment of the present invention, the anti-CD39 the antibody contains a V _L CDR1, CDR2 and/or CDR3 (e.g., according to Kabat numbering) of a light chain variable region having the amino acid sequence of SEQ ID NO: 20.

1-396 VH:

EVQLQQSGAELVKPGASVKLSCIVSGFNIKDTYINWVKQRPEQGLEWIGRIDPANGNTKYD IPKFQGKATMTSDTSSNTAYLHLSSLTSDDSAVYYCARWGYDDEEADYFDSWGQGTTLTV SS (SEQ ID NO: 19).

I-396 VL:

DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSG VPARFSGSGSGTDFSLNILPMEEVDAAMYFCHQSKEVPWTFGGGTKLEIK (SEQ ID NO: 20).

The Ahtu-CD39 antibody may, for example, comprise HCDR1 containing the amino acid sequence DTYIN (SEQ ID NO: 21) or a sequence of at least 4 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; HCDR2 comprising the amino acid sequence RIDPANGNTKYDPKFQG (SEQ ID NO: 22) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid ; HCDR3 comprising the amino acid sequence WGYDDEEADYFDS (SEQ ID NO: 23) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid ; LCDR1 region containing the amino acid sequence RASESVDNYGISFMN (SEQ ID NO: 24) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids may be replaced another amino acid; an LCDR2 region containing the amino acid sequence AASNQGS (SEQ ID NO: 25) or a sequence of at least 4, 5 or 6 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; and/or an I-396 LCDR3 region comprising the amino acid sequence HQSKEVPWT (SEQ ID NO: 26) or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids, where optionally one or more of these amino acids may be removed or replaced with another amino acid. The positions of the CDR areas can be in accordance with the Kabat numbering.

Another example of an anti-CD39 antibody with a V _H and V _L pair in accordance with the present invention is the antibody I-399, the amino acid sequence of the variable region of the heavy chain of which is indicated below (SEQ ID NO: 27), and the amino acid sequence of the variable region of the light chain of which is also listed below (SEQ ID NO: 28). The CDR regions in SEQ ID NOs: 27 and 28 are underlined in accordance with Kabat's numbering. Optionally, VH and VL comprise (eg, modified to include) human acceptor framework regions. In one embodiment of the present invention, the anti-CD39 antibody comprises a V _H CDR1, CDR2, and/or CDR3 (e.g., Kabat numbering) heavy chain variable region having the amino acid sequence of SEQ ID NO: 27.

In one embodiment of the present invention, the anti-CD39 antibody comprises a VL CDR1, CDR2 and/or CDR3 (e.g., Kabat numbering) of a light chain variable region having the amino acid sequence of SEQ ID NO: 28.

1-399 VH:

PVQLQQPGAEWMPGASVKLSCKASGYTFTSFWMNWMRQRPGQGLEWIGEIDPSDFYTN SNQRFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCARGDFGWYFDVWGTGTSVTVSS (SEQ ID NO: 27).

1-399 VL:

EIVLTQSPTTTMTSSPGEKITFTCSASSSINSNYLHWYQQKPGFSPKLLIYRTSNLASGVPTRF

SSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSLPRTFGGGTKLEIK (SEQ ID NO: 28).

The Ahtu-CD39 antibody may, for example, comprise HCDR1 containing the amino acid sequence SFWMN (SEQ ID NO: 29) or a sequence of at least 4 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; HCDR2 containing the amino acid sequence EIDPSDFYTNSNQRFKG (SEQ ID NO: 30) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid ; HCDR3 comprising the amino acid sequence GDFGWYFDV (SEQ ID NO: 31) or a sequence of at least 4, 5 or 6 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; an LCDR1 region containing the amino acid sequence SASSSINSNYLH (SEQ ID NO: 32) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; an LCDR2 region containing the amino acid sequence RTSNLAS (SEQ ID NO: 33) or a sequence of at least 4, 5 or 6 contiguous amino acids, where optionally one or more of these amino acids may be replaced by another amino acid; and/or an I-399 LCDR3 region comprising the amino acid sequence QQGSSLPRT (SEQ ID NO: 34) or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids, where optionally one or more of these amino acids may be removed or replaced with another amino acid. CDR provisions may correspond to Kabat numbering.

In any of the antibodies I-394, I-395, I-396 and I-399, the sequences HCDR1, 2, 3 and LCDR1, 2, 3 (each CDR region independently or all CDRs) may be indicated according to the Kabat numbering system (as indicated in the V _H and V L sequences by underlining), Chotia numbering system, IMGT numbering system or any other suitable numbering system.

In any aspect of the present invention, said variable regions, framework regions 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 sequence modifications). In one embodiment of the present invention, the replacement is a conservative modification.

In another aspect of the present invention, the anti-CD39 compound contains a _VH domain that has at least about 60, 70, or 80% amino acid sequence identity, optionally at least about 85, 90, 95, 97, 98, or 99% amino acid sequence identity. the sequence of the V _H domain of the antibody disclosed herein. In another aspect of the present invention, an anti-CD39 antibody comprises a VL domain that has at least about 60, 70, or 80% amino acid sequence identity, optionally at least about 85, 90, 95, 97, 98, or 99% amino acid sequence identity. the VL domain of the antibody disclosed herein.

Optionally, in any embodiment of the present invention, the anti-CD39 antibody can be characterized as a functionally conserved variant of any of the antibodies, heavy and/or light chains, CDRs or variable regions thereof described herein. Functionally conserved variants are variants in which a given amino acid residue in a protein or enzyme has been changed without changing the overall conformation and function of the polypeptide, including, but not limited to, substitution of an amino acid with an amino acid having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic properties, etc.). Amino acids other than those indicated as conserved may vary throughout the protein such that the percentage similarity of protein or amino acid sequence between any two proteins of similar function may vary and range from, for example, 70 to 99%, as determined in accordance with a matching scheme, such as a cluster method, where similarity is based on the MEGALIGN algorithm. A functionally conserved variant also includes a polypeptide that is at least 60% identical in amino acid sequence, as determined by the BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, even more preferably at least 90% and even more preferably at least 95% and which has the same or substantially

- 18 045378 similar properties or functions as the native or parent protein (eg, heavy or light chains, or CDRs, or variable regions) to which it is compared. In one embodiment of the present invention, the antibody includes a heavy chain variable region that is a functionally conserved variant of the antibody heavy chain variable region I-394, I-395, I-396, I-397, I-398, or I-399, and a light chain variable region that is a functionally conserved variant of the light chain variable region of antibody I-394, I-395, I-396, I-397, I-398, or I-399, respectively. In one embodiment of the present invention, the antibody includes a heavy chain that is a functionally conserved variant of the antibody heavy chain variable region I-394, I-395, I-396, I-397, I-398, or I-399 fused to the constant region a human heavy chain disclosed herein, optionally, a constant region of any of SEQ ID NOs: 44-47, as well as a light chain that is a functionally conserved variant of the light chain variable region of the corresponding antibody I-394, I-395, I- 396, I-397, I-398, or I-399 fused to the human kappa light chain constant region.

Production/receipt of antibodies.

Ahtu-CD39 antibodies can be prepared by any of a variety of methods known in the art. Typically, they are obtained by immunizing a non-human animal, such as a mouse, with an immunogen containing a CD39 polypeptide, respectively, or by screening a gene library for CD39 polypeptide binding domains. The CD39 polypeptide may comprise the full length sequence of a human CD39 polypeptide, respectively, or a fragment or derivative thereof, typically an immunogenic fragment, i.e. part of a polypeptide containing an epitope exposed on the surface of cells expressing the CD39 polypeptide. Such fragments typically contain at least about 7 contiguous amino acids in the mature polypeptide sequence, even more preferably at least about 10 contiguous amino acids. The fragments are typically derived substantially from the extracellular domain of the receptor. In one embodiment of the present invention, the immunogen comprises a wild-type human CD39 polypeptide in a lipid membrane, typically on the surface of a cell. In a specific embodiment of the present invention, the immunogen contains intact cells, in particular intact human cells, optionally subjected to any treatment or lysis. In another embodiment of the present invention, the polypeptide is a recombinant CD39 polypeptide.

Immunization of a non-human mammal with antigen can be accomplished by any method well known in the art for stimulating antibody production in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the full description of which is incorporated herein by reference). The immunogen is suspended or dissolved in a buffer, optionally with an adjuvant such as Freund's complete or incomplete adjuvant. Methods for determining the amount of immunogen, types of buffers and amount of adjuvant are well known to those skilled in the art and are not limiting. These parameters may vary for different immunogens, but are easy to establish.

Likewise, the scope and frequency of immunization sufficient to stimulate antibody production is also well known in the art. In a typical immunization protocol, non-human animals are given antigen intraperitoneally on day 1 and again approximately one week later. This is followed by repeated antigen injections around day 20, optionally together with an adjuvant such as Freund's incomplete adjuvant. Secondary injections are given intravenously and can be repeated for several days in a row. This is followed on day 40 by an intravenous or intraperitoneal booster injection, usually without an adjuvant. Following this protocol results in the production of antigen-specific antibody-producing B cells in approximately 40 days. Other protocols may also be used provided they result in the production of B cells expressing an antibody directed to the antigen used for immunization.

To produce monoclonal antibodies, splenocytes are isolated from an immunized non-human mammal and then inserted into immortalized cells to form an antibody-producing hybridoma. Obtaining splenocytes from a non-human mammal is well known in the art and typically involves removing the spleen from an anesthetized non-human mammal, cutting it into small pieces, and extruding the splenocytes from the spleen capsule through a nylon mesh cell strainer into an appropriate buffer to obtain a single cell suspension. . The cells are washed, centrifuged and resuspended in buffer, which lyses any red blood cells. The solution is centrifuged again and the remaining precipitated lymphocytes are finally resuspended in fresh buffer.

Once the lymphocytes have been isolated and placed in a single cell suspension, they can be combined with an immortal cell line. Typically, this is a mouse myeloma cell line, although many other immortal cell lines are known in the art for use in producing hybridomas. Murine myeloma lines include, but are not limited to, lines derived from mouse tumors MOPC-21 and MPC-11, available from the Salk Institute Cell Distribution Center, San Diego, USA, and X63 Ag8653 and SP-2 cells, available in the American Type Culture Collection, Rockville,

- 19 045378 Maryland, USA. Fusion is carried out using polyethylene glycol or the like. The resulting hybridomas are then grown in selective media that contain one or more substances that inhibit the growth or survival of unfused parent myeloma cells. For example, if the parent myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then the hybridoma culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

Hybridomas are usually grown on a macrophage feeder layer. Macrophages are preferably taken from littermates of non-human mammals used to obtain splenocytes, and are typically primed with incomplete Freund's adjuvant or the like several days before inoculation with the hybridoma. Fusion methods are described in Goding Monoclonal Antibodies: Principles and Practice, p. 59103 (Academic Press, 1986), which is incorporated herein by reference.

The cells are allowed to grow in the selection medium for a period of time sufficient to form colonies and produce antibodies, usually 7 to 14 days.

The hybridoma colonies are then assayed for the production of antibodies that specifically bind to the CD39 polypeptide gene products. The assay is usually an enzyme immunoassay with a colorimetric detection method, although any assay that can be adapted to the wells in which the hybridomas are grown can be used. Other types of analysis include radioimmunoassay or fluorescent cell sorting. Wells suitable for the desired antibody production are examined for the presence of one or more colonies. If more than one colony is present, the cells can be re-cloned and grown to ensure that only one cell will give rise to a colony that will produce the desired antibody. Typically, antibodies are also tested for their ability to bind to CD39 polypeptides, for example, cells expressing CD39.

Hybridomas that have been confirmed to produce a monoclonal antibody can be grown in large quantities in a suitable medium such as DMEM or RPMI-1640. Alternatively, hybridoma cells can be grown in vivo as ascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, the growth medium containing the monoclonal antibody (or ascitic fluid) is separated from the cells and the monoclonal antibody present therein is purified. Purification is typically accomplished by gel electrophoresis, dialysis, Protein A or Protein G chromatography, Sepharose, or anti-mouse Ig bound to a solid support such as agarose or Sepharose beads (all described in e.g. Antibody Purification Handbook, Biosciences, Publication No. 18-1037-46, Edition AC, which is hereby incorporated by reference).

Bound antibody is typically eluted from protein A/G columns using low pH buffers (glycine or acetate buffers pH 3.0 or lower) with immediate neutralization of antibody-containing fractions. These fractions are pooled, dialyzed and concentrated as needed.

The single visible colony from the positive wells is usually recloned and reassayed to ensure that only one monoclonal antibody is detected and produced.

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

Identification of one or more antibodies that bind to the antigen of interest, i.e. CD39, in particular or substantially the same region, determinant or epitope as monoclonal antibody I-394, I-395, I-396 or I-399, can be easily performed using any of a variety of immunological screening assays, during which antibody competition can be 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 is specifically incorporated herein by reference).

For example, if the test antibodies to be analyzed are from different animals or even have a different Ig isotype, a simple competition assay can be used, where the control (I-394, I-395, I-396 or I-399) and the test antibodies mixed (or pre-adsorbed) and applied to a sample containing CD39 polypeptides. Protocols based on Western blotting and analysis using BIACORE systems are suitable for such competitive assays.

In some embodiments, control antibodies (e.g., I-394, I-395, I-396, or I-399) are premixed with, for example, varying amounts of test antibodies (e.g., at a ratio of about 1:10 or about 1: 100) some time before applying the CD39 antigen to the sample. In other embodiments of the present invention, control antibodies and test antibodies in different amounts can simply be mixed during exposure of the CD39 antigen sample. As long as it is possible to distinguish bound from free antibodies (at

- 20 045378 example, by separation or washing method to remove free antibodies), as well as I-394 from test antibodies (for example, using species-specific or isotype-specific secondary antibodies, or by specifically labeling I-394, I-395, I-396 or I-399 detectable label), it can be determined whether the test antibodies reduce the degree of binding of I-394, I-395, I-396 or I-399 to antigens. The binding of (labeled) control antibodies in the absence of a completely irrelevant antibody can serve as a high control value. A low control value can be obtained by incubating labeled (I-394, I-395, I-396 or I-399) antibodies with unlabeled antibodies of exactly the same type (I-394, I-395, I-396 or I-399) , during which competition will arise and the binding of labeled antibodies will weaken. In a test assay, a significant decrease in the reactivity of labeled antibodies in the presence of the test antibody indicates that the test antibody cross-reacts or competes with the labeled antibody (I-394, I-395, I-396, or I-399). Any test antibody may be selected that reduces the binding of I-394, I-395, I-396, or I-399 to CD39 antigens by at least about 50%, such as at least about 60%, or more preferably at least by about 80 or 90% (eg, about 65-100%), at any ratio of I-394, I-395, I-396, or I-399 to test antibody from 1:10 to 1:100. In one embodiment of the present invention, such a test antibody will reduce the binding of I-394, I-395, I-396, or I-399 to the CD39 antigen by at least about 90% (eg, about 95%).

Competition can also be assessed, for example, using flow cytometry. In such an assay, cells bearing a given CD39 polypeptide can be first incubated, for example, with I-394 and then with a fluorochrome- or biotin-labeled test antibody. An antibody is considered to compete with I-394 if the binding resulting from preincubation with a limiting amount of I-394 is about 80%, preferably about 50%, about 40% or less (e.g., about 30, 20, or 10% ) on the degree of binding (measured by fluorescence) of the antibody to I-394 without prior incubation. Alternatively, an antibody is considered to compete with I-394 if binding to (fluorochrome or biotin) labeled I-394 antibody on cells preincubated with a limiting amount of the test antibody is about 80%, preferably about 50%, about 40%, or less (eg, approximately 30, 20, or 10%) of the extent of binding without prior incubation with the test antibody.

Determining whether an antibody binds to an epitope region can be done by methods known to one of ordinary skill in the art. An example of such mapping/characterization methods is the determination of the epitope region for an anti-CD39 antibody by footprinting using chemical modification of exposed amines/carboxyls in the corresponding CD39 protein. A specific example of such a footprint method is hydrogen-deuterium exchange detected by mass spectrometry (HXMS), in which hydrogen-deuterium exchange occurs between amide protons during receptor-protein and ligand-protein interactions, binding and reverse exchange, with In this case, the main amide groups involved in binding to proteins are protected from reverse exchange and will therefore remain deuterated. At this stage, relevant regions can be identified using peptic proteolysis, fast microcolumn HPLC separation, and/or electrospray ionization mass spectrometry. See, for example, Ehring H. Analytical Biochemistry, Vol. 267(2), p. 252-259 (1999), Engen, J.R. and Smith, D.L. (2001), Anal. Chem. 73, 256A-265A. Another example of a suitable epitope identification technique is nuclear magnetic resonance (NMR) with epitope mapping, which typically compares the signal positions in the two-dimensional NMR spectra of a free antigen and an antigen-antigen-binding peptide complex, such as an antibody. Typically, the antigen is 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. The antigen signals from the amino acids involved in the interaction with the antigen-binding peptide usually change their 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), p. 61-67 (1998); and Saito and Patterson, Methods. June 1996; 9(3):516-24.

Epitope mapping/characterization can also be performed using mass spectrometry. See, for example, Downard, J. Mass Spectrom. April 2000; 35(4):493-503 and Kiselar and Downard, Anal Chem. May 1, 1999; 71(9):1792–1801. Protease digestion techniques may also be useful in the context of epitope mapping and identification. Antigenic determinant regions/sequences can be determined by protease digestion, for example using trypsin at a ratio of approximately 1:50 relative to CD39, or o/n digestion at pH 7-8 followed by mass spectrometric analysis to identify peptides. Peptides protected from trypsin digestion by the anti-CD39 binder can subsequently be identified by comparing samples digested with trypsin and samples incubated with an

- 21 045378 antibody and then digested with, for example, trypsin (thus revealing a binding recognition region). Other enzymes such as chymotrypsin, pepsin, etc. may also, or alternatively, be used in similar epitope characterization methods. In addition, enzymatic digestion may be a rapid way to analyze whether a potential antigenic determinant sequence is located within a region of the CD39 polypeptide that is not exposed to the surface and therefore is not likely to be related to immunogenicity/antigenicity.

Site-directed mutagenesis is another method for determining the epitope to bind. For example, in an alanine scan, each residue in a protein segment is replaced by an alanine residue and the implications for binding affinity are determined. 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 to the unfolded protein) can be used to confirm that the alanine substitution does not affect the overall foldability of the protein. See, for example, Clackson and Wells, Science 1995; 267:383-386 and Wells, Proc. Natl. Acad. Sci. USA 1,996; 93:1-6.

Electron microscopy can also be used to determine the region of epitope recognition. For example, Wang et al. (Wang et al., Nature, 1992; 355:275-278) used cryo-electron microscopy, 3D image reconstruction, and X-ray crystallography to determine the physical imprint of the Fab fragment on the surface of the native cowpea mosaic virus capsid.

Other marker-free assays for epitope assessment include surface plasmon resonance (SPR, BIACORE) and reflectometric interference spectroscopy (RIS). See, for example, Fagerstam et al., Journal of Molecular Recognition, 1990; 3:208-14; Nice et al., J. Chromatogr. 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.

It should also be noted that an antibody that binds to the same or substantially the same epitope as the antibody can be identified by one or more of the competition assays described herein as examples.

Typically, an anti-CD39 antibody provided herein has an affinity for the corresponding CD39 polypeptide in the range of about 10 ⁴ to about 10 ¹¹ M ^-1 (eg, about 10 8 to about 10 ¹⁰ M ^-1 ). For example, a particular aspect of the present invention provides an anti-CD39 antibody that has an average dissociation constant (KD) of less than 1 x 10 ⁹ M relative to CD39 as measured, for example, by SPR (performed, for example, on a BIAcore™ assay device). In a more specific illustrative aspect of the present invention, anti-CD39 antibodies are provided with a KD of about 1x10 ^-8 to about 1x10 ^-10 M or from about 1x10 ^-9 to about 1x10 ^-11 M relative to CD39, respectively. In one embodiment of the present invention, the binding is a monovalent binding. In one embodiment of the present invention, the binding is a bivalent binding.

Antibodies may have, for example, an average Kd value of no more than (i.e., better affinity than) about 100, 60, 10, 5 or 1 nmol, preferably subnanomolar, or optionally no more than about 500, 200, 100 or 10 pmol. K _d can be determined, for example, by immobilizing recombinantly produced human CD39 proteins on the surface of the chip, followed by application of the test antibody in solution. In one embodiment of the present invention, the method further includes the step of selecting antibodies from step (b) that are capable of competing for binding to CD39 with antibody I-394.

In one aspect of any embodiment of the present invention, the antibodies produced in accordance with the described methods are monoclonal antibodies. In another aspect of the present invention, the non-human animal used to produce antibodies in accordance with the methods described herein is a mammal, such as a rodent, bovine, pig, bird, camel, horse, rabbit, goat or sheep.

DNA encoding an antibody that binds to an epitope present on the surface of the CD39 polypeptide is isolated from the hybridoma and placed into an appropriate expression vector for transfection into a suitable host. The host is then used to recombinantly produce the antibody or variants thereof, eg, a humanized version of the monoclonal antibody, active antibody fragments, chimeric antibodies comprising the antigen-binding portion of the antibody, or versions containing a detectable amount thereof.

DNA encoding monoclonal antibodies of the present invention, such as antibody I394, can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding mouse antibody heavy and light chains). Once isolated, the DNA can be placed into ex

- 22 045378 compression vectors that are then transfected into host cells, such as E. coli cells, SV-40 virus (COS) transformed monkey kidney cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not otherwise produce the protein immunoglobulin, for 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, such as to humanize antibodies, produce fragments or derivatives, or to modify the antibody sequence, for example, at the antigen binding site so as to optimize binding specificity. antibody. In one embodiment, the present invention provides an isolated nucleic acid sequence encoding the light chain and/or heavy chain of an antibody, as well as a recombinant host cell containing (eg, in its genome) such nucleic acid. Recombinant expression in bacteria of DNA encoding an antibody is well known in the art (see, for example, Skerra et al., Curr. Opinion in Immunol., 5, p. 256 (1993) and Pluckthun, Immunol. 130, p. 151 (1992).

Antibody fragments and derivatives (which are encompassed by the term antibody or antibodies as used herein unless otherwise indicated or otherwise clearly contradicted by context) can be prepared by methods known in the art. The fragments include part of an intact antibody, typically the binding site or variable region of an antigen. 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 (herein referred to as a single-chain antibody fragment or single-chain polypeptide), including but not limited to (1) single-chain Fv molecules, (2) single-chain polypeptides , containing only one light chain variable domain, or a fragment thereof, which contains three CDRs of a light chain variable domain, without an associated heavy chain fragment, and (3) single chain polypeptides having only one heavy chain variable region, or a fragment thereof, containing three CDRs of a variable heavy chain regions without an associated light chain moiety; as well as multispecific (eg, bispecific) antibodies formed from antibody fragments. Also, but not limited to, a nanobody, domain antibody, single domain antibody, or dAb.

In one aspect of the present invention, the agent is an antibody selected from a fully human antibody, a humanized antibody, and a chimeric antibody.

In one aspect of the present invention, the agent is an antibody fragment comprising a constant domain selected from IgG1, IgG2, IgG3 and IgG4. In one aspect of the present invention, the agent is an antibody fragment selected from a Fab fragment, Fab' fragment, Fab'-SH fragment, F(ab)2 fragment, F(ab')2 fragment, Fv fragment, heavy chain Ig (Llama Ig or camel), Vhh fragment, single domain Fv and single chain antibody fragment. In one aspect of the present invention, the agent is a molecule derived from a synthetic or semi-synthetic antibody selected from scFV, dsFV, mini-antibody, diabody, tribody, kappa body, IgNAR; as well as a multispecific antibody. In one aspect of the present invention, the antibody is in at least partially purified form. In one aspect of the present invention, the antibody is in a substantially isolated/isolated form.

Ahtu-CD39, such as an antibody, can be included in a pharmaceutical composition at a concentration of 1 to 500 mg/ml, wherein the composition has a pH of 2.0 to 10.0. The composition may additionally contain a buffer system, preservative(s), tonicity regulator(s), chelator(s), stabilizers and surfactants. In one embodiment of the present invention, the pharmaceutical composition is an aqueous composition, i.e. composition containing water. Such a composition is usually a solution or suspension. In another embodiment of the present invention, the pharmaceutical composition is an aqueous solution.

The term aqueous composition is defined as a composition containing at least 50% by weight water. Likewise, the term aqueous solution is defined as a solution containing at least 50% by weight water, and the term aqueous suspension is defined as a suspension containing at least 50% by weight water.

In another embodiment of the present invention, the pharmaceutical composition is a lyophilized composition to which solvents and/or diluents are added by the physician or patient prior to use.

In another embodiment of the present invention, the pharmaceutical composition is a dry composition (eg, lyophilized or spray dried) ready for use without any prior dilution.

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

In another embodiment of the present invention, the pH of the composition is in a range selected from about 2.0 to about 10.0, about 3.0 to about

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9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, and about 5.5 to about 7.5.

In another embodiment of the present invention, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium phosphate, sodium phosphate, as well as tris-(hydroxymethyl)aminomethane, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these buffers represents an alternative embodiment of the present invention.

In another embodiment of the present invention, the composition further comprises a pharmaceutically acceptable preservative. In another embodiment of the present invention, the composition further comprises an isotonic agent. In another embodiment of the present invention, the composition also contains a chelator. In a further embodiment of the present invention, the composition further comprises a stabilizer. In another embodiment of the present invention, the composition further comprises a surfactant. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th ed., 1995.

Other ingredients may be present in the peptide pharmaceutical composition of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, fillers, tonicity regulators, chelators, metal ions, oil carriers, proteins (e.g., human serum albumin, gelatin, or proteins), and zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical composition of the present invention.

Administration of the pharmaceutical compositions of the present invention can be carried out in several ways, for example, intravenously. Suitable antibody compositions 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, for example, Rituxan (rituximab), Herceptin (trastuzumab), Xolair (omalizumab), Bexar (tositumomab), Campat (alemtuzumab), Zevalin, Oncolym and other similar drugs can be used with antibodies according to the present invention.

Also provided are kits that contain a pharmaceutical composition containing an anti-CD39 antibody, as well as an agent that causes the release of ATP from tumor cells, and a pharmaceutically acceptable carrier in a therapeutically effective amount selected for use in the previous methods. The kits may also optionally contain instructions, for example containing a schedule of administration, that will allow a user (eg, a physician, nurse, or patient) to administer the composition contained in the kit to a patient having cancer (eg, a solid tumor). The kit may also include a syringe.

Optionally, the kits contain multiple single-dose packages of pharmaceutical compositions, each of which contains an effective amount of an anti-CD39 antibody or agent that causes the release of ATP from tumor cells for a single administration according to the methods described above. Instruments or devices necessary for administering the pharmaceutical composition(s) may also be included in the kit. For example, the kit may include one or more pre-filled syringes containing an amount of anti-CD39 antibody and an agent that causes the release of ATP from tumor cells.

In one embodiment, the present invention provides a kit for treating cancer in a human patient, which includes:

(a) a dose of an anti-CD39 antibody that neutralizes the activity of sCD39, where optionally the antibody comprises a hypervariable region (e.g., CDR1, CDR2, and CDR3 domains) of the heavy chain variable region of antibody I-394, and a hypervariable region (e.g., CDR1, CDR1, CDR2 and CDR3) of the light chain variable region of antibody I-394;

(b) a dose of an agent that causes the release of ATP from tumor cells; and (c) optionally, instructions for use of an anti-CD39 antibody and an agent that causes the release of ATP from tumor cells in any of the methods described herein.

Diagnosis, prognosis and treatment of malignant tumors.

Described herein are methods used to diagnose, prognosis, monitor, treat and prevent cancer in an individual by using anti-CD39 antibodies to enhance the activity of an agent that causes the extracellular release of ATP from tumor cells.

Extracellular ATP is released from tumor cells in case of stress (mechanical, hypotonic or hypoxic) or in case of cell death. Necrosis promotes passive ATP release through the release of general cellular contents, whereas apoptosis promotes ATP release through activation of caspases 3 and 9, which cleave and activate panexin-1 (ATP transporter). Agents that induce extracellular ATP release from tumor cells may include chemotherapy, radiation therapy, and, in general, agents that induce

- 24 045378 apoptosis and therefore promote the release of ATP. Agents that induce extracellular ATP release have been shown to cause immunogenic cell death. For example, significant ATP release can be caused by anthracyclines, oxaliplatin, cisplatin, x-rays, PARP inhibitor, taxanes, anthracyclines, DNA damaging agents, camptothecins, epothilones, mitomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamicins, duocarmycins , tubulisins , dolastatins, auristatins, enediines, amatoxins, pyrrolebenzodiazepines, ethylene imines, radioactive isotopes, therapeutic proteins or peptides, as well as toxins or fragments thereof.

ATP may also be released by administration together with depleting antibodies that bind to antigens expressed on the surface of cancer cells (eg, tumor antigen), for example, antibodies associated with a chemotherapeutic agent that causes ATP release, antibodies that are capable of mediating apoptosis, or antibodies that direct ADCC to cancer cells (for example, when the antibody has the Fc domain of the human IgG1 isotype to mediate ADCC).

Anthracyclines include, for example, daunorubicin, doxorubicin, epirubicin or idarubicin, optionally liposomal formulations thereof, for example liposomal daunorubicin such as DaunoXome™ or Vyxeos™ or CPX-351 (a combination of cytarabine and daunorubicin). Anthracyclines are widely used to treat solid and hematologic malignancies, including, for example, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), and Kaposi's sarcoma. Doxorubicin and its derivative, epirubicin, are used for breast cancer, pediatric solid tumors, soft tissue sarcoma, and aggressive lymphoma. Daunorubicin is used to treat acute lymphoblastic or myeloblastic leukemia, and its derivative, idarubicin, is used for multiple myeloma, non-Hodgkin's lymphoma and breast cancer. Nemorubicin is used for the treatment of hepatocellular carcinoma, and pixantrone is used for the second-line treatment of non-Hodgkin's lymphomas. Sabarubicin is used to treat non-small cell lung cancer, hormone-resistant metastatic prostate cancer, and ovarian cancer that is resistant to taxane or platinum-based drugs. Valrubicin is used for topical treatment of bladder cancer.

Platinum-based drugs include, for example, oxaliplatin, cisplatin, carboplatin, nedaplatin, phenanthriplatin, picoplatin, satraplatin.

Taxanes include, for example, paclitaxel (Taxol) and docetaxel (Taxotere).

DNA damaging agents include, for example, DNA intercalating agents, for example, an agent that inserts into the DNA structure of a cell and binds to the DNA, thereby causing DNA damage (eg, daunorubicin). Compounds include topoisomerase inhibitors, chemical compounds that block the action of topoisomerases (topoisomerases I and II). Such compounds are used for the treatment of a wide range of solid tumors and hematological malignancies, especially lymphomas. Topoisomerase I inhibitors include camptothecins, such as irinotecan (approved for the treatment of colon cancer), topotecan (approved for the treatment of ovarian and lung cancer), camptothecin, lamellarin D, indenoisoquinoline, indimitecan. Other campthecins include silatecan, cositecan, exatecan, lurtothecan, hymatecan, belotecan and rubitecan. Topoisomerase II inhibitors include, for example, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, and HU-331, a quinolone synthesized from cannabidiol.

PARP inhibitors are known to change the type of cell death from necrosis to caspase-independent apoptosis in cancer cells. Inhibition of PARP leads to an increase in the amount of intracellular ATP, which in turn leads to extracellular ATP release during apoptosis. The poly(ADP-ribose) polymerase (PARP) family of enzymes transforms NAD+ (nicotinamide adenine dinucleotide) into nicotinamide and ADP-ribose to form long and branched (ADP-ribose) polymers on glutamic acid residues of a number of acceptor proteins typically associated with chromatin. PARP-1, the most common PARP, is a nuclear enzyme that catalyzes the formation of poly(ADP-ribose) on target proteins using NAD+ as a substrate. PARP inhibitors typically contain benzoxazole or benzamide moieties as key pharmacophores, and various benzamide derivatives have also been reported. Approved PARP inhibitors that can be used in accordance with the present invention include Olaparib (AZD-2281, Lynparza® from Astra Zeneca, approved for the treatment of ovarian cancer, also effective against breast cancer, prostate cancer and colorectal cancer), Rucaparib (PF -01367338, Rubraca® from Clovis Oncology, approved for the treatment of ovarian cancer), Niraparib (MK-4827, Zejula® from Tesaro, approved for the treatment of epithelial ovarian cancer, ovarian cancer, fallopian tube cancer and primary peritoneal cancer). Also PARP inhibitors that can be used in accordance with the present invention include Talazoparib (BMN-673, BioMarin Pharmaceutical Inc., Pfizer) against hematological and advanced or recurrent solid tumors, Veliparib (Veliparib,

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ABT-888, developed by AbbVie) against ovarian cancer, triple negative breast cancer, non-small cell lung cancer, melanoma, CEP 9722 against non-small cell lung cancer,

E7016 (developed by Eisai) against melanoma, as well as Pamiparib (Pamiparib,

BGB-290), developed by Beigene for the treatment of various solid tumors.

Epothilones include, for example, epothilone B and its various analogues, for example ixabepilone (BMS-247550) is approved for the treatment of breast cancer. Vinca alkaloids include, for example, vinblastine, vincristine, vindesine and vinorelbine. Nitrogen mustards include, for example, cyclophosphamide, chlorambucil, uramustine, ifosfamide, melphalan and bendamustine. Maytansinoids include, for example, Ansamitocin or mertansine (DM1), or DM4, developed by Immunogen Inc.

The agents may be in any suitable configuration or dosage form, including, for example, as a free compound or as part of a conjugate, in a nanoparticle dosage form, in a capsule (e.g., in a liposome), in each case, optionally also in combination with additional pharmaceutically active agents. Conveniently, the agents can be conjugated to a targeting moiety, such as an immunoconjugate. The terms immunoconjugate and antibody conjugate are used interchangeably and refer to an antigen-binding agent, such as an antibody-binding protein or antibody conjugated to another moiety (eg, a cytotoxic agent, a chemotherapeutic agent that causes the release of ATP, as described herein). An immunoconjugate containing an antigen-binding agent conjugated to a cytotoxic substance may also be called an antibody-drug conjugate or CALP.

Although the treatment regimens and methods described herein are particularly useful for the treatment of solid tumors, the treatment regimens and methods described herein can also be used in a variety of hematologic cancers. The methods and compositions of the present invention are used, for example, for the treatment of various types of cancer and other proliferative diseases, including, but not limited to, cancer of the bladder, breast, colon, kidney, liver, lung, ovary, uterus, prostate, pancreas, stomach, cervix, thyroid, head and neck (squamous cell carcinoma of the head and neck), skin (eg, melanoma); hematoblastoses of lymphoid origin, including leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia and Burkitt's lymphoma, multiple myeloma; hematoblastoses of myeloid origin, including acute and chronic myeloid leukemia, promyelocytic leukemia and myelodysplastic syndrome; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma; other tumors including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and neuroma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, and follicular thyroid cancer.

The combination therapy for the treatment of cancer provided herein includes the use of an anti-CD39 neutralizing agent (eg, an antibody) and an agent that induces extracellular ATP release to treat subjects suffering from cancer. In one embodiment, the present invention provides an anti-CD39 antibody and therapy (eg, agent) that induces extracellular ATP release, for use in combination to treat subjects having a solid tumor (eg, solid tumor, advanced refractory solid tumor), or subjects with a hematological tumor. In one embodiment, the present invention provides an anti-CD39 antibody (e.g., having additional characteristics described herein) for use in treating an individual having cancer, wherein the treatment comprises administering to the individual an anti-CD39 antibody in combination with an agent for promoting apoptosis in cancer cells (for example, to induce extracellular ATP release in cancer cells). In one embodiment, the present invention provides an anti-CD39 antibody (eg, having additional characteristics described herein) for use in treating an individual having cancer, wherein the treatment comprises administering to the individual an anti-CD39 antibody in combination with a pharmaceutical composition comprising (a ) an agent (eg, an agent or therapeutic agent) for promoting apoptosis of cancer cells; and (b) a pharmaceutically acceptable carrier. In one embodiment, the present invention provides an anti-CD39 antibody (eg, having additional characteristics described herein) for use in treating an individual having cancer, wherein the treatment comprises administering to the individual an anti-CD39 antibody in combination with a pharmaceutical composition comprising (a ) an agent (eg, an agent or therapeutic agent) for promoting extracellular ATP release in cancer cells; and (b) a pharmaceutically acceptable carrier. In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with a platinum-based drug (eg,

- 26 045378 oxaliplatin, cisplatin, carboplatin, nedaplatin, phenanthriplatin, picoplatin, satraplatin or combination therapy including a platinum-based drug) for the treatment of an individual having a solid tumor. In one embodiment of the present invention, the solid tumor is lung cancer, squamous cell lung cancer, non-small cell lung cancer, ovarian cancer, carcinoma, head and neck squamous cell cancer, colorectal cancer, urothelial cancer, bladder cancer, cervical cancer, gastric cancer, cancer esophagus or breast cancer. In one embodiment of the present invention, the combination therapy comprising a platinum-based drug is FOLFOX (folinic acid, 5-fluorouracil and oxaliplatin). In one embodiment of the present invention, the combination therapy comprising a platinum-based drug also includes carboplatin and a taxane (eg, paclitaxel).

In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with a taxane-based drug (e.g., paclitaxel (Taxol™) or docetaxel (Taxotere™)) for the treatment of an individual having a solid tumor, e.g., ovarian cancer, breast cancer glands.

In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with gemcitabine for the treatment of an individual having a solid tumor, such as ovarian cancer.

In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with an anthracycline-based drug (eg, daunorubicin, doxorubicin, epirubicin, or idarubicin) for the treatment of an individual having a solid tumor, such as ovarian cancer, breast cancer, non-small cell cancer lung, colorectal cancer, prostate cancer, soft tissue sarcoma or bladder cancer. In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with an anthracycline-based drug (eg, daunorubicin, doxorubicin, epirubicin, or idarubicin) for the treatment of an individual having a hematologic tumor, such as acute myelocytic leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), lymphoma, acute lymphoblastic leukemia, myeloblastic leukemia, multiple myeloma or non-Hodgkin's lymphoma.

In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with a PARP inhibitor (eg, a PARP-1 inhibitor, olaparib, rucaparib, niraparib, talazoparib, veliparib, CEP 9722, E7016, or pamiparib) for treating an individual having a solid tumor eg epithelial cancer, ovarian cancer, breast cancer, prostate cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, lung cancer, non-small cell lung cancer or melanoma.

The combination treatment described herein may be particularly effective in the treatment of cancers characterized by high expression of CD39 (with or without the prior step of assessing CD39 expression or levels in the individual), including, but not limited to, ovarian cancer, gastric cancer, and esophageal cancer.

In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with a platinum-based drug (eg, carboplatin) and, optionally, in combination with gemcitabine for the treatment of an individual having ovarian cancer.

In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with a platinum-based drug (eg, oxaliplatin), optionally wherein the anti-CD39 antibody is used in combination with FOLFOX (folinic acid, 5-fluorouracil, and oxaliplatin) for the treatment of an individual having stomach cancer or esophageal cancer.

In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with a platinum-based drug (e.g., carboplatin) and, optionally, in combination with a taxane (e.g., paclitaxel) for treating an individual having non-small cell lung cancer (NSCLC), optionally, wherein the NSCLC is squamous cell lung cancer.

In one embodiment, the present invention provides an anti-CD39 antibody for use in combination with FOLFOX.

In one embodiment of the present invention, the solid tumor is ovarian cancer and the individual is treated with an anti-CD39 antibody in combination with oxaliplatin or carboplatin, optionally additionally in combination with gemcitabine. Optionally, in any embodiment of the present invention, the ovarian cancer is platinum-resistant ovarian cancer. An individual having platinum-resistant ovarian cancer may, for example, be characterized as having cancer that has progressed, relapsed, or failed to respond to prior therapy with a platinum-based drug that does not contain an anti-CD39 antibody. In another embodiment of the present invention, ovarian cancer can be characterized as ovarian cancer that is sensitive to the action of platinum-based drugs.

Combination therapy including an anti-CD39 antibody may be effective in enhancing the effect of an agent or therapy that induces extracellular ATP release from tumors.

- 27 045378 left cells and/or tumor cell death. This may be useful, for example, in an individual having cancer (eg, lung cancer, ovarian cancer, colorectal cancer, gastric cancer, esophageal cancer) that is resistant to an agent or therapy that causes extracellular release of ATP from tumor cells and/or death of tumor cells. An individual having a cancer that is resistant to an agent or therapy may, for example, be characterized as having a cancer that has progressed, relapsed, or failed to respond to prior treatment with such agent or therapy (or a treatment regimen including such agent or therapy where the regimen does not includes anti-CD39 antibody).

For example, an individual who has cancer (eg, breast cancer, ovarian cancer, colorectal cancer, gastric cancer, esophageal cancer) that is resistant to a particular type of agent or therapy that causes extracellular release of ATP from tumor cells (eg, taxane, drugs platinum-based, PARP inhibitor or combination therapy including the above) can be treated with an anti-CD39 antibody in combination with the specified type of agent or therapy that causes extracellular ATP release from tumor cells. For example, in one embodiment of the present invention, an anti-CD39 antibody can be used in combination with a taxane to treat an individual having taxane-resistant cancer. In another example, an anti-CD39 antibody can be used in combination with a platinum-based drug to treat an individual having cancer that is resistant to the platinum-based drug. In another example, an anti-CD39 antibody can be used in combination with a PARP inhibitor to treat an individual who has PARP inhibitor-resistant cancer.

In another embodiment of the present invention, an individual having cancer that is resistant to a particular agent or therapy that causes extracellular ATP release from tumor cells (eg, a taxane, a platinum-based drug, a PARP inhibitor, or a combination therapy including the above) may treat with an anti-CD39 antibody in combination with another type of agent or therapy that causes extracellular ATP release from tumor cells. For example, in one embodiment of the present invention, an anti-CD39 antibody can be used in combination with a taxane to treat an individual having platinum-resistant cancer. In another embodiment of the present invention, an anti-CD39 antibody can be used in combination with a platinum-based drug to treat an individual having taxane-resistant cancer. In another embodiment of the present invention, an anti-CD39 antibody can be used in combination with a PARP inhibitor to treat an individual having taxane- and/or platinum-resistant cancer.

In one embodiment, the present invention provides an antibody that is capable of binding to soluble human CD39 extracellular domain protein and inhibiting the ATPase activity of soluble human CD39 extracellular domain protein for use in the treatment or prevention of cancer in an individual, wherein the treatment includes the following:

a) determining whether the individual has a poor prognosis for response to treatment with an agent that causes extracellular release of ATP from tumor cells, and

b) after determining that the individual has a poor prognosis for response to treatment with an agent that causes extracellular release of ATP from tumor cells, administering to the individual an antibody that is capable of binding to human CD39 protein and inhibiting the ATPase activity of human CD39 protein in the presence of exogenously added ATP.

Optionally, the individual has cancer that is resistant to a platinum, taxane, or PARP inhibitor. Optionally, the step of determining whether an individual has a poor prognosis for response to treatment with an agent that causes extracellular ATP release from tumor cells includes assessing whether immune effector cells in a biological sample collected from the individual are characterized by one or more markers of immune suppression and/or exhaustion, wherein the presence of and/or elevated levels of immune effector cells characterized by one or more markers of immune suppression and/or exhaustion indicates a poor prognosis of response to treatment with an agent that causes extracellular ATP release from tumor cells.

The benefit of using an anti-CD39 antibody to enhance the effect of an agent or therapy that causes extracellular ATP release from tumor cells and/or tumor cell death can also be used to achieve improved outcomes in an individual having cancer (eg, lung cancer, ovarian cancer, colorectal cancer, gastric cancer, esophageal cancer) that is sensitive (eg, predicted or determined to be sensitive) to an agent or therapy that causes extracellular ATP release from tumor cells and/or tumor cell death. For example, in one embodiment of the present invention, an anti-CD39 antibody can be used in combination with a taxane to treat an individual having a taxane-sensitive cancer. In another example, an anti-CD39 antibody can be used in combination with a platinum-based drug to treat an individual having platinum-sensitive cancer. In another example, an anti-CD39 antibody can be used in combination with a PARP inhibitor to treat an individual having a PARP inhibitor-sensitive cancer.

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In accordance with the present invention, adjunctive or combined administration (simultaneous administration) includes simultaneous administration of compounds in the same or different dosage forms or separate administration of compounds (eg, sequential administration). Thus, an anti-CD39 antibody and an agent that causes extracellular ATP release can be administered simultaneously in a single composition. Alternatively, the anti-CD39 antibody and the agent that causes extracellular ATP release may be designed to be administered separately and administered in parallel or sequentially.

A patient with cancer can be treated with an anti-CD39 agent and an agent that induces extracellular ATP release with or without a prior detection step to assess tumor cell ATPase activity, tumor ATP (eg, intratumoral ATP concentration), and/or CD39 expression on cells. Optionally, the methods of treatment may include the step of detecting a CD39 nucleic acid or polypeptide in a biological sample of a tumor (eg, on the surface of a tumor or a tumor-infiltrating cell) taken from an individual.

Optionally, the methods of treatment may include the step of detecting a CD39 nucleic acid or polypeptide in a biological sample taken from an individual. Biological samples include any suitable biological fluid (eg, serum, lymph, blood), cell sample, or tissue sample. Any determination that cells in a biological sample (e.g., cancer cells, lymphocytes, e.g., regulatory T cells, B cells, T cells) express CD39 at high levels, or that a large number of cells in the sample are CD39 positive, or exhibit a high degree of anti-CD39 antibody staining (compared to a reference sample), may indicate that the individual has a cancer that is best treated with an agent that inhibits CD39 in combination with an agent that causes ATP release from tumor cells. In one embodiment of the present invention, the methods of treatment may include the step of detecting a CD39 nucleic acid or polypeptide in a biological sample of a tumor (eg, on the surface of a tumor-infiltrating cell) taken from an individual.

In the proposed methods of treatment, the anti-CD39 antibody and the agent or therapy that causes extracellular ATP release can be administered separately, together or sequentially, or as a cocktail (if necessary). In some embodiments of the present invention, an agent or therapy that causes extracellular ATP release is administered prior to administration of the anti-CD39 antibody. In preferred embodiments of the present invention, the anti-CD39 antibody is administered before or simultaneously with the administration of an agent or therapy that causes extracellular ATP release. In one preferred embodiment of the present invention, the anti-CD39 antibody is administered concurrently with or 0-15 days prior to a course or cycle of treatment with an agent or therapy that causes extracellular ATP release. For example, an anti-CD39 antibody can be administered approximately 0-15 days prior to administration of an agent or therapy that causes extracellular ATP release. In some embodiments, the anti-CD39 antibody is administered at least 1, 12, 24, or 48 hours before administration of an agent or therapy that causes extracellular ATP release. In some embodiments, the anti-CD39 antibody is administered at least 1, 12, 24, or 48 hours before administration of an agent or therapy that causes extracellular ATP release, but no more than 1 week before administration of an agent or therapy that causes extracellular release of ATP. In some embodiments, the anti-CD39 antibody is administered between 0 and 48 hours or between 1 and 48 hours before administration of an agent or therapy that causes extracellular ATP release. In some embodiments of the present invention, the anti-CD39 antibody is administered over a period of from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 hour to about 2 weeks, from about 1 hour to about 1 week, from about 1 to about 2 hours, about 2 to about 4 hours, about 4 to about 6 hours, about 6 to about 8 hours, about 8 hours to 1 day, about 24 or 48 hours to about 5, 6 or 7 days, from about 1 hour to about 5, 6 or 7 days, from about 1 hour to about 15 days, from about 24 or 48 hours to about 15 days, from about 3 to 7 days, or from about 1 in 5 days before administration of an agent or therapy that causes extracellular ATP release. In some embodiments of the present invention, the anti-CD39 antibody is administered concomitantly with an agent or therapy that causes extracellular ATP release. In some preferred embodiments of the present invention, an agent or therapy that causes extracellular ATP release is administered at least twice within 15 days or approximately two weeks after administration of the anti-CD39 antibody. In other embodiments of the present invention, the anti-CD39 antibody is administered after an agent or therapy that causes extracellular ATP release. In some embodiments of the present invention, the anti-CD39 antibody is administered for about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 to about 24, 36 or 48 hours, from about 1 to about 2 hours, from about 2 to about 4 hours, about 4 to about 6 hours, about 6 to about 6 hours

- 29 045378 about 8 hours, from about 8 hours to 1 day, or from about 1 to about 2, 3, 4 or 5 days after administration of an agent or therapy that causes extracellular release of ATP.

An agent or therapy that causes extracellular release of ATP can be used in such amounts and treatment regimens as are typically used for that agent or therapy in monotherapy for a particular disease or condition.

An example of a suitable amount of anti-CD39 antibody is 1 to 20 mg/kg body weight. In one embodiment of the present invention, said amount is administered to an individual weekly, every two weeks, monthly, or every two months.

In one embodiment, the present invention provides a method of treating a human having cancer that consists of at least one cycle of application of an effective amount of an anti-CD39 antibody of the present invention and an effective amount of an agent or therapeutic agent that causes extracellular ATP release. In one embodiment of the present invention, the cycle is a period of eight weeks or less (eg, 2, 4, 8 weeks). In one embodiment of the present invention, one, two, three or four doses of anti-CD39 antibody are administered for each of at least one cycle, optionally at a dosage of 1-20 mg/kg body weight. In one embodiment of the present invention, the anti-CD39 antibody is administered by intravenous infusion. In one embodiment of the present invention, one, two, three, or four doses of an agent or therapeutic that causes extracellular ATP release are administered for each of at least one cycle.

As shown herein, the most potent antitumor responses were observed when repeated administration of the chemotherapeutic agent in the presence of saturating concentrations of anti-CD39 antibody allowed ATP accumulation and suppression of adenosine production for the standard two-week period required to initiate an effective antitumor immune response. Accordingly, in one preferred embodiment of the present invention, treatment according to the present invention includes at least two sequential administrations of an agent or therapy that causes extracellular release of ATP.

In one embodiment of the present invention, an agent or therapy that causes extracellular ATP release is administered at least twice within 15 days or approximately two weeks after administration of the anti-CD39 antibody. The Ahtu-CD39 antibody can be administered in an amount and/or schedule such that concentrations of the anti-CD39 antibody in the bloodstream and/or tissue of interest (eg, tumor tissue) inhibit the ATPase activity of CD39 (eg, at a concentration that saturates the CD39 protein) at each of two successive administrations of an agent or therapy that causes extracellular release of ATP. For example, in one preferred therapeutic regimen, the anti-CD39 antibody is administered concurrently with or at least 1-48 hours prior to administration of the ATP-releasing chemotherapeutic agent. In one preferred therapeutic regimen, the anti-CD39 antibody is administered at least 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the ATP-releasing chemotherapeutic agent.

In one embodiment, the present invention provides an antibody that is capable of binding to human CD39 protein (NTPDasei) and inhibiting the ATPase activity of human CD39 protein when used in the treatment of a human tumor, which treatment comprises administering to the individual an effective amount of both an anti-CD39 antibody and and an agent or therapeutic agent that causes extracellular release of ATP from tumor cells, wherein the agent or therapeutic agent that causes extracellular release of ATP from tumor cells is administered at least twice (for example, in a first and second sequential administration) and wherein antu-CD39 the antibody is administered in an amount and/or schedule effective to achieve and/or maintain a saturating concentration of anti-CD39 antibody between said two administrations of an agent or therapeutic agent that causes extracellular release of ATP from tumor cells. Ahtu-CD39 antibody can be administered, for example, once or twice.

In one embodiment of the present invention, two administrations of an agent or therapeutic agent that causes extracellular ATP release from tumor cells are separated by two weeks or less (eg, daily, weekly, every other week). In one embodiment of the present invention, an agent or therapeutic agent that causes extracellular release of ATP from tumor cells is administered at least 2, 3, or 4 times over a two-week period. In one embodiment of the present invention, the anti-CD39 antibody can be administered in an amount that results in a concentration that is at least the minimum concentration required to substantially completely (e.g., 90%, 95%) fill (saturate) CD39 protein.

In one embodiment of the present invention, the anti-CD39 antibody can be administered in an amount that results in a concentration that is at least the minimum concentration required to substantially completely (e.g., 90, 95%) saturate the antibody with protein CD39 between said two administrations of an agent or therapeutic that causes extracellular release of ATP from tumor cells.

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Typical protocols for treating a human with an aHTu-CD39 antibody include, for example, administering to the patient an effective amount of both an anti-CD39 antibody and an agent or therapeutic agent that causes extracellular release of ATP from tumor cells, wherein the method includes at least one cycle of administration in which at least one dose of an anti-CD39 antibody and two doses of an agent or therapeutic agent that causes extracellular ATP release from tumor cells, where two doses of an agent or therapeutic agent that causes extracellular ATP release are administered at an interval of two weeks or less. In one embodiment of the present invention, the application cycle is from 2 to 8 weeks. In one embodiment of the present invention, the anti-CD39 antibody can be administered in an amount and/or schedule such that the concentration of the anti-CD39 antibody in the bloodstream and/or tissue of interest (eg, tumor tissue) inhibits the ATPase activity of CD39. Optionally, the anti-CD39 antibody is administered in an amount that provides a concentration at which substantially complete (eg, 90, 95%) CD39 loading is achieved. In one embodiment of the present invention, the anti-CD39 antibody is administered simultaneously with or 1-48 hours before the administration of an agent or therapeutic agent that causes extracellular ATP release from tumor cells.

In one embodiment, the present invention provides an antibody that is capable of binding to human CD39 protein (NTPDase1) and inhibiting the ATPase activity of human CD39 protein for use in the treatment of a human tumor, wherein the treatment comprises administering to the individual an amount and amount of an anti-CD39 antibody schedule effective to achieve and/or maintain a saturating concentration of anti-CD39 antibody for at least one week, optionally at least two weeks (e.g., two, three, four weeks or more), and (b) an agent or therapeutic agent causing extracellular release of ATP from tumor cells, wherein the anti-CD39 antibody is administered simultaneously or 1-48 hours before administration of an agent or therapeutic agent that causes extracellular release of ATP from tumor cells. Optionally, an agent or therapeutic agent that causes extracellular release of ATP from tumor cells is administered at least twice during the two-week period after administration of the anti-CD39 antibody, for example, an agent or therapeutic agent that causes extracellular release of ATP from tumor cells can be administered at least once a week.

In any embodiment of the present invention, the anti-CD39 antibody can be used in an amount that provides a concentration that is at least the minimum concentration required to substantially completely (e.g., 90, 95%) saturate the antibody with CD39 protein during one week. In one embodiment of the present invention, the anti-CD39 antibody can be used in an amount that provides a concentration that is at least the minimum concentration required to substantially completely (e.g., 90, 95%) saturate the antibody with the protein CD39 for two weeks.

Compositions with anti-CD39 antibodies in combination with an agent or therapeutic agent that causes extracellular ATP release may optionally be combined (further combined) with treatment with one or more other therapies or therapeutic agents, including agents typically used for the particular therapeutic purpose for which antibody is used. The additional therapeutic agent is typically administered in amounts and on a schedule typically used for that agent in monotherapy for the particular disease or condition being treated. In one embodiment of the present invention, the additional therapeutic agent is an agent (eg, an antibody) that inhibits the CTLA-4 or PD-1 axis (ie, inhibits PD-1 or PD-L1). Antibodies that bind to CTLA-4, PD1 or PD-L1 can be used, for example, at the doses and/or frequency with which such agents are used as monotherapy, for example, as described below. In one embodiment of the present invention, the additional therapeutic agent is an agent (eg, an antibody) that binds to the human CD73 protein and neutralizes the 5'-ectonucleotidase activity of the human CD73 protein (eg, soluble CD73 protein, cell-expressed CD73 protein). Human CD73, also known as ecto-5'-nucleotidase, 5'-ribonucleotide phosphohydrolase, EC 3.1.3.5, encoded by the NT5E gene, exhibits 5'-nucleotidase, particularly AMP, NAD and HMN nucleosidase activity. CD73 catalyzes the conversion of purine 5' mononucleotides to nucleosides at neutral pH, with AMP being the preferred substrate. The enzyme consists of a dimer of 2 identical 70 kDa subunits linked by a glycosyl-phosphatidylinositol anchor to the outer surface of the plasma membrane. The amino acid sequence of the human CD73 precursor protein (monomer), including the signal sequence in the region of amino acids 1-26, is shown in Genbank under accession number NP 002517 and is incorporated herein by reference in its entirety:

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MCPRAARAPA TLLLALGAVL WPAAGAWELT ILHTNDVHSR LEQTSEDSSK CVNASRCMGG VARLFTKVQQ IRRAEPNVLL LDAGDQYQGT IWFTVYKGAE VAHFMNALRY DAMALGNHEF DNGVEGLIEP LLKEAKFPIL SANIKAKGPL ASQISGLYLP YKVLPVGDEV VGIVGYTSK E TPFLSNPGTN LVFEDEITAL QPEVDKLKTL NVNKIIALGH SGFEMDKLIA QKVRGVDVW GGHSNTFLYT GNPPSKEVPA GKYPFIVTSD DGRKVPWQA YAFGKYLGYL KIEFDERGNV ISSHGNPILL NSSIPEDPSI KADINKWRIK LDNYSTQELG KTIVYLDGSS QSCRFRECNM GNLIC DAMIN NNLRHTDEMF WNHVSMCILN GGGIRSPIDE RNNGTITWEN LAAVLPFGGT FDLVQLKGST LKKAFEHSVH RYGQSTGEFL QVGGIHWYD LSRKPGDRW KLDVLCTKCR VPSYDPLKMD EVYKVILPNF LANGGDGFQM IKDELLRHDS GDQDINWST YISKMKVIYP AVEGRIKFST GSHCHGSFSL IFLSLWAVIF VLYQ (SEQ ID NO: 48).

As used herein, the terms inhibit, inhibit, neutralize, or neutralize a CD73 polypeptide (e.g., neutralize CD73, neutralize CD73 activity, or neutralize CD73 enzymatic activity, etc.) refer to a process in which 5'-nucleotidase activity (5' -ectonucleotidase) CD73 is inhibited. This includes, in particular, inhibition of CD73-mediated adenosine formation, i.e. inhibition of CD73-mediated catabolism of AMP to adenosine. This can be determined, for example, by a cell-free assay that measures the ability of a test compound to inhibit the conversion of AMP to adenosine, directly or indirectly. In one embodiment of the present invention, the antibody formulation causes at least a 50% reduction in the conversion of AMP to adenosine, at least a 70% reduction in the conversion of AMP to adenosine, or at least an 80% reduction in the conversion of AMP to adenosine, by reference, for example, to the assays described herein.

Examples

Ways.

Formation of mutant forms of CD39.

Mutant forms of CD39 were generated by PCR. Amplified sequences were run on an agarose gel and purified using the Macherey Nagel PCR Clean-Up Gel Extraction kit (#740609). Purified PCR products obtained for each mutant were then ligated into an expression vector using the ClonTech InFusion system. Vectors containing the mutated sequences were prepared as minipreps and sequenced. After sequencing, vectors containing the mutated sequences were prepared as Midipreps using the Promega PureYield™ Plasmid Midiprep System. HEK293T cells were grown in Dulbecco's modified medium (DMEM) (Invitrogen), transfected with vectors using Lipofectamine 2000 from Invitrogen, and incubated at 37°C in a CO ₂ incubator for 48 hours before testing for transgene expression. . Mutant forms were transfected in HEK293T cells as shown in the table. The target amino acid mutations in the table are shown using SEQ ID NO: 1.

Mutant Substitutions 1 V77G H79Q Q444K G445D 2A V81S E82A R111A V115A 2B E11OA R113T E114A 3 R118A S119A Q120K Q122H E123A 4 D150A E153S R154A S157K N158A L278F 5 Q96A N99A E143A R147E 6 K188R Replacement of residues 190-207 with the KTPGGS linker 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 N371K L372K E375A K376G Insert 377V V377S 12 K388N Q392K P393S E396A 13 А402Р G403A K405A E406A 15 K87A E100A D107A 16 0323A Q324A Q327A E331K 17 N334A S336A Y337G N346A 18 Q228A I230S D234A Q238A 19 R138A M139A E142K

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Cloning, production and purification of soluble human CD39.

Molecular biology.

Human CD39 protein was cloned from human PBMC (peripheral blood mononuclear cell) cDNA using the following primers:

TACGACTCACAAGCTTGCCGCCACCATGGAAGATACAAAGGAGTC (SEQ ID NO: 35) (forward primer) and

CCGCCCCGACTCTAGATCACTTGTCATCGTCATCTTTGTAATCGACATAGGTGGAGTGGGAGAG (SEQ ID NO: 36) (reverse primer).

The purified PCR product was then cloned into an expression vector using the InFusion cloning system. The M2 marker (FLAG marker underlined in SEQ ID NO: 39) was added to the C-terminal region of the protein for the purification step; It is understood that the CD39 extracellular domain protein (eg, SEQ ID NO: 39) in any embodiment of the present invention may optionally be designated as lacking the M2 marker.

Expression and purification of human CD39 protein.

After validation of the cloned sequence, CHO cells were nucleofected and the production population was then subcloned to generate a human CD39 protein-producing cell clone. The supernatant from the roller-grown human CD39 clone was collected and purified using an M2 chromatography column and eluted using M2 peptide. The purified proteins were then loaded onto an S200 size exclusion chromatography column. The purified protein corresponding to the monomer was prepared in Tris-buffered saline (TBS) pH 7.5. The amino acid sequence of the recombinant CD39-M2 extracellular domain protein without the M2 marker was as follows:

MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGSSHTSLYIY

KWPAEKENDTGWHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERAREVIPRSQHQETPV YLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGK FSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQTIESPDNALQFRLYGKDYNVY THSFLC YGKDQALWQKLAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLP FQQFEIQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFGAFSAFYFVMKFLNL TSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADS WEHI HFIGKIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHSTYV (SEQ ID NO: 2).

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

MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGSSHTSLYIY

KWPAEKENDTGWHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERAREVIPRSQHQETPV YLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGK FSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQTIESPDNALQFRLYGKDYNVY THSFLC YGKDQALWQKLAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLP FQQFEIQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFGAFSAFYFVMKFLNL TSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADS WEHI HFIGKIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHSTYVDYKDDDDK (SEQ ID NO: 39).

Inhibition of enzymatic activity of soluble CD39.

Antibody inhibition of the enzymatic activity of the resulting soluble CD39 protein was assessed using the CellTiter Glo™ system (Promega, no. G7571), which allows the assessment of ATP hydrolysis by using a reagent that generates a luminescent signal proportional to the amount of ATP present. In this way, the inhibition of ATP hydrolysis mediated by soluble CD39 can be assessed. Briefly, dose ranges of anti-CD39 antibodies from 100 to 6x10 ^-3 μg/ml were incubated together with 400 ng/ml soluble recombinant human CD39 protein having the amino acid sequence described in the Methods section (SEQ ID NO: 39) 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 (CellTiter Glo™). 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.

Antibody inhibition of CD39 enzymatic activity in CD39-expressing cells was assessed using the CellTiter Glo™ system (Promega, no. G7571), which allows the assessment of ATP hydrolysis by using a reagent that generates a luminescent signal proportional to the amount of ATP present. Therefore, the assay was designed to evaluate the inhibition of ATP hydrolyzed by CD39 in cell culture supernatant. Briefly, 5 × 10 ⁴ human Ramos lymphoma cells, as well as 5 × 10 ³ CHO cells expressing human CD39, cynomolgus CD39, and mouse CD39 were incubated for 1 h at 37°C with 30 to 5 × 10 anti-CD39 antibodies ^{. 4} µg/ml. Cells were then further incubated with 20 μM ATP for 1 h at 37°C. The plates were centrifuged for 2 min at 400 g, after which 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 production: Immunization and screening of mice.

To generate anti-human CD39 antibodies, Balb/c mice were immunized with the above-described recombinant human CD39-M2 extracellular domain protein. Mice first received a primary immunization with an emulsion of 50 μg of CD39 protein and complete Freund's adjuvant intraperitoneally, then a second immunization with an emulsion of 50 μg of CD39 protein and incomplete Freund's adjuvant intraperitoneally, and finally a booster of 10 μg of CD39 protein intravenously. Spleen immune cells were fused 3 days after boost with immortalized X63.Ag8.653 B cells and cultured in the presence of irradiated spleen cells. Hydridomes were seeded in semisolid medium containing methylcellulose, and growing clones were selected using a Clonepix 2 apparatus (from Molecular Devices).

Example 1. Mapping of epitopes of known monoclonal antibodies that neutralize CD39.

To understand how antibodies can inhibit the enzymatic (ATPase) activity of cellular CD39, we examined the epitopes associated with antibodies that are known to inhibit the ATPase activity of CD39 in cell-based assays: BY40, disclosed in PCT Publication No. WO 2009/095478 .

To define the epitopes of anti-CD39 antibodies, we developed mutant forms of CD39 defined by amino acid substitutions exposed on the molecular surface above the surface of CD39. Mutant forms were transfected into HEK-293T cells as shown in the table 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 studied on 20 resulting mutants using flow cytometry. BY40 antibodies showed both complete loss of binding to cells expressing CD39 mutant 5 and 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. Monoclonal antibodies known to neutralize CD39 are not able to inhibit the ATPase activity of recombinant soluble CD39 protein.

Two antibodies (BY40 and BY12) known to inhibit CD39 ATPase activity from cell assays 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 prepared as described above was assessed using CellTiter Glo™ (Promega, #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 novel antibodies identified herein.

Example 3 Screening for new monoclonal antibodies to block sCD39 activity.

A series of immunizations was performed to detect antibodies that neutralize the ATPase activity of soluble sCD39. To obtain anti-huCD39 antibodies, animals were immunized with the recombinant human CD39-M2 extracellular domain protein described above. A total of 15 series of immunizations were carried out using different protocols and different animals, including different strains of mouse, rat and rabbit.

In the initial immunization protocols, primary screening included analysis of the supernatant of growing clones by flow cytometry using wild-type CHO cells and CHO cells expressing huCD39. Cells were stained with 0.1 and 0.005 μM carboxyfluorescein succinimidyl ester (CFSE), respectively. For flow cytometry screening, all cells were mixed equally and the presence of reactive antibodies in the supernatants was detected by an APC-labeled polyclonal goat anti-mouse antibody. For antibodies that bind to human CD39, supernatants were then monitored for inhibition of soluble CD39 enzymatic activity using the screening assay developed and described above (see Methods).

The results showed that although many specific CD39-binding antibodies could be obtained, none of the antibodies from any of these immunizations demonstrated

- 34 045378 any inhibition of the enzymatic activity of soluble CD39. One possibility is that the dominant epitopes on the surface of CD39 do not include any epitopes suitably located near this CD39 catalytic site. Due to the small number of antibodies available that inhibit cellular CD39 and the known difficulties in inhibiting the catalytic sites of enzymes using antibodies, the absence of antibodies that neutralize soluble CD39 may indicate that it is not possible to obtain antibodies that inhibit soluble (extracellular domain) CD39. Other possibilities involve nonfunctional screening assays and/or misfolded or functional soluble CD39 protein, particularly since the lack of any antibody capable of inhibiting soluble CD39 makes validation of soluble CD39 blocking assays difficult.

Due to the lack of antibodies capable of inhibiting soluble CD39, an additional immunization screening protocol was performed designed to obtain antibodies that bind to the active site of CD39, as identified by the BY40 antibody epitope. Briefly, the primary screen involved analysis of the supernatant of growing clones by flow cytometry using wild-type CHO cells and CHO cells expressing human CD39 cell lines as in previous immunizations, followed by screening for loss of binding with HEK293T cells expressing the mutant form 5 CD39 compared to wild-type CD39, as shown in the table. Mutant 5 has substitutions at residues Q96, N99, E143, and R147. However, again the results showed that although many specific CD39-binding antibodies could be generated that showed loss of binding to mutant 5, none of the antibodies from any of the initial immunizations demonstrated any inhibition of the enzymatic activity of soluble CD39.

Example 4 Identification of a first antibody that inhibits the activity of soluble CD39 as part of an epitope-directed screen.

The present inventors sought to identify anti-CD39 antibodies that do not bind to the region of Q96, N99, E143 and R147 (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, can be used in pharmacological studies of antibodies that inhibit cellular CD39 that binds to the binding site of BY40, for example, to detect and quantify free CD39 proteins on the surface of cells in the presence of BY40 or BY40-like antibodies that inhibit cellular CD39.

Starting from the immunization results of Example 3, where hybridomas were monitored for loss of binding to CD39 mutant 5, a hybridoma was selected that was not among those that showed loss of binding to CD39 mutant 5. This hybridoma (I-394) was among the broader population due to inconclusive data indicating possible partial loss of binding to mutant 5, but not loss of binding to mutant 5, and was therefore not initially retained.

In the context of ongoing screening of supernatants for further immunizations to inhibit the enzymatic activity of soluble CD39, antibody I-394, which had been cloned and produced, 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 (see Methods).

Antibody I-394 was obtained with constant regions of the human IgG1 isotype with a modified Fc domain having mutations L234A/L235E/G237A/A330S/P331S (Kabat numbering), which leads to a lack of binding to Fcγ receptors CD16A, CD16B, CD32A, CD32B and human CD64. Briefly, the _VH and _Vk sequences of antibody I-394 ( _VH and _Vk variable regions shown in SEQ ID NO: 3 and 4, respectively) were cloned into expression vectors containing huIgG1 constant domains having the above mutations and the huCk constant domain, respectively. . The two resulting vectors were co-transfected into the CHO cell line. The resulting cell population was used to produce antibodies in CHO medium. The antibody was then purified using Protein A. The amino acid sequences of the corresponding heavy and light chain variable domains of I-394 are shown below (Kabat CDR regions are underlined).

I-394 heavy chain variable domain sequence:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGRTLEWIGYIVPIjNGGSTFNQKFKGRA

TLTVNTSSRTAYMELRSLTSEDSAAYYCARGGTRFAYWGQGTLVTVSA (SEQ ID NO: 3).

I-394 light chain variable domain sequence:

DIVLTQSPASLAVSLGQRATISCRASESVDNFGVSEblYWFQQKPGQPPNLLIYGASNQGSGVPARFRG

SGSGTDFSLNIHPMEADDTAMYFCQQTKEVPYTFGGGTKLEIK (SEQ ID NO: 4).

The sequences of the heavy and light chains of I-394 with human IgG1 constant regions with substitutions L234A/L235E/G237A/A330S/P331S (retaining X297-linked glycosylation) are shown below:

I-394 heavy chain sequence:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYNMHWVKQSHGRTLEWIGYIVPIjNGGSTFNQKFKGRA

TLTVNTSSRTAYMELRSLTSEDSAAYYCARGGTRFAYWGQGTLVTVSAASTKGPSVFPLAPSS

KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPSSIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 37).

I-394 light chain sequence:

DIVLTQSPASLAVSLGQRATISCRASESVDNFGVSFMYWFQQKPGQPPNLLIYGASNQGSGVPARFRG

SGSGTDFSLNIHPMEADDTAMYFCQQTKEVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT

ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC (SEQ ID NO: 38).

Antibody I-394 was then tested for loss of binding to CD39 mutants, determined by amino acid substitutions exposed on the molecular surface above the surface of CD39. Mutants were transfected into HEK293T cells as shown in the table using SEQ ID NO: 1. Dosage ranges of I-394 antibodies were tested on 20 mutants using flow cytometry. As shown in FIG. 3B, I-394 showed complete loss of binding to cells expressing the 19 CD39 mutant. Mutant 19 includes substitutions at residues R138, M139, and E142. Thus, the core epitope I-394 includes one or more (or all) of residues R138, M139, and E142.

Unlike the previous 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, while strongly reducing binding to mutant 5 (but some residual binding to mutant 5 remains). Interestingly, residues from mutant 19 are in close proximity or adjacent to residues from mutant 5, so that compared to BY40, I-394 may represent a shift in epitope position. Thus, antibody I-394 represents a valuable new epitope for anti-CD39 antibodies that allows inhibition of the ATPase activity of the soluble CD39 protein. It also provides a specific positive control that allows validation and screening assays to identify additional antibodies that neutralize the ATPase activity of soluble CD39 protein.

Example 5. Non-epitope-directed screening of monoclonal antibodies that neutralize soluble CD39.

Based on the results of Example 4 showing that antibody-mediated inhibition of soluble CD39 is possible, combinations of different immunizations using different protocols from Example 3 were revised to detect antibodies that neutralize the ATPase activity of soluble CD39.

Different screening approaches for ATPase inhibition were then evaluated. In one experiment, antibody I-394 was used to isolate supernatants from the immunization hybridomas of Example 3, which were negative for the ability to inhibit the ATPase activity of soluble CD39. 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, whereby the present inventors observed that the purified antibody I-394 was again able to inhibit ATPase activity and recover.

Based on the above results, new immunization and screening protocols were developed where growing clones from new and past immunization series were screened by flow cytometry using wild-type CHO cells and human CD39-expressing CHO cells without assessing inhibition of soluble CD39 activity or ATPase activity of cellular CD39 and without disruption of epitope screening. While data regarding 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 results in the ATPase activity blocking assay. Hybridomas that bind to CD39 were selected and cloned and then purified using protein A according to the following protocol:

10 μl of protein A granules were added to 300 μl of hybridoma supernatant;

adjusted with NaCl to a final concentration of 1.5 M;

rotated the tubes for 3-4 hours at 4°C;

centrifuged for 1 min at 1500 rpm;

- 36 045378 the supernatant was drained and washed three times using 1 ml of TBS;

drained the entire fuel pump after the third flush;

added 50 μl of 0.1 M citrate with pH 3, homogenized and incubated at room temperature for 5 min;

centrifuge the granules for 1 min at 1500 rpm;

collected 50 μl of eluent and quickly added 450 μl of TBS;

stored at 4°C.

The resulting antibodies were then comparatively screened for their ability to inhibit CD39 ATPase activity to the same extent as I-394. The assay methods used to inhibit the enzymatic activity of soluble and cellular CD39 were the same as described above (see Methods section). Surprisingly, among the typical antibodies produced in this manner, some have demonstrated inhibition of soluble CD39 (as well as inhibition of cellular CD39).

In fig. 1 shows a representative screen result showing antibodies I-397, I-398 and I-399 compared to the positive control antibody I-394. Similarly, antibodies I-395 and I-396 from different immunization series inhibited the enzymatic activity of soluble CD39 protein.

In fig. 2A and 2B show results for antibodies I-395 and I-396, where more antibodies were available for additional neutralization testing of both soluble and cellular CD39. In fig. Figure 2A shows that, compared to antibodies BY40 and I-394, antibodies I-395 and I-396 inhibit cell membrane-associated CD39, with both I-394 and I-395 demonstrating greater activity and maximum inhibition of cellular CD39 compared from BY40. In fig. 2B shows that antibodies I-395 and I-396 inhibit soluble CD39 compared to antibodies BY40 and I-394. Although BY40 does not inhibit soluble CD39 at any concentration, antibodies I-394, I-395 and I-396 all inhibit soluble CD39, with I-394 showing the greatest activity, followed by I-395 and then I-396 with the least activity.

These results raise the possibility that due to a factor(s) in hybridoma supernatants, ATP is rapidly hydrolyzed in both cell culture and soluble CD39 assays such that the ATP signal would not be detected in conventional antibody screening. The soluble factor may be CD39 or some other enzyme, for example, produced by the fusion partner.

The antibodies were then cloned with modification to have constant regions with the Fc domain of human IgG1 with mutations L234A/L235E/G237A/A330S/P331S (EU Kabat numbering), which results in a lack of binding to the Fcγ receptors CD16A, CD16B, CD32A , human CD32B and CD64, the same as shown for I-394. The resulting antibodies can then be titrated and then their activity assessed in more detail as described in Examples 7-9 (titration, inhibition of ATPase activity) to determine the EC ₅₀ and IC ₅₀ to rank the antibodies according to their activity.

Example 6. Epitope mapping of monoclonal antibodies that neutralize soluble CD39.

As shown in Example 4, antibody 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 number of mutants CD39 shown in example 1 and table. The mutants were transfected into HEK-293T cells as shown in the table using SEQ ID NO: 1. Test antibodies in the dose range (10 -2.5 - 0.625 - 0.1563 - 0.0391 - 0.0098 - 0, 0024 - 0.0006 μg/ml) were analyzed on 20 generated mutants using flow cytometry.

The results showed that the antibodies selected in Example 5 for their ability to inhibit soluble CD39 presented several different epitopes. Among the antibodies that demonstrated 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 includes substitutions at residues R138, M139, and E142. Thus, the major surface epitope of 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, which has substitutions at residues R138, M139, and E142, but showed no reduction or loss of binding to mutant 5, which has 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 showed no loss of binding to either mutant 5 or 19, suggesting that soluble CD39 may also be inhibited by binding to other sites on the surface of soluble CD39 . For some antibodies, loss of binding to one of the 20 mutants in the table allowed the binding site to be localized to CD39, while for others the binding site remained to be determined because they did not lose binding to any of the 20 mutants.

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Among the antibodies inhibiting 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 major epitope on the CD39 surface 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 (referred to as Box 377V in the table), without loss of binding to any of the other 20 mutants. Thus, the primary CD39 surface epitope 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. Figure 3B shows the binding results of various antibodies to mutants 5, 15 and 19.

Thus, the results indicate that it is possible to generate antibodies that inhibit soluble CD39 to different 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 loses binding to mutant 5), epitopes that are defined by one or more residues of mutant 19 and some of mutant 5, indicating a possible smaller shift compared to BY40 or BY40-like antibodies, epitopes defined by one or more residues of mutant 19 and residues of non-mutant 5, as well as other epitopes, e.g., that are defined by one or more residues of mutant 11 or one or more residues of mutant 15, or other antibodies that do not have reduced binding to any of mutants 5, 15 or 19, for which the localization of epitopes is to be determined.

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

Antibody I-394 was tested in duplicate for binding to human CD39-expressing CHO cells, macaca flavicularis CD39-expressing CHO cells, murine CD39-expressing CHO cells, and human Ramos lymphoma cells (ATCC™, no. CRL- 1596). Cells were incubated with various concentrations of unlabeled anti-CD39 antibody, from 30 to 5x10' ⁴ μg/ml, for 30 min at 4°C. After washing, the cells were incubated with a marker-labeled goat anti-mouse H+L secondary antibody for 30 min at 4°C.

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

Example 8 Determination of IC ₅₀ for inhibition of cellular ATPase activity.

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

The results are shown in Fig. 5. I-394 has a high ability to block the enzymatic activity of CD39 in tumor cells (Ramos) and is more effective than all other antibodies tested. I-394 also blocks CD39 enzymatic activity in cells expressing human CD39 (CHO-huCD39) and in cells expressing cynomolgus CD39 (CHO-cyCD39). Cells expressing mouse CD39 (CHO-moCD39) are shown as a negative control. The calculated IC ₅₀ value (50% inhibition of CD39 enzymatic activity expressed by 50,000 Ramos cells) is 0.05 μg/ml. The maximum inhibition achieved was 81.6%. Isotype control had no effect.

Example 9. Determination of IC ₅₀ 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 as described above (under Methods). The results are shown in Fig. 6. I-394 inhibits 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 value is 0.003 µg/ml. The maximum inhibition achieved was 74.9%.

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

Antibody I-394 was tested for binding to recombinant human CD39 isoforms (rec-huCD39 isoforms) having the amino acid sequences shown below. The antibody was placed overnight in a 96-well plate in PBS 1X in an amount of 500 ng/ml or 1 μg/ml at 4°C. Wells were washed in Tween 20 TBS and further saturated for 2 hours at room temperature in TBS blocking buffer. The first antibody in the dose range was incubated in TBS blocking buffer

- 38 045378 for 2 hours at room temperature. Wells were washed in Tween 20 TBS. Second antibody (GAM-HRP or GAH-HRP in TBS blocking buffer) was incubated for 1 h at room temperature and detected with trimethylbenzene (TMB). Optical density was measured at

Enspire™ at OD=450 nm.

Amino acid sequence of cloned human CD39 (vascular isoform):

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

MAGKVRSLLP PLLLAAAGLA GLLLLCVPTR DVREPPALKY GIVLDAGSSH TSMFIYKWPA 61 DKENDTGIVG QHSSCDVPGG GISSYADNPS GASQSLVGCL EQALQDVPKE RHAGTPLYLG 121 ATAGMRLLNL TNPEASTSVL MAVTHTLTQY PFDFRGARIL SGQEEGVFGW VTANYLLE NF 181 IKYGWVGRWF RPRKGTLGAM DLGGASTQIT FETTSPAEDR ASEVQLHLYG QHYRVYTHSF 241 LCYGRDQVLQ RLLASALQTH GFHPCWPRGF STQVLLGDVY QSPCTMAQRP QNFNSSARVS 301 LSGSSDPHLC RDLVSGLFSF SSCPFSRCSF NGVFQPPVA G NFVAFSAFFY TVDFLRTSMG 361 LPVATLQQLE AAAVNVCNQT WAQQLLSRGY GFDERAFGGV IFQKKAADTA VGWALGYMLN 421 LTNLIPADPP GLRKGTDFSS WWLLLLFAS ALLAALLVLL RQVHSAKLPS TI (SEQ ID NO: 40).

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

MKKGIRYETS RKTSYIFQQP QHGPWQTRMR KISNHGSLRV AKVAYPLGLC VGVFIYVAYI 61 KWHRATATQA FFSITRAAPG ARWGQQAHSP LGTAADGHEV FYGIMFDAGS TGTRVHVFQF 121 TRPPRETPTL THETFKALKP GLSAYADDVE KSAQGIRELL DVAKQDIPFD FWKATPLVLK 181 ATAGLRLLPG EKAQKLLQKV KEVFKASPFL VGDDCVSIMN GTDEGVSAWI TINFLTGSLK 241 TPGGSSVGML DLGGGSTQIA FLPRVEGTLQ ASPPGYLTAL RMFNRTYKLY SYSYLGLGLM 301 SARLAILGGV EGQPAKDGKE LVSPCLSPSF KGEWEHAEVT Y RVSGQKAAA SLHELCAARV 361 SEVLQNRVHR TEEVKHVDFY AFSYYYDLAA GVGLIDAEKG GSLWGDFEI AAKYVCRTLE 421 TQPQSSPFSC MDLTYVSLLL QEFGFPRSKV LKLTRKIDNV ETSWALGAIF HYIDSLNRQK 481 SPAS (SEQ ID NO: 41).

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

MFTVLTRQPC EQAGLKALYR TPTIIALWL LVSIWLVSI TVIQIHKQEV LPPGLKYGIV 61 LDAGSSRTTV YVYQWPAEKE NNTGWSQTF KCSVKGSGIS SYGNNPQDVP RAFEECMQKV 121 KGQVPSHLHG STPIHLGATA GMRLLRLQNE TAANEVLESI QSYFKSQPF D FRGAQIISGQ 181 EEGVYGWITA NYLMGNFLEK NLWHMWVHPH GVETTGALDL GGASTQISFV AGEKMDLNTS 241 DIMQVSLYGY VYTLYTHSFQ CYGRNEAEKK FLAMLLQNSP TKNHLTNPCY PRDYSISFTM 301 GHVFDSLCTV DQRPESYNPN DVITFEGTG D 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: 42).

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

MATSWGTVFF MLWSCVCSA VSHRNQQTWF EGIFLSSMCP INVSASTLYG IMFDAGSTGT 61 RIHVYTFVQK MPGQLPILEG EVFDSVKPGL SAFVDQPKQG AETVQGLLEV AKDSIPRSHW 121 KKTPWLKAT AGLRLLPEHK AKALLFEVKE IFRKSPFLVP KGSVSIMDGS D EGILAWVTV 181 NFLTGQLHGH RQETVGTLDL GGASTQITFL PQFEKTLEQT PRGYLTSFEM FNSTYKLYTH 241 SYLGFGLKAA RLATLGALET EGTDGHTFRS ACLPRWLEAE WIFGGVKYQY GGNQEGEVGF 301 EPCYAEVLRV VRGKLHQPEE VQRGSFYAFS YYYDR AVDTD MIDYEKGGIL KVEDFERKAR 361 EVCDNLENFT SGSPFLCMDL SYITALLKDG FGFADSTVLQ LTKKVNNIET GWALGATFHL 421 LQSLGISH (SEQ ID NO: 43).

I-394 is associated with CD39, but not with any of the CD39-L1, -L2, -L3, or -L4 isoforms. Isotype control (IC) antibodies did not bind to any CD39 or CD39-L molecules. The results are shown in Fig. 7.

Example 11. Activation of dendritic cells.

Although ATP has proinflammatory effects, CD39-mediated ATP catabolism is thought to be capable of impairing dendritic cell (DC) activation, thereby altering in turn the broader adaptive immune response to tumor antigen. To evaluate whether blocking CD39 using anti-CD39 antibodies can help overcome the CD39-mediated alteration in dendritic cell (DC) activation in the presence of ATP, we incubated monocyte-derived DCs (moDCs) with anti-CD39 antibodies in the presence of ATP.

Briefly, human monocytes were isolated from healthy human blood and differentiated into moDCs in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 6 days. MoDC were then activated in the presence of ATP (Sigma, 0.25-1 mM) for 24 h, and DC activation was assessed by analyzing the expression of CD80, CD83 and HLA-DR by flow cytometry. In some cases, moDC were preincubated for 1 h in the presence of CD39 inhibitor: ARL6716 (Tocris, 250 μM), CD73 inhibitor: APCP (Tocris 50 μM), anti-CD39 blocking antibody I-394 or BY40 (for BY40 see WO 2009 /095478) or anti-CD73 blocking antibodies. Liposaccharide (Invivogen, 10 ng/ml) was used as a positive control. To evaluate the end result of ATP-mediated DC activation on CD4 T cell activation, ATP-activated DCs were washed and then incubated with allogeneic CD4 T cells (at a ratio of 1 moDC/4 T cells) to perform a mixed lymphocyte reaction (MSL) in within 5 days. T cell activation and proliferation were analyzed by CD25 expression and by dilution using the Cell kit

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Trace Violet by flow cytometry (Fig. 8).

The results are shown in Fig. 9-11. In the presence of a negative control (medium), moDC activation was observed in the presence of 1 mM ATP, but ATP at 0.125, 0.25, or 0.5 mM did not activate moDC. Addition of chemical CD39 inhibitors, which are thought to completely block CD39 enzymatic activity by binding to the active site, resulted in moDC activation at each of the concentrations of 0.125, 0.25, or 0.5 mM. However, anti-CD39 antibodies, such as BY40, or anti-CD73 antibodies, were unable to stimulate ATP-induced dendritic cell (DC) activation, meaning that the antibodies were unable 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 essentially completely blocks the ATPase activity of CD39 and therefore may allow ATP to accumulate, allowed the activation of moDC, as confirmed by the results of assessing HLA expression -DR or CD83 at each of concentrations of 0.125, 0.25 or 0.5 mM (Fig. 9 and 10). Interestingly, moDC activated in the presence of ATP are able to induce better T cell activation and proliferation during RSL. Additionally, enhancing ATP-mediated moDC activation with the anti-CD39 blocking antibody I-394 resulted in higher T cell proliferation and activation (Figure 11).

Assessing the ability of CD39 inhibitors to activate DCs in the presence of ATP is a way to identify and evaluate anti-CD39 antibodies that are 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 enhance the adaptive immune response to antigens, especially to tumor cells. In addition, such anti-CD39 antibodies may be of particular interest when used to enhance the immunogenic effects of chemotherapeutic agents. Numerous chemotherapeutic agents that cause tumor cell necrosis are capable of inducing ATP; combination use with anti-CD39 antibodies may be particularly useful in enhancing the antitumor response in such settings.

Example 12 In vivo combination therapy with anti-CD39 antibodies of ATP releasing agents.

1x10 ⁶ mouse MCA205 (sarcoma) tumor cells were transplanted subcutaneously into the right flank of mice genetically modified to express human CD39 (KI mouse CD39). Mice (n=9-13) were treated with control drug oxaliplatin (10 mg/kg, intraperitoneal, on days 5 and 12 or 14), mouse anti-CD39 antibody I-394 (20 mg/kg for the first injection, then 10 mg/kg). kg, intravenously, twice a week for 3 or 4 weeks, starting on the 4th day) or a combination thereof. Tumor size was measured twice a week using calipers (L - length and w - width), and tumor volume was calculated using the formula (Lxw2)/2. Mice were sacrificed when tumor volume exceeded 1800 mm ³ or when tumors were severely necrotic. Human CD39 KI mice were transplanted with MCA205 tumor cells on day 0. Mouse I-394 antibody was generated using a non-glycosylated mouse IgG1 isotype (with a substitution of Kabat numbering heavy chain residue #297) to prevent mouse FcR from binding to complement and so that the only effect observed would be due to the blocking property of the antibody rather than ADCC or ADCC. CDC lysis of CD39+ immunosuppressive cells or CD39+ endothelial cells.

In the first series of trials, mice were treated on day 5 after tumor cell engraftment with either control (group 1) PBS or oxaliplatin chemotherapy (group 2). In parallel, one group of mice treated with oxaliplatin was treated with anti-CD39 antibody twice weekly, with anti-CD39 antibody therapy starting just one day before oxaliplatin therapy (on day 4). This ensured that oxaliplatin induced ATP release in a tumor environment where CD39 was already completely inhibited, and thus provided optimal prevention of ATP degradation by intratumoral CD39. In this trial, a delay in tumor growth and survival in mice could be observed with the combination of oxaliplatin and the I-394 antibody group, but the delay was considered to be relatively small, despite the fact that there was one complete response in this group, compared to a complete response in the control group or the group with oxaliplatin alone was not observed. The average survival time for control was 20 days, oxaliplatin - 25 days, antibody I-394 in combination with oxaliplatin - 31 days. The results are shown in Fig. 12.

In the second series of trials (one representative experiment of 2 is shown), the oxaliplatin injection was repeated one week after the first oxaliplatin injection, again just one day after I-394 antibody therapy to ensure optimal inhibition of ATP degradation. Antibody I-394, administered alone, had only a minor effect on tumor growth and survival in mice. Oxaliplatin as a single agent with repeated (twice) injections did produce some reduction in tumor volume and increased survival in mice. However, the combination of repeated injections of oxaliplatin with antibody I-394 administered before oxaliplatin caused regression of tumor volume in all mice with three complete responses (CRs).

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Claims (3)

compared with two in the oxaliplatin alone group and six partial responses versus three PRs in the oxaliplatin alone group. The combination also improved survival in mice 40 days after tumor engraftment in 4/13 tumor-free mice compared with 2/12 in the oxaliplatin-only group. The results are shown in Fig. 13. It is believed that a second booster injection of oxaliplatin in this situation allows the accumulation of ATP and adenosine in the presence of anti-CD39 blocking antibody over the standard two-week period required for the maturation of an effective antitumor immune response. Therefore, in humans, an improved treatment regimen may include repeat chemotherapy at least twice to achieve the most potent combined effect with the anti-CD39 blocking antibody. In addition, the anti-CD39 antibody can ideally be administered at least 1 to 48 hours before the ATP-releasing chemotherapeutic agent to ensure complete inhibition of intratumoral CD39 and complete inhibition of ATP degradation to adenosine at the time the chemotherapeutic agent causes ATP release . All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference in their entirety as if each reference had been cited individually, specifically indicated for inclusion by reference, and had been fully set forth herein (to the maximum extent possible). to the extent permitted by law), regardless of any specific document included elsewhere herein. Unless otherwise indicated, all precise values presented herein represent the corresponding approximate values (e.g., all precise values in the examples given for a particular indicator or measurement may also be considered the corresponding approximate measurement readings due to the approximate designation inserted where appropriate). If approximately is used in conjunction with a number, it means that values within the range of ±10% of the stated number are also implied. The description herein of any aspect or embodiment of the present invention using terms such as consisting of, having, including or containing with reference to a component or components is intended to establish a similar aspect or embodiment of the present invention which consists of, consists essentially of or essentially includes that particular component or components unless otherwise indicated or clearly inconsistent with the context (e.g., a composition described herein as containing a specific component should also be understood to include a description of a composition consisting of that component in unless otherwise stated or clearly inconsistent with the context). The use of any and all examples or introductory words before an example (eg, such as) presented herein is merely intended to better highlight the present invention and does not constitute any limitation on the scope of the invention unless otherwise stated. No term in the description should be construed as indicating any element not stated as essential to the practice of the present invention. CLAIM 1. The use of an antibody that is capable of binding to human CD39 protein (NTPDase1) and inhibiting the ATPase activity of human CD39 protein (NTPDase1) in the treatment of cancer, wherein said antibody is administered in combination with a platinum-based drug, and wherein said antibody contains HCDR1, containing the amino acid sequence DYNMH (SEQ ID NO: 5); HCDR2 containing the amino acid sequence YIVPLNGGSTFNQKFKG (SEQ ID NO: 6); HCDR3 containing the amino acid sequence GGTRFAY (SEQ ID NO: 7); LCDR1 containing the amino acid sequence RASESVDNFGVSFMY (SEQ ID NO: 8); LCDR2 containing the amino acid sequence GASNQGS (SEQ ID NO: 9); and LCDR3 containing the amino acid sequence QQTKEVPYT (SEQ ID NO: 10). 2. The use of an antibody according to claim 1, characterized in that said antibody is capable of binding to a soluble human CD39 extracellular domain protein and inhibiting the ATPase activity of a soluble human CD39 extracellular domain protein, wherein optionally said antibody is capable of causing a decrease in the ATPase activity of said CD39 extracellular domain protein human in solution by more than 70%, wherein the antibody inhibits the ATPase activity of soluble human CD39 extracellular domain protein in the presence of exogenously added ATP, optionally the concentration of the added ATP being 20 μM. 3. The use of an antibody according to any one of claims 1 or 2, characterized in that the antibody that is capable of binding to CD39 and inhibiting the ATPase activity of CD39 in the presence of exogenously added ATP is an antibody that is capable of causing an increase in the expression of an activation marker on the cell surface in monocyte-derived dendritic cells (moDCs), where such moDCs are incubated in vitro with said antibody and ATP, optionally providing exogenously added ATP at a concentration of 0.125, 0.25 or 0.5 mM. -