WO2025233266A1 - Use of cd39 blocking agents in combination with anti-cd20 x cd3 t cell engagers - Google Patents

Abstract

The present invention relates to agents that inhibit the enzymatic activity of human CD39, for use in enhancing the activity of T-cell engager therapies in the treatment of disease, notably B cell Non-Hodgkin Lymphoma.

Description

USE OF CD39 BLOCKING AGENTS IN COMBINATION WITH ANTI-CD20 X CD3 T CELL ENGAGERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/643,443 filed on May 7, 2024; which is incorporated herein by reference in its entirety; including any drawings.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “CD39-11 WO. xml”. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to agents that inhibit the enzymatic activity of human CD39, for use in enhancing the activity of T-cell engager therapies in the treatment of disease.

BACKGROUND

Rituximab has become widely used in the treatment of B cell lymphomas and is effective in many patients. Ritixumab is believed to have multiple modes of action, including inter alia, the ability to mediate ADCC toward malignant CD20-expressing cells. Natural killer (NK) cells mediate ADCC and are believed to have an important role in the anti-tumor immunity of rituximab. The addition of rituximab into combination therapies for DLBCL have greatly improved patient outcomes. However, patients with refractory DLBCL following treatment under the current standards of care still have a dire prognosis, with no curative treatment options (Flowers et al. 2010 CA: A Cancer Journal for Clinicians 60(6): 393-408). In particular, patients with DLBCL who relapse after, or are refractory to, first-line therapy have a poor prognosis.

Recently, T cell engagers (TCEs) have been developed to treat B cell lymphomas. The TCEs bind, via a first antigen binding domain, to CD20 on the malignant cells and, via a second antigen binding domain, to CD3 on effector T cells. TCEs, via their ability to mediate tumor cell killing by effector T cells, are promising in tumors who are in relapse or are refractory (R/R) after rituximab treatment.

The impressive anti-tumor activity of TCEs have nevertheless been hampered by the significant adverse events, notably cytokine release syndrome (CRS). CRS is triggered by on- target effects induced by binding of a TCE to its antigen and by subsequent activation of bystander immune and non-immune cells. CRS is associated with high circulating concentrations of several pro-inflammatory cytokines, including interleukins, interferons, tumor necrosis factors, colony-stimulating factors, and transforming growth factors. Advances have been made to prevent and control CRS, for example protocols for administration of agents such as steroids and inhibitors of IL-6 activity, as well as IL-1 , IFN-y, TNF-a, and IL-2 inhibitors for unresponsive patients. However risk CRS remains a downside of TCE therapy. One potential option for mitigating CRS risk is to use reduced doses of the TCE.

Despite the progress made with these novel approved therapies and high complete response rates being observed, there is still a sizeable portion of patients with B cell lymphoma that will relapse or who are refractory or will not be eligible for treatment with TCEs. There is therefore a need for treatments with increased efficacy, response and/or duration, as well as decreased risk of cytokine release syndrome or serious adverse effects in patients.

SUMMARY OF THE INVENTION

The present disclosure relates to the use of an agent that neutralizes the enzymatic activity of CD39 to enhance the anti-tumor activity of a TCE. As shown herein, tumor cells derived from patients can be targeted in a highly effective manner with TCEs in combination with an agent that neutralizes CD39. The addition of the agent that blocks CD39 to the TCE enhanced the efficacy of the TCE. Further, as shown herein, the extent to which individual samples benefited from combinations compared to TCE monotherapy was quantified in order to identify potential indicators for the efficacy of the combination treatment. The benefit from adding anti-CD39 to anti-CD20 TCE was higher in DLBCL compared to FL. Also, the benefit from adding anti-CD39 to anti-CD20 TCE was higher in patients at initial diagnosis compared to patients at relapse. Results showed that a higher proportion of exhausted T-cells correlated with greater benefits of the TCE/anti-CD39 combination treatment. Furthermore, adding anti- CD39 enhanced the anti-tumor activity of the TCE at sub-optimal concentrations of TCE, suggesting that the combination may permit high anti-tumor activity with lower doses of TCE.

The disclosure thus provides in one aspect a method of treating a disease (e.g. a disease caused or characterized by unwanted proliferating cells; a tumor or cancer) and/or preventing the recurrence of a disease (e.g. a tumor or cancer) in a patient in need thereof, comprising administering an CD39 neutralizing agent (e.g. anti-CD39 antibody) in combination with a TCE. In one aspect the disclosure provides a method of eliminating a target cell (e.g. in a patient in need thereof), comprising administering an CD39 neutralizing agent (e.g. anti- CD39 antibody) in combination with a TCE, wherein the TCE specifically binds to the target cell with a first antigen binding domain and to an effector T cell with a second antigen binding domain. In one embodiment the target cell is a proliferating and/or a cell involved in or causing disease. In one embodiment the target cell is a proliferating B cell. In one embodiment the target cell is a CD20-expressing cell. In one embodiment the target cell is a tumor cell. In one embodiment, the TCE comprises a first antigen binding domain that specifically binds to an antigen present at the surface of a malignant B cell (e.g. CD20) and a second antigen binding domain that specifically binds to an antigen present at the surface of an effector T cells (e.g. CD3). In one embodiment, the disease (e.g. tumor or cancer) is a B cell proliferative disorder. In one embodiment, the tumor or cancer is a B-NHL, optionally a DLBCL. In one embodiment, the tumor or cancer is a previously untreated disease, for example an untreated NHL or a treatment naive NHL (e.g., previously untreated DLBCL or treatment naive DLBCL). In one embodiment, the method does not comprise the administration in combination with further anti-cancer agents.

In one embodiment, the B-NHL is Diffuse Large B Cell Lymphoma (DLBCL). In one embodiment, the B-NHL is High-grade B-cell lymphoma (HGBCL). In one embodiment, the B- NHL is Primary mediastinal large B-cell lymphoma (PMBCL). In one embodiment, the B-NHL is Follicular Lymphoma (FL). In one embodiment, the B-NHL is Mantle cell lymphoma (MCL). In one embodiment the B-NHL is marginal zone lymphoma (MZL) (nodal, extranodal or splenic).

In one embodiment, the patient has not received prior systemic treatment (e.g. anticancer agent) for the disease. In one embodiment, the patient has exhausted T cells, as determined by assessing expression of markers of exhaustion on T cells, optionally wherein the marker is CD39. In any embodiment, a method of treatment can comprise a step of assessing expression of markers of exhaustion on T cells from a patient, optionally wherein the marker is CD39. In one embodiment, provided is a method for the treatment or prevention of a proliferative disease (e.g. cancer) in an individual in need thereof, the method comprising: (a) detecting whether the individual has exhausted T cells, as determined by assessing expression of one or more markers of T cell exhaustion, and (b) upon a determination the individual has exhausted T cells, administering to the individual a TCE and an agent that neutralizes CD39.

These aspects are more fully described in, and additional aspects, features, and advantages will be apparent from, the description provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows the flow cytometry-based ex vivo model of TCE (embodied as a CD20- and CD3-binding bispecific antibody, referred to also as BsAb) treatment used herein, based on autologous culture model of nodal B-cell lymphoma. Figure 1 B-F shows results from lymph node-derived lymphocytes incubated with or without (w/o) a maximum of four concentrations (C1-C4) of a CD20- and CD3- binding TCE (BsAb) and/or a maximum of two concentrations of anti-CD39 blocking antibodies, anti-CD73 blocking antibodies, or anti-CD39 blocking antibodies + anti-CD73 blocking antibodies, as indicated. After seven days, cells (B-E) or supernatants (F) were analyzed by quantitative flow cytometry or a bead-based immunoassay, respectively. Shown are the percentages based on the absolute numbers of viable B cells normalized to w/o (B, D), the x-fold expansion based on the absolute numbers of viable T cells normalized to w/o (C, E) or the cytokine levels in pg/pl (F) in n = 27 biologically independent samples. G) Lymph node-derived lymphocytes were incubated with or without the anti-CD39 blocking antibody at two different concentrations and then exposed to ATP at a concentration of 20 pM. Shown are ATP levels of n = 4 biologically independent samples normalized to untreated control (w/o) after 60 minutes. BG) P values were calculated between w/o and every other condition using the two-sided Wilcoxon’s test and corrected for multiple testing using the Benjamini-Hochberg procedure. Only p values < 0.05 are shown.

Figure 2A-B shows Box plots illustrating the associations of patient characteristics with benefit from three different combination treatments. P values were calculated using the Wilcoxon-test. Figure 2C) Shown is the expression of CD39 and CD73 at protein level based on publicly available single-cell data of in n = 51 biologically independent lymph node samples. Figure 2D) Overview of fourteen T-cell subsets defined by existing single-cell RNA and epitope sequencing in n = 51 biologically independent lymph node samples. Figure 2E) Shown is the expression of CD39 and CD73 at protein level based on previously mentioned T-cell reference data. Figure 2F) The proportion of fourteen specific T-cell subsets, defined in panel A (y axis), was correlated with the benefit from three different combination therapies (x axis) compared to BsAb monotherapy. Only p values < 0.05 are shown. Figure 2G) The protein expression of six different exhaustion markers (x axis) across three effector memory T-cells, defined in panel A, was correlated with the benefit when combining anti-CD39 blocking antibody with BsAb compared to BsAb monotherapy. Only values < 0.05 are shown.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Where "comprising" is used, this can optionally be replaced by "consisting essentially of' or by "consisting of".

Whenever "treatment of cancer" or the like (e.g. "treatment of B-NHL") is mentioned with reference to anti-CD39 binding agent (e.g., antibody), this can include: (a) method of treatment of cancer, said method comprising the step of administering (for at least one treatment) an anti-CD39 binding agent, (preferably in a pharmaceutically acceptable carrier material) to an individual, a mammal, especially a human, in need of such treatment, in a dose that allows for the treatment of cancer, (a therapeutically effective amount), preferably in a dose (amount) as specified herein; (b) the use of an anti-CD39 binding agent for the treatment of cancer, or an anti-CD39 binding agent, for use in said treatment (especially in a human); (c) the use of an anti-CD39 binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer, a method of using an anti-CD39 binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer, optionally comprising admixing an anti-CD39 binding agent with a pharmaceutically acceptable carrier, or a pharmaceutical preparation comprising an effective dose of an anti-CD39 binding agent that is appropriate for the treatment of cancer; or (d) any combination of a), b), and c), in accordance with the subject matter allowable for patenting in a country where this application is filed.

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. The agent can be specified as being a "therapeutic agent", which refers to an agent that has biological activity.

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

The term “antibody,” as used herein, can include polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as lgG1 , lgG2, lgG3, lgG4, and the like. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain ( L) and variable heavy chain ( H) refer to these light and heavy chains respectively. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed “alpha,” “delta,” “epsilon,” “gamma” and “mu,” respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG are the exemplary classes of antibodies employed herein because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. Optionally the antibody is a monoclonal antibody. Particular examples of antibodies are humanized, chimeric, human, or otherwise-human-suitable antibodies. “Antibodies” also includes any fragment or derivative of any of the herein described antibodies.

The term “specifically binds to” means that an agent (e.g. antibody, TCE) can bind preferably in a competitive binding assay to the binding partner, e.g., CD20, CD3 or CD39, as assessed using either recombinant forms of the proteins, epitopes therein, or native proteins present on the surface of isolated target cells. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

When an antibody is said to “compete with” a particular agent or antibody, it means that the antibody competes with the agent or antibody in a binding assay using either recombinant antigen molecules or surface expressed antigen molecules. For example, if a test antibody reduces the binding of a reference antibody to a CD39 polypeptide or CD39- expressing cell in a binding assay, the antibody is said to “compete” respectively with the reference antibody.

The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] I [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant K_a is defined by 1/Kd. Methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device).

For the purposes herein, a “humanized” antibody refers to an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g., the CDR, of an animal immunoglobulin. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody.

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity-determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991) and/or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H 1), 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 for determining essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as “Kabat position”, "variable domain residue numbering as in Kabat" and "according to 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 a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

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

The terms "Fc domain," "Fc portion," and "Fc region" refer to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of human y (gamma) heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., a, 5, E and p for human antibodies), or a naturally occurring allotype thereof. Unless otherwise specified, the commonly accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et al. (1991 ) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

The terms “isolated”, “purified” or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

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

Within the context herein, the term antibody that “binds” a polypeptide or epitope designates an antibody that binds said determinant with specificity and/or affinity.

The term “identity” or “identical”, when used in a relationship between the sequences of two or more polypeptides, refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, 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).

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

Treatment of cancer

The disclosure generally relates to methods for treating a cancer in a patient using a CD39-neutralizing agent in combination with a T cell engager. The methods are particularly advantageous in the treatment of B-cell proliferative disorders, particularly non-Hodgkins Lymphoma (NHL), particularly B-NHL, particularly aggressive B-NHL, and particularly DLBCL. The methods are thus particularly advantageous in the treatment of tumor or cancers when used as a first line therapy and/or in patients who have T cells (e.g. a T cell subset or population) characterized by a cellular marker of exhaustion.

As used herein, combined administration (co-administration) includes simultaneous administration of the compounds in the same or different dosage form, or separate administration of the compounds (e.g., sequential administration). Typically the CD39- neutralizing agent and TCE can be formulated for separate administration and are administered concurrently or sequentially. However, the CD39-neutralizing agent and TCE may alternatively be simultaneously administered in a single formulation.

Non-Hodgkin’s lymphoma (“NHL”) is a heterogeneous malignancy originating from lymphocytes. NHL is characterized by a clonal proliferation of lymphocytes that accumulate in the lymph nodes, blood, bone marrow and spleen, although any major organ may be involved. The current classification system used by pathologists and clinicians is the World Health Organization (WHO) Classification of Tumours, which organizes NHL into precursor and mature B-cell or T-cell neoplasms. The combination treatments of the disclosure are particularly suited to treat B-cell NHL, referred to as B-NHL. In any embodiment herein, a NHL can be specified to be a B-NHL. The NHL or B-NHL may optionally be specified as being indolent or aggressive. NHL may be divided into indolent or aggressive. The indolent NHL group is comprised primarily of follicular subtypes, small lymphocytic lymphoma, MALT (mucosa-associated lymphoid tissue), and marginal zone. Indolent NHL includes many newly diagnosed B-cell NHL patients. Aggressive NHL includes patients with histologic diagnoses of primarily diffuse large B cell lymphoma (DLBL, “DLBCL”, or DLCL) (40% of all newly diagnosed patients have diffuse large cell lymphoma), Burkitt's, and mantle cell lymphoma (“MCL”).

In some embodiments, the treatment of the disclosure can be administered to patients based on the individual IPI score and/or age. IPI scores to predict outcome or survival include the National Comprehensive Cancer Network IPI (NCCN-IPI) or Age-adjusted IPI (aalPI). In DLBCL, subgroups of patients that can be treated according to the disclosure include for example: elderly patients (>60 years, aalPI=0-3), young patients with low risk (<60 years, aalPI=0-1) and young patients with high risk (<60 years, aalPI=2-3).

In one embodiment, disclosed is a method of reducing or inhibiting tumor growth in a subject in need thereof. A treatment can be specified as achieving at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastasis or tumor growth. Positive therapeutic effects in cancer can be measured in a number of ways. I n some embodiments, response to a combination treatment of the disclosure is assessed using RECIST 1.1 criteria.

In some embodiments, the treatment achieved by a combination treatment of the disclosure is any of a partial response (PR), a complete response (CR), progression free survival (PFS), disease free survival (DFS), objective response (OR) or overall survival (OS). In some embodiments, the combination treatment of the disclosure provides a method of increasing the rate or likelihood of a PR, PR or OR, or a method for increasing PFS, DFS or OS.

In one embodiment, a treatment method of the disclosure comprises administering to the subject a therapeutically effective amount of each of a T cell engager and a CD39- neutralizing agent.

The methods and regimens herein can be specified to be used for example for treating and/or preventing the recurrence of a tumor or cancer in a patient, for preventing metastases or the recurrence of metastases, for preventing the progression or growth of a tumor or cancer, for reducing the rate of (or preventing) cancer cell infiltration into peripheral organs, for improving or increasing survival, for improving or increasing time to recurrence, and/or for improving or increasing recurrence-free survival.

The methods and regimens herein can also be useful for example for using an CD39 to increase or potentiate an anti-tumor immune response mediated by a TCE. The increase or potentiate an anti-tumor immune response can be specified as being mediated by inhibiting the enzymatic activity of CD39 (e.g. in a lymph node or extranodal tumor site and/or in circulation), increasing the intratumoral concentration of ATP, decreasing the intratumoral concentration of adenosine, or increasing the activity and/or number of T cells, tumor infiltrating T cells and/or dendritic cells.

In some embodiments, the treatment regimen of the disclosure can be specified as being with or without combined treatment with a further therapeutic agent.

In some embodiments, the treatment regimen of the disclosure can be specified as being without combined treatment with CHOP, R-CHOP. R-CHOP refers to rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone) and CHOP refers to cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone.

In one example, the treatment regimen of the disclosure can be used to treat a cancer (e.g., B-NHL, DLBCL) in an individual who is not eligible for a chemotherapy (e.g. CHOP, CHOP-R, etc), for example an individual for whom CHOP is expected to involve excessive toxicity.

In some embodiments, the treatment regimen of the disclosure can be specified as being for first line treatment, for example for administration to patients not having received prior systemic treatment regimen for the disease.

A response to treatment (e.g. in an individual) can be assessed using any endpoint indicating a benefit to the subject, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., progression of a CD20-positive B cell proliferative disorder, e. g., a non-Hodgkin's lymphoma (NHL)); including slowing down and complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e. , reduction, slowing down or complete stopping) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (5) relief, to some extent, of one or more symptoms associated with the disease (e.g., a CD20-positive B cell proliferative disorder; a B cell proliferative disorder; (6) increase or extend in the length of survival, including overall survival and progression-free survival; and/or (9) decreased mortality at a given point of time following treatment.

A “complete response” or “CR” refers to disappearance of all target lesions. In one embodiment, standard NHL response criteria are assessed for determining CR. (Lugano Criteria, Cheson et al. J Clin Oncol. 2014 Sep. 20; 32(27): 3059-3067). CR can be determined by PET-CT (“complete metabolic response” or “CMR”) or CT (“complete radiologic response”). In some embodiments, complete response (CR) can be used interchangeably with “complete metabolic response” or “CMR”.

“Duration of complete response” (DOCR) is the time from the initial occurrence of a documented CR until documented disease progression or death due to any cause, whichever occurs first. In one embodiment, DOCR is assessed based on the Lugano Classification (Cheson et al. J Clin Oncol. 2014 Sep. 20; 32(27): 3059-3067).

“Duration of objective response” (DOR) is the first occurrence of a documented, objective response until the time of disease progression, relapse or death from any cause. In one embodiment, DOR is assessed based on the Lugano Classification (Cheson et al. J Clin Oncol. 2014 Sep. 20; 32(27): 3059-3067).

As used herein, “objective response rate” refers to the sum of patients with a complete response (CR), patients with a partial response (PR) and patients with stable disease (SD) in a patient population. In one embodiment, objective response rate is evaluated based on the Lugano Classification (Cheson et al. J Clin Oncol. 2014 Sep. 20; 32(27): 3059-3067).

The “overall response rate” (ORR) is as the sum of partial response (PR) rate and complete response (CR) rate. In one embodiment, overall response is evaluated based on the Lugano Classification (Cheson et al. J Clin Oncol. 2014 Sep. 20; 32(27): 3059-3067).

“Progression-free survival” (PFS) is the time from the first treatment with the anti- CD20/anti-CD3 bispecific antibody to the first occurrence of disease progression or death from any cause, whichever occurs first. In one embodiment, PFS is assessed based on the Lugano Classification (Cheson et al. J Clin Oncol. 2014 Sep. 20; 32(27): 3059-3067).

“Overall survival” (OS) is the time from the first treatment to the date of death from any cause.

In certain aspects an anti-CD39 agent combined with a TCE can be used to treat a cancer (e.g., B-NHL, DLBCL) in an individual having a poor disease prognosis for response to an anti-cancer agent used (e.g. approved) in the treatment of such cancer. An individual having a poor disease prognosis can be, for example, at a higher risk of disease progression, based on one or more predictive factors. For example a poor prognosis can be evidenced by any one or combination of: I PI score, agent, one or more biological markers indicative of lack of a sufficient anti-tumor immune response, one or more biological markers indicative of immune exhaustion, and/or one or more biological markers indicative of immunosuppression. In one embodiment, the predictive factor(s) comprises possessing or lacking a mutation in one or more genes. In one embodiment, the predictive factor(s) comprises level(s) of expression of one or more genes or proteins (e.g., a gene signature). In one embodiment, a predictive factor(s) comprises ATPase activity or adenosine accumulation.

An individual having a cancer can be treated with a treatment of the disclosure with or without a prior detection step to assess tumoral ATPase activity, 5’-ectonucleotidase activity, tumoral or lymph node adenosine accumulation (e.g., adenosine concentration), and/or CD39 and/or CD73 expression on cells (e.g., circulating and/or lymph node or extranodal tissueinfiltrating leukocytes, effector T cells, regulator T (Treg) cells, B cells and/or on tumor cells). Optionally, the treatment methods can comprise a step of detecting ATPase activity and/or adenosine accumulation (e.g., elevated intratumor or intra lymph node adenosine concentration) in a biological sample from an individual (e.g., a blood sample, or a sample comprising lymph node or extranodal tumor tissue and/or tumor adjacent tissue). A determination that a biological sample has elevated ATPase activity or adenosine accumulation (e.g., intratumoral adenosine concentration), for example compared to a reference, indicates that the individual has a cancer that may have a strong benefit from treatment with a combination treatment of the disclosure. In certain aspects the combination treatment of the disclosure can be used to treat a cancer in an individual having immune effector cells characterized by one or more markers of exhaustion (e.g. T cell exhaustion) and/or immunosuppression.

In one example, the individual treated with a combination treatment of the disclosure may have poor prognosis for response to an agent that binds CD20 (e.g. rituximab) or without combined treatment with CHOP.

In one example, the individual treated with a combination treatment of the disclosure may have poor prognosis for response to a TCE. In one embodiment, the individual is a nonresponder, or has experienced a partial or an incomplete response to treatment with a TCE, or whose disease has progressed following treatment with a TCE. In one embodiment, the individual has a substantially number of immune effector cells characterized by one or more markers of exhaustion and/or immunosuppression. In one embodiment, the exhaustion marker is CD39. In one embodiment the individual has a substantially number of effector T cells characterized that express CD39.

In another example, the individual treated with a combination treatment of the disclosure may be suitable for treatment with a TCE and/or have good prognosis for response to a TCE, optionally the individual does not have substantial amount of immune effector cells characterized by marker(s) of exhaustion (e.g. T cell exhaustion) and/or immunosuppression. In the case of an individual that is suitable for treatment with a TCE and/or has good prognosis for response to a TCE, the combination treatment of the disclosure can enhance the efficacy of the anti-cancer treatment compared to treatment with the TCE alone.

In another example, the individual is susceptible to experience immune-mediated toxicity from a TCE, and the individual is treated with a combination treatment of the disclosure in which the TCE is administered at a decreased dose and/or frequency (compared to an approved (e.g. FDA-approved) reference regimen). Susceptibility to immune-mediated toxicity from a TCE can be for example as determined by marker(s) indicative of susceptibility to immune-mediated toxicity or from prior experience of immune-mediated toxicity (for example upon administration of a TCE or other immunotherapy). In one embodiment, immune- mediated toxicity comprises cytokine release syndrome.

In some aspects, an individual having a disease (e.g. proliferative disease; B cell proliferative disease; cancer) can be treated by a method comprising: (a) detecting whether the individual has exhausted T cells, as determined by assessing expression of one or more markers of T cell exhaustion, and (b) upon a determination the individual has exhausted T cells, administering to the individual a TCE and an agent that neutralizes CD39. In one embodiment assessing expression of one or more markers of T cell exhaustion comprises detecting, in a biological sample obtained from the individual that comprises T cells (e.g. one or more of the subset of T cells referred to herein, e.g. in the Examples), one or more biological markers (e.g., proteins, nucleic acids, cell surface proteins) that indicate that the T cells (optionally a subset of T cells) are exhausted (e.g. that the sample comprises a significant level of exhausted T cells).

An individual having a cancer can be treated with a combination treatment of the disclosure with or without a prior detection step to assess expression of CD73 on cells in the tumor microenvironment (e.g. on tumor cells, CD4 T cells, CD8 T cells, B cells).

In one embodiment, a predictive factor(s) for poor disease prognosis comprises presence (e.g., numbers) of cells in circulation or in the tumor environment expressing CD73 polypeptide, and/or expression levels of CD73 on cells in circulation or in the tumor environment; in one embodiment, the cells are malignant cells (e.g. B-NHL cells); in one embodiment the cells are leukocytes, e.g., B cells, regulatory T cells (Treg). Presence of elevated expression of CD73 polypeptide, and/or elevated levels of CD73-expressing cells can indicate an individual has a poor prognosis for response to treatment with a TCE.

Determining whether an individual has a cancer characterized by cells that express a CD73 polypeptide can for example comprise obtaining a biological sample (e.g. by obtaining a blood sample or by performing a biopsy) from the individual that comprises cells from the cancer environment (e.g. lymph node or extranodal tumor or tumor adjacent tissue), bringing said cells into contact with an antibody that binds an CD73 polypeptide, and detecting whether the cells express CD73 on their surface. Optionally, determining whether an individual has cells that express CD73 comprises conducting an immunohistochemistry assay.

In one embodiment, the disclosure provides a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting CD73 polypeptide (e.g. cell membrane-bound CD73 and/or soluble CD73 polypeptide) in circulation and/or in the tumor environment (e.g. within the tumor and/or within adjacent tissue; on T cells; on effector T cells), and b) upon a determination that tumor environment comprises CD73 polypeptide, optionally at a level that is increased compared to a reference level, administering to the individual a combination treatment of the disclosure.

In one embodiment, an individual having a cancer can be treated with a combination treatment of the disclosure with or without a prior detection step to assess expression of CD39 on cells in the tumor microenvironment (e.g. on tumor cells, CD4 T cells, CD8 T cells, B cells).

In one embodiment, a predictive factor(s) poor disease prognosis comprises presence (e.g., numbers) of cells in circulation or in the tumor environment expressing CD39 polypeptide, and/or expression levels of CD39 on cells in circulation or in the tumor environment; in one embodiment, the cells are malignant cells (e.g. B-NHL cells); in one embodiment the cells are leukocytes, e.g., B cells, regulatory T cells (Treg). Presence of elevated expression of CD39 polypeptide, and/or elevated levels of CD39-expressing cells can indicate an individual has a poor prognosis for response to treatment with a TCE.

Determining whether an individual has a cancer characterized by cells that express a CD39 polypeptide can for example comprise obtaining a biological sample (e.g. by obtaining a blood sample or by performing a biopsy) from the individual that comprises cells from the cancer environment (e.g. lymph node or extranodal tumor or tumor adjacent tissue), bringing said cells into contact with an antibody that binds an CD39 polypeptide, and detecting whether the cells express CD39 on their surface. Optionally, determining whether an individual has cells that express CD39 comprises conducting an immunohistochemistry assay.

In one embodiment, the disclosure provides a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting CD39 polypeptide (e.g. cell membrane-bound CD39 and/or soluble CD39 polypeptide) in circulation and/or in the tumor environment (e.g. within the tumor and/or within adjacent tissue; on T cells; on effector T cells; on Treg cells), and b) upon a determination that tumor environment comprises CD39 polypeptide, optionally at a level that is increased compared to a reference level, administering to the individual a treatment of the disclosure.

In one embodiment, the disclosure provides a method for the treatment or prevention of a cancer in an individual in need thereof, the method comprising: a) detecting ATPase activity and/or adenosine accumulation, and b) upon a determination that lymph node or extranodal tumor environment comprises ATPase activity and/or adenosine accumulation, optionally at a level that is increased compared to a reference level, administering to the individual a treatment of the disclosure.

CD39 neutralizing agents

Eight different ENTPD genes encode members of the NTPDase protein family. The individual NTPDase subtypes differ in cellular location and functional properties. Plasma membrane-bound nucleoside triphosphate diphosphohydrolases control nucleotide levels at the cell surface by hydrolyzing the c and b phosphates of nucleotides. NTPDase 1 (ectonucleoside triphosphate diphosphohydrolasel), also known as CD39/ENTPD1 or vascular CD39, functions together with another enzyme, CD73 (ecto-5‘-nucleotidase), to hydrolyze extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to generate adenosine, which binds to adenosine receptors and inhibits T-cell and natural killer (NK)-cell responses, thereby suppressing the immune system. The generation of adenosine via the CD73/CD39 pathway is recognized as a major mechanism of regulatory T cell (Treg) immunosuppressive function. The number of CD39⁺ Tregs is increased in some human cancers, and the importance of CD39⁺ Tregs in promoting tumor growth and metastasis has been demonstrated using several in vivo models. However, CD39 is also expressed by tumor cells and CD39⁺ tumor cells can mediate immunosuppression via the adenosine pathway. CD39 in cancer cells displays ATPase activity and, together with CD73, generates adenosine. CD73⁺CD39⁺ cancer cells inhibited the proliferation of CD4 and CD8 T cells and the generation of cytotoxic effector CD8 T cells (CTL) in a CD39- and adenosine-dependent manner.

Human CD39, also known as “vascular” CD39, NTPdasel , ENTPD1 , ATPDase and vascular ATP diphosphohydrolase, exhibits ATPase activity. CD39 hydrolyzes extracellular ATP and ADP to AMP, which is further converted to adenosine by another enzyme, 5-prime nucleotidase. The amino acid sequence of the “vascular” human CD39 mature polypeptide chain is shown in Genbank under accession number P49961 , the entire disclosure of which is incorporated herein by reference, and as follows: MEDTKESNVK TFCSKNILAI LGFSSI IAVI ALLAVGLTQN KALPENVKYG IVLDAGSSHT SLYIYKWPAE KENDTGVVHQ VEECRVKGPG ISKFVQKVNE IGIYLTDCME RAREVIPRSQ HQETPVYLGA TAGMRLLRME SEELADRVLD VVERSLSNYP FDFQGARI IT GQEEGAYGWI TINYLLGKFS QKTRWFSIVP YETNNQETFG ALDLGGASTQ VTFVPQNQTI ESPDNALQFR LYGKDYNVYT HSFLCYGKDQ ALWQKLAKDI QVASNEILRD PCFHPGYKKV VNVSDLYKTP CTKRFEMTLP FQQFEIQGIG NYQQCHQSIL ELFNTSYCPY SQCAFNGI FL PPLQGDFGAF SAFYFVMKFL NLTSEKVSQE KVTEMMKKFC AQPWEEIKTS YAGVKEKYLS EYCFSGTYIL SLLLQGYHFT ADSWEHIHFI GKIQGSDAGW TLGYMLNLTN MIPAEQPLST PLSHSTYVFL MVLFSLVLFT VAIIGLLI FH KPSYFWKDMV (SEQ ID NO: 1).

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

The anti-CD39 antibodies used herein are antibodies that neutralize CD39. “Neutralize” or neutralizing”, when referring to the CD39 polypeptide (e.g., “neutralize CD39”, “neutralize the activity of CD39” or “neutralize the enzymatic activity of CD39”), refers to a process in which the ATP hydrolysis (ATPase) activity of CD39 is inhibited. Such antibodies have been reported in several publications, supra, the disclosures of amino acid sequences of which are incorporated herein by reference. Some antibodies may neutralize membranebound CD39 by inhibiting the domain motion of membrane-bound CD39 (memCD39), however without similarly affecting the activity of the soluble CD39 protein (sCD39). It has been reported that memCD39 occurs as a homo-multimer while sCD39 is a monomer, and moreover that the transmembrane domains in memCD39 undergo dynamic motions that underlie a functional relationship with the active site. Consequently, unlike sCD39, memCD39 may present a setting that makes antibody-mediated neutralization possible. One possibility is that use of a bivalent antibody that binds simultaneously to two memCD39 molecules (e.g., within a memCD39 homo-multimer) is required for functional neutralization.

It will be appreciated that any suitable CD39-neutralizing agent can be used in accordance with the invention. For example, any of a wide range of known antibody agents that neutralize human CD39 have been reported and several of them are being investigated in human clinical trials, including: ES-002 (Elpiscience); ES-014 (Elpiscience); SRF-617 (Surface Oncology; Coherus Biosciences); TTX-030 (Abbvie; Trishula Therapeutics); IPH5201 (Innate Pharma); AB598 (Arcus Biosciences); JS019 (Suzhou Kebo Ruijun Biotechnology Co., Ltd); and PLIR001 (Purinomia Biotech, Inc.).

Other exemplary antibody CD39-neutralizing agents are known, for example CDRs and variable regions of anti-CD39 antibodies are described in PCT publication nos. WO20 18/167267, WO2019/178269, WO2019/127935, WO2019/243252, W02021/037037 and WO2023164872, the disclosures of which are incorporated herein by reference. An anti- CD39 antibody may be specified as comprising the heavy and light chain CDRs or heavy and light chain variable regions of any of such antibodies.

In another aspect, an anti-CD39 antibody may be a function-conservative variant of any of the antibodies, or of an antibody having their heavy and/or light chains, CDRs or variable regions thereof. “Function-conservative variants” are those in which a given amino acid residue in a protein or antibody has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A “functionconservative variant” also includes a polypeptide which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein (e.g. heavy or light chains, or CDRs or variable regions thereof) to which it is compared. In one embodiment, the antibody comprises a heavy chain variable region that is a functionconservative variant of the heavy chain variable region of antibody I-394, I-395, I-396 or I-399 provided in PCT publication no. WO2018/167267, and a light chain variable region that is a function-conservative variant of the light chain variable region of the respective antibody I-394, I-395, I-396 or I-399 antibody. In one embodiment, the antibody comprises a heavy chain that is a function-conservative variant of the heavy chain variable region of antibody I-394, I-395, I-396 or I-399 fused to a human heavy chain constant region disclosed herein, optionally a human lgG4 constant region, optionally a modified IgG (e.g. lgG1) constant region, e.g. a constant region of any of SEQ ID NOS: [93-96], and a light chain that is a function-conservative variant of the light chain variable region of the respective antibody I-394, I-395, I-396 or I-399 antibody fused to a human Ckappa light chain constant region.

The anti-CD39 antibodies can for example bind an epitope present on human CD39 protein expressed at the surface of cells (e.g. they can compete with an anti-CD39 antibody having the VH and VL of SEQ ID NOS: 8 and 9 for binding to an epitope on CD39), including tumor cells and potently inhibit the enzymatic (ATPase activity) activity of the cell membrane bound CD39 enzyme (CD39 as expressed at the surface of cells), and the anti-CD39 antibodies described herein further inhibit the enzymatic (ATPase activity) activity of soluble (extracellular domain) human CD39 protein. The antibodies thereby mediate strong neutralization of CD39 activity in an individual by neutralizing both membrane-bound and soluble CD39 protein, including soluble CD39 released or shed from tumor cells, thereby reducing immunosuppression, e.g., for the treatment of cancer and/or infectious disease.

In one aspect, the anti-CD39 antibody comprises (a) a HCDR1 , HCDR2 and HCDR3 of SEQ ID NOS: 2, 3 and 4, respectively, and (b) an LCDR1 , LCDR2 and LCDR3 sequence of SEQ ID NOS: 5, 6 and 7, respectively.

In one aspect, the anti-CD39 antibody can be characterized as comprising: a HCDR1 comprising an amino acid sequence: DYNMH (SEQ ID NO: 2), or a sequence of at least 3 or 4 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR2 comprising an amino acid sequence: YIVPLNGGSTFNQKFKG (SEQ ID NO: 3), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid, optionally wherein the asparagine at Kabat position 61 is substituted, optionally wherein the lysine at Kabat position 65 is substituted; a HCDR3 comprising an amino acid sequence: GGTRFAY (SEQ ID NO: 4), or a sequence of at least 4, 5 or 6 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR1 comprising an amino acid sequence: RASESVDNFGVSFMY (SEQ ID NO: 5), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid, optionally wherein the arginine at Kabat position 24 is substituted; a LCDR2 region comprising an amino acid sequence: GASNQGS (SEQ ID NO: 6) or a sequence of at least 4, 5 or 6 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; and/or a LCDR3 region comprising an amino acid sequence: QQTKEVPYT (SEQ ID NO: 7), or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be deleted or substituted by a different amino acid.

In any embodiment, CDR positions may be specified as being according to Kabat numbering.

In one aspect, the anti-CD39 antibody comprises the hypervariable region of, optionally the CDRs of, the antibody having the VH and VL of SEQ ID NOS: 8 and 9, shown below.

Anti-CD39

VH:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWIGYIVPLNGGST FNQKFKGRATITVDTSARTAYMELSSLRSEDTAVYYCARGGTRFAYWGQGTLVTVSS (SEQ ID NO: 8).

Anti-CD39 VL:

DIVMTQSPDSLAVSLGERATINCRASESVDNFGVSFMYWFQQKPGQPPKLLIYGASNQGS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTKEVPYTFGGGTKVEIK

(SEQ ID NO: 9).

In one embodiment, an anti-CD39 antibody comprises a heavy chain variable region (VH) comprising CDR1 , CDR2 and CDR3 having the respective amino acid sequences shown in SEQ ID NOS: 2, 3 and 4 and human frameworks (e.g., FR1 , FR2, FR3 and FR4 of human origin); and a light chain variable region (VL) CDR1 , CDR2 and CDR3 comprising the respective amino acid sequences shown in SEQ ID NOS: 5, 6 and 7, and human frameworks (e.g., FR1 , FR2, FR3 and FR4 of human origin), wherein the (VH) comprises an amino acid sequence at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NOS: 8, and a light chain variable region (VL) comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9.

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

In one embodiment, an anti-CD39 antibody comprises a heavy chain comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10, and a light chain comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11.

Anti-CD39 Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWIGYIVPLNGGST FNQKFKGRATITVDTSARTAYMELSSLRSEDTAVYYCARGGTRFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

Anti-CD39 Light chain

DIVMTQSPDSLAVSLGERATINCRASESVDNFGVSFMYWFQQKPGQPPKLLIYGAS NQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTKEVPYTFGGGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

In one embodiment, an anti-CD39 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a light chain comprising the amino acid sequence of SEQ ID NO: 11. In one embodiment, the anti-CD39 antibody is IPH5201. Another exemplary anti-CD39 VH and VL pair is that of antibody 31905, the amino acid sequence of the heavy chain variable region of which is listed below (SEQ ID NO: 12), and the amino acid sequence of the light chain variable region of which is listed below (SEQ ID NO: 13). The CDRs according to Kabat numbering are underlined in SEQ ID NOS: 12 and 13. The VH and VL can be specified as comprising human acceptor frameworks. In one embodiment, an anti-CD39 antibody of the disclosure comprises the VH CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 12. In one embodiment, an anti-CD39 antibody of the disclosure comprise the VL CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the light chain variable region having the amino acid sequence of SEQ ID NO: 13.

31905 VH:

QVQLVQSGAEVKKPGSSVKVSCKASGGTFPSNAISWVRQAPGQGLEWMGGIGFGTANYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGAKYARTYGMDVWGQGTTVTV SS

(SEQ ID NO: 12).

31905 VL:

DIVMTQSPDSLAVSLGERATINCKSSKSVLYSNNNKNYLAWYQQKPGQPPKLLIYWASTR QSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYLLYPLTFGGGTKVEIK (SEQ ID NO: 13).

An anti-CD39 antibody may for example comprise: a HCDR1 comprising an amino acid sequence: SNAIS (SEQ ID NO: 14), or a sequence of at least 4 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR2 comprising an amino acid sequence: GIGFGTANYAQKFQG (SEQ ID NO: 15), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR3 comprising an amino acid sequence: GGAKYARTYGMDV (SEQ ID NO: 16), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR1 comprising an amino acid sequence: KSSKSVLYSNNNKNYLA (SEQ ID NO: 17), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR2 region comprising an amino acid sequence: WASTRQS (SEQ ID NO: 18) or a sequence of at least 4, 5 or 6 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; and/or a LCDR3 region comprising an amino acid sequence: QQYLLYPLT (SEQ ID NO: 19), or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be deleted or substituted by a different amino acid. CDR positions may be according to Kabat numbering.

Another exemplary anti-CD39 VH and VL pair is that of antibody 31901 , the amino acid sequence of the heavy chain variable region of which is listed below (SEQ ID NO: 20), and the amino acid sequence of the light chain variable region of which is listed below (SEQ ID NO: 21). The CDRs according to Kabat numbering are underlined in SEQ ID NOS: 20 and 21 . The VH and VL can be specified as comprising human acceptor frameworks. In one embodiment, an anti-CD39 antibody of the disclosure comprises the VH CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 20. In one embodiment, an anti-CD39 antibody of the disclosure comprise the VL CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the light chain variable region having the amino acid sequence of SEQ ID NO: 21.

31901 VH:

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSLPISWVRQAPGQGLEWMGGIGFGTANYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGAKYAGRYGMDVWGQGTTVTV SS

(SEQ ID NO: 20).

31901 VL:

GIVMTQSPDSLAVSLGERATINCKSSQSVLFSSNNKNYLAWYQQKPGQPPKLLIYWASTR ASGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYLYPLTFGGGTKVEIK

(SEQ ID NO: 21).

An anti-CD39 antibody may for example comprise: a HCDR1 comprising an amino acid sequence: SLPIS (SEQ ID NO: 22), or a sequence of at least 4 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR2 comprising an amino acid sequence: GIGFGTANYAQKFQ (SEQ ID NO: 23), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR3 comprising an amino acid sequence: GGAKYAGRYGMDV (SEQ ID NO: 24), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR1 comprising an amino acid sequence: KSSQSVLFSSNNKNYLA (SEQ ID NO: 25), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR2 region comprising an amino acid sequence: WASTRAS (SEQ ID NO: 26) or a sequence of at least 4, 5 or 6 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; and/or a LCDR3 region comprising an amino acid sequence: QQYYLYPLT (SEQ ID NO: 27), or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be deleted or substituted by a different amino acid. CDR positions may be according to Kabat numbering.

Another exemplary anti-CD39 VH and VL pair is that of antibody 31873, the amino acid sequence of the heavy chain variable region of which is listed below (SEQ ID NO: 28), and the amino acid sequence of the light chain variable region of which is listed below (SEQ ID NO: 29. The CDRs according to Kabat numbering are underlined in SEQ ID NOS: 28 and 29. The VH and VL can be specified as comprising human acceptor frameworks. In one embodiment, an anti-CD39 antibody of the disclosure comprises the VH CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 28. In one embodiment, an anti-CD39 antibody of the disclosure comprise the VL CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the light chain variable region having the amino acid sequence of SEQ ID NO: 29.

31873 VH:

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSKYGISWVRQAPGQGLEWMGSIIPEFGIANY AQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESGGYRDHRLGVWGQGTMVTV SS

(SEQ ID NO: 28).

31873 VL:

EIVMTQSPATLSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGASTRASGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYLLWPLTFGGGTKVEIK (SEQ ID NO: 29).

An anti-CD39 antibody may for example comprise: a HCDR1 comprising an amino acid sequence: KYGIS (SEQ ID NO: 30), or a sequence of at least 4 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR2 comprising an amino acid sequence: SIIPEFGIANYAQKFQG (SEQ ID NO:31), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR3 comprising an amino acid sequence: ESGGYRDHRLGV (SEQ ID NO: 32), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR1 comprising an amino acid sequence: RASQSVGSNLA (SEQ ID NO: 33), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR2 region comprising an amino acid sequence: GASTRAS (SEQ ID NO: 34) or a sequence of at least 4, 5 or 6 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; and/or a LCDR3 region comprising an amino acid sequence: QQYLLWPLT (SEQ ID NO: 35), or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be deleted or substituted by a different amino acid. CDR positions may be according to Kabat numbering.

Another exemplary anti-CD39 VH and VL pair is that of antibody 31414, the amino acid sequence of the heavy chain variable region of which is listed below (SEQ ID NO: 36), and the amino acid sequence of the light chain variable region of which is listed below (SEQ ID NO: 37). The CDRs according to Kabat numbering are underlined in SEQ ID NOS: 36 and 37. The VH and VL can be specified as comprising human acceptor frameworks. In one embodiment, an anti-CD39 antibody of the disclosure comprises the VH CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 36. In one embodiment, an anti-CD39 antibody of the disclosure comprise the VL CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the light chain variable region having the amino acid sequence of SEQ ID NO: 37.

31414 VH:

QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGST WYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRNWGQGTL VTVSS

(SEQ ID NO: 36).

31414 VL:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHSYITFGGGTKVEIK (SEQ ID NO: 37). An anti-CD39 antibody may for example comprise: a HCDR1 comprising an amino acid sequence: SYEMH (SEQ ID NO: 38), or a sequence of at least 4 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR2 comprising an amino acid sequence: RINPSVGSTWYAQKFQG (SEQ ID NO: 39), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR3 comprising an amino acid sequence: GKREGGTEYLRN (SEQ ID NO: 40), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR1 comprising an amino acid sequence: RASQSVSSSYLA (SEQ ID NO: 41), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR2 region comprising an amino acid sequence: GASSRAT (SEQ ID NO: 42) or a sequence of at least 4, 5 or 6 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; and/or a LCDR3 region comprising an amino acid sequence: QQYHSYIT (SEQ ID NO: 43), or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be deleted or substituted by a different amino acid. CDR positions may be according to Kabat numbering.

Another exemplary anti-CD39 VH and VL pair is that of antibody 31895, the amino acid sequence of the heavy chain variable region of which is listed below (SEQ ID NO: 44), and the amino acid sequence of the light chain variable region of which is listed below (SEQ ID NO: 45). The CDRs according to Kabat numbering are underlined in SEQ ID NOS: 44 and 45. The VH and VL can be specified as comprising human acceptor frameworks. In one embodiment, an anti-CD39 antibody of the disclosure comprises the VH CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 44. In one embodiment, an anti-CD39 antibody of the disclosure comprise the VL CDR1 , CDR2 and/or CDR3 (e.g., according to Kabat numbering) of the light chain variable region having the amino acid sequence of SEQ ID NO: 45.

31895 VH:

QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGST WYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTL VTVSS

(SEQ ID NO: 44).

31895 VL: EIVLTQSPGTLSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIK

(SEQ ID NO: 45).

An anti-CD39 antibody may for example comprise: a HCDR1 comprising an amino acid sequence: SYEMH (SEQ ID NO: 46), or a sequence of at least 4 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR2 comprising an amino acid sequence: RINPSVGSTWYAQKFQG (SEQ ID NO: 47), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a HCDR3 comprising an amino acid sequence: GKREGGTEYLRK (SEQ ID NO: 48), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR1 comprising an amino acid sequence: RASQSVASSYLA (SEQ ID NO: 49), or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; a LCDR2 region comprising an amino acid sequence: GASNRHT (SEQ ID NO: 50) or a sequence of at least 4, 5 or 6 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be substituted by a different amino acid; and/or a LCDR3 region comprising an amino acid sequence: QQYHNAIT (SEQ ID NO: 51), or a sequence of at least 4, 5, 6, 7 or 8 contiguous amino acids thereof, optionally wherein one or more of these amino acids may be deleted or substituted by a different amino acid. CDR positions may be according to Kabat numbering.

An exemplary 31895 antibody may have a heavy chain comprising the amino acid sequence of SEQ ID NO 52, shown below, and a light chain comprising the amino acid seuqnce of SEQ ID NO: 53, shown below.

31895 heavy chain: QVQLVQSGAEVKKPGASVKVSCKASGYTFKSYEMHWVRQAPGQGLEWMGRINPSVGST WYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGKREGGTEYLRKWGQGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 52) 31895 light chain: EIVLTQSPGTLSLSPGERATLSCRASQSVASSYLAWYQQKPGQAPRLLIYGASNRHTGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHNAITFGGGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO: 53).

Another exemplary anti-CD39 VH and VL pair is that of antibody SRF 367-C, the amino acid sequence of the heavy chain variable region of which is listed below (SEQ ID NO: 54), and the amino acid sequence of the light chain variable region of which is listed below (SEQ ID NO: 55).

SRF 367-C VH: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSEGISWVRQAPGQGLEWMGSILPIFGTANY AQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREAGYYRYRYFDLWGKGTLVTVSS (SEQ ID NO: 54).

SRF 367-C VL: EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQHALWPLTFGGGTKVEIK (SEQ ID NO: 55).

An anti-CD39 antibody may thus for example comprise: a HCDR1, 2 and 3 of SEQ ID NOS 56, 57 and 58 and the LCDR1 of SEQ ID NOS: 59, LCDR2 having the amino sequence GAS and LCDR3 of SEQ ID NO : 60, respectively, as shown below:

HCDR1 GGTFSSEG (SEQ ID NO: 56)

HCDR2 ILPIFGTA (SEQ ID NO: 57)

HCDR3 AREAGYYRYRYFDL (SEQ ID NO: 58)

LCDR1 QSVSSN (SEQ ID NO: 59)

LCDR2: GAS

LCDR3 QQHALWPLT (SEQ ID NO: 60)

Another exemplary anti-CD39 VH and VL pair and set of CDRs according to the disclosure is that of antibody SRF 360, the amino acid sequence of the heavy chain variable region of which is listed below (SEQ ID NO: 61), and the amino acid sequence of the light chain variable region of which is listed below (SEQ ID NO: 62).

SRF 360 VH:

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYRMNWVRQAPGKGLEWVSSISSSSSSIWY ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKGPRYDSSGYRWRYGMDVWGQ

GTTVTVSS (SEQ ID NO: 61)

SRF 360 VL: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQLYVDPPWTFGGGTKVEIK (SEQ ID NO: 62).

Another exemplary anti-CD39 VH and VL pair and set of CDRs is that of antibody 19, having the amino acid sequence of the HCDR1, 2 and 3 of SEQ ID NOS: 63, 64 and 65 and the LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 66, 67 and 68, respectively, as shown below:

HCDR1 : GFSLTSYFTQ (SEQ ID NO: 63)

HCDR2 : FIRSGGSTQYNSAFKS (SEQ ID NO: 64)

HCDR3 : DRPDYHDGTYGVLDV (SEQ ID NO: 65)

LCDR1 : LASEDIDSDLA (SEQ ID NO: 66)

LCDR2 : NGNALQN (SEQ ID NO: 67)

LCDR3 : QQYINYPYT (SEQ ID NO: 68).

Another exemplary anti-CD39 VH and VL pair and set of CDRs is that of antibody 31, having the amino acid sequence of the HCDR1, 2 and 3 of SEQ ID NOS 69, 70 and 71 and the LCDR1, LCDR2 and LCDR3 of SEQ ID NOS: 72-76, as shown below:

HCDR1 : GDSITAYFMH (SEQ ID NO: 69)

HCDR2 : RIDPDHERTKYAEKFKN (SEQ ID NO: 70)

HCDR3 : ERVPGFETDY (SEQ ID NO: 71)

LCDR1 : RTNQTVDHYGTTYMN (SEQ ID NO: 72), RTNPTVDHYGTTYMN (SEQ ID NO: 73), or RTQQTVDHYGTTYMN (SEQ ID NO: 74)

LCDR2 : LASSLAS (SEQ ID NO: 75)

LCDR3 : QQSRNLWT (SEQ ID NO: 76).

Another exemplary anti-CD39 VH and VL pair and set of CDRs is that of antibody 39, having the amino acid sequence of the HCDR1, 2 and 3 of SEQ ID NOS 77, 78 and 79, and an LCDR1 of SEQ ID NOS: 80, 81 and 82, respectively, as shown below:

HCDR1 : GFTFNNYGMN (SEQ ID NO: 77)

HCDR2 : SFSSSSTYISYADTVKG (SEQ ID NO: 78)

HCDR3 : HGRYGGYGGFYFDY (SEQ ID NO: 79)

LCDR1 : RASEGVKSYMH (SEQ ID NO: 80) LCDR2: KASNLAS (SEQ ID NO: 81)

LCDR3 QQSWNDPT (SEQ ID NO: 82).

Another exemplary anti-CD39 VH and VL pair and set of CDRs is that of antibody 23, having the amino acid sequence of the HCDR1, 2 and 3 of SEQ ID NOS 85, 86 and 87, and an LCDR1 of SEQ ID NOS: 88, 89 and 90, respectively, as shown below, and the VH and VL as shown below in SEQ ID NOS 83 and 84: hu23H5L5 VH:

QVQLVQSGAEVKKPGASVKVSCKASGYNLKDTFLHWVRQAPGQGLEWMGRIDPANGNIK YDPKFQGRVTITADTSANTAYMELISLRSEDTAVYYCANSPYYYGSGYRIFDVWGQGTTVT VSS (SEQ ID NO: 83) hu23H5L5 VL:

QIVLTQSPATLSLSPGERATLSCSAFSSVNYMHWYQQKPGQAPRLLIYTTSNLASGIPTR FSGSGSGTSYTLTISSLEPEDFAVYYCQQRSTYPFTFGQGTKLEIK (SEQ ID NO: 84)

HCDR1 : DTFLH (SEQ ID NO: 85)

HCDR2 : RIDPANGNIKYDPKFQ (SEQ ID NO: 86)

HCDR3 : SPYYYGSGYRIFDV (SEQ ID NO: 87)

LCDR1 : SAFSSVNYMH (SEQ ID NO: 88)

LCDR2: TTSNLAS (SEQ ID NO: 89)

LCDR3 QQRSTYPFT (SEQ ID NO: 90).

In any of the I-394, I-395, I-396, I-399, 19, 23, 31, 39, SRF-360, SRF-617-C, 31414, 31895, 31873, 31901 or 31905 antibodies, the HCDR1 , 2, 3 and LCDR1 , 2, 3 sequences (each CDR independently, or all CDRs) can be specified as being those of the Kabat numbering system, (as indicated in in the VH and VL sequences by underlining), those of the Chothia numbering system, or, those of the IMGT numbering system, or any other suitable numbering system. In any embodiment, an anti-CD39 antibody can be characterized as comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO providing its VH, and a light chain variable region (VL) comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: providing its VL. In any aspect, the specified variable region, FR and/or CDR sequences may comprise one or more sequence modifications, e.g., a substitution (1 , 2, 3, 4, 5, 6, 7, 8 or more sequence modifications). In one embodiment the substitution is a conservative modification.

In any embodiment, an anti-CD39 antibody can be characterized as binding to and inhibiting or neutralizing the ATPase activity of a soluble CD39 protein (sCD39). In one embodiment the sCD39 protein lacks the two transmembrane domains (i.e. the transmembrane domains near the N- and C-terminal ends) found in membrane bound CD39. In one embodiment, sCD39 is a non-membrane bound sCD39 protein found in circulation, e.g., in a human individual. In one embodiment, the CD39-derived sequence of a sCD39 protein comprises or consists of the Thr38-Val478 fragment of CD39. Thr38-Val478 protein with C-terminal His tag is available commercially from R&D Systems, Inc., (product number 4397-EN). In one embodiment, the protein, antibody or antibody fragment inhibits the ATPase activity of sCD39 when incubated with sCD39 in solution, e.g. in tumor cell supernatants. In one embodiment, the protein, antibody or antibody fragment specifically binds the human CD39 protein, both in soluble (extracellular domain protein) and in membrane-bound form.

The anti-CD39 antibody described herein comprises a human Fc domain that is modified to have decreased or substantially lack binding to a human Fey receptor, e.g., one or more (or all of) human CD16, CD32a, CD32b and CD64. The antibody does not depend on ADCC-, CDC- or toxin- mediated depletion of CD39-expressing cells for their CD39 inhibitory activity. Such antibodies are thus used as “pure” CD39 blockers, with an immunomodulatory activity.

Anti-CD39 antibodies can be prepared such that they do not have substantial binding to human Fey receptors, e.g., any one or more of CD16A, CD16B, CD32A, CD32B and/or CD64). Such antibodies may comprise constant regions of various heavy chains that are known to lack or have low binding to Fey receptors. Alternatively, antibody fragments that do not comprise (or comprise portions of) constant regions, such as F(ab’)2 fragments, can be used to avoid Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including for example testing binding of an antibody to Fc receptor protein in a BIACORE assay. Also, generally any antibody IgG isotype can be used in which the Fc portion is modified (e.g., by introducing 1 , 2, 3, 4, 5 or more amino acid substitutions) to minimize or eliminate binding to Fc receptors (see, e.g., WO 03/101485, the disclosure of which is herein incorporated by reference). Assays such to assess Fc receptor binding are well known in the art, and are described in, e.g., WO 03/101485.

In one embodiment, the anti-CD39 antibody can comprise one or more specific mutations in the Fc region that result minimal interaction with effector cells. Silenced effector functions can be obtained by mutation in the Fc region of the antibodies and have been described in the art: N297A mutation, the LALA mutations, (Strohl, W., 2009, Curr. Opin. Biotechnol. Vol. 20(6):685-691); and D265A (Baudino et al., 2008, J. Immunol. 181 : 6664-69) see also Heusser et al., WO2012/065950, the disclosures of which are incorporated herein by reference. In one embodiment, an antibody comprises one, two, three or more amino acid substitutions in the hinge region. In one embodiment, the antibody is an lgG1 or lgG2 and comprises one, two or three substitutions at residues 233-236, optionally 233-238 (Ell numbering). In one embodiment, the antibody is an lgG4 and comprises one, two or three substitutions at residues 327, 330 and/or 331 (Ell numbering). Examples of silent Fc lgG1 antibodies are the LALA mutant comprising L234A and L235A mutation in the IgG 1 Fc amino acid sequence. Another example of an Fc silent mutation is a mutation at residue D265, or at D265 and P329 for example as used in an lgG1 antibody as the DAPA (D265A, P329A) mutation (US 6,737,056). Another silent IgG 1 antibody comprises a mutation at residue N297 (e.g., N297A, N297S mutation), which results in aglycosylated/non-glycosylated antibodies. Other silent mutations include: substitutions at residues L234 and G237 (L234A/G237A); substitutions at residues S228, L235 and R409 (S228P/L235E/R409K,T,M,L); substitutions at residues H268, V309, A330 and A331 (H268Q/V309L/A330S/A331S); substitutions at residues C220, C226, C229 and P238 (C220S/C226S/C229S/P238S); substitutions at residues C226, C229, E233, L234 and L235 (C226S/C229S/E233P/L234V/L235A; substitutions at residues K322, L235 and L235 (K322A/L234A/L235A); substitutions at residues L234, L235 and P331 (L234F/L235E/P331S); substitutions at residues 234, 235 and 297; substitutions at residues E318, K320 and K322 (L235E/E318A/K320A/K322A); substitutions at residues (V234A, G237A, P238S); substitutions at residues 243 and 264; substitutions at residues 297 and 299; substitutions such that residues 233, 234, 235, 237, and 238 defined by the EU numbering system, comprise a sequence selected from PAAAP, PAAAS and SAAAS (see WO2011/066501).

In one embodiment, the anti-CD39 antibody can comprise one or more specific mutations in the Fc region. For example, such an antibody can comprise an Fc domain of human lgG1 origin, comprises a mutation at Kabat residue(s) 234, 235, 237, 330 and/or 331. One example of such an Fc domain comprises substitutions at Kabat residues L234, L235 and P331 (e.g., L234A/L235E/P331S or (L234F/L235E/P331S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A/L235E/G237A/P331S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237, A330 and P331 (e.g., L234A/L235E/G237A/A330S/P331 S). In the shorthand notation used here, the format is: Wild type residue: Position in polypeptide: Mutant residues, wherein residue positions are indicated according to Ell numbering according to Kabat.

In one embodiment, an anti-CD39 antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235 and 331 (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEA EGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91).

In one embodiment, an anti-CD39 antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235 and 331 (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEF EGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92).

In one embodiment, an anti-CD39 antibody comprises a heavy chain constant region comprising the amino acid sequence below, or an amino acid sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237, 330 and 331 (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEA EGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93).

In one embodiment, an anti-CD39 antibody comprises a heavy chain constant region comprising the amino acid sequence below, or a sequence at least 90%, 95% or 99% identical thereto but retaining the amino acid residues at Kabat positions 234, 235, 237 and 331 (underlined):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEA EGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 94).

T-Cell Engagers

T cell engager proteins (TCEs) are proteins that mediate the engagement of T cells against target cells that are sought to be depleted. The TCEs comprise at least two antigen binding domains (ABDs) that bind to different antigens, wherein at least one ABD binds to an antigenic determinant on the target cells (e.g. malignant cells; B-NHL cells) and at least one ABD binds to an antigenic determinant on the T cells. Binding to the antigenic determinant on T cells can then result in T-cell activation. Most TCEs bind to CD3, in particular the CD3 epsilon chain (CD3E) present on T cells. Numerous T cell engager proteins (TCEs) have been developed to mediate the engagement of T cells against malignant B cells, thereby treating B- NHL. The TCEs that activate endogenous T cells to recognize tumour targets have been most widely exemplified using TCEs that bind CD20 or CD19 on malignant B cells and CD3 on the T cells. In different CD20+ hematologic malignancies, this approach was initially exemplified by blinatumomab, a TCE that binds CD19 and CD3 (Bargou et al., Science (2008) 321 , 974- 977). Targeting CD19 has been proposed to have the advantage that B cell lymphomas that may down-regulate CD20 expression remain positive for CD19.

In one embodiment, a TCE is a CD20- and CD3-binding TCE. A TCE that binds CD20 and CD3 can be specified as being an agent or protein (e.g. an anti-CD20/anti-CD3 bispecific antibody) that targets CD20 expressed on B cells and CD3 (preferably CD3 epsilon chain (CD3E)) present on T cells. Simultaneous binding leads to T-cell activation and T-cell mediated killing of B cells. In the presence of CD20+ B cells, whether circulating or tissue resident, pharmacologically active doses of anti-CD20/anti-CD3 bispecific antibody will trigger T-cell activation and associated cytokine release. The TCE can be specified as comprising a first ABD that binds to CD3, and a second ABD that binds to CD20. The first and/or second ABD can be for example a Fab (e.g. a conventional Fab in natural format or a crossover-Fab) or an scFv. A “crossover” Fab refers to a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1 , in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in N- to C-terminal direction.

The TCE may further comprise an Fc domain of human origin, for example an Fc domain of human lgG4 isotype or preferably any human gamma isotype (e.g. lgG1 , lgG2, lgG3, lgG4). Optionally, the the Fc domain is modified to reduce or abolish binding to one or more human Fey receptors (e.g. CD16A, CD64).

In one embodiment, the TCE as used herein can optionally be specified as comprising a first antigen binding domain that binds CD3 comprising a heavy chain variable region (VHCDS) and a light chain variable region (VLCDS), and a second antigen binding domain that binds CD20 comprising a heavy chain variable region (VHCD2O) and a light chain variable region (VLCD20).

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities and “knob-in-hole” engineering. Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules; by cross-linking of two or more antibodies or fragments such as by using leucine zippers to produce bi-specific antibodies; through use of diabody technology for making bispecific antibody fragments; through use of single-chain Fv (sFv) dimers; through use of “Crossmab” antibodies (see e.g. WO 2009/080251 , WO 2009/080252, W02009/080253, or W02009/080254); and BiTE® antibodies (see, e.g., W02004/106381 , W02005/061547, W02007/042261 , and W02008/119567), based on two antibody variable domains arranged on a single polypeptide, typically comprising two Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between the two domains.

The “knob-into-hole” technology is described e.g. in U.S. Pat. Nos. 5,731 ,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine orthreonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, (2001), J Immunol Methods 248, 7-15).

Many TCEs that bind CD20 and CD3 have been reported and developed in human clinical trials, including several that have been recently approved by the FDA. Examples of TCEs that bind CD20 and CD3 are plamotamab, FBTA05 (Lymphomun), Mosunetuzumab, Odronextamab, glofitamab and epcoritamab, or a generic, biosimilar or non-comparable biologic version of an antibody of any thereof. Further examples of TCEs that bind CD20 and CD3 are described in PCT publication no. WO2016/020309, WO2018/114748 or in WO 2015/095392, the disclosure of which are incorporated herein by reference.

In one embodiment, TCE comprises at least one antigen binding domain that specifically binds to CD20, comprising a heavy chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region sequence of the CD20-binding domain of any of the TCEs referred to herein, and a light chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region sequence of the CD20-binding domain of any of the TCEs referred to herein. In any embodiment, an antigen binding domain can be a function-conservative variant of any of said known CD3 binding domains.

In one embodiment, TCE comprises at least one antigen binding domain that specifically binds to CD3, comprising a heavy chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region sequence of the CD3-binding domain of any of the TCEs referred to herein, and a light chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region sequence of the CD3-binding domain of any of the TCEs referred to herein; and a comprises at least one antigen binding domain that specifically binds to CD20, comprising a heavy chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region sequence of the CD20-binding domain of any of the TCEs referred to herein, and a light chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region sequence of the CD20-binding domain of any of the TCEs referred to herein.

In one embodiment, TCE comprises at least one antigen binding domain that specifically binds to CD3 derived from a known CD3 antigen binding domain. The term “CD3” refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CD3 as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, e.g., splice variants or allelic variants. In one embodiment, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3E). The amino acid sequence of human CD3E is shown in UniProt (www.uniprot.org) accession no. P07766.

Amino acid sequences of an exemplary VH and VL pair that form and ABD that binds to CD3 are shown below:

VH:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO: 95)

VL:

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYR FSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 96).

In one embodiment, an antigen binding domain that specifically binds to CD3 comprises a heavy chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region sequence of the CD3- binding domain of any of the TCEs referred to herein, and a light chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region sequence of the CD3-binding domain of any of the TCEs referred to herein. In any embodiment, an antigen binding domain can be a function-conservative variant of any of said known CD3 binding domains.

“CD20” refers to B-lymphocyte antigen CD20, also known as B-lymphocyte surface antigen B1 or Leukocyte surface antigen Leu-16, and includes any native CD20 from any vertebrate source, including mammals such as primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The amino acid sequence of human CD20 is shown in Uniprot accession no. P11836. CD20 is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD expressed on pre-B and mature B lymphocytes. The corresponding human gene is membrane-spanning 4-domains, subfamily A, member 1 , also known as MS4A1. This gene encodes a member of the membrane-spanning 4A gene family. Members of this nascent protein family are characterized by common structural features and similar intron/exon splice boundaries and display unique expression patterns among hematopoietic cells and nonlymphoid tissues. This gene encodes the B-lymphocyte surface molecule which plays a role in the development and differentiation of B-cells into plasma cells. This family member is localized to 11 q12, among a cluster of family members. Alternative splicing of this gene results in two transcript variants which encode the same protein. The term “CD20” encompasses “full- length,” unprocessed CD20 as well as any form of CD20 that results from processing in the cell. The term also encompasses naturally occurring variants of CD20, e.g., splice variants or allelic variants.

A wide range of anti-CD20 antibodies have been reported and/or developed through clinical trials, including for example rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921 , ublituximab, HI47 lgG3 (ECACC, hybridoma), 2C6 lgG1 (as disclosed in WO 2005/103081), 2F2 lgG1 (as disclosed in WO 2004/035607 and WO 2005/103081) and 2H7 lgG1 (as disclosed in WO 2004/056312).

Amino acid sequences of an exemplary VH and VL pairs that form and ABD that binds to CD20 are shown below, with reference to the TOE or antibody that contains such ABD:

GA101 :

VH:

QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPG DGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS (SEQ ID NO: 97)

VL:

DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMS NLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK (SEQ ID NO: 98)

Ofatumumab:

VH:

EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIG YADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVT VSS (SEQ ID NO: 99)

VL:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK (SEQ ID NO: 100)

Ocrelizumab: VH:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTS YNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVT VSS (SEQ ID NO: 101)

VL:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIK (SEQ ID NO: 102)

Epcoritamab:

VH:

EVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMHWVRQAPGKGLEWVSTISWNSGTIG YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVT VSS (SEQ ID NO: 103)

VL:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK (SEQ ID NO: 104)

Plamotamab:

VH:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGNGDT SYNQKFQGRVTITADKSISTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGAGTLVT VSS (SEQ ID NO: 105)

VL:

QIVLTQSPSSLSASVGDRVTITCRASSSVSYIHWFQQKPGKSPKPLIYATSNLASGVPVRFS GSGSGTDYTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 106)

Mosunetuzumab:

VH:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTS YNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVT VSS (SEQ ID NO: 107)

VL:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIK (SEQ ID NO: 108)

In one embodiment, an antigen binding domain that specifically binds to CD20 comprises a heavy chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region sequence of the CD20- binding domain of any of the TCEs referred to herein, and a light chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region sequence of the CD20-binding domain of any of the TCEs referred to herein. In one embodiment, TCE comprises at least one antigen binding domain that specifically binds to CD20, comprising a heavy chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region sequence of the CD20-binding domain of any of the TCEs referred to herein, and a light chain variable region sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the light chain variable region sequence of the CD20-binding domain of any of the TCEs referred to herein. In any embodiment, an antigen binding domain can be a function-conservative variant of any of said known CD20 binding domains.

Administration of combination therapy

In the treatment methods herein, the TCE and the CD39-neutralizing agent can be administered separately, together or sequentially, or in a cocktail. In some embodiments, the CD39-neutralizing agent is administered prior to the administration of the TCE. For example, the CD39-neutralizing agent can be administered approximately 0 to 30 days prior to the administration of the TCE. In some embodiments, a CD39-neutralizing agent is administered from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day, or from about 1 to 5 days prior to the administration of the TCE. In some embodiments, a CD39-neutralizing agent is administered concurrently with the administration of the TCE. In some embodiments, a CD39-neutralizing agent is administered after the administration of the TCE. For example, a CD39-neutralizing agent can be administered approximately 0 to 30 days after the administration of the TCE. In some embodiments, a CD39-neutralizing agent is administered from about 30 minutes to about 2 weeks, from about 30 minutes to about 1 week, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day, or from about 1 to 5 days after the administration of the TCE.

In some embodiments the TCE will be administered according to its approved dosing regimen. For example, TCEs can be advantageously administered at a dose of 1 pg/kg to 10 mg/kg body weight, optionally 1 pg/kg to 1 mg/kg body weight, optionally 0.1 - 1 mg/kg body weight, optionally 0.1 - 2 mg/kg body weight, optionally 0.1 - 3 mg/kg body weight, optionally 0.1 - 4 mg/kg body weight, optionally 0.1 - 5 mg/kg body weight, or optionally at a flat dose of between 10 mg and 200 mg, optionally between 30 mg and 160 mg, optionally between 30 mg and 60 mg, optionally 30 mg, 60 mg, 48 mg, 80 mg or 160 mg. The administration can be specified to be following a step-up dosing protocol during which two or more successive administrations occur at doses that are lower than the full dose. The TCE can be advantageously administered 1-4 times per month, preferably 1-2 times per month. Optionally, administration is by intravenous infusion of subcutaneous administration.

Optionally, in some embodiments the TCE protein will be administered according to a reduced dose regimen, in which the amount administered (e.g. per dose, for one or more doses, or all doses) and/or frequency of administration is reduced compared to its approved dosing regimen.

The CD39-neutralizing agent can be specified as being administered at a dose and frequency suitable to substantially neutralize the enzymatic activity of CD39. An anti-CD39 antibody can for example be administered in an amount effective to achieve, and/or to maintain between two successive administrations of the anti-CD39 antibody, a concentration in blood (serum) or an extravascular tissue (e.g., lymph node or extranodal tumor environment) that corresponds to at least the EC50 (e.g., an EC50 between 0.01 and 0.5 pg/ml), optionally the EC70 or optionally the EC100, for neutralization of the enzymatic activity of CD39 (e.g., an EC100 between 0.05 and 1 pg/ml, between 0.1 and 1 pg/ml) . The antibody can for example be administered in an amount to achieve and/or maintained a concentration in circulation or in an extravascular tissue (e.g., tumor environment) of at least about 0.1 pg/ml, 0.5 pg/ml, 1 pg/ml or 2 pg/ml). For example, to achieve a concentration in an extravascular tissue of between 0.05 and 1 pg/ml, or between 0.1 and 1 pg/ml, the anti-CD39 antibody is administered in amounts effective to achieve a concentration in circulation of the anti-CD39 antibody of between 0.5 and 10 pg/ml, or between 1 and 10 pg/ml. Optionally, the anti-CD39 antibody is administered at least twice and in amounts effective to maintain the concentration of the anti-CD39 antibody at least the aforementioned concentration for at least 1 week, 2 weeks, 3 weeks, 4 weeks, between two successive administrations of the anti-CD39 antibody and/or throughout the administration cycle.

The ECso, EC70 or the EC100 can be assessed for example in a cellular assay for neutralization of the enzymatic activity of CD39, e.g., neutralization of ATPase activity in B cells by quantifying hydrolysis of ATP to AMP (or ATP to downstream adenosine), see Examples, Methods. "EC50" with respect to neutralization of the enzymatic activity of CD39, refers to the efficient concentration of anti-CD39 antibody which produces 50% of its maximum response or effect with respect to neutralization of the enzymatic activity. "EC70" with respect to neutralization of the enzymatic activity of CD39, refers to the efficient concentration of anti- CD39 antibody which produces 70% of its maximum response or effect. "EC100" with respect to neutralization of the enzymatic activity of CD39, refers to the efficient concentration of anti- CD39 antibody which produces its substantially maximum response or effect with respect to such neutralization of the enzymatic activity. In one embodiment, the amount of anti-CD39 antibody is between 1 and 40 mg/kg body weight, optionally between 10 and 40 mg/kg body weight optionally between 10 and 20 mg/kg body weight, optionally a fixed dose of between of between 100 mg and 3000 mg, optionally between 750 mg and 3000 mg, optionally between 1000 mg and 3000 mg, optionally 2250 or 3000 mg. The anti-CD39 antibody can be advantageously administered 1-4 times per month, preferably 1-2 times per month. Optionally, administration is by intravenous infusion of subcutaneous administration.

Thus, when the anti-CD39 antibody and the TCE are used in combination, a treatment can thus comprise:

(a) administering one or more administrations of anti-CD39 antibody at a dose of between 10 and 40 mg/kg body weight or a fixed dose of between of between 750 mg and 3000 mg; and

(b) administering one or more administrations of TCE at a dose of between optionally 0.1 - 5 mg/kg body weight, optionally at a flat dose of between 10 mg and 200 mg.

It will be appreciated that the anti-CD39 antibody and the TCE can each be incorporated in a pharmaceutical formulation in a suitable concentration (e.g. from 1 mg/ml to 500 mg/ml, wherein said formulation has a pH from 2.0 to 10.0). The anti-CD39 antibody and the TCE can be comprised in the same or separate pharmaceutical formulations. The formulation may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e., formulation comprising water. Such formulation is typically a solution or a suspension. In a further embodiment, the pharmaceutical formulation is an aqueous solution. The term “aqueous formulation” is defined as a formulation comprising at least 50 %w/w water. Likewise, the term “aqueous solution” is defined as a solution comprising at least 50 %w/w water, and the term “aqueous suspension” is defined as a suspension comprising at least 50 %w/w water.

Optionally, the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use. Optionally, the pharmaceutical formulation is a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.

In a further aspect, the pharmaceutical formulation comprises an aqueous solution of such an antibody, and a buffer, wherein the antibody is present in a concentration from 1 mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0. Optionally, the pH of the formulation is in the range selected from the list consisting of from about 2.0 to about 10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, and about 5.5 to about 7.5. In a further embodiment, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.

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

It is possible that other ingredients may be present in the pharmaceutical formulation of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a 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 formulation of the present invention.

Administration of pharmaceutical compositions according to the invention may be through any of several routes of administration, for example, intravenous. Suitable antibody formulations can also be determined by examining experiences with other already developed therapeutic monoclonal antibodies.

In one aspect, provided herein are kits, for example kits which include a pharmaceutical composition containing a CD39-neutralizing agent, and a pharmaceutical composition containing a TCE comprising a first antigen binding domain that binds CD3 and a second antigen binding domain that binds CD20. In one aspect, provided is a kit which includes: a pharmaceutical composition containing an anti-CD39 antibody such as an anti- CD39 antibody comprising a VH and VL having respectively the amino acid sequences of the heavy and light chains CDRs as disclosed herein, and a pharmaceutical composition containing a TCE comprising a first antigen binding domain that binds CD3 comprising a heavy chain variable region (VHCDS) and a light chain variable region (VLCDS), and a second antigen binding domain that binds CD20 comprising a heavy chain variable region (VHCD2O) and a light chain variable region (VLCD2O).

A pharmaceutical composition may optionally be specified as comprising a pharmaceutically-acceptable carrier. The kits optionally also can include instructions, e.g., comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein to a patient having cancer (e.g., a B- NHL). In any embodiment, a kit optionally can include instructions to administer said anti-CD39 antibody simultaneously, separately, or sequentially with said TCE. The instructions can optionally further specify administration of said anti-CD39 antibody and said TCE antibody simultaneously, separately, or sequentially). The kit also can include a syringe.

A kit can be specified as comprising one or more containers (e.g. single use vials or pre-filled syringes) comprising the specified pharmaceutical composition or antibody.

A specified amount or dose (e.g. disclosed herein) can be specified as being provided in a plurality of vials.

Optionally, the kits include multiple packages of the single-dose pharmaceutical compositions each containing an effective amount of the anti-CD39 antibody, and/or the TCE, for a single administration in accordance with the methods provided above. Instruments or devices necessary for administering the pharmaceutical composition(s) also may be included in the kits. For instance, a kit may provide one or more pre- filled syringes containing an amount of the anti-CD39 antibody or TCE.

EXAMPLES

Example 1 : Blocking the CD39/CD73 pathway synergistically augments response to anti-CD20/anti-CD3 TCE (BsAb)

T cell engagers (TCEs) are designed to overcome some of the challenges associated with targeting tumor-associated antigens (TAAs) by linking two antibody fragments that recognize distinct epitopes on the TAAs and on the T cell surface. By bridging the T cell and tumor cell, TCEs trigger a cascade of events that lead to T cell activation and subsequent tumor cell lysis, and TCEs have shown strong anti-tumor activity in human clinical trials in B- NHL. However, there is still a sizeable portion of patients with B-NHL that will relapse or who are refractory or will not be eligible for treatment with TCEs. One possibility is that T-cell exhaustion and an immunosuppressive microenvironment may contribute to treatment failure and early relapses in a subset of patients receiving TCEs.

We sought to investigate whether, despite the already potent T cell activation mediated by TCEs, blocking the CD39/CD73 pathway would enhance the anti-tumor activity of anti-CD20 TCEs in B-NHL. We used a flow cytometry-based ex vivo model of bispecific antibody (BsAb) treatment described in Roider, T., et al., An autologous culture model of nodal B-cell lymphoma identifies ex vivo determinants of response to bispecific antibodies. Blood Adv, 2021. 5(23): p. 5060-5071. An overview of the approach used is shown in Figure 1A. Materials and Methods

Flow cytometric ex vivo assays

Cryopreserved lymph node cell suspensions were thawed at 37°C using a water bath and resuspended in RPMI 1640 cultural medium supplemented with 10% heat- inactivated human AB serum (Sigma Aldrich, St. Louis, MO). To remove cell debris, the cell suspension was kept on a rotating mixer for 3 h. Cells were strained using a 70 pm strainer. Unsorted cells were plated at 200,000 cells/well in a 96-well flat bottom plate containing 200 pl cell medium per well. The following antibodies were added: CD20xCD3 BsAb (BSFV-H226, Creative Biolabs, Shirley, NY) at 10 ng/ml (C1), 100 ng/ml (C2), 1.000 ng/ml (C3), or 10.000 ng/ml (C4). The monoclonal anti-CD39 (heavy and light chain amino acid sequences of SEQ ID NOS : 10 and 11 , respectively) and anti-CD73 antibodies were added at 1 pg/ml or 10 pg/ml. Cells were cultured at 37°C and 5% CO2 for 7 days. Afterwards, cells were washed and stained for flow cytometric analysis.. Samples were analysed using a FACSSymphony™ A3 (BD Biosciences, Heidelberg, Germany) and BD FACSDiva™ software (BD Biosciences)

Data analysis

Flow cytometric data was analysed with FlowJo™ (BD Biosciences). Count Beads (Biolegend, San Diego, CA) were used to quantify absolute cell numbers. The benefit from combination treatments was quantified by the difference in normalized number of B-cells between monotherapy and combination therapy. Therefore, lower/negative numbers indicate stronger benefit from combinations.

Descriptive statistics were performed using R version 4.3.1 (R Foundation for Statistical Computing, Vienna, Austria). Correlation between continuous variables was calculated using Spearman’s rank correlation. Means of independent variables were compared using the unpaired Wilcoxon signed-rank test. P values were adjusted for multiple testing using the Benjamini-Hochberg method.

Cytokine assays

Supernatants from lymph node assays were analyzed. Granzyme B, perforin, IFN-y, IL-10 and TNF-a levels were measured with a LEGENDplex™ assay kit as per manufacturer’s protocol. Flow cytometric analysis was performed by using a LSRFortessa™ (BD Biosciences) and the LEGENDplex™ Data Analysis Software v8.0 (BioLegend).

ATP assay

The effect of CD39 blockade on ATP levels in lymph node cell cultures was analysed using the CellTiterGlo™ assay (Promega, Walldorf, Germany), as previously described in Perrot et al. 2019 Cell Rep. 27(8): 2411-2425. Lymph node samples were processed as described above and incubated with anti-CD39 antibody at 10 pg/ml for 60 minutes. Next, ATP was added at a concentration of 20 pM. After 60 minutes, supernatants were analysed using an EnSight™ multimode plate reader (PerkinElmer, Waltham, MA). Inhibition of enzymatic activity was calculated as follows: anti — CD39: 100

Results

Using a flow cytometry- based ex vivo model of TCE treatment, we quantified responses to treatment with a CD20- and CD3-binding TCE in 27 primary lymph node samples from patients diagnosed with follicular (FL) or diffuse large B-cell lymphoma (DLBCL). The CD20- and CD3-binding TCE is also referred to in these Examples interchangeably as BsAb (bispecific antibody) or anti-CD20 BsAb. As most patient samples are included in reference data sets of B-NHL, transcriptomic and epitope profiles at the single-cell level were available. Consistent with previous data, anti-CD20 TCE (TCE that binds CD20 and CD3, also referred to as BsAb) successfully induced concentration-dependent killing of B-cells with a median reduction of 73.3 % (Figure 1 B) and a median T cell expansion of 13.6-fold at the highest concentration (Figure 1C). Anti-CD39 or anti-CD73 blocking antibodies at two different concentrations (1 pg/ml, 10 pg/ml) were combined with anti-CD20 BsAb at 0.1 pg/ml. Blockade of CD39, CD73 or both significantly enhanced BsAb-induced lysis of B-cells (Figure 1 D). Likewise, combination treatments dramatically augmented T-cell expansion in all tested conditions compared to single agent treatment (Figure 1 E). Using anti-CD39 blockade as representative example, we observed significantly higher levels of Granzyme B, perforin, IL- 10, IFN-y, and TNF-a compared to BsAb alone (Figure 1 F). Next, we incubated primary lymph node samples with anti-CD39 blocking antibody at a concentration of 1 pg/ml or 10 pg/ml and ATP at 20 pM. Indeed, we found that the addition of anti-CD39 antibody significantly increases ATP levels (Figure 1G).

Results are shown in Figure 1 B-F. Lymph node-derived lymphocytes were incubated with or without (w/o) a maximum of four concentrations of a CD20-BsAb (C1-C4) and/or a maximum of two concentrations of anti-CD39, anti-CD73, anti-CD39/CD73 blocking antibodies, as indicated. After seven days, cells (B-E) or supernatants (F) were analyzed by quantitative flow cytometry or a bead-based immunoassay, respectively. Shown are the percentages based on the absolute numbers of viable B cells normalized to w/o (B, D), the x- fold expansion based on the absolute numbers of viable T cells normalized to w/o (C, E) or the cytokine levels in pg/pl (F) in n = 27 biologically independent samples. G) Lymph node- derived lymphocytes were incubated with or without the anti-CD39 blocking antibody at two different concentrations and then exposed to ATP at a concentration of 20 pM. Shown are ATP levels of n = 4 biologically independent samples normalized to untreated control (w/o) after 60 minutes. BG) P values were calculated between w/o and every other condition using the two-sided Wilcoxon’s test and corrected for multiple testing using the Benjamini-Hochberg procedure. Only p values < 0.05 are shown.

Example 2: Identification of indicators for the efficacy of treatment involving anti-CD39 or anti-CD73 antibodies by single cell sequencing

To identify potential indicators for the efficacy of combined treatment with TCEs and anti-CD39 or anti-CD73, we quantified the extent to which individual samples benefited from combinations compared to TCE monotherapy.

We harnessed existing single-cell data associated with the samples used in the ex- vivo tumor model of Example 1 to test the combination of TCE and CD39/CD73 blockade, and sought to identify potential indicators to identify settings where the combination treatment would have the highest efficacy. The materials and methods are as described in Example 1 ; the overview of the approach used is shown in Figure 1A.

Results are shown in Figure 2. Abbreviations: Tp_r: Proliferating T-cells; TH: Helper T- cells; TFH: Follicular helper T-cells; TREG: Regulatory T-cells; TTOX: Cytotoxic T-cells; TDN: Double negative T-cells; CM: Central memory; EM: Effector memory; LN: Lymph node; TF: Transcription factor. Figure 2A-B shows Box plots illustrating the associations of patient characteristics with benefit from three different combination treatments. P values were calculated using the Wilcoxon-test. Figure 2C shows the expression of CD39 and CD73 at protein level based on publicly available single-cell data of in n = 51 biologically independent lymph node samples. Figure 2D shows an overview of fourteen T-cell subsets defined by existing single-cell RNA and epitope sequencing in n = 51 biologically independent lymph node samples. Figure 2E shows the expression of CD39 and CD73 at protein level based on previously mentioned T-cell reference data. Figure 2F shows the proportion of fourteen specific T-cell subsets, defined in panel A (y axis), was correlated with the benefit from three different combination therapies (x axis) compared to BsAb monotherapy. Only p values < 0.05 are shown. Figure 2G shows the protein expression of six different exhaustion markers (x axis) across three effector memory T-cells, defined in panel A, was correlated with the benefit when combining anti-CD39 blocking antibody with anti-CD20 BsAb compared to BsAb monotherapy. Only values < 0.05 are shown.

Results showed that the benefit from adding anti-CD39, anti-CD73, or both to anti- CD20 BsAb was higher in DLBCL (Figure 2A) and patients at initial diagnosis (Figure 2B) compared to FL and patients at relapse, respectively. Moreover, we used single-cell data of these samples to examine whether baseline data of T- and B-cells are associated with ex vivo response to combination treatment. In non-malignant B-cells, we detected both CD39 and CD73, while malignant B-cells spanning all investigated lymphoma entities exhibited a notably lower expression (Figure 2C). Across 14 multimodally defined T-cell subsets described in Roider et al. 2022 BioRxiv p.2022.11.04.514366, we found the highest levels of CD39 in Treg subsets and exhausted T-cells (Figure 2D). Likewise, we observed CD73 to be present in T- cells at moderate, but - in contrast to CD39 - relatively consistent levels across all cell types (Figure 2C, E). Merging single-cell data with ex vivo responses to CD39/CD73 blocking antibodies revealed that a higher proportion of exhausted T-cells (alias PD1+ TIM3+ TTOX effector memory cells), as previously defined at single-cell level (Roider et al. 2022 BioRxiv p.2022.11.04.514366), correlated with greater benefits of the anti-CD20 BsAb I anti-CD39 combination treatment (Figure 2F). No such association was observed with other T-cell subsets or when using anti-CD73 blocking antibody (Figure 2F). In B-NHL, T-cell exhaustion is considered to contribute to treatment failure and early relapses in a subset of patients receiving BsAb (Philipp et al. 2022 Blood 140(10): 1104-1118). Therefore, these patients could benefit the most from targeting CD39 and/or CD73. Furthermore, we examined the expression of various exhaustion markers, namely PD1 , TIM3, LAG3, CD43, TIGIT, and CD39 in lymph node-derived effector memory T-cells. Interestingly, we noted increased levels of CD39 in effector T-cells, including those displaying an exhaustion phenotype, were linked to pronounced benefits from the combination treatment of anti-CD39 and BsAb (Figure 2G). In contrast, higher levels of PD1 were associated with an inferior ex vivo response (Figure 2G).

Clinical studies investigating blocking antibodies against CD39 and CD73 are limited to advanced stages of solid cancers. However, our findings identify their application in B-NHL in combination with BsAb.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar references are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate). The description herein of any aspect or embodiment herein using terms such as

“comprising”, “having,” “including,” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or embodiment herein that “consists of”, “consists essentially of’, or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Claims

1. An agent that neutralizes CD39, for use in the treatment of a B-NHL, wherein the agent is used in combination with a T-cell engager (TCE) comprising an antigen binding domain (ABD) that binds human CD20 and an ABD that binds human CD3.

2. A method of treating a CD20+ B cell proliferative disorder and/or of eliminating unwanted CD20+ cells in a patient in need thereof, the method comprising administering to the patient a treatment regimen comprising: (a) an agent that neutralizes CD39, and (b) a TCE comprising an antigen binding domain (ABD) that binds human CD20 and an ABD that binds human CD3.

3. The agent or method of claims 1 or 2, wherein the disease is a CD20+ B cell proliferative disorder.

4. The agent or method of any one of claims 1-3, wherein the disease is a DLBCL.

5. The agent or method of any one of claims 1-4, wherein the patient has not received prior systemic treatment for the disease.

6. The agent or method of any one of the above claims, wherein the patient has exhausted T cells, as determined by assessing expression of markers of exhaustion on T cells, optionally wherein the marker is CD39.

7. The agent or method of any one of the above claims, wherein the treatment comprises administering to the patient (a) a TCE and (b) an antibody that neutralizes CD39.

8. The agent or method of any one of the above claims, wherein the TCE is selected from the group consisting of: plamotamab, FBTA05, mosunetuzumab, odronextamab, glofitamab and epcoritamab.

9. The agent or method of any one of the above claims, wherein the agent that neutralizes CD39 is an antibody that specifically binds human CD39.

10. A method of decreasing the likelihood of cancer relapse or recurrence in a patient receiving treatment with a TCE, the method comprising administering to the patient an agent that neutralizes CD39, wherein the agent that neutralizes CD39 is administered as a combination therapy with the TCE.

11. A method for the treatment or prevention of a CD20+ B cell proliferative disorder in an individual in need thereof, the method comprising: (a) detecting whether the individual has exhausted T cells, as determined by assessing expression of one or more markers of T cell exhaustion, and (b) upon a determination the individual has exhausted T cells, administering to the individual a TCE and an agent that neutralizes CD39.

12. The method of claim 11 , wherein detecting whether the individual has exhausted T cells comprises assessing expression of CD39 by T cells.

13. The method of claims 11-12, wherein the disorder is a B-NHL or a DLBCL.

14. The method of claims 11-13, wherein the patient has not received prior systemic treatment for the cancer.

15. A kit for treating a CD20+ B cell proliferative disorder in a human patient, the kit comprising:

(a) one or more vials comprising an anti-CD39 antibody; and

(b) one or more vials comprising a TCE; and

(c) optionally, instructions for using said anti-CD39 antibody and/or TCE.

16. The kit of claim 15, wherein the disorder is a B-NHL or a DLBCL, optionally wherein the instructions specify that the patient has not received prior systemic treatment for the cancer.

17. The method, agent or kit of any one of the above claims, wherein the TCE and the CD39-neutralizing agent are formulated for separate administration and are administered concurrently or sequentially.

18. The method, agent or kit of any one of the above claims, wherein tthe anti- CD39 antibody comprises (a) a HCDR1 , HCDR2 and HCDR3 of SEQ ID NOS: 2, 3 and 4, respectively, and (b) an LCDR1 , LCDR2 and LCDR3 sequence of SEQ ID NOS: 5, 6 and 7, respectively.

19. An agent that neutralizes CD39, for use in the treatment of a B-NHL, wherein the agent is used in combination with a T-cell engager (TCE) comprising (a) an ABD that binds a protein present at the surface of a malignant B cell and (b) an ABD that binds human CD3.