ANTI-CD39 ANTIBODIES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. US 62/404,782 filed 6 October 2016; 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 "CD39-5_ST25", created 28 September 2017, which is 43 KB in size. 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 antigen-binding compounds (e.g., antibodies) that inhibit CD39. The invention also relates to cells producing such compounds; methods of making such compounds, and antibodies, fragments, variants, and derivatives thereof; pharmaceutical compositions comprising the same; methods of using the compounds to diagnose, treat or prevent diseases, e.g., cancer.

BACKGROUND

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. Antibodies that bind and inhibit CD39 antibodies are disclosed in WO2009/095478. Hayes et al. (2015) Am. J. Transl. Res. 7(6):1 181 -1 188 makes use of an anti-CD39 that binds FcyR and has effector function but it stated to also be blocking.

Consequently, CD39 expression on different cell types, including immune cells and tumor cells, combined with use of antibodies that either do not actually block CD39 or are not pure blockers, create a complex setting for evaluation of the underlying activity of antibodies. Blocking enzymatic active sites using protein agents such as antibodies has generally known to be difficult. There is therefore a need to understand whether and how antibodies can inhibit the ATPase activity of CD39, and how to design improved molecules.

SUMMARY OF THE INVENTION

The present invention arises from the solution of the co-crystal structure of the glycosylated CD39 protein with antibodies that potently inhibit the enzymatic (ATPase activity) activity of cell surface CD39, thereby identifying key structural features underlying the mechanism of action of the antibodies and in turn permitting the design of new V _L and V _H domains and modification and/or improvement of existing V _L and V _H domains.

The inventors found that antibodies that potently inhibit the enzymatic (ATPase activity) activity of the CD39 enzyme appear to do so by immobilizing the enzyme in one of its conformations thereby preventing it from hydrolyzing its substrate. The antibodies achieve this via a V _H A _L pair capable of binding to both C- and N-terminal domains of CD39 at the same time. The antibodies bind CD39 in its form "A", also referred to as the open conformation of CD39. The antibodies can thus be considered to be capable of acting as allosteric inhibitors, e.g. they inhibit the activity of the human CD39 polypeptide without a requirement for binding to the enzymatic active site of the polypeptide.

The inventors observed that the positioning of the V _H CDR3 for binding to CD39 occurs via a surprising mechanism involving a V _L CDR / V _H CDR3 / CD39 binding matrix in which VH CDR3 is trapped between the VL CDRs and CD39. The V _L CDRs form a paratope that binds to the CDR3 of the V _H . The V _H CDR3 comprises numerous aromatic residues, and while one of the aromatic residues in V _H CDR3 interacts with CD39, other aromatic amino acids within V _H CDR3 interact with the residues of the V _L . The V _H CDR3 comprises multiple aromatic residues which form pi-interactions with the V _L on one face, and CD39 on another face, resulting in a matrix of pi-interactions structure that traps the V _H CDR3 between the V _L CDRs and CD39 and permits a large binding area across the surface of CD39. Further surprisingly, the structure appears to have arisen in the context of certain framework residues, as both the V _H CD39 interaction and the V _H CDR3-V _L interaction involve framework residues (according to Kabat numbering), such that the overall contact residues involved a limited set of Kabat CDR residues combined with Kabat FR residues.

The numerous aromatic residues of the CDR3 however increase hydrophobicity of the antibody, causing a reduction in stability in solution. Solution of the co-crystal structure of the glycosylated CD39 protein together with antibodies permitted the design of antibodies with modified CDRs and/or FR. Among the different modifications, the number of aromatic residues in V _H CDR3 was decreased, resulting in a reduction of the hydrophobicity of the antibody.

Interestingly, the antibodies can bind to both C- and N-terminal domains of CD39 via their V _H CDRs (there is no need for the V _L CDRs to bind CD39 at all). Binding to the N- terminal domain (domain 1 ) of CD39 is achieved by two of the CDRs (CDR1 and CDR3, as well as a first segment of Kabat CDR2, while binding to the C-terminal domain (domain 2) is achieved through a single CDR (CDR2) in combination with an extended segment of FR3. Not only is the contribution of CDR2 surprising, but additionally the CDR2 cooperates with FR3 in binding to CD39. Furthermore, the binding by CDR2-FR to CD39 involves the glycan at N292 of CD39 (the N292 glycan covers a significant amount of the apical surface of the C- terminal domain). The resulting binding by the V _H to both domains 1 and 2 is believed to be important in immobilizing the CD39 enzyme so as to prevent substrate hydrolysis. The antibodies thus have a V _H CDR2 that binds to the N-terminal domain of CD39 via a first portion of the CDR2 and to the C-terminal domain of CD39 (including the glycan at N292) via a second portion of the CDR2, in combination with FR3 residues.

The overall structure permits the V _H to bind N-terminal domain of CD39 (e.g., at residues V95, Q96, K97, L137, E140, L144 of CD39, with reference to the CD39 amino acid sequence of SEQ ID NO: 1 ) on one side of the enzymatic active site, and the C-terminal domain of CD39 (e.g., at residues S294, K298, as well as to the glycan linked to N292 of CD39, of CD39) on the opposite side of the enzymatic active site.

These findings permit a wide range of CD39-binding polypeptides to be generated that retain the mechanism of action and permits desired amino acid sequences and features to be incorporated. Antibodies and other V _H A _L -containing proteins can be designed which include, inter alia, the V _H CDR2 segment that binds the N-terminal domain, the V _H CDR2-FR3 segment capable of binding the C-terminal domain (and, notably, the N292-linked glycosylation) and the V _H CDR3 that binds the C-terminal domain and gives rise to the V|_CDR - V _H CDR3 - CD39 matrix. Amino acid sequences of V _L domains can be selected in the context of the desired V _H sequence, e.g., by introducing substitutions that maintain the overall V _H or V _L domain structure without affecting V domain interactions, and where V domain interactions would be modified, making amino acid substitutions pair-wise in both V _H and V|_ at the respective contact positions. The resulting protein binds to CD39 essentially or exclusively via the V _H domain(s), and the V _L domain(s) binds to V _H CDR3 but is not involved in binding to CD39. A wide range of both V _L CDR amino acid sequences can be employed in such protein; optionally V _L CDR residues and the respective V _H residues can be chosen and substituted as pairs such that the V _H A _L CDR contact is maintained. Similarly, a wide range of human (or non-human) V _H and V _L framework sequences can be selected as acceptor frameworks that bear resides at the specified positions that maintain the V _H CDR-V _L CDR contact and the V _H CDR-CD39 contact.

Consequently, in one embodiment, provided is an anti-CD39 antigen binding domain, or an antigen-binding protein that comprises the antigen binding domain (e.g., an antibody or antibody fragment, a protein that comprises a single anti-CD39 VHA L pair, a multispecific binding protein (that comprises, e.g. one or two anti-CD39 VHA/L pair(s), a bispecific antibody, etc.), comprising complementary determining regions (CDR) and framework regions (FR). The antigen binding domains can be designed or modified so as to provide desired and/or improved properties.

In one embodiment, provided is an anti-CD39 antigen-binding protein is capable of binding to and inhibiting the activity of a human CD39 polypeptide, the antigen-binding protein comprising a VH and a VL that each comprise a framework (e.g. a framework having an amino acid sequence of human origin) and a CDR1 , CDR2 and CDR3, wherein the antigen-binding protein is capable of binding to the N-terminal domain of CD39 and the C- terminal domain of CD39. In one embodiment, the antigen-binding protein restricts the domain movement of CD39 when bound to CD39. In one embodiment, the antigen binding protein is an allosteric inhibitor of CD39. In one embodiment, the antigen binding protein neutralizes the ATPase activity of CD39 without inhibiting the enzymatic active site. In one embodiment, the antigen binding protein binds to the "A" or "open" conformation of CD39. Optionally, the VH and/or VL framework (e.g. FR1 , FR2, FR3 and/or FR4) is of human origin. In one embodiment, the V _H comprises a first CDR (or antigen binding domain) that is capable of binding to the N-terminal domain of CD39 and a second CDR (or antigen binding domain) that that is capable of binding to amino acid residues of the C-terminal domain of CD39.

In one aspect of the invention, provided is an anti-CD39 binding molecule or antigen- binding fragment thereof capable of binding to and inhibiting the activity of CD39, comprising a V _H and a V _L , wherein the molecule or fragment differs in its CDRs and/or frameworks from a reference anti-CD39 antibody and has desirable pharmacological properties, e.g. compared to known anti-CD39 binding molecules or antibodies, or compared to a reference antibody or to a parental antibody that has been modified (e.g. a murine parental antibody, a humanized antibody not having the improved pharmacological properties). In one embodiment, the V _H comprise an amino acid modification in a Kabat CDR1 , CDR2 and/or CDR3. In one embodiment, the V _L comprises an amino acid modification in a Kabat CDR1 , CDR2 and/or CDR3.

Exemplary complementarity-determining region (CDR) residues and/or sites for amino acid substitutions in framework region (FR) and/or of such V _H and V _L to produce antibodies and/or other VH- and VL-containing proteins having improved properties such as, e.g., lower immunogenicity, improved antigen-binding (e.g. greater binding affinity for CD39) or other functional properties, and/or improved physicochemical properties such as, e.g., better stability, are provided.

In one aspect of the invention (e.g. in any aspect herein), provided is a binding molecule or antigen-binding fragment thereof capable of binding to and inhibiting the activity of CD39, comprising a V _H and a V _L , wherein the Kabat CDR1 of the V _H binds to amino acid residues in the N-terminal domain of CD39, the Kabat CDR2 (optionally together with the FR3) of the V _H binds to amino acid residues and/or to the N292-linked glycan in the C- terminal domain of CD39, and the Kabat CDR3 of the V _H binds to amino acid residues the N- terminal domain of CD39. Optionally the CDR2 of the V _H comprises a first amino acid segment that binds to the N-terminal domain of CD39 together and an amino acid segment that binds, together with FR3 residues, to the C-terminal domain of CD39. Optionally, the CDR3 comprises an aromatic residue (e.g. a tyrosine) that is capable of binding an amino acid residue in the N-terminal domain of CD39 and a second aromatic amino acid residue (e.g., a tyrosine, a phenylalanine) that is capable of contacting an amino acid residue in the V|_. Optionally, the binding molecule or antigen-binding fragment comprises a V _L that binds, via a residue in a Kabat CDR, to the Kabat CDR3 of the V _H .

In one embodiment, the CDR1 comprises a residue that is capable of contacting amino acid residues in the N-terminal domain of CD39. Optionally a CD39 contact residue is at Kabat position 33, optionally at both positions 31 and 33. In one embodiment, the Kabat FR1 comprise a CD39 contact residue at Kabat position 30, optionally wherein the residue is a threonine.

In one embodiment, the CDR2 comprises a segment of residues in Kabat positions 50-56, optionally residues 50, 52, 52a, 53 and/or 56, that is capable of contacting amino acid residues in CD39 (e.g. in the N-terminal domain of CD39), optionally wherein the residue at position 53 aromatic is an amino acid residue. In one embodiment, the CDR2 comprises residues 50, 52, 52a, 53 and/or 56, that are capable of contacting amino acid residues in CD39. In one embodiment, the CDR2 comprises residues at Kabat position 50-56 having the formula Xi X ₂ X3 X ₄ X5 Χβ X7 Xs, wherein Xi represents tryptophan, X ₂ represents any amino acid, optionally an isoleucine, X ₃ represents asparagine or optionally glutamine, X ₄ represents threonine, X ₅ represents tyrosine or optionally phenylalanine, or optionally a non- aromatic residue, optionally glutamic acid, X ₆ represents any amino acid, optionally threonine, optionally serine, optionally asparagine, alanine or glycine, optionally a residue other than a large or hydrophobic residue, X ₇ represents any amino acid, optionally glycine, optionally alanine, serine, threonine, asparagine or glutamine, optionally a residue other than aspartic acid or glutamic acid, optionally a residue other than lysine or arginine, X ₈ represents glutamic acid, optionally aspartic acid. In one embodiment, any one or more of Xi X2 X ₃ X4 X ₅ X ₆ X ₇ or X ₈ are substituted by an amino acid residue as set out in Example 2 as a possible substitution at the particular Kabat position corresponding to the position in the formula for the particular CDR or FR.

In one embodiment, the CDR2 and FR3 comprise a segment of residues within Kabat positions 59-71 , optionally within 59-72b, that is capable of contacting amino acid residues in the C-terminal domain of CD39, optionally further wherein residues within Kabat positions 59-71 contact the glycan at residue N292 of CD39. For example, the Kabat CDR2 and FR3 can comprise residues at Kabat positions 59, 65, 67, 68, 69, 70 and/or 71 , and optionally further at residue 72, 72a and/or 72b that are capable of contacting the C-terminal domain of CD39, e.g. including amino acid resides in CD39 and the glycan at N292 of the CD39 polypeptide.

In one embodiment, the CDR2 (e.g., Kabat CDR2-FR3 segment) comprises residues at Kabat position 59-71 having the formula Xi X ₂ X3 X ₄ X5 Χβ X7 Xs X9 X10 X11 X12 X13, wherein Xi represents a tyrosine, each of X ₂ , X3, X ₄ , X5 and X ₆ each represent any amino acid, X ₇ represents glycine or another residue which does not introduce steric hindrance that reduces antigen binding, X ₈ represents any amino acid, X ₉ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity, X ₁₀ represents alanine or valine, or optionally leucine, optionally a hydrophobic residue, Xn represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity and X12 represents serine, optionally further wherein and X13 represents any amino acid, optionally leucine, optionally alanine, valine or threonine. In one embodiment, the V _H FR3 residues at Kabat positions 72, 72a and 72b have the formula Xi X ₂ X3 , wherein Xi represents aspartic acid, glutamic acid or alanine, X ₂ represents any amino acid, optionally alanine or threonine, or a conservative substitution thereof, and X ₃ represents serine, optionally alanine, or a conservative substitution thereof. In one embodiment, the V _H comprises a leucine residue at Kabat position 71 (FR3). Human or humanized antibodies can advantageously have the FR3 signature sequence FVFSL at Kabat positions 67-71 , present in certain human V _H FR domains. Thus in one aspect of any embodiment herein, the segment of residues at Kabat positions 67-71 comprises the amino acid sequence: FVFSL.

In another embodiment, a V _H comprises a Kabat CDR2-FR3 segment that binds to both the N- and C- terminal domain of CD39 (e.g., across the N- and C- domain surface, across the substrate cleft, groove entry or active site) the CDR2-FR3 segment comprising residues (e.g. at Kabat position 50-71 ) having the formula Xi X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 Xi3 Xi4 Xi5 X16 Xi7 X18 Xi9 X20 X21 X22 X23, wherein represents tryptophan, X ₂ represents any amino acid, optionally an isoleucine, X ₃ represents asparagine or optionally glutamine, X ₄ represents threonine, X ₅ represents tyrosine or optionally phenylalanine, or optionally a non-aromatic residue, optionally glutamic acid, X ₆ represents any amino acid, optionally threonine, optionally serine, optionally asparagine, alanine or glycine, optionally residues other than large or hydrophobic resides, X ₇ represents any amino acid, optionally glycine, optionally alanine, serine, threonine, asparagine or glutamine, optionally a residue other than aspartic acid or glutamic acid, optionally a residue other than lysine or arginine, X ₈ represents glutamic acid, optionally aspartic acid, X ₉ represents any amino acid, optionally proline, Xi ₀ represents any amino acid, optionally threonine, optionally serine, asparagine, glutamine, histidine, glutamic acid, aspartic acid, arginine, lysine, alanine or tyrosine, optionally any residue other than a hydrophobic residue or proline, Xn represents a tyrosine, each of X12, X13, Xi4, X15 and Xi ₆ each represent any amino acid, X17 represents glycine or another residue which does not introduce steric hindrance that reduces antigen binding, X ₁₈ represents any amino acid, optionally arginine, X19 represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity, X ₂₀ represents alanine or valine, or optionally leucine, optionally a hydrophobic residue, X21 represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity, X ₂₂ represents serine, and wherein and X ₂₃ represents any amino acid, optionally leucine, optionally alanine, valine or threonine. In one embodiment, the CDR2-FR3 segment further comprises residues at Kabat positions 72, 72a and 72b having the formula X ₂₄ X ₂₅ X ₂₆ , wherein X ₂₄ represents aspartic acid, glutamic acid or alanine, X ₂₅ represents any amino acid, optionally alanine or threonine, or a conservative substitution thereof, and X ₂₆ represents serine, optionally alanine, or a conservative substitution thereof.

In one embodiment, the CDR3 comprises a first aromatic amino acid residue that is capable of contacting an amino acid residue in CD39 and a second aromatic amino acid residue that is capable of contacting an amino acid residue in the V _L , wherein the first and second aromatic residues are at any of Kabat positions 100, 100b, 100c, 100d, 100e and/or 10Of. In one embodiment the Kabat CDR3 (e.g. Kabat positions 100 to 10Of, to the extent residues are present at these positions) comprises a sequence of amino residues having the formula (Xi X ₂ X ₃ X ₄ X5), wherein any three or more of Xi, X ₂ , X ₃ , X ₄ and X ₅ represent an aromatic amino acid.

In one embodiment, provided is an antigen-binding protein capable of binding to and inhibiting the activity of a human CD39 polypeptide, the protein comprising a V _H that binds CD39 and a V _L (e.g., a V _L that binds the CDR3 of the V _H ), wherein the V _H comprises:

(a) a CDR1 capable of contacting the N-terminal domain of CD39, optionally comprising a residue at Kabat position 33, optionally at both positions 31 and 33, that is capable of contacting amino acid residues in CD39;

(b) a CDR2-FR3 domain comprising:

a. a segment comprising amino acid residues capable of contacting the N- terminal domain of CD39, optionally wherein the segment comprises residues within Kabat positions 50-56, optionally wherein the segment comprises one, two, three, four or more of (or all of) the residues at Kabat positions 50, 52, 52a, 53 and/or 56, optionally wherein the residue at position 53 aromatic is an amino acid residue;

b. a segment comprising amino acid residues capable of contacting the C- terminal domain of CD39, optionally wherein the segment comprises residues within Kabat positions 59-71 , optionally wherein the segment comprises one, two, three, four or more of (or all of) the residues at Kabat positions 59, 65, 67, 68, 69, 70 and/or 71 , optionally further the residue at Kabat position 54, optionally further the residues at Kabat positions 72, 72a and/or 72b; and

(c) a CDR3 (e.g. according to Kabat) capable of contacting the N-terminal domain of CD39, optionally capable of contacting the N-terminal domain of CD39 and the V _L , optionally comprising a first aromatic amino acid residue that is capable of contacting an amino acid residue in CD39 and a second aromatic amino acid residue that is capable of contacting an amino acid residue in the V _L , wherein the first and second aromatic residues are at any of Kabat positions 100, 100b, 100c, 100d, 100e and/or 100f (to the extent a residue is present at the particular Kabat position).

Optionally, the CDR3 further comprises a further (third) aromatic amino acid residue, and optionally further a fourth aromatic amino acid residue, wherein the third aromatic residue (and fourth aromatic residue, where present) is capable of contacting an amino acid residue in the V _L , wherein the third aromatic residue (and fourth residue, where present) is at any of Kabat positions 100, 100b, 100c, 100d, 100e and/or 100f.

In one embodiment, provided is an antigen-binding protein capable of binding to and inhibiting the activity of a human CD39 polypeptide, the protein comprising a V _H that binds CD39 and a V _L (e.g., a V _L that binds the CDR3 of the V _H ), wherein the V _H comprises:

(a) a CDR1 (e.g. according to Kabat) capable of contacting the N-terminal domain of CD39, optionally wherein the residues at Kabat position 31 , 32 and 33 have the formula Xi X ₂ X3, wherein X-i represents any amino acid, optionally a histidine or asparagine, or optionally a conservative substitution thereof, X ₂ represents any amino acid, optionally an aromatic residue, optionally a tyrosine or a conservative substitution thereof, or optionally an amino acid residue other than a proline or glycine, and X ₃ represents glycine, or another amino acid that avoids steric hindrance;

(b) a CDR2-FR3 segment (e.g. according to Kabat) capable of contacting the C- terminal domain of CD39, optionally wherein the residues at Kabat position 50-71 having the formula Xi X ₂ X3 X ₄ X5 Χβ X7 Xs X9 X10 X11 X12 X13 Xi ₄ X15 X16 X17 X18 X19 X20 X21 X22 X23, wherein represents tryptophan, X ₂ represents any amino acid, optionally an isoleucine, X ₃ represents asparagine or optionally glutamine, X ₄ represents threonine, X ₅ represents tyrosine or optionally phenylalanine, or optionally a non-aromatic residue, optionally glutamic acid, X ₆ represents any amino acid, optionally threonine, optionally serine, optionally asparagine, alanine or glycine, optionally a residue other than a large or hydrophobic residue, X ₇ represents any amino acid, optionally glycine, optionally alanine, serine, threonine, asparagine or glutamine, optionally residues other than aspartic acid or glutamic acid, optionally a residue other than lysine or arginine, X ₈ represents glutamic acid, optionally aspartic acid, X ₉ represents any amino acid, optionally proline, X10 represents any amino acid, optionally threonine, optionally serine, asparagine, glutamine, histidine, glutamic acid, aspartic acid, arginine, lysine, alanine or tyrosine, optionally any residue other than a hydrophobic residue or proline, Xn represents a tyrosine, each of Xi ₂ , X13, Χι ₄ , X15 and Xi ₆ each represent any amino acid, X-I7 represents glycine or another residue which does not introduce steric hindrance that reduces antigen binding, X ₁₈ represents any amino acid, optionally arginine, X19 represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity, X ₂₀ represents alanine or valine, or optionally leucine, optionally a hydrophobic residue,

X ₂ i represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity and X22 represents serine, optionally further wherein and X23 represents any amino acid, optionally leucine, optionally alanine, valine or threonine, optionally wherein the CDR2-FR3 segment further comprises residues at Kabat positions 72, 72a and 72b having the formula X ₂₄ X25 X26 , wherein X ₂₄ represents aspartic acid, glutamic acid or alanine, X25 represents any amino acid, optionally alanine or threonine, or a conservative substitution thereof, and X ₂ 6 represents serine, optionally alanine, or a conservative substitution thereof; and

(c) a CDR3 (e.g. according to Kabat) capable of contacting the N-terminal of CD39, optionally capable of contacting the N-terminal domain of CD39 and the V _L , optionally wherein the residues at Kabat position 95-102 have the formula Χι X2 X3 X ₄ Xs X6 X7 Χβ Χθ Xio X9 X10 X11 X12 Χ-ΐβ Χ-Η, wherein

Xi represents arginine or lysine, or optionally a conservative substitution thereof,

- X ₂ represents any amino acid, optionally arginine, optionally lysine or alanine, or optionally a conservative substitution thereof,

X ₃ represents any amino acid residue, optionally a residue comprising an aromatic ring, optionally a tyrosine,

X ₄ represents any amino acid, optionally glutamic acid or tyrosine, or optionally a conservative substitution thereof, or an amino acid residue other than proline or glycine,

X ₅ represents glycine, optionally arginine, or optionally a conservative substitution thereof,

X ₆ represents any amino acid, optionally asparagine, serine or tyrosine, or optionally a conservative substitution thereof,

X ₇ represents any amino acid, optionally tyrosine, asparagine or aspartic acid, serine, or optionally a conservative substitution thereof, optionally an amino acid residue other than proline or glycine, optionally a residue other than an aromatic residue (e.g. other than a tyrosine),

- X ₈ represents valine or optionally alanine, isoleucine or leucine, optionally an aromatic amino acid, optionally tyrosine,

X ₉ represents any amino acid, optionally an aromatic amino acid, optionally phenylalanine, optionally tyrosine, optionally valine or a conservative substitution thereof,

- X10 represents tyrosine, optionally phenylalanine, optionally methionine, or optionally a conservative substitution thereof, Xii is absent or represents any amino acid, optionally tyrosine, optionally phenylalanine, optionally tryptophan, or optionally a conservative substitution thereof, optionally an amino acid residue other than P, G, E or D, or other than a small hydrophobic residue (e.g. T, S),

- X-I2 is absent or represents any amino acid, optionally phenylalanine, or optionally a conservative substitution thereof,

Xi ₃ represents any amino acid, optionally aspartic acid, or optionally a conservative substitution thereof, optionally a serine, optionally a threonine, optionally a glutamic acid, optionally an asparagine, optionally a residue other than a large and hydrophobic residue, and

X-I4 represents any amino acid, optionally tyrosine or optionally a conservative substitution thereof, optionally an aromatic amino acid, optionally a non-aromatic amino acid.

In one aspect of any embodiment herein, any one or more of the amino acid residues are substituted by an amino acid residue as set out in Example 2 as a possible substitution at the particular Kabat position corresponding to the position in the formula for the particular CDR or FR. In one aspect of any embodiment herein, any amino acid residue in a V _H or V _L can be specified to be a residue that maintains V domain (e.g. V _H or V _L respectively) domain structure integrity. In one aspect of any embodiment herein, an amino acid residue in a V _H or V|_ can that contacts an antigen or binding partner (e.g. CD39, a V _L or a V _L ) can be specified to be a residue which does not introduce steric hindrance that reduces antigen or binding partner binding.

In one aspect of any embodiment, a V _H Kabat CDR1 , CDR2 and/or CDR3 comprises at least one, two or three amino acid modifications, optionally substitutions, compared to a parental V _H .

In one embodiment, a parental V _H Kabat CDR1 comprises or consists of the amino acid sequence HYGMN; in one embodiment, a parental V _H Kabat CDR2 comprises or consists of the amino acid sequence WINTYTGEPTYADDFKG; in one embodiment, a parental V _H Kabat CDR3 comprises or consists of the amino acid sequence RRYEGNYVFYYFDY.

In another embodiment, a parental V _H Kabat CDR1 comprises or consists of the amino acid sequence NYGMN. In one embodiment, a parental V _H Kabat CDR2 comprises or consists of the amino acid sequence WINTYTGEPTYADDFKG. In one embodiment, a parental V _H Kabat CDR3 comprises or consists of the amino acid sequence KAYYGSNYYFDY. In another embodiment, a parental V _H Kabat CDR1 comprises or consists of the amino acid sequence HYGMN. In one embodiment, a parental V _H Kabat CDR2 comprises or consists of the amino acid sequence WINTYTGELTYADDFKG. In one embodiment, a parental V _H Kabat CDR3 comprises or consists of the amino acid sequence RAYYRYDYVMDY.

In one aspect of any embodiment, a V _L Kabat CDR1 , CDR2 and/or CDR3 comprises at least one, two or three amino acid modifications, optionally substitutions, compared to a parental V _L .

In one embodiment, a parental V _L Kabat CDR1 comprises or consists of the amino acid sequence RASENIYSYFS. In one embodiment, a parental V _L Kabat CDR2 comprises or consists of the amino acid sequence TAKTLAE. In one embodiment, a parental V _L Kabat CDR3 comprises or consists of the amino acid sequence QHHYVTPYT.

In another embodiment, a parental V _L Kabat CDR1 comprises or consists of the amino acid sequence KASQDVSTAVA. In one embodiment, a parental V _L Kabat CDR2 comprises or consists of the amino acid sequence SASYRYT. In one embodiment, a parental V|_ Kabat CDR3 comprises or consists of the amino acid sequence QQHYTTPPYT.

In another embodiment, a parental V _L Kabat CDR1 comprises or consists of the amino acid sequence KASHNVGTNVA. In one embodiment, a parental V _L Kabat CDR2 comprises or consists of the amino acid sequence SASYRYS. In one embodiment, a parental V _L Kabat CDR3 comprises or consists of the amino acid sequence HQYNNYPYT.

In one aspect, the binding molecule or antigen-binding fragment thereof (e.g., an antibody, or a V _H , V _L , FR or CDR sequence thereof) has an advantageous or improved pharmacological property selected from decreased hydrophobicity, increased physical stability, e.g., in pharmaceutical formulations, decreased or low aggregation propensity under conditions found in pharmaceutical formulations, increased human amino acid content (e.g. humanized antibodies comprising V _H and V _L framework regions of human origin; antibodies comprising one or more substitutions to introduce residues present at a corresponding position in a human CDR into a CDR of non-human origin), decreased potential for human anti-mouse antibodies (HAMA) response in humans, maintenance or increase in binding affinity to CD39, and/or maintenance of increase in potency in inhibition of ATPase activity of CD39.

In one aspect, the binding molecule or antigen-binding fragment comprises a V _H and/or V _L having a reduced number of aromatic residues (e.g. tyrosines), for example, resulting in a reduction of the hydrophobicity of the antibody (to improve physical stability), optionally further in combination with reduced potential for immunogenicity in humans (e.g. reduced potential for giving rise to human anti-mouse antibodies). In one embodiment, an aromatic residue is substituted by a non-aromatic residue, optionally further by a residue that is present at the particular position in the particular CDR or FR in a human variable region acceptor sequence. For example, parental antibodies having five, six or seven aromatic residues in V _H CDR3 can be modified so as to have less than five, six or seven aromatic residues, e.g. V _H CDR3 can be modified to have three, four, five or six aromatic residues. In one embodiment, a V _H CDR2 comprises a substitution in which an aromatic residue is replaced by a non-aromatic residue. In one embodiment, a V _H CDR3 comprises a substitution in which an aromatic residue is replaced by a non-aromatic residue. In one embodiment, a V _L CDR1 comprises a substitution in which an aromatic residue is replaced by a non-aromatic residue. In one embodiment, a V _L CDR2 comprises a substitution in which an aromatic residue is replaced by a non-aromatic residue. In one embodiment, a V _L CDR3 comprises a substitution in which an aromatic residue is replaced by a non-aromatic residue.

In any embodiment herein, a V _H may comprise a non-aromatic or non-tyrosine residue, at Kabat position 53, 100 and/or 100a, e.g., the V _H comprises a Y53 (e.g. Y53E) substitution in CDR2, a Y100 substitution in CDR3 and/or a Y100a substitution in CDR3 (e.g. Y(100a)S), wherein the aromatic residue is replaced by any amino acid, optionally a non- aromatic residue, optionally, where the antibody is a humanized antibody, wherein the aromatic residue is substituted by a residue present at the corresponding portion in the human acceptor sequence.

In any embodiment herein, a V _H may comprise a non-aspartic acid residue, optionally a glutamine, at Kabat position 61 , e.g., the V _H comprises a D61 Q substitution in CDR2.

In any embodiment herein, a V _L may comprise a non-aromatic or non-tyrosine residue, at Kabat position 27, 28, 30, 56, 92 and/or 92. In one embodiment, an aromatic residue at Kabat position 30 and/or 92 is replaced by any amino acid, optionally a non- aromatic residue). Optionally, where the antibody is a humanized antibody, the aromatic residue can be substituted by a residue present at the corresponding portion in the human acceptor sequence.

In any embodiment herein, a V _L may comprise a non-glutamic acid residue, optionally a glutamine, at Kabat position 27, e.g., the V _L comprises a E27Q substitution in CDR1 .

In any embodiment herein, a V _L may comprise a non-asparagine residue, optionally a serine, at Kabat position 28, e.g., the V _L comprises a N28S substitution in CDR1.

In any embodiment herein, a V _L may comprise a non-aromatic or non-tyrosine residue, optionally a serine, at Kabat position 30, optionally wherein the aromatic residue is replaced by any amino acid, optionally a non-aromatic residue e.g., the V _L comprises a Y30S substitution in CDR1. In any embodiment herein, a V _L may comprise a non-glutamic acid residue, optionally a serine, at Kabat position 56, e.g., the V _L comprises a E56S substitution in CDR2.

In any embodiment herein, a V _L may comprise a non-aromatic residue (e.g. a residue other than a tyrosine), optionally a serine, at Kabat position 92, optionally wherein the aromatic reside at 92 is replaced by a non-aromatic residue, e.g., the V _L comprises a Y92S substitution in CDR3.

In any embodiment herein, a V _L may comprise a non-valine residue, optionally a threonine, at Kabat position 93, e.g., the V _L comprises a V93T substitution in CDR3.

In one aspect, the binding molecule or antigen-binding fragment thereof comprises human framework regions, e.g. the molecule comprises a V _H comprising human V _H FR-i , FR ₂ , FR ₃ and FR ₄ amino acid sequences (optionally comprising one or more amino acid substitutions), and/or a V _L comprising human V _L FR-i , FR ₂ , FR ₃ and FR ₄ amino acid sequences (optionally comprising one or more amino acid substitutions). Provided are methods for modifying CDR and FR residues so as to maintain an active antibody (and V _H and V _L domain structure) while making use of any of a range of human V _H and V _L acceptor frameworks.

In one aspect, the invention provides human or humanized V _H and/or V _L domains (and antibodies or other proteins comprising the V _H and V _L ) in which at least a portion of a Kabat CDR (e.g. a contiguous sequence of 2, 3 4, 5 or more amino acids) is identical to the corresponding portion in the human acceptor sequence. In one aspect, the invention provides V _H and/or V _L domains (and antibodies or other proteins comprising the V _H and V _L ) comprising an amino acid substitution in a Kabat CDR, wherein the CDR residue is substituted by a residue that is present at the corresponding portion in the human acceptor sequence. In one embodiment, the human acceptor framework sequence does not comprise any amino acid substitutions or back-mutations. In another embodiment, the human framework sequence comprises at least one amino acid substitution.

In one aspect, the binding molecule or antigen-binding fragment comprises CDRs having human amino acid sequences, for example, one or more CDRs comprising a residue present at the particular position in a human. For example, a V _H may comprise a partially or fully human V _H CDR1 , CDR2 and/or CDR3. A V _L may comprise a partially or fully human V _L CDR1 , CDR2 and/or CDR3. CDRs can be defined according to Kabat numbering. For example, a V _H may comprise a residue present at the Kabat position 53, 61 and/or 100a that is identical to, or alternatively that differs from, the corresponding portion in the human acceptor sequence. For example, a V _L may comprise a residue present at the Kabat position 27, 28, 30, 56, 92 and/or 93 that is identical to, or alternatively that differs from, the corresponding portion in the human acceptor sequence. Where a CDR residue(s) at a V _H -V _L CDR contact position is substituted by another amino acid residue(s), the substitution(s) in the V _H and/or V _L CDR can be made such that the particular interaction between the V _L CDR residue and its partner residue in the V _H CDR3 is maintained.

In one embodiment, the binding molecule or antigen-binding fragment comprises a human Fc domain. In one embodiment, the Fc domain is a human Fc domain or fragment thereof comprising one or more amino acid modifications compared to a wild-type Fc domain. Optionally, the Fc domain is a human Fc domain or fragment thereof comprising one or more amino acid modifications to decrease binding to a human Fey receptor (e.g. compared to a parental Fc domain, to a wild-type Fc domain).

In one embodiment, the CDR2 comprises an aromatic amino acid residue that contacts any of amino acid residues V95, Q96 and/or L137 of CD39. In one embodiment, the aromatic amino acid residue is an aromatic residue, e.g. a tyrosine, optionally a phenylalanine.

In one embodiment, the V _H comprises a FR3 comprising an amino acid residue that contacts CD39, optionally wherein the residue in the V _H is a leucine at Kabat position 71 .

In one embodiment, the V _H comprises an amino acid residue in FR1 at Kabat position 19 that contacts CD39, optionally wherein the residue is a lysine.

In one embodiment, the V _L comprises a FR2 comprising an aromatic amino acid residue that contacts the CDR3 of the V _H , optionally wherein the residue in the V _L is at Kabat position 49. Optionally the residue at V _L Kabat position is an aromatic residue, optionally further wherein a V _H comprises an aromatic residue (e.g. at Kabat 100e) capable of forming a pi stacking interaction therewith.

In one embodiment, the V _H CDR3 comprises an amino acid residue(s) comprising an aromatic ring (optionally tyrosine, histidine, tryptophan or phenylalanine), capable of forming a pi interaction (e.g., attractive, noncovalent interactions between an aromatic ring and an binding partner, for example in an amide pi-stacked interaction, a pi-pi stacked interaction or a pi-donor interaction) with an amino acid residue (e.g. an aromatic residue) in the V _L polypeptide. In one embodiment, the residue(s) in the CDR3 comprising an aromatic ring is a tyrosine, e.g. at Kabat position 100e. In one embodiment, the residue(s) in the V _L comprising an aromatic ring is a tyrosine, e.g. a tyrosine at Kabat position 49 in the V _L .

In one embodiment, the V _H CDR3 comprises an amino acid residue(s) comprising an aromatic ring (optionally tyrosine, histidine, tryptophan or phenylalanine), capable of forming a pi interaction (e.g., attractive, noncovalent interactions between an aromatic ring and an binding partner, for example in an amide pi-stacked interaction) with an amino acid residue in the CD39 polypeptide (e.g. at residue Q96 in CD39 of SEQ ID NO: 1 ). In one embodiment, the residue comprising an aromatic ring is a phenylalanine.

In one embodiment, the V _H CDR3 comprises (e.g. at Kabat position 100e) an amino acid residue comprising an aromatic ring (e.g. a tyrosine, optionally a phenylalanine), which forms or is capable of forming a pi-pi stacking interaction with an amino acid residue comprising an aromatic ring in the V _L , e.g. the tyrosine at Kabat framework (FR2) position 49 in the V _L . In one embodiment, the V _H CDR3 comprises (e.g. at Kabat position 10Of) an amino acid residue comprising an aromatic ring (e.g. a phenylalanine, optionally a tyrosine), which is capable of a pi-donor interaction with an amino acid residue comprising an aromatic ring in the V|_, e.g. a glutamine at Kabat CDR3 position 89 in the V _L .

In one embodiment, the V _H CDR3 comprises (i) a first amino acid residue comprising an aromatic ring, which is capable of forming a pi-stacking interaction with an amino acid residue in the V _L , (ii) a second an amino acid residue comprising an aromatic ring, which is capable of forming a pi interaction (e.g. an amide pi-stacked interaction) with an amino acid residue comprising an aromatic ring in the CD39 polypeptide. In one embodiment, the first and/or second residue in the CDR3 comprising an aromatic ring is a tyrosine.

In one embodiment, the V _H CDR3 comprises (i) a first and second amino acid residue comprising an aromatic ring (optionally a tyrosine or phenylalanine), which has formed or is capable of a pi (e.g. pi-stacked) interaction with an amino acid residue in the V _L ; (ii) a third amino acid residue comprising an aromatic ring (optionally a tyrosine), which has formed or is capable of a pi interaction with an amino acid residue in the CD39 polypeptide. In one embodiment, one of the first and second residue comprising an aromatic ring in the V _H CDR3 is capable of or has formed a pi-stacking interaction with an amino acid residue comprising an aromatic ring in the V _L . In one embodiment, the first residue in the CDR3 comprising an aromatic ring is a tyrosine and the second residue in the CDR3 comprising an aromatic ring is a phenylalanine. Optionally, the third amino acid residue comprising an aromatic ring is a tyrosine.

An exemplary V _L can comprise:

a CDR1 comprising a residue, e.g. at one, two, three of four of Kabat positions 31 , 32, 33 and/or 34, capable of contacting the CDR3 of the V _H ;

a FR2 comprising an aromatic residue, optionally a tyrosine, at Kabat position 49 capable of contacting the CDR3 of the V _H ; and/or

a CDR3 comprising a residue, e.g. at Kabat positions 89 and/or 91 capable of contacting the CDR3 of the V _H . In one embodiment, the invention provides a binding molecule or antigen-binding fragment thereof capable of binding to and inhibiting the activity of CD39, comprising a V _H of any of the embodiment herein, and a V _L , wherein the V _L comprises:

a CDR1 comprising a residue at Kabat positions 31 , 32, 33 and/or 34 capable of contacting the CDR3 of the V _H ;

a FR2 comprising an aromatic residue, optionally a tyrosine, at Kabat position 49; and/or

a CDR3 comprising a residue at Kabat positions 89 and/or 91 capable of contacting the CDR3 of the V _H .

In one embodiment, the invention provides a binding molecule or antigen-binding fragment thereof capable of binding to and inhibiting the activity of CD39, comprising an antibody V _H and an antibody V _L ,

wherein the V _H comprises the amino acid sequence of Formula I: [FR ₁ ]CDR1 [FR ₂ ]CDR2[FR ₃ ]CDR3[FR ₄ ] (Formula I) wherein [FR-i], [FR ₂ ], [FR ₃ ] and [FR ₄ ] represent V _H framework regions and CDR1 , CDR2 and CDR3 represent V _H CDRs, wherein:

• CDR1 comprises a residue, optionally at Kabat positions 31 and/or 33, that is capable of contacting the N-terminal domain of CD39,

• CDR2 comprises residues capable of contacting CD39, optionally the N- terminal domain of CD39, optionally wherein two, three, four or five of Kabat positions 50, 52, 52a, 53 and 56 are capable of contacting CD39, optionally in the N-terminal domain, optionally wherein the residue at position 53 comprises an aromatic ring, optionally tyrosine, optionally further wherein a residue in the Kabat CDR2 (e.g. at one, two or three of Kabat positions 57, 59 and/or 65), in combination with residues in the Kabat FR3 (e.g. at one, two or three of Kabat positions 67, 68, 69, 70 and/or 71 ) are capable of contacting the C-terminal domain of CD39,

· CDR3 comprises an aromatic residue capable of contacting CD39, optionally in the N-terminal domain of CD39, optionally wherein the CDR3 further comprises an aromatic residue capable of contacting the V _L , optionally wherein the aromatic residue(s) is/are at any of Kabat positions 100, 100b, 100c, 100d, 100e and/or 10Of (to the extent residues are present at the particular position), optionally wherein the aromatic residue capable of contacting the V _L is a tyrosine or a phenylalanine and optionally wherein the aromatic residue capable of contacting CD39 is a tyrosine or a phenylalanine; and

wherein the V _L comprises the amino acid sequence of Formula II: [FR ₁ ]CDR1 [FR ₂ ]CDR2[FR ₃ ]CDR3[FR ₄ ] (Formula II) wherein [FR-i], [FR ₂ ], [FR ₃ ] and [FR ₄ ] represent V _L framework regions and CDR1 , CDR2 and CDR3 represent V _L CDRs, wherein:

• CDR1 comprises a residue, optionally at Kabat positions 31 , 32, 33 and/or 34, capable of contacting the CDR3 of the V _H ;

• FR2 comprises a residue, optionally an aromatic residue at Kabat position 49, capable of contacting the CDR3 of the V _H ; and

• CDR3 comprises a residue, optionally at Kabat positions 89 and/or 91 , capable of contacting the CDR3 of the V _H .

In any embodiment herein, the V _H CDR1 contacts the N-terminal domain of CD39. In any embodiment herein, the V _H CDR2 contacts the C-terminal domain of CD39. In any embodiment herein, the V _H CDR3 contacts the N-terminal domain of CD39.

In one embodiment, the V _H of formula I comprises a segment of residues within Kabat positions 59-71 (CDR2-FR3), optionally within 59-72b, that are capable of contacting amino acid residues in the C-terminal domain of CD39, optionally further wherein residues within Kabat positions 59-71 contact the glycan at residue N292 of CD39. For example, the Kabat CDR2 and FR3 can comprise residues at Kabat positions 59, 65, 67, 68, 69, 70 and/or 71 , and optionally further at residue 72, 72a and/or 72b that are capable of contacting the C- terminal domain of CD39, e.g. including amino acid resides in CD39 and the glycan at N292 of the CD39 polypeptide.

In one embodiment, the V _L comprises a CDR1 wherein the residues at Kabat positions 31 -34 have the formula Xi X ₂ X ₃ X ₄ , wherein represents a serine, optionally a threonine, alanine or asparagine, or optionally a residue other than lysine, arginine, isoleucine or leucine, optionally a residue other than phenylalanine, tyrosine or tryptophan, X ₂ represents a tyrosine, or optionally alanine or asparagine, X ₃ represents phenylalanine or optionally leucine, isoleucine, methionine, valine, tryptophan, optionally a residue other than aspartic acid, glutamic acid, asparagine or lysine, and X ₄ represents serine, optionally alanine, optionally a small residue (e.g. alanine, threonine, glycine, asparagine or histidine), optionally other than a large hydrophobic residue. In one embodiment, any one or more of Xi X ₂ X ₃ or X ₄ are substituted by an amino acid residue as set out in Example 2 as a possible substitution at the particular Kabat position corresponding to the position in the formula for the particular CDR or FR.

In one embodiment, the V _L comprises a CDR1 wherein the residues at Kabat positions 31 -34 have the formula Xi X ₂ X3 X ₄ , wherein represents a threonine or a conservative substitution thereof, X ₂ represents alanine or asparagine, or a conservative substitution thereof, X ₃ represents valine or a conservative substitution thereof, and X ₄ represents alanine or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR1 wherein the residues at Kabat position 24 to 34 have the formula Xi X ₂ X ₃ X ₄ X ₅ X6 X? Xs Xg X10 9 X10 X11 , wherein

Xi represents any amino acid, optionally arginine or lysine,

X ₂ represents any amino acid, optionally alanine,

X ₃ represents any amino acid, optionally serine,

X ₄ represents any amino acid, optionally glutamic acid, glutamine, or histidine,

X ₅ represents any amino acid, optionally asparagine or aspartic acid, optionally serine,

X ₆ represents any amino acid, optionally isoleucine or valine,

X ₇ represents any amino acid, optionally tyrosine, serine or glycine,

X ₈ represents serine or threonine,

X ₉ represents tyrosine, alanine or asparagine, X1 ₀ represents phenylalanine or valine, and

X11 represents serine or alanine.

In one embodiment, the V _L comprises a CDR2 wherein the residues at Kabat position 50-56 have the formula Xi X ₂ X ₃ X ₄ X ₅ X ₆ X7, wherein

Xi represents threonine or serine, or a conservative substitution thereof,

X ₂ represents any amino acid, optionally alanine,

X ₃ represents any amino acid, optionally lysine or serine,

X ₄ represents any amino acid, optionally threonine or tyrosine,

X ₅ represents any amino acid, optionally leucine or arginine,

X ₆ represents any amino acid, optionally alanine or tyrosine, and

X ₇ represents any amino acid, optionally glutamic acid, threonine or serine.

In one embodiment, the V _L comprises a CDR2 wherein the residues at Kabat position 50-56 have the formula Xi X ₂ X ₃ X ₄ X ₅ X ₆ X7, wherein:

Xi represents serine, or a conservative substitution thereof,

X ₂ represents alanine, or a conservative substitution thereof,

X ₃ represents serine, or a conservative substitution thereof,

X ₄ represents tyrosine, or a conservative substitution thereof, X ₅ represents arginine, or a conservative substitution thereof,

X ₆ represents tyrosine, or a conservative substitution thereof, and

X ₇ represents threonine or serine, glutamic acid, or a conservative substitution thereof.

In one embodiment, any one or more of the V _L CDR2 residues are substituted by an amino acid residue as set out in Example 2 as a possible substitution at the particular Kabat position corresponding to the position in the formula for the particular CDR.

In one embodiment, the V _L comprises a FR2 comprising a tyrosine, or optionally a phenylalanine, at Kabat position 49.

In one embodiment, the V _L comprises a CDR3 wherein the residues at Kabat position

89-91 have the formula Xi X ₂ X3, wherein X-i represents any amino acid, optionally a glutamine, optionally a histidine, X ₂ represents any amino acid, optionally a glutamine or histidine, and X ₃ represents histidine, or optionally tyrosine, or optionally asparagine, or optionally a residue other than a large or hydrophobic residue.

Optionally, in one embodiment, the V _L comprises residues at any one, two, three or four of Kabat positions 94, 95, 96 and 97 (Kabat CDR3) that contact the V _H domain framework.

In one embodiment, the V _L comprises a CDR3 wherein the residues at Kabat position 89 is a glutamine or histidine, or a conservative substitution thereof, the residue at position 91 is a tyrosine or histidine, or a conservative substitution thereof, the residue at position 95 is a proline, or a conservative substitution thereof, and the residue at position 96 is a tyrosine, or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR3 wherein the residues at Kabat position 89-97 have the formula Xi X ₂ X ₃ X ₄ X ₅ Xe X? Xs X9X10, wherein

Xi represents glutamine or histidine,

X ₂ represents any amino acid, optionally glutamine or histidine,

X ₃ represents tyrosine or histidine,

X ₄ represents any amino acid, optionally tyrosine or asparagine, or a non-aromatic residue, optionally a serine,

X ₅ represents any amino acid, optionally valine, threonine or asparagine,

X ₆ represents any amino acid, optionally threonine or tyrosine,

X ₇ represents any amino acid, optionally proline,

X ₈ is absent or represents any one or more amino acids, optionally a proline, X ₉ represents any amino acid, optionally tyrosine, and

X10 represents any amino acid, optionally threonine. In one embodiment, any one or more of the V _L CDR3 residues are substituted by an amino acid residue as set out in Example 2 as a possible substitution at the particular Kabat position corresponding to the position in the formula for the particular CDR or FR.

In one embodiment, the V _L comprises:

- a CDR1 wherein the residues at Kabat position 31 , 32, 33 and 34 have the formula Xi

X ₂ X ₃ X ₄ , wherein represents a threonine, X ₂ represents alanine or asparagine, X ₃ represents valine, and X ₄ represents alanine;

a FR2 comprising an aromatic residue, optionally a tyrosine, at Kabat position 49; and a CDR3 wherein the residues at Kabat position 89 is a glutamine or histidine, the residue at position 91 is a tyrosine or histidine, optionally wherein the residue at position 95 is a proline, optionally wherein the residue at position 96 is a tyrosine. It will be appreciated that the crystal structures disclosed can be used to guide design of FR and CDR amino acid sequences while retaining the desired functional properties.

Specified variable region and CDR sequences of exemplary V _H and V _L domains may thus optionally comprise 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. A conservative sequence modification refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are typically those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Specified variable region and CDR sequences may comprise one, two, three, four or more amino acid insertions, deletions or substitutions. Where substitutions are made, preferred substitutions will be conservative modifications. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the properties set forth herein) using the assays described herein. In one aspect, provided are antibodies that bind an epitope present on human CD39 expressed at the surface of cells, including tumor cells and/or leukocytes, and that potently inhibit the enzymatic (ATPase activity) activity of the CD39 enzyme.

In one embodiment, the VH comprises a human VH acceptor framework and the VL comprises a human VL acceptor framework. In one embodiment, the VH segment of the VH human acceptor framework is from the human IGHV7 gene, optionally IGHV7-4, optionally IGHV7-4-1 (e.g., IGHV7-4-1 ^* 1 , IGHV7-4-1 ^* 2) optionally a human framework gene (e.g. a IGHV7 gene) other than IGHV7-4-1 , other than IGHV7-4-1 ^* 1 or other than IGHV7-4-1 ^* 2. In one embodiment, the J-segment is from IGHJ6 or a J-segment other than IGHJ6. In one embodiment, the VH human acceptor framework is from IGHV7-4-1 ^* 01 . In one embodiment, the CDR1 , 2 and 3 of the VH are from the murine IGHV9 gene, optionally the IGHV9-3-1 gene, optionally from IGHV9-3-1 ^* 01. In one embodiment, the VL domain human acceptor framework is from IGKJ4 or IGKJ2, a human framework other than IGKJ4, or a human framework other than IGKJ2. Optionally the VL domain human acceptor framework is from IGKJ4 ^* 01 or a human framework other than IGKJ4 ^* 01 . In one embodiment, the CDR1 , 2 and 3 of the VL are from the murine IGKV12-44 gene, optionally from I GKV 12-44 ^* 01. In one embodiment, the V _H comprises a human acceptor framework or portion thereof (e.g. an FR3 domain) that naturally comprises the amino acid sequence FVFSL at Kabat positions 67-71 .

In one embodiment, the human heavy chain acceptor framework comprises no framework mutations compared to a naturally occurring human VH segment. In one embodiment, the human light chain acceptor framework comprises no framework mutations compared to a naturally occurring human VL segment.

In one embodiment, a V _H comprises an amino acid sequence of SEQ ID NO: 6. In one embodiment, a V _H comprises an amino acid sequence of SEQ ID NO: 7. In one embodiment, a V _H comprises an amino acid sequence of SEQ ID NO: 8. In one embodiment, a V _H comprises an amino acid sequence of SEQ ID NO: 9.

In one embodiment, a V _L comprises an amino acid sequence of SEQ ID NO: 10. In one embodiment, a V _L comprises an amino acid sequence of SEQ ID NO: 1 1 . In one embodiment, a V _L comprises an amino acid sequence of SEQ ID NO: 12. In one embodiment, a V _L comprises an amino acid sequence of SEQ ID NO: 13. In one embodiment, a V _L comprises an amino acid sequence of SEQ ID NO: 14. In one embodiment, a V _L comprises an amino acid sequence of SEQ ID NO: 15. In one embodiment, a V _L comprises an amino acid sequence of SEQ ID NO: 16.

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

(a) an antibody binding domain comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 10, 1 1 , 12, 13, 14, 15 or 16;

(b) an antibody binding domain comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 10, 1 1 , 12, 13, 14, 15 or 16;

(c) an antibody binding domain comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 10, 1 1 , 12, 13, 14, 15 or 16;

(d) an antibody binding domain comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 10, 1 1 , 12, 13, 14, 15 or 16; and

(e) an antibody binding domain comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 6, 7, 8 or 9 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 5.

Also provided is a V _L CDR - V _H CDR3 - CD39 binding complex, optionally isolated and/or crystallized, comprising a CD39 polypeptide (optionally comprising N292 linked glycosylation) and an antibody or antibody fragment comprising a V _H and a V _L of the disclosure.

The CD39 binding proteins (e.g. antibodies) are particularly advantageous when used as pure inhibitors of CD39 ATPase activity (e.g., lacking effector function and/or as "naked" antibodies that are not conjugated to a cytotoxic drug). However, it will be appreciated that the antibodies can alternatively be used in a form that can mediate ADCC and/or CDC (e.g. an Fc domain that has effector function, binds to human Fey receptors), and/or the antibodies can be conjugated to a moiety of interest (e.g. a cytotoxic moiety, a detectable moiety).

In one embodiment of any aspect herein, an antibody comprises a modified human lgG1 Fc domain comprising N-linked glycosylation at Kabat residue N297 and comprising an amino acid substitution at Kabat residue(s) 234 and 235, optionally further at Kabat residue 331 , optionally at Kabat residues 234, 235, 237 and at Kabat residues 330 and/or 331 , optionally wherein the Fc domain comprises a L234A L235E/P331 S substitution, L234F/L235E/P331 S substitution, L234A/L235E/G237A/P331 S substitution, or L234A/L235E/G237A/A330S/P331 S substitution.

Provided also are methods for treating an individual, the method comprising administering to an individual (e.g., an individual having a disease, an infectious disease, a cancer, etc.) a therapeutically active amount of any of the anti-CD39 antigen binding compounds described herein.

Also provided are nucleic acids encoding a V _H , a V _L or an anti-CD39 antigen binding compound described herein, a vector comprising such a nucleic acid(s), a cell comprising such a vector, and a method of producing an anti-CD39 antigen binding compound described herein, comprising culturing such a cell under conditions suitable for expression of the anti- CD39 antigen binding compound. The disclosure also relates to compositions, such as pharmaceutically acceptable compositions and kits, comprising such proteins, nucleic acids, vectors, and/or cells and typically one or more additional ingredients that can be active ingredients or inactive ingredients that promote formulation, delivery, stability, or other characteristics of the composition (e.g., various carriers). The disclosure further relates various new and useful methods making and using such protein and antibodies, nucleic acids, vectors, cells, organisms, and/or compositions, such as in the modulation of CD39- mediated biological activities, for example in the treatment of diseases related thereto, notably cancers and/or infectious disease (e.g. viral or bacterial infections).

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

Figures 1 and 2 show results from a study of anti-CD39/CD39 complexes by X-ray diffraction. The 3-dimensional structure is illustrated, showing that binding of the neutralizing anti-CD39 to the target antigen CD39 entirely relies on the heavy chain variable domain; the anti-CD39 antibody light chain does not contact the antigen directly.

Figure 3 shows results from a study of anti-CD39/CD39 complexes by X-ray diffraction. The anti-CD39 heavy chain binds to both the CD39 N-terminal domain 1 and C- terminal domain 2 of CD39). The anti-CD39 binding site is located at the apex of the two CD39 domains and at the entry of the catalytic cleft.

Figure 4 shows results from a study of anti-CD39/CD39 complexes by X-ray diffraction. The human CD39/anti-CD39 frozen conformation perfectly superimposes with rat CD39 form A of the pdb crystal 3ZX3. Binding of the antibody to both domains at the same time thus likely inhibits domain motion and block the enzyme in a given frozen status.

Figures 5 and 6 show the three residues D72, T72a and S72b of the heavy chain Kabat framework (FR3) are bound by three H-bonds, which in turn positions the D72 in an orientation that shapes the CD39 binding surface, as shown in Figure 5 (panel A) and Figure 6. If S72b is substituted with a residue that results in the loss of the 72-72b interaction, the side chain of aspartic acid at position 72 may be affected such that CD39 binding is diminished as shown in Figure 5 (panel B).

Figure 7 shows the positions of the CDR mutations made on the antibody surface to enhance the pharmacological properties of the antibody.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the specification, "a" or "an" may mean one or more. As used in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.

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

Human CD39, also known as "vascular" CD39, NTPdasel , ENTPD1 , ATPDase and vascular ATP diphosphohydrolase, EC 3.6.1.5, 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 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:

1 MEDTKESNVK TFCSKNILAI LGFSSIIAVI ALLAVGLTQN KALPENVKYG IVLDAGSSHT 61 SLYIYKWPAE KENDTGWHQ VEECRVKGPG ISKFVQKVNE IGIYLTDCME RAREVIPRSQ

121 HQETPVYLGA TAGMRLLRME SEELADRVLD WERSLSNYP FDFQGARI IT GQEEGAYGWI 181 TINYLLGKFS QKTRWFSIVP YETNNQETFG ALDLGGASTQ VTFVPQNQTI ESPDNALQFR 241 LYGKDYNVYT HSFLCYGKDQ ALWQKLAKDI QVASNEILRD PCFHPGYKKV VNVSDLYKTP 301 CTKRFEMTLP FQQFEIQGIG NYQQCHQSIL ELFNTSYCPY SQCAFNGIFL PPLQGDFGAF 361 SAFYFVMKFL NLTSEKVSQE KVTEMMKKFC AQPWEEIKTS YAGVKEKYLS EYCFSGTYIL

421 SLLLQGYHFT ADSWEHIHFI GKIQGSDAGW TLGYMLNLTN MIPAEQPLST PLSHSTYVFL

481 MVLFSLVLFT VAI IGLLIFH KPSYFWKDMV (SEQ I D NO: 1 )

In the context herein, "inhibit", "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", etc.), 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. 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 example, disappearance of ATP and/or generation of AMP can be assessed, as described herein. 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).

Whenever within this whole specification "treatment of cancer" or the like is mentioned with reference to anti-CD39 binding agent (e.g., antibody), there is meant: (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, 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 "antibody," as used herein, refers to 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 1 10 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _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 antibody can bind preferably in a competitive binding assay to the binding partner, e.g., 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 monoclonal antibody, it means that the antibody competes with the monoclonal antibody in a binding assay using either recombinant CD39 molecules or surface expressed CD39 molecules. For example, if a test antibody reduces the binding of a reference antibody to a CD39 polypeptide or 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] / [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).

Within the context herein a "determinant" designates a site of interaction or binding on a polypeptide.

The term "epitope" refers to an antigenic determinant, and is the area or region on an antigen to which an antibody binds. A protein epitope may comprise amino acid residues directly involved in the binding as well as amino acid residues which are effectively blocked by the specific antigen binding antibody or peptide, i.e., amino acid residues within the "footprint" of the antibody. It is the simplest form or smallest structural area on a complex antigen molecule that can combine with e.g., an antibody or a receptor. Epitopes can be linear or conformational/structural. The term "linear epitope" is defined as an epitope composed of amino acid residues that are contiguous on the linear sequence of amino acids (primary structure). The term "conformational or structural epitope" is defined as an epitope composed of amino acid residues that are not all contiguous and thus represent separated parts of the linear sequence of amino acids that are brought into proximity to one another by folding of the molecule (secondary, tertiary and/or quaternary structures). A conformational epitope is dependent on the 3-dimensional structure. The term 'conformational' is therefore often used interchangeably with 'structural'.

The term "deplete" or "depleting", with respect to CD39-expressing cells, means a process, method, or compound that results in killing, elimination, lysis or induction of such killing, elimination or lysis, so as to negatively affect the number of such CD39-expressing cells present in a sample or in a subject.

The term "internalization", used interchangeably with "intracellular internalization", refers to the molecular, biochemical and cellular events associated with the process of translocating a molecule from the extracellular surface of a cell to the intracellular surface of a cell. The processes responsible for intracellular internalization of molecules are well-known and can involve, inter alia, the internalization of extracellular molecules (such as hormones, antibodies, and small organic molecules); membrane-associated molecules (such as cell- surface receptors); and complexes of membrane-associated molecules bound to extracellular molecules (for example, a ligand bound to a transmembrane receptor or an antibody bound to a membrane-associated molecule). Thus, "inducing and/or increasing internalization" comprises events wherein intracellular internalization is initiated and/or the rate and/or extent of intracellular internalization is increased.

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 term "therapeutic agent" refers to an agent that has biological activity.

For the purposes herein, a "humanized" or "human" 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. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered" to produce specific human antibodies in response to antigenic challenge (see, e.g., Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun 6:579, the entire teachings of which are herein incorporated by reference). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated in their entirety by reference). A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

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; 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)) and/or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1 ), 50-52 (L2) and 91 -96 (L3) in the light-chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901 -917), or a similar system for determining essential amino acids responsible for antigen binding. 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. Another suitable numbering system is the Abnum system. Unless otherwise specified, the Kabat amino acid numbering nomenclature for immunoglobulins is used to refer to positions in the VH and VL domains. Sequence numbering using the Abnum system (see Abhinandan and Martin, (2008) Molecular Immunology 45: 3832-3839, the disclosure of which is incorporated by reference) can also be automatically generated at http://www.bioinfo.org.uk/abs/abnum. However it will be appreciated that the person of skill in the art can use an alternative numbering system and identify positions corresponding to Kabat numbering. Phrases such as "Kabat position", "Kabat numbering" and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains. 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 (Kabat numbering) of human γ (gamma) heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., α, δ, ε and μ for human antibodies), or a naturally occurring allotype thereof.

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, 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.

Production of binding compounds

The present invention is based, in part, on the discovery of amino acid resides in

CDR and FR of V _H and V _L of anti-CD39 antibodies that define the function of the antibodies. Based on the antibody:CD39 crystal structure new CD39-binding compounds of the disclosure can be constructed. For example, a new CD39-binding compounds can be exemplified by a protein that binding to both C- and N-terminal domains of CD39 at the same time, and that thereby upon binding to CD39 immobilizes CD39 in one of its conformations thereby preventing it from hydrolyzing its substrate. The protein may interact with amino acid residue(s) in the active site region of CD39 (e.g. residues V95, Q96 and/or L137 of CD39), wherein the protein comprises a first protein domain (e.g. a V _H ) and second protein domain (e.g., a V _L ), wherein the first protein domain is capable of binding to both CD39 and to the second protein domain (e.g., the V _L ), and wherein the second protein domain binds to the first protein domain so as to position the first protein domain for binding to CD39. The first protein domain (e.g. the V _H ) is capable of binding via a first binding face to the second protein domain (e.g., the V _L ) and via a second binding face to CD39. The first protein domain (the domain that is capable of binding to both CD39 and to the second protein domain) can comprise or consist of a V _H CDR3 domain. A protein can be designed using a CDR3 that comprises a first and a second aromatic residue (e.g., tyrosine residue(s)), wherein one aromatic residue interacts with a residue(s) in a CDR of the V _L domain and the second aromatic residue interacts with CD39. A CDR3 can comprise one, two or more amino acid residue having an aromatic ring (e.g., tyrosine, histidine, tryptophan or phenylalanine), that is capable of pi interactions (attractive, noncovalent interactions between an aromatic ring and a second group, e.g. an aromatic group whereby the two aromatic groups interact via pi- stacking) with an amino acid residue in the second protein domain, for example within or adjacent to a CDR2 of a V _L , for example the residue at Kabat position 49 and/or 89 in a V _L ; and an amino acid residue having an aromatic ring, that is capable of pi interaction with an amino acid residue in the CD39 polypeptide.

For example, a V _H can comprise a CDR1 and a CDR2 that comprise amino acid residues that contact CD39, optionally wherein the CDR1 contacts the N-terminal domain, wherein the CDR2 binds across the N- and C- terminal domains (including, e.g. the N292 glycan and/or across or to a residue within the substrate cleft (or groove entry), and a CDR3 that comprises multiple aromatic residues, some of which interact with residues in the V _L and other(s) which interact with residues in CD39, e.g. by forming pi interactions.

A V _H may thus be characterized by presence in the Kabat CDR3 of a plurality of aromatic amino acid residues, one (or more) of which may interact via pi-interactions with residues within CD39 and some of which (e.g. 1 or 2) may interact via pi-interactions with residues in the V _L (e.g. a Kabat CDR of the V _L ).

A V|_, in turn, can comprise one or more CDRs (e.g. a CDR1 , a CDR2 and/or a CDR3) that comprise amino acid residues that contact the CDR3 of the V _H . Optionally the V _L further comprises a FR2 that includes an aromatic residue at Kabat residue 49 (e.g. a tyrosine), e.g., that is able to form pi-stacked interactions with Kabat residue 100e in CDR3 of the V _H . The CDRs of the V _L can be chosen so that they interact with a partner amino acid in the CDR3 of the V _H . It will therefore be appreciated that the particular amino acid residues present at the contact positions in V _L CDR1 , CDR2 and CDR3 can vary, as a function of the nature of the complementary residue present at the respective contact position in V _H CDR3. The V _L CDR1 can comprise residues at Kabat positions 31 , 32, 33 and 34 that contact the V _H CDR3. The V|_ CDR3 can comprise residues at Kabat positions 89 and 91 that contact the V _H CDR3, and optionally the V _L CDR3 can further comprise residues at Kabat positions 94, 95, 96 and/or 97 that contact the framework of the V _H .

The interaction of the VL CDRs (and optionally further FR2) with the VH CDR3 positions the VH CDRs for binding, such that at least one CDR binds the N-terminal domain and at least one CDR (optionally in combination with framework residues) binds the C- terminal domain of CD39. Optioanlly, further the VL and VH CDR3 position the VH such that the VH CDR2 contacts CD39 within the region of the enzymatic active site and/or that the FR3 binds to the N292 glycan of CD39 in the C-terminal domain. The CDR2 of the VH can therefore comprise one more residues that contact the active site region so as to inhibit the enzymatic activity of the CD39 protein. For example, CDR2 can comprise one or more residues, for example at Kabat positions 52, 52a and/or 53 (e.g., an aromatic residue at position 53), that contact a residue in the CD39 active site or N-terminal domain (domain 1 ) groove entry (e.g. residues V95, Q96 and/or L137 of the CD39 of SEQ ID NO: 1 ).

The V _H can comprise various further contact residues within the FR1 , CDR1 , CDR2 and FR3 that contact CD39. Contact residues can be, for example, at Kabat positions 31 , 32, and/or 33 in CDR1 , at Kabat positions 50, 52, 52a, 54 and/or 56 (in addition to 53) in CDR2. Optionally an aromatic residue (e.g., a histidine) is present at Kabat position 31 that forms pi- stacked interactions with CD39 (residue E140 and/or L144 in CD39 of SEQ ID NO: 1 ), and a residue at Kabat position 33 that contacts CD39. Optionally, in any embodiment herein, the V _H comprises a Kabat FR1 comprising a residue at Kabat position 30, optionally where the residue is a threonine or an arginine, that contacts residue Q96 of CD39. Optionally in any embodiment herein the V _H comprises a Kabat FR1 comprising a residue at Kabat position 19, optionally where the residue is a lysine, that contacts the N292 glycan of CD39. Optionally the V _H further comprises a FR3 comprising residue(s) (e.g., at Kabat positions 67- 71 ) that contacts CD39, e.g. at an amino acid residue or the N292 glycan of CD39.

V _H and V|_ domains with human acceptor framework sequences can thus be produced that have high human amino acid content, physicochemical stability and high binding affinity and potency in neutralization of the enzymatic activity of human CD39.

A CD39-binding compound can be readily produced according to any of several different approaches. For example, a binding compound can be produced using the structures and sequences disclosed herein, optionally incorporating further amino acid modifications, and tested for binding to CD39, inhibition of CD39, internalization, effector function, and/or any further property of interest (e.g. pharmacological properties).

When incorporating further amino acid modifications, a suitable parental CD39 V _H A _L having the amino acid sequence features and/or other properties disclosed herein can be used as a starting point and modified. A starting (parental) binding protein may for example comprise a murine V _H and V _L , a humanized V _H and V _L each comprising murine CDR1 , 2, 3 and human frameworks, or any of the V _H and a V _L of any of antibodies mAbl to mAb39 having the V _H and V _L sequences of Table 1 for further modification.

V _H and/or V _L domains (and, e.g., Ig heavy and light chains comprising them) may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. Nucleic acids encoding them can be may be chemically synthesized by commercial suppliers using known oligonucleotide synthesis methodology. Site-directed (or oligonucleotide-mediated) mutagenesis and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Mutagenesis protocols, kits, and reagents are commercially available, e.g. QuikChange® Multi Site-Direct Mutagenesis Kit (Stratagene, La Jolla, CA). Single mutations are also generated by oligonucleotide directed mutagenesis using double stranded plasmid DNA as template by PCR based mutagenesis (Sambrook and Russel, (2001 ) Molecular Cloning: A Laboratory Manual, 3rd edition).

In one approach for generating a CD39-binding compound, a set of candidate anti- CD39 binding compounds are generated and those having the V _H A _L amino acid sequences and/or other characteristics described herein are selected. A set of candidate compounds may be generated by mutagenesis of V _H and V _L sequence disclosed herein, or by a variety of other techniques known in the art. For example a set of compounds (e.g. antibodies or other non-immunoglobulin scaffolds) can be generated as a library (e.g. as generated from phage display library), or antibodies can be generated by immunization of a non-human animal, preferably a mouse, with an immunogen comprising a CD39 polypeptide, preferably a human CD39 polypeptide. The CD39 polypeptide may comprise the full length sequence of a human CD39 polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, i.e., a portion of the polypeptide comprising an epitope exposed on the surface of cells expressing a CD39 polypeptide. Such fragments typically contain at least about 7 consecutive amino acids of the mature polypeptide sequence, even more preferably at least about 10 consecutive amino acids thereof. Fragments typically are essentially derived from the extra-cellular domain of the receptor. In one embodiment, the immunogen comprises a wild-type human CD39 polypeptide in a lipid membrane, typically at the surface of a cell. In a specific embodiment, the immunogen comprises intact cells, particularly intact human cells, optionally treated or lysed. In another embodiment, the polypeptide is a recombinant CD39 polypeptide, optionally a CD39 polypeptide comprising glycosylation at residue N292.

The step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is herein incorporated by reference). Antibodies may also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in (Ward et al. Nature, 341 (1989) p. 544, the entire disclosure of which is herein incorporated by reference). Where human antibodies (having human FR and CDR sequences) are desired, a XenoMouse (Abgenix, Fremont, CA) or similar mouse can be used for immunization. A XenoMouse is an example of a murine host according that has had its immunoglobulin genes replaced by functional human immunoglobulin genes. Thus, antibodies produced by this mouse or in hybridomas made from the B cells of this mouse, are already humanized. The XenoMouse is described in United States Patent No. 6,162,963, which is herein incorporated in its entirety by reference. A range of other animals that have been engineered to express a human antibody repertoire can be used in similar fashion (see, e.g., Jakobovitz et al., Nature 362 (1993) 255), PCT patent publication nos. W0201 1/004192, WO2009/143472 and WO2010/039900 describing mice that express human antibodies; PCT publication no. WO2008/151081 ; and US patent publication no. US2010/122358 describing mice producing heavy-chain-only binding proteins, which are herein incorporated in their entirety by reference).

Accordingly, in one embodiment, provide is a method of preparing a CD39-binding protein from a library of candidates proteins. For example, a method of identifying, producing and/or testing an antibody which binds and neutralizes the enzymatic activity of CD39, can comprise the steps of:

(a) providing a plurality of candidate antigen-binding proteins comprising a V _H and a

V|_ (e.g. antibodies), and, optionally, determining the amino acid sequence of the V _H (and optionally further the V _L ), e.g. by determining the nucleic acid sequence that encodes them or by directly determining the amino acid sequence, and

(b) selecting (e.g. for further evaluation, for further processing, production of a quantity of, for use in treatment) an antigen-binding protein (or a V _H and a V _L thereof) that comprises a V _H and/or a V _L as described herein; and, optionally,

(c) introducing an amino acid substitution in a CDR of a V _H and/or a V _L selected in step (b), optionally wherein the substitution comprises replacing an aromatic amino acid residue with a non-aromatic amino acid residue; optionally wherein the substitution comprises replacing an amino acid residue present in a murine V gene with an amino acid residue present in a human V gene at the position. In one embodiment, the method comprises selecting a V _H that comprises a V _H CDR3 comprises at least two aromatic amino acid residues, optionally tyrosines, optionally further within the segment at Kabat residues 100-1 OOf (to the extent a residue is present at the particular position). In one embodiment, the method comprises selecting a V _H that comprises an amino acid sequence of formula I described herein and/or a V _L amino acid sequence of formula II described herein. In one embodiment, the method comprises selecting a V _H and/or V _L that comprises a human framework as described herein, e.g. a human V _H FR3 segment capable of binding the C- terminal domain (and, notably, the N292-linked glycosylation), optionally a FR3 (e.g., as a CDR2-FR3 segment) having the residues described herein. In one embodiment, the step of providing a plurality of candidate antigen-binding proteins (e.g. antibodies) comprises immunizing a non-human mammal with an immunogen comprising a CD39 polypeptide (e.g. of human origin) comprising N292 glycosylation. In one embodiment, the non-human animal is a rodent (e.g. mouse, rat) comprising native (e.g. mouse, rat) immunoglobulin V gene segments. In one embodiment, the non-human animal is a rodent (e.g. mouse, rat) comprising human immunoglobulin V gene segments. In one embodiment, the step of providing a plurality of candidate antigen-binding proteins (e.g. antibodies) comprises generating a phage display library encoding V _H and/or V _L domains.

Optionally, the method further comprises introducing one or more amino acid modifications (e.g. substitutions) in a CDR1 , 2 and/or CDR3, and/or in a FR1 , FR2, FR3 and/or FR4 of the V _H or V _L . Optionally, a modification confers decreased hydrophobicity, increased physical stability, e.g., in pharmaceutical formulations, decreased or low aggregation propensity under conditions found in pharmaceutical formulations, increased human amino acid content, and/or increased binding affinity for CD39.

Optionally, the method further comprises selecting a V _H comprising (or introducing into a VH) a human acceptor framework comprising residues at Kabat positions 59, 65, 67, 68, 69, 70 and/or 71 , and optionally further at residue 72, 72a and/or 72b that are capable of contacting the C-terminal domain of CD39. Optionally, the method comprises selecting a V _H human acceptor framework comprises residues at Kabat position 66-71 having the formula Xi X ₂ X3 X ₄ X5 Χβ, wherein represents any amino acid, X ₂ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity, X ₃ represents alanine or valine, or optionally leucine, optionally a hydrophobic residue, X ₄ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity and X ₅ represents serine, optionally further wherein and X ₆ represents any amino acid, optionally leucine, optionally alanine, valine or threonine.

Optionally, in any aspect, the method further comprises: bringing each of the antibodies selected in step into contact with CD39-expressing cells, optionally human B cells, optionally Ramos human lymphoma cells; and assessing production of AMP and/or disappearance of ADP, wherein a decrease in AMP generated and/or an decrease in disappearance of ADP indicates neutralization of ATPase activity; and selecting an antibody that results in a decrease of AMP generated and/or an decrease in disappearance of ADP (e.g. by at least 70%, optionally 80% or optionally 90%).

Optionally, in any aspect, the method further comprises: bringing each of said antigen-binding proteins (e.g. antibodies) into contact with a mutant CD39 polypeptide, optionally a CD39 polypeptides comprising a mutation at 1 , 2, 3 or 4 residues selected from the group consisting of Q96, N99, E143 and R147 (with reference to SEQ ID NO: 1 ), and assessing binding between the antigen-binding protein and the mutant CD39 polypeptide, relative to binding between the antigen-binding protein and a wild-type CD39 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 , and selecting an antigen-binding protein that has reduced binding to the mutant CD39 polypeptide, relative to binding between the antigen-binding protein and a wild-type CD39 polypeptide comprising the amino acid sequence of SEQ ID NO: 1 .

Optionally, in any aspect, the method further comprises: bringing each of said antigen-binding proteins (e.g. antibodies) into contact with a CD39 polypeptide in the presence of a control antigen binding protein (e.g. an antigen binding protein described herein, e.g. a parental murine antibody or any of mAbl to mAb39), and assessing binding between the antigen-binding protein and the CD39 polypeptide, and selecting an antigen- binding protein from that is capable of competing for binding to CD39 with control antigen binding protein.

Where an antibody is produced from mice (or other non-human) retaining murine variable region gene segments, the V _H and V _L may be humanized. The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is important to reduce antigenicity. Typically, according to the so-called "best-fit" method, the sequence of the variable domain of an antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the mouse (or other non-human) is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol. 151 , pp. 2296 (1993); Chothia and Lesk, J. Mol. 196, 1987, pp. 901 ). However, advantageously, acceptor frameworks can be used in the V _H and/or V _L that are not the closest to that of the mouse (or other non-human) variable- domain sequences of the particular murine (or other non-human) V _H or V _L , as long as the acceptor framework retains the amino acid residues enabling the V _H and/or V _L to retain the properties described herein. Similarly, amino acid modifications (e.g. substitutions) can be made in the highest similarity (or less than highest similarity) V _H and/or V _L acceptor frameworks as long as the acceptor framework retains the properties described herein.

One example of murine anti-CD39 V _H and V _L domain pairs that can be used as parental or starting V _H and V _L domains for humanization and/or other modifications, e.g. by making amino acid modifications in CDRs and/or FRs (e.g. substituting an FR) include the V _H shown below:

QIQLVQSGPEVKKPRETVKISCKASGYTFTHYGMNWVKQAPGKGLKWMGWINTYT GEPTYADDFKGRFAFSLEASASTAYLQINNLKNEDTATYFCARRRYEGNYVFYYFDYWGQ G TTLTVSS (SEQ ID NO: 2) and V|_ shown below:

DIQMTQSPASLSASVGETVTITCRASENIYSYFSWYQQKQGKSPQLLVYTAKTLAE GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYVTPYTFGGGTKLEIK

(SEQ ID NO: 3).

Another example of murine anti-CD39 V _H and V _L domain pairs that can be used as parental or starting V _H and V _L domains for humanization and/or other modifications, e.g. by making amino acid modifications in CDRs and/or other modifications, e.g. by making amino acid modifications in CDRs and/or FRs (e.g. substituting an FR) include the V _H shown below:

QVQLVQSGSELKKPGASVKVSCKASGYTFTHYGM ^* NWVRQAPGQGLEWMGWINT YTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARRRYEGNYVFYYFDYW G QGTTVTVSS (SEQ ID NO: 4)

and V|_ shown below:

DIQMTQSPSSLSASVGDRVTITCRASENIYSYFSWYQQKPGKAPKLLIYTAKTLAEGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVTPYTFGGGTKVEIK (SEQ ID NO: 5).

Another example of murine anti-CD39 V _H and V _L domain pairs that can be used as parental or starting V _H and V _L domains for humanization and/or other modifications, e.g. by making amino acid modifications in CDRs and/or other modifications, e.g. by making amino acid modifications in CDRs and/or FRs (e.g. substituting an FR) include the V _H shown below:

QIQLVQSGPELKKPGETVKISCKASGYTFRNYGMNWVKQAPGKGLKWMGWINTYT GEPTYADDFKGRFAFSLATSASTAYLQISNLKNEDTATYFCARKAYYGSNYYF DYWGQGTTLTVSS (SEQ ID NO: 17)

and V|_ shown below:

DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYT GVPDRFTGSGSGTDFTFTISTVQAEDLAVYYCQQHYTTPPYTFGGGTKLEIK (SEQ ID NO: 18).

A further example of an anti-CD39 V _H and V _L domain pairs that can be used as parental or starting V _H and V _L domains for humanization and/or other modifications, e.g. by making amino acid modifications in CDRs and/or FRs (e.g. substituting an FR) include the V _H shown below:

QVQLVQSGSELKKPGASVKISCKASGYTFTHYGMNWVRQAPGQGLEWMGWINTY TGELTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARRAYYRYDYVMDYWGQG T LVTVSS (SEQ ID NO: 19)

and V|_ shown below:

DIQMTQSPSSLSASVGDRVTITCKASHNVGTNVAWFQQKPGKAPKSLIYSASYRYS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYNNYPYTFGQGTKLEIK

(SEQ ID NO: 20). For example, the CDRs and/or FRs of these V _H and V _L according to Kabat numbering can serve as starting CDRs and/or FRs into which amino acid modifications can be introduced. Optionally, in any embodiment, an antibody of V _H or V _L thereof can be characterized as having decreased hydrophobicity, increased physical stability, e.g., in pharmaceutical formulations, decreased or low aggregation propensity under conditions found in pharmaceutical formulations, increased human amino acid content, and/or increased binding affinity for CD39 compared to a parental or reference antibody (or V _H or V _L thereof), e.g. compared to the antibody having the respective V _H and V _L (or CDRs thereof) of SEQ ID NOS: 2 and 3, SEQ ID NOS: 17 and 18, or SEQ ID NOS: 19 and 20.

Human V _H acceptor frameworks having the properties described herein can be selected such that they retain the ability to bind CD39 and/or the CDRs of the V _L via their Kabat FR regions as described herein.

Optionally, the method further comprises selecting a V _H comprising (or introducing into a VH) a human acceptor framework comprising residues at Kabat positions 67, 68, 69, 70 and/or 71 , and optionally further at residue 72, 72a and/or 72b that are capable of contacting the C-terminal domain of CD39. Optionally, the method comprises selecting a V _H human acceptor framework comprises residues at Kabat position 66-71 having the formula Χι X2 X ₃ X ₄ X ₅ X ₆ , wherein represents any amino acid, X ₂ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity, X ₃ represents alanine or valine, or optionally leucine, optionally a hydrophobic residue, X ₄ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity and X ₅ represents serine, optionally further wherein and X ₆ represents any amino acid, optionally leucine, optionally alanine, valine or threonine.

In one embodiment, the V _H FR3 (according to Kabat) comprises residues at Kabat positions 72, 72a and 72b having the formula Xi X ₂ X3 , wherein represents aspartic acid, glutamic acid or alanine, X ₂ represents any amino acid, optionally alanine or threonine, or a conservative substitution thereof, and X ₃ represents serine, optionally alanine, or a conservative substitution thereof.

In one embodiment, the V _H comprises the FR3 signature sequence FVFSL at Kabat positions 67-71 . In one embodiment, the V _H comprises a human acceptor framework or portion thereof (e.g. an FR3 domain) that naturally comprises the amino acid sequence FVFSL at Kabat positions 67-71. In another embodiment, the V _H comprises a human acceptor framework or portion thereof (e.g. an FR3 domain) that is comprises one or more amino acid modifications (e.g., one or more substitution(s) at Kabat positions 67-71 ) and comprises the amino acid sequence FVFSL at Kabat positions 67-71 . Optionally, the method further comprises selecting a V _H comprising (or introducing into a VH) a human acceptor framework FR1 comprising a residues at Kabat position 30 capable of contacting the N-terminal domain of CD39, optionally wherein the residue is a threonine or optionally a serine, or optionally an arginine.

Human V _L acceptor frameworks having the properties described herein can be selected such that they retain the ability to bind CDR3 of the V _H via their Kabat FR region (e.g. the residue at Kabat position 49) as described herein. For example, a human light chain acceptor framework (e.g. Kabat FR2) is selected that has an aromatic amino acid residue, optionally a tyrosine, optionally a phenylalanine, at Kabat position 49.

Exemplary anti-CD39 V _H and V _L amino acid sequences comprising amino acid modifications in CDRs to improve their pharmacological properties are incorporated into antibodies mAbl to mAb28, as well as mAbs 36-38, whose sequence (by reference to the respective SEQ ID NOS) is provided below in Table 1 :

Table 1

Antibody VH SEQ ID VL SEQ ID

NO NO

mAbl 4 10

mAb2 4 1 1

mAb3 4 12

mAb4 4 13

mAb5 4 14

mAb6 4 15

mAb7 4 16

mAb8 6 10

mAb9 6 1 1

mAb10 6 12

mAb1 1 6 13

mAb12 6 14

mAb13 6 15

mAb14 6 16

mAb15 7 10

mAb16 7 1 1

mAb17 7 12

mAb18 7 13

mAb19 7 14 mAb20 7 15

mAb21 7 16

mAb22 8 10

mAb23 8 1 1

mAb24 8 12

mAb25 8 13

mAb26 8 14

mAb27 8 15

mAb28 8 16

mAb29 9 10

mAb30 9 1 1

mAb31 9 12

mAb32 9 13

mAb33 9 14

mAb34 9 15

mAb35 9 16

mAb36 6 5

mAb37 7 5

mAb38 8 5

mAb39 9 5

An exemplary binding molecule or antigen-binding fragment thereof capable of binding to and inhibiting the activity of CD39 may comprise a V _H and a V _L , wherein the V _H comprises:

optionally, a FR1 comprising a residue that is capable of contacting CD39, optionally wherein the residue is at Kabat position 30, optionally wherein the residue is a threonine, a CDR1 comprising a residue at Kabat position 33, optionally at both positions 31 and 33, that is capable of contacting CD39;

a CDR2 comprising a residue at any 1 , 2, 3, 4, 5 of 6 of Kabat positions 50, 52, 52a, 53, 54 and 56 that are capable of contacting CD39, optionally wherein the residue at position 53 comprises an aromatic ring, optionally tyrosine;

a FR3 comprising a residue at any 1 , 2, 3, 4 of 5 of Kabat positions 67, 68, 69, 70 and 71 capable of contacting with CD39, optionally wherein FR3 further comprises a residue at any 1 , 2 of 3 of Kabat positions 72, 72a and 72b capable of contacting with CD39, and a CDR3 comprising a residue at any 1 , 2 or more of Kabat positions 100, 100b, 100c, 100d, 100e and/or 10Of (to the extent a residue is present at the particular position) that is capable of contacting CD39, wherein the residue(s) comprise an aromatic ring, optionally tyrosine. Optionally, the CDR3 comprises a residue at any 1 , 2 or more of Kabat positions 100, 100b, 100c, 100d, 100e and/or 100f (to the extent a residue is present at the particular position) that is capable of contacting the V _L , wherein the residue comprises an aromatic ring.

Heavy chain CDR1 (and FR1 )

An exemplary CDR1 (optionally together with one or more residues in the FR1 ) binds the N-terminal domain of CD39. A V _H can for example have CD39 contact residues at Kabat positions 30 (within the Kabat FR1 , adjacent to the CDR1 ); residue 30 may contact residue Q96 of CD39. In one embodiment, a V _H can for example have CD39 contact residues at Kabat position 31 (within the Kabat CDR1 ). In one embodiment, a V _H can for example have CD39 contact residues at Kabat position 32 (within the Kabat CDR1 ). In one embodiment, a V _H can for example have CD39 contact residues at Kabat position 33 (within the Kabat CDR1 ).

In one embodiment, a V _H comprises a CDR1 wherein the residues at Kabat position

31 , 32 and 33 have the formula Χι X2 X ₃ , wherein represents any amino acid, optionally a histidine or asparagine, or optionally a conservative substitution thereof, X ₂ represents any amino acid, optionally an aromatic residue, optionally a tyrosine or a conservative substitution thereof, or optionally an amino acid other than proline or glycine, and X ₃ represents glycine. Optionally, the residues at Kabat positions 32, 34 and/or 35 are identical to the corresponding residue in the human acceptor sequence of the V _H .

In one embodiment, the V _H comprises a CDR1 wherein the residues at Kabat position 31 to 35 have the formula Xi X ₂ X3 X ₄ X5, wherein represents histidine or asparagine, or a conservative substitution thereof, X ₂ represents any amino acid, optionally a tyrosine, or a conservative substitution thereof, X ₃ represents glycine, or a conservative substitution thereof, X ₄ represents any amino acid, optionally a methionine, or a conservative substitution thereof, and X ₅ represents any amino acid, optionally an asparagine, or a conservative substitution thereof. In one embodiment, the CDR1 comprises an amino acid sequence HYGMN (SEQ ID NO: 21 ), optionally comprising one or two amino acid substitutions. In one embodiment, the Kabat positions 31 -35 have an amino acid sequence that differs (e.g. by one or more amino acid residues) from the amino acid sequence HYGMN.

Heavy chain CDR2-FR3 segment

A CDR2 (e.g. according to Kabat) can be capable of contacting the N-terminal domain of CD39 (e.g. via residues within the segment of Kabat positions 50-56), and can further comprise residues capable of contacting the C-terminal domain of CD39 (together with residues of the Kabat FR3 domain, e.g. within the segment of Kabat positions 59-71 or optionally 59-72b).

For example, a CDR2 can have a residue at Kabat position 50 capable of contacting CD39. In one embodiment, a CDR2 can have a residue at Kabat position 52 capable of contacting CD39. In one embodiment, a CDR2 can have a residue at Kabat position 52a capable of contacting CD39. In one embodiment, a CDR2 can have a residue at Kabat position 53 capable of contacting CD39, optionally an aromatic residue. In one embodiment, CDR2 can have a residue at Kabat position 54 capable of contacting CD39. In one embodiment, a CDR2 can have a residue at Kabat position 56 capable of contacting CD39.

In one embodiment, the V _H comprises a CDR2 wherein the residues at Kabat position

50-56 have the formula Xi X ₂ X3 X ₄ X5 Χβ X7 Xs, wherein represents tryptophan, X ₂ represents any amino acid, optionally an isoleucine, X ₃ represents asparagine or optionally glutamine, X ₄ represents threonine, X ₅ represents tyrosine or optionally phenylalanine, or optionally a non-aromatic residue, optionally glutamic acid, X ₆ represents any amino acid, optionally threonine, optionally serine, optionally asparagine, alanine or glycine, optionally residues other than large or hydrophobic resides, X ₇ represents any amino acid, optionally glycine, optionally alanine, serine, threonine, asparagine or glutamine, optionally residues other than aspartic acid or glutamic acid, optionally residues other than lysine or arginine, and X ₈ represents glutamic acid, optionally aspartic acid. Optionally, the residues at Kabat positions 51 , 55, 57, 58, 59, 60, 61 , 62, 63, 64 and/or 65 are identical to the corresponding residue in the human acceptor sequence of the V _H . Optionally, the residues at Kabat positions 57 to 65 are at least 60%, 70%, 80%, 90% identical, or 100% identical, to the corresponding residue in the human acceptor sequence of the V _H . Optionally, the residues at Kabat positions 57 to 65 comprise one, two or three amino acid differences compared to the human acceptor sequence of the VH. In one embodiment, the Kabat positions 50-56 have an amino acid sequence WINTYTGE (SEQ ID NO: 22), optionally comprising one or two amino acid substitutions. In one embodiment, the residue at Kabat position 61 is a glutamine (Q). In one embodiment, the residue at Kabat position 61 is an aspartic acid (D). In one embodiment, the Kabat positions 50-65 have an amino acid sequence WINTX ₁ TGEPTYAX ₂ DFKG (SEQ ID NO: 23), WINT X ₁ TGELTYAX ₂ DFKG (SEQ ID NO: 24), wherein X-i is an amino acid residue other than tyrosine, optionally a non-aromatic residue, optionally a serine, and wherein X ₂ is an amino acid residue other than aspartic acid, optionally a glutamine, optionally wherein the positions 50-65 further comprising one or two amino acid substitutions. In another embodiment, the Kabat positions 50-65 have an amino acid sequence that differs (e.g. by one or more amino acid residues) from the amino acid sequence WINTYTGEPTYADDFKG (SEQ ID NO: 25) and/or WINTYTGELTYADDFKG (SEQ ID NO: 26).

In one embodiment, the V _H FR3 (according to Kabat) comprises residues that are capable of contacting amino acid residues in the C-terminal domain of CD39, optionally further wherein residues within Kabat positions 59-71 contact the glycan at residue N292 of CD39. In one embodiment, the V _H Kabat FR3 comprises residues at Kabat positions 67, 68, 69, 70 and/or 71 , and optionally further at residue 72, 72a and/or 72b that are capable of contacting the C-terminal domain of CD39, e.g. including amino acid resides in CD39 and/or the glycan at N292 of the CD39 polypeptide.

In one embodiment, the Kabat positions 50-71 have an amino acid sequence

YADDFKGRFAFSL (SEQ ID NO: 27) or YADDFKGRFVFSL (SEQ ID NO: 28), optionally comprising one or two amino acid substitutions, or YAX-i DFKGRFAFSL (SEQ ID NO: 29) or YAXi DFKGRFVFSL (SEQ ID NO: 30) wherein Xi is an amino acid residue other than aspartic acid, optionally a glutamine.

For example, a CDR2 can have a residue at Kabat position 67 capable of contacting the C-terminal domain of CD39. In one embodiment, a CDR2 can have a residue at Kabat position 68 capable of contacting CD39. In one embodiment, a CDR2 can have a residue at Kabat position 69 capable of contacting CD39. In one embodiment, a CDR2 can have a residue at Kabat position 70 capable of contacting CD39. In one embodiment, a CDR2 can have a residue at Kabat position 71 capable of contacting CD39.

In one embodiment, FR3 (e.g. the N-terminal segment of the Kabat FR3) comprises residues at Kabat position 66-71 having the formula Χι X2 X ₃ X4 X ₅ X ₆ , wherein represents any amino acid, X ₂ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity, X ₃ represents alanine or valine, or optionally leucine, optionally a hydrophobic residue, X ₄ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity and X ₅ represents serine, optionally further wherein and X ₆ represents any amino acid, optionally leucine, optionally alanine, valine or threonine.

In one embodiment, the V _H FR3 (according to Kabat) comprises residues at Kabat positions 72, 72a and 72b having the formula Xi X ₂ X3 , wherein represents aspartic acid, glutamic acid or alanine, X ₂ represents any amino acid, optionally alanine or threonine, or a conservative substitution thereof, and X ₃ represents serine, optionally alanine, or a conservative substitution thereof.

In one embodiment, the V _H comprises the FR3 signature sequence FVFSL at Kabat positions 67-71 . In one embodiment, the V _H comprises a human acceptor framework or portion thereof (e.g. an FR3 domain) that naturally comprises the amino acid sequence FVFSL at Kabat positions 67-71. In another embodiment, the V _H comprises a human acceptor framework or portion thereof (e.g. an FR3 domain) that is comprises one or more amino acid modifications (e.g., one or more substitution(s) at Kabat positions 67-71 ) and comprises the amino acid sequence FVFSL at Kabat positions 67-71 .

Heavy chain CDR3

In one embodiment, the V _H comprises a CDR3 (e.g. according to Kabat) capable of contacting the N-terminal of CD39, optionally capable of contacting the N-terminal domain of CD39 and the V _L , optionally wherein the CDR3 comprises an aromatic residue (e.g. a tyrosine, a phenylalanine) that is capable of binding an amino acid residue in the N-terminal domain of CD39 and a second aromatic amino acid residue (e.g. a tyrosine, a phenylalanine) that is capable of contacting an amino acid residue in the V _L .

For example, a CDR3 can have a residue at Kabat position 95 capable of contacting CD39. In one embodiment, a CDR3 can have a residue at Kabat position 99 capable of contacting CD39. In one embodiment, a CDR3 can have a residue at Kabat position 100b capable of contacting CD39. In one embodiment, a CDR3 can have a residue at Kabat position 100d capable of contacting CD39, optionally an aromatic residue. In one embodiment, CDR3 can have a residue at Kabat position 100f capable of contacting CD39.

Optionally the residues at Kabat position 95-102 have the formula Xi X2 X3 X4 X5 X6 X7 Xe Xg X10 X9 X10 X11 X12 X13 X14! wherein!

- Xi represents arginine or lysine, or optionally a conservative substitution thereof,

X ₂ represents any amino acid, optionally arginine, optionally lysine or alanine, or optionally a conservative substitution thereof,

X ₃ represents any amino acid residue, optionally a residue comprising an aromatic ring, optionally a tyrosine,

X ₄ represents any amino acid, optionally glutamic acid or tyrosine, or optionally a conservative substitution thereof, or amino acid residues other than proline or glycine,

X ₅ represents glycine, optionally arginine, or optionally a conservative substitution thereof,

X ₆ represents any amino acid, optionally asparagine, serine or tyrosine, or optionally a conservative substitution thereof,

X ₇ represents any amino acid, optionally tyrosine, asparagine or aspartic acid, or optionally a conservative substitution thereof, optionally serine, optionally an amino acid residue other than proline or glycine, optionally a residue other than an aromatic residue (e.g. other than a tyrosine), X ₈ represents valine or optionally alanine, isoleucine or leucine, optionally an aromatic amino acid, optionally tyrosine,

X ₉ represents any amino acid, optionally an aromatic amino acid, optionally phenylalanine, optionally tyrosine, optionally valine or a conservative substitution thereof,

X-io represents tyrosine, optionally phenylalanine, optionally methionine, or optionally a conservative substitution thereof,

Xii is absent or represents any amino acid, optionally tyrosine, optionally phenylalanine, optionally phenylalanine, or optionally a conservative substitution thereof, optionally an amino acid residue other than P, G, E or D, or other than a small hydrophobic residues (e.g. T, S),

X-I2 is absent or represents any amino acid, optionally phenylalanine, or optionally a conservative substitution thereof,

Xi ₃ represents any amino acid, optionally aspartic acid, or optionally a conservative substitution thereof, optionally a serine, optionally a threonine, optionally a glutamic acid, optionally an asparagine, optionally a residue other than a large and hydrophobic residue, and

X-I4 represents any amino acid, optionally tyrosine or optionally a conservative substitution thereof, optionally an aromatic amino acid, optionally a non-aromatic amino acid.

Optionally, the residues at Kabat positions 96, 97, 98, 100, 100a, 100c, 100e, 100f, 101 and/or 102 are identical to the corresponding residue in the human acceptor sequence of the V _H . In one embodiment, the Kabat positions 95-1 OOf are present and have an amino acid sequence RRYEGNYVFYYF (SEQ ID NO: 31 ) or RRYEGNSVFYYF (SEQ ID NO: 32), optionally comprising one, two, three, four or five amino acid substitutions. In one embodiment, the Kabat positions 95-1 OOd are present and have an amino acid sequence KAYYGSNYYF (SEQ ID NO: 33) or RAYYRYDYVM (SEQ ID NO: 34), optionally comprising one, two, three, four or five amino acid substitutions. In one embodiment, the residue at Kabat position 101 is an aspartic acid (D). In another embodiment, the Kabat positions 95- 102 have an amino acid sequence that differs (e.g. by one or more amino acid residues) from the amino acid sequence RRYEGNYVFYYFDY, KAYYGSNYYFDY and/or RAYYRYDYVMDY.

An exemplary V _H can comprise:

(a)a CDR1 (e.g. according to Kabat) capable of contacting the N-terminal domain of CD39, optionally wherein the residues at Kabat position 31 , 32 and 33 have the formula Xi X ₂ X3, wherein X-i represents any amino acid, optionally a histidine or asparagine, or optionally a conservative substitution thereof, X ₂ represents any amino acid, optionally an aromatic residue, optionally a tyrosine or a conservative substitution thereof, or optionally an amino acid other than proline or glycine, and X ₃ represents glycine;

(b) a CDR2 (e.g. according to Kabat) capable of contacting the C-terminal domain of

CD39, optionally wherein the residues at Kabat position 50-56 having the formula X2 X3 X4 X5 X6 X7 X8, wherein Xi represents tryptophan, X ₂ represents any amino acid, optionally an isoleucine, X ₃ represents asparagine or optionally glutamine, X ₄ represents threonine, X ₅ represents tyrosine or optionally phenylalanine, or optionally a non-aromatic residue, optionally glutamic acid, X ₆ represents any amino acid, optionally threonine, optionally serine, optionally asparagine, alanine or glycine, optionally residues other than large or hydrophobic resides, X ₇ represents any amino acid, optionally glycine, optionally alanine, serine, threonine, asparagine or glutamine, optionally residues other than aspartic acid or glutamic acid, optionally residues other than lysine or arginine, and X ₈ represents glutamic acid, optionally aspartic acid;

(c) optionally, an FR3 comprising residues at Kabat position 66-71 having the formula

X ₂ X3 X ₄ X ₅ Χβ, wherein Xi represents any amino acid, X ₂ represents phenylalanine or another hydrophobic residue capable of maintaining the beta- strand position and V _H domain structure integrity, X ₃ represents alanine or valine, or optionally leucine, optionally a hydrophobic residue, X ₄ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity and X ₅ represents serine, optionally further wherein and X ₆ represents any amino acid, optionally leucine, optionally alanine, valine or threonine; and

(d) a CDR3 (e.g. according to Kabat) capable of contacting the N-terminal of CD39, optionally capable of contacting the N-terminal domain of CD39 and the V _L , optionally wherein the CDR3 comprises an aromatic residue (e.g. a tyrosine, a phenylalanine) that is capable of binding an amino acid residue in the N-terminal domain of CD39 and a second aromatic amino acid residue (e.g. a tyrosine, a phenylalanine) that is capable of contacting an amino acid residue in the V _L , optionally wherein the residues at Kabat position 95-102 have the formula Χι X2 X3 X4 X5 X6 X7 Χβ Χθ Xio X9 X10 X11 X12 X13 X14, wherein

Xi represents arginine or lysine, or optionally a conservative substitution thereof, X ₂ represents any amino acid, optionally arginine, optionally lysine or alanine, or optionally a conservative substitution thereof,

X ₃ represents any amino acid residue, optionally a residue comprising an aromatic ring, optionally a tyrosine,

- X ₄ represents any amino acid, optionally glutamic acid or tyrosine, or optionally a conservative substitution thereof, or amino acid residues other than proline or glycine,

X ₅ represents glycine, optionally arginine, or optionally a conservative substitution thereof,

- X ₆ represents any amino acid, optionally asparagine, serine or tyrosine, or optionally a conservative substitution thereof,

X ₇ represents any amino acid, optionally tyrosine, asparagine or aspartic acid, or optionally a conservative substitution thereof, optionally amino acid residues other than proline or glycine, optionally serine, optionally a residue other than an aromatic residue (e.g. other than a tyrosine),

X ₈ represents valine or optionally alanine, isoleucine or leucine, optionally an aromatic amino acid, optionally tyrosine,

X ₉ represents any amino acid, optionally an aromatic amino acid, optionally phenylalanine, optionally tyrosine, optionally valine or a conservative substitution thereof,

X-io represents tyrosine, optionally phenylalanine, optionally methionine, or optionally a conservative substitution thereof,

Xii is absent or represents any amino acid, optionally tyrosine, optionally phenylalanine, optionally phenylalanine, or optionally a conservative substitution thereof, optionally an amino acid residue other than P, G, E or D, or other thana small hydrophobic residues (e.g. T, S),

X-I2 is absent or represents any amino acid, optionally phenylalanine, or optionally a conservative substitution thereof,

Xi ₃ represents any amino acid, optionally aspartic acid, or optionally a conservative substitution thereof, optionally a serine, optionally a threonine, optionally a glutamic acid, optionally an asparagine, optionally a residue other than a large and hydrophobic residue, and

Xi ₄ represents any amino acid, optionally tyrosine or optionally a conservative substitution thereof, optionally an aromatic amino acid, optionally a non-aromatic amino acid. Optionally, the residue at Kabat position 30 (FR1 ) is a threonine. Another exemplary VH can comprise:

(a) a CDR1 (e.g. according to Kabat) capable of contacting the N-terminal domain of CD39, optionally wherein the residues at Kabat position 31 , 32 and 33 have the formula Xi X ₂ X3, wherein X-i represents any amino acid, optionally a histidine, optionally a conservative substitution thereof, X ₂ represents any amino acid, optionally an aromatic residue, optionally a tyrosine, or a conservative substitution thereof, or optionally an amino acid residue other that proline or glycine, and X ₃ represents glycine;

(b) a CDR2 (e.g. according to Kabat) capable of contacting the C-terminal domain of CD39, optionally wherein the residues at Kabat position 50-56 having the formula

X2 X3 X4 X5 X6 X7 X8, wherein Xi represents tryptophan, X ₂ represents any amino acid, optionally an isoleucine, X ₃ represents asparagine or optionally glutamine, X ₄ represents threonine, X ₅ represents tyrosine or optionally phenylalanine, or optionally a non-aromatic residue, optionally glutamic acid, X ₆ represents any amino acid, optionally threonine, optionally serine, optionally asparagine, alanine or glycine, optionally a residue other than large or hydrophobic resides, X ₇ represents any amino acid, optionally glycine, optionally alanine, serine, threonine, asparagine or glutamine, optionally a residue other than aspartic acid or glutamic acid, optionally a residue other than lysine or arginine, and X ₈ represents glutamic acid, optionally aspartic acid;

(c) optionally, an FR3 comprising residues at Kabat position 66-71 having the formula

X ₂ X3 X ₄ X ₅ Χβ, wherein Xi represents any amino acid, X ₂ represents phenylalanine or another hydrophobic residue capable of maintaining the beta- strand position and V _H domain structure integrity, X ₃ represents alanine or valine, or optionally leucine, optionally a hydrophobic residue, X ₄ represents phenylalanine or another hydrophobic residue capable of maintaining the beta-strand position and V _H domain structure integrity and X ₅ represents serine, optionally further wherein and X ₆ represents any amino acid, optionally leucine, optionally alanine, valine or threonine; and

(d) a CDR3 (e.g. according to Kabat) capable of contacting the N-terminal of CD39, optionally capable of contacting the N-terminal domain of CD39 and the V _L , optionally wherein the CDR3 comprises an aromatic residue (e.g. a tyrosine, a phenylalanine) that is capable of binding an amino acid residue in the N-terminal domain of CD39 and a second aromatic amino acid residue (e.g. a tyrosine, a phenylalanine) that is capable of contacting an amino acid residue in the V _L , optionally wherein the residues at Kabat position 95-102 have the formula Χι X2 X3 X4 X5 X6 X7 Χβ Χθ Xio X9 X10 X11 X12 X13 X14, wherein

Xi represents arginine or lysine, or optionally a conservative substitution thereof,

- X ₂ represents any amino acid, optionally arginine, optionally lysine, or optionally a conservative substitution thereof,

X ₃ represents any amino acid residue, optionally a residue comprising an aromatic ring, optionally a tyrosine,

X ₄ represents any amino acid, optionally glutamic acid, or optionally a conservative substitution thereof, or an amino acid residue other than proline or glycine,

X ₅ represents glycine, or optionally a conservative substitution thereof, X ₆ represents any amino acid, optionally asparagine, or optionally a conservative substitution thereof,

- X ₇ represents any amino acid, optionally tyrosine, asparagine or aspartic acid, or optionally a conservative substitution thereof, optionally serine, optionally an amino acid residue other than proline or glycine, optionally a residue other than an aromatic residue (e.g. other than a tyrosine),

X ₈ represents valine or optionally alanine, isoleucine or leucine, - X ₉ represents any amino acid, optionally an aromatic amino acid, optionally phenylalanine, optionally tyrosine,

X10 represents tyrosine, optionally phenylalanine,

X11 or represents any amino acid, optionally tyrosine, optionally phenylalanine, optionally phenylalanine, or optionally a conservative substitution thereof, optionally an amino acid residue other than P, G, E or D, or other than a small hydrophobic residue (e.g. T, S),

X12 or represents any amino acid, optionally phenylalanine, or optionally a conservative substitution thereof,

Xi ₃ represents any amino acid, optionally aspartic acid, or optionally a conservative substitution thereof, optionally a serine, optionally a threonine, optionally a glutamic acid, optionally an asparagine, optionally a residue other than a large and hydrophobic residue, and

X-I4 represents any amino acid, optionally tyrosine or optionally a conservative substitution thereof, optionally an aromatic amino acid, optionally a non-aromatic amino acid.

An exemplary V _L can comprise: a CDR1 comprising a residue at Kabat positions 31 , 32, 33 and/or 34 capable of contacting the CDR3 of the V _H ;

a FR2 comprising an aromatic residue, optionally a tyrosine, at Kabat position 49; and a CDR3 comprising a residue at Kabat positions 89 and/or 91 capable of contacting the CDR3 of the V _H .

Light chain CDR1

In one embodiment, the V _L comprises a CDR1 wherein the residues at Kabat positions 31- 34 have the formula Χι X2 X ₃ X4, wherein represents a serine or a threonine, or a conservative substitution thereof, X ₂ represents a tyrosine, alanine or asparagine, or a conservative substitution thereof, X ₃ represents phenylalanine or valine, and X ₄ represents serine or alanine, or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR1 wherein the residues at Kabat positions 31-34 have the formula Xi X ₂ X3 X ₄ , wherein represents a threonine or a conservative substitution thereof, X ₂ represents alanine or asparagine, or a conservative substitution thereof, X ₃ represents valine or a conservative substitution thereof, and X ₄ represents alanine or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR1 wherein the residues at Kabat position 24 to 34 have the formula Xi X2 X3 X4 X5 X6 X? XsXg X10 9 X10 X11 , wherein

Xi represents any amino acid, optionally arginine or lysine, or a conservative substitution thereof,

X ₂ represents any amino acid, optionally alanine, or a conservative substitution thereof,

X ₃ represents any amino acid, optionally serine, or a conservative substitution thereof,

X ₄ represents any amino acid, optionally glutamic acid, glutamine, or histidine, or a conservative substitution thereof,

X ₅ represents any amino acid, optionally asparagine, serine or aspartic acid, or a conservative substitution thereof,

X ₆ represents any amino acid, optionally isoleucine or valine, or a conservative substitution thereof,

X ₇ represents any amino acid, optionally tyrosine, serine or glycine, or a conservative substitution thereof,

X ₈ represents serine or threonine, or a conservative substitution thereof,

X ₉ represents tyrosine, alanine or asparagine, or a conservative substitution thereof, X1 ₀ represents phenylalanine or valine, or a conservative substitution thereof, and X11 represents serine or alanine, or a conservative substitution thereof. Optionally, the residues at Kabat positions 24, 25, 26, 27, 28, 29 and/or 30 are identical to the corresponding residue in the human acceptor sequence of the V _L . Optionally, the residues at Kabat positions 24 to 30 are at least 60%, 70%, 80%, 90% identical, or 100% identical, to the corresponding residue in the human acceptor sequence of the V _L . Optionally, the residues at Kabat positions 24 to 30 comprise one, two or three amino acid differences compared to the human acceptor sequence of the V _L . In one embodiment, the Kabat positions 24 to 30 have an amino acid sequence RASENIY (SEQ ID NO: 35), RASQSIY (SEQ ID NO: 36), RASQNIY (SEQ ID NO: 37), RASENIS (SEQ ID NO: 38), RASQSIS (SEQ ID NO: 39), RASESIY (SEQ ID NO: 40), KASQDVS (SEQ ID NO: 41 ) or KASHNVG (SEQ ID NO: 42), optionally comprising one, two or three amino acid substitutions. In one embodiment, the Kabat positions 31 -34 have an amino acid sequence SYFS, TAVA or TNVA. In one embodiment, the Kabat positions 24-34 have an amino acid sequence RASENIYSYFS (SEQ ID NO: 43), RASQSIYSYFS (SEQ ID NO: 44), RASQNIYSYFS (SEQ ID NO: 45), RASENISSYFS (SEQ ID NO: 46), RASQSISSYFS (SEQ ID NO: 47), RASESIYSYFS (SEQ ID NO: 48), KASQDVSTAVA (SEQ ID NO: 49) or KASHNVGTNVA (SEQ ID NO: 50), optionally comprising one, two or three amino acid substitutions. In another embodiment, the Kabat positions 24-34 have an amino acid sequence that differs (e.g. by one or more amino acid residues) from the amino acid sequence RASENIYSYFS KASQDVSTAVA and/or KASHNVGTNVA.

Light chain CDR2

In one embodiment, the V _L comprises a CDR2 that comprises a Kabat FR residue. In one embodiment, the residue at Kabat position 49 is an aromatic amino acid, optionally a tyrosine. In one embodiment, the residue at Kabat position 50 is a serine or threonine or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR2 wherein the residues at Kabat position

49-56 have the formula Xi X ₂ X3 X ₄ X5 Χβ X7 Xs, wherein

Xi represents an aromatic amino acid residue, optionally a tyrosine,

X ₂ represents threonine or serine, or a conservative substitution thereof,

X ₃ represents any amino acid, optionally alanine, or a conservative substitution thereof,

X ₄ represents any amino acid, optionally lysine or serine, or a conservative substitution thereof,

X ₅ represents any amino acid, optionally threonine or tyrosine, or a conservative substitution thereof,

X ₆ represents any amino acid, optionally leucine or arginine, or a conservative substitution thereof, X ₇ represents any amino acid, optionally alanine or tyrosine, or a conservative substitution thereof, and

X ₈ represents any amino acid, optionally glutamic acid, threonine or serine, or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR2 wherein the residues at Kabat position

50-56 have the formula Xi X2 X3 X4 X5 X6 X7, wherein:

Xi represents serine, or a conservative substitution thereof,

X ₂ represents alanine, or a conservative substitution thereof,

X ₃ represents serine, or a conservative substitution thereof,

X ₄ represents tyrosine, or a conservative substitution thereof,

X ₅ represents arginine, or a conservative substitution thereof,

X ₆ represents tyrosine, or a conservative substitution thereof, and

X ₇ represents threonine or serine, or a conservative substitution thereof. Optionally, the V|_ further comprises an aromatic residue, e.g. a tyrosine, at Kabat residue 49.

Optionally, the residues at Kabat positions 51 , 52, 53, 54, 55 and/or 56 are identical to the corresponding residue in the human acceptor sequence of the V _L . Optionally, the CDR comprises an amino acid substitution at Kabat position 56. Optionally, the residue at Kabat position 56 is a serine (S). Optionally, the residues at Kabat positions 51 to 56 are at least 60%, 70%, 80%, 90% identical, or 100% identical, to the corresponding residue in the human acceptor sequence of the V _L . Optionally, the residues at Kabat positions 51 to 56 comprise one, two or three amino acid differences compared to the human acceptor sequence of the V|_. In one embodiment, the Kabat positions 49 to 51 have an amino acid sequence YTA or YSA. In one embodiment, the Kabat positions 49 to 56 have an amino acid sequence YTAKTLAE (SEQ ID NO: 51 ), YTAKTLAS (SEQ ID NO: 52), YSASYRYT (SEQ ID NO: 53) or YSASYRYS (SEQ ID NO: 54), optionally comprising one, two or three amino acid substitutions. In another embodiment, the Kabat positions 50-56 have an amino acid sequence that differs (e.g. by one or more amino acid residues) from the amino acid sequence TAKTLAE, YSASYRYT and/or YSASYRYS.

Light chain CDR3

In one embodiment, the V _L comprises a CDR3 wherein the residues at Kabat position

89-91 have the formula Xi X ₂ X3, wherein represents any amino acid, optionally a glutamine or histidine, or a conservative substitution thereof, X ₂ represents any amino acid, optionally a glutamine or histidine, or a conservative substitution thereof, and X ₃ represents tyrosine or histidine, or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR3 wherein the residues at Kabat position

89 is a glutamine or histidine, or a conservative substitution thereof, the residue at position 91 is a tyrosine or histidine, or a conservative substitution thereof, the residue at position 95 is a proline, or a conservative substitution thereof, and the residue at position 96 is a tyrosine, or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR3 wherein the residues at Kabat position

89-97 have the formula Xi X ₂ X ₃ X ₄ X ₅ Xe X? Xs XgX-ιο, wherein

Xi represents glutamine or histidine, or a conservative substitution thereof,

X ₂ represents any amino acid, optionally glutamine or histidine, or a conservative substitution thereof,

X ₃ represents tyrosine or histidine, or a conservative substitution thereof,

X ₄ represents any amino acid, optionally tyrosine, serine or asparagine, or a conservative substitution thereof,

X ₅ represents any amino acid, optionally valine, threonine or asparagine, or a conservative substitution thereof,

X ₆ represents any amino acid, optionally threonine or tyrosine, or a conservative substitution thereof,

X ₇ represents any amino acid, optionally proline, or a conservative substitution thereof,

X ₈ is absent or represents any one or more amino acids, optionally a proline, or a conservative substitution thereof,

X ₉ represents any amino acid, optionally tyrosine, or a conservative substitution thereof, and

X-io represents any amino acid, optionally threonine, or a conservative substitution thereof.

In one embodiment, the V _L comprises a CDR3 wherein the residues at Kabat position 89-97 have the formula QXiHXzXsTPYT (SEQ ID NO: 55), wherein

Xi represents any amino acid, optionally glutamine or histidine, or a conservative substitution thereof,

X ₂ represents any amino acid, optionally serine or tyrosine, or a conservative substitution thereof, and

X ₃ represents any amino acid, optionally valine or threonine, or a conservative substitution thereof.

Optionally, the residues at Kabat positions 90, 92, 93, 94 and/or 97 are identical to the corresponding residue in the human acceptor sequence of the V _L . Optionally, the CDR comprises an amino acid substitution at Kabat position 92 and/or 93. Optionally, the residue at Kabat position 92 is a serine (S). Optionally, the residue at Kabat position 92 is a threonine (T). In one embodiment, the Kabat positions 89-97 have an amino acid sequence QHHYVTPYT (SEQ ID NO: 56), QHHSVTPYT (SEQ ID NO: 57), QHHSTTPYT (SEQ ID NO: 58), QHHYTTPYT (SEQ ID NO: 59), QQHYTTPPYT (SEQ ID NO: 60), HQYNNYPYT (SEQ ID NO: 61 ), optionally comprising one, two or three amino acid substitutions. In another embodiment, the Kabat positions 89-97 have an amino acid sequence that differs (e.g. by one or more amino acid residues) from the amino acid sequence QHHYVTPYT, QQHYTTPPYT and/or HQYNNYPYT.

In one embodiment, the V _L comprises:

a CDR1 wherein the residues at Kabat position 31 , 32, 33 and 34 have the formula Xi X2 X ₃ X4, wherein represents a threonine or a conservative substitution thereof, X ₂ represents alanine or asparagine, or a conservative substitution thereof, X ₃ represents valine or a conservative substitution thereof, and X ₄ represents alanine or a conservative substitution thereof;

a FR2 comprising an aromatic residue, optionally a tyrosine, at Kabat position 49; a CDR2 wherein the residue at Kabat position 50 is a serine or threonine or a conservative substitution thereof; and

a CDR3 wherein the residues at Kabat position 89 is a glutamine or histidine, or a conservative substitution thereof, the residue at position 91 is a tyrosine or histidine, or a conservative substitution thereof, optionally wherein the residue at position 95 is a proline, or a conservative substitution thereof, optionally wherein the residue at position 96 is a tyrosine, or a conservative substitution thereof.

In one aspect, provided is a CD39-binding protein comprising a V _H and a V _L domain, wherein the V _H comprises human heavy chain framework sequences and a heavy chain CDR1 , CDR2 and CDR3 (as defined by Kabat numbering) of any of mAbl to mAb28 or mAb36 to mAb39 as set forth in Table 1 ; and wherein the V _L comprises human light chain framework sequences and a light chain CDR1 , CDR2 and CDR3 (as defined by Kabat numbering) of the respective mAbl to mAb39 as set forth in Table 1 . In one aspect, provided is an antibody that binds a human CD39 polypeptide, wherein the antibody comprises: a heavy chain CDR1 , CDR2 and CDR3 (as defined by Kabat numbering) of any of mAbl to mAb28 or mAb36 to mAb39 as set forth in Table 1 ; a light chain CDR1 , CDR2 and CDR3 (as defined by Kabat numbering) of the respective mAbl to mAb39 as set forth in Table 1 , and human heavy and light chain framework sequences.

In another aspect, the anti-CD39 compound comprises a V _H domain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity) to the VH domain of any one of SEQ ID NOS: 6 to 9. In another aspect, the anti- CD39 antibody comprises a V _L domain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity) to the VL domain of any one or SEQ ID NOS: 10 to 16.

In one aspect, provided is a CD39-binding compound comprising a V _H and a V _L domain, wherein V _H comprises an amino acid sequence having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity) to the V _H of any of mAbl to mAb39 as set forth in Table 1 ; and wherein the V _L comprises an amino an acid sequence having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99%) to the V _L the respective mAbl to mAb28 or mAb36 to mAb39 as set forth in Table 1 .

The SEQ ID NOS corresponding to the amino acid sequences of the heavy chain variable region and the light chain variable region for each of the antibodies mAbl to mAb28 or mAb36 to mAb39 are listed in Table 1. In any of the embodiments herein, the antibodies mAbl to mAb28 or mAb36 to mAb39 can be characterized by the amino acid sequences and/or nucleic acid sequences encoding it. In one embodiment, the monoclonal antibody comprises the Fab or F(ab') ₂ portion of the any of mAbl to mAb28 or mAb36 to mAb39. Also provided is a monoclonal antibody that comprises the heavy chain variable region of the antibodies mAbl to mAb28 or mAb36 to mAb39 and the light chain variable region of the respective mAbl to mAb28 or mAb36 to mAb39 antibody. Optionally, provided is an antibody where any of the light and/or heavy chain variable regions comprising part or all of an antigen binding region of mAbl to mAb28 or mAb36 to mAb39 are fused to an immunoglobulin constant region of the human IgG type, optionally a human constant region, optionally a human lgG1 , lgG2, lgG3 or lgG4 isotype, optionally further comprising an amino acid substitution in the Fc domain, e.g. to add a functionality, to increase or decrease effector function (e.g. binding to human Fey receptors).

DNA encoding a V _H and/or a V _L of CD39-binding compound (e.g. antibody) can be prepared and placed in an appropriate expression vector for transfection into an appropriate host. The host is then used for the recombinant production of the compound, antibody, or variants thereof, such as a humanized version of that monoclonal antibody, active fragments of the antibody, chimeric antibodies comprising the antigen recognition portion of the antibody, or versions comprising a detectable moiety.

DNA encoding the compound can be readily isolated and sequenced using conventional procedures (e. g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). In one aspect, provided is a nucleic acid encoding a heavy chain or a light chain variable region of any embodiment herein. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. As described elsewhere in the present specification, such DNA sequences can be modified for any of a large number of purposes, e.g., for humanizing antibodies, producing fragments or derivatives, or for modifying the sequence of the antibody, e.g., in the antigen binding site in order to optimize the binding specificity of the antibody. In one embodiment, provided is an isolated nucleic acid sequence encoding a light chain and/or a heavy chain of an antibody, as well as a recombinant host cell comprising (e.g., in its genome) such nucleic acid. Recombinant expression in bacteria of DNA encoding the antibody is well known in the art (see, for example, Skerra et al., Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun, Immunol. 130, p. 151 (1992).

Once antibodies are identified that are capable of binding CD39 and/or having other desired properties, they will also typically be assessed, using methods such as those described herein, for their ability to bind to other polypeptides, including unrelated polypeptides. Ideally, the antibodies bind with substantial affinity only to CD39, and do not bind at a significant level to unrelated polypeptides, or other polypeptides of the NTPDase family. However, it will be appreciated that, as long as the affinity for CD39 is substantially greater (e.g., 10x, 100x, 500x, 1000x, 10,000x, or more) than it is for other, unrelated polypeptides), then the antibodies are suitable for use in the present methods.

Typically, an anti-CD39 antibody provided herein has an affinity for a CD39 polypeptide (e.g., a monomeric CD39 polypeptide as produced in the Examples herein) in the range of about 10 ⁴ to about 10 ¹¹ M ^"1 (e.g., about 10 ⁸ to about 10 ¹⁰ M ^"1 ). For example, in a particular aspect the disclosure provides Anti-CD39 antibody that have an average disassociation constant (K _D ) of less than 1 x 10 ^"9 M with respect to CD39, as determined by, e.g., surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device). In a more particular exemplary aspect, the disclosure provides anti- CD39 antibodies that have a KD of about 1 x 10 ^"8 M to about 1 x 10 ^"10 M, or about 1 x 10 ^"9 M to about 1 x 10 ^"11 M, for CD39.

Antibodies can be characterized for example by a mean K _D of no more than about (i.e. better affinity than) 100, 60, 10, 5, or 1 nanomolar, preferably sub-nanomolar or optionally no more than about 500, 200, 100 or 10 picomolar. K _D can be determined for example for example by immobilizing recombinantly produced human CD39 proteins on a chip surface, followed by application of the antibody to be tested in solution. In one embodiment, the method further comprises a step of selecting antibodies from that are capable of competing for binding to CD39 with control antibody.

Where the test antibodies have modifications in their V _H and/V _L , or are obtained from different source animals, a simple competition assay may be employed in which the control (any of mAbl to mAb28, for example) and test antibodies are admixed (or pre-adsorbed) and applied to a sample containing CD39 polypeptides. Protocols based upon western blotting and the use of BIACORE analysis are suitable for use in such competition studies.

In certain embodiments, one pre-mixes the control antibodies with varying amounts of the test antibodies (e.g., about 1 :10 or about 1 :100) for a period of time prior to applying to the CD39 antigen sample. In other embodiments, the control and varying amounts of test antibodies can simply be admixed during exposure to the CD39 antigen sample. As long as one can distinguish bound from free antibodies (e. g., by using separation or washing techniques to eliminate unbound antibodies) and control antibody from the test antibodies (e. g., by using species-specific or isotype-specific secondary antibodies or by specifically labelling control antibody with a detectable label) one can determine if the test antibodies reduce the binding of control antibody to the antigens, indicating that the test antibody recognizes substantially the same epitope as control antibody. The binding of the (labelled) control antibodies in the absence of a completely irrelevant antibody can serve as the control high value. The control low value can be obtained by incubating the labelled control antibodies with unlabelled antibodies of exactly the same type, where competition would occur and reduce binding of the labelled antibodies. In a test assay, a significant reduction in labelled antibody reactivity in the presence of a test antibody is indicative of a test antibody that recognizes substantially the same epitope, i.e., one that "cross-reacts" or competes with the labelled control antibody. Any test antibody that reduces the binding of control antibody to CD39 antigens by at least about 50%, such as at least about 60%, or more preferably at least about 80% or 90% (e. g., about 65-100%), at any ratio of control antibody:test antibody between about 1 :10 and about 1 :100 is considered to be an antibody that binds to substantially the same epitope or determinant as control antibody. Preferably, such test antibody will reduce the binding of control antibody to the CD39 antigen by at least about 90% (e.g., about 95%).

Competition can also be assessed by, for example, a flow cytometry test. In such a test, cells bearing a given CD39 polypeptide can be incubated first with control antibody, for example, and then with the test antibody labelled with a fluorochrome or biotin. The antibody is said to compete with control antibody if the binding obtained upon preincubation with a saturating amount of control antibody is about 80%, preferably about 50%, about 40% or less (e.g., about 30%, 20% or 10%) of the binding (as measured by mean of fluorescence) obtained by the antibody without preincubation with control antibody. Alternatively, an antibody is said to compete with control antibody if the binding obtained with a labelled control antibody antibody (by a fluorochrome or biotin) on cells preincubated with a saturating amount of test antibody is about 80%, preferably about 50%, about 40%, or less (e.g., about 30%, 20% or 10%) of the binding obtained without preincubation with the test antibody.

A simple competition assay in which a test antibody is pre-adsorbed and applied at saturating concentration to a surface onto which a CD39 antigen is immobilized may also be employed. The surface in the simple competition assay is preferably a BIACORE chip (or other media suitable for surface plasmon resonance analysis). The control antibody (e.g., any of mAbl to mAb28 for example) is then brought into contact with the surface at a CD39- saturating concentration and the CD39 and surface binding of the control antibody is measured. This binding of the control antibody is compared with the binding of the control antibody to the CD39-containing surface in the absence of test antibody. In a test assay, a significant reduction in binding of the CD39-containing surface by the control antibody in the presence of a test antibody indicates that the test antibody recognizes substantially the same epitope as the control antibody such that the test antibody "cross-reacts" with the control antibody. Any test antibody that reduces the binding of control antibody to a CD39 antigen by at least about 30% or more, preferably about 40%, can be considered to be an antibody that binds to substantially the same epitope or determinant as control antibody. Preferably, such a test antibody will reduce the binding of the control antibody to the CD39 antigen by at least about 50% (e. g., at least about 60%, at least about 70%, or more). It will be appreciated that the order of control and test antibodies can be reversed: that is, the control antibody can be first bound to the surface and the test antibody is brought into contact with the surface thereafter in a competition assay. Preferably, the antibody having higher affinity for the CD39 antigen is bound to the surface first, as it will be expected that the decrease in binding seen for the second antibody (assuming the antibodies are cross-reacting) will be of greater magnitude. Further examples of such assays are provided in, e.g., Saunal (1995) J. Immunol. Methods 183: 33-41 , the disclosure of which is incorporated herein by reference.

Determination of whether an antibody binds within an epitope region can be carried out in ways known to the person skilled in the art. As one example of such mapping/characterization methods, an epitope region for an anti-CD39 antibody may be determined by epitope "foot-printing" using chemical modification of the exposed amines/carboxyls in the CD39 protein. One specific example of such a foot-printing technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry) wherein a hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange occurs, wherein the backbone amide groups participating in protein binding are protected from back exchange and therefore will remain deuterated. Relevant regions can be identified at this point by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and/or electrospray ionization mass spectrometry. See, e. g., Ehring H, Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999) Engen, J. R. and Smith, D. L. (2001 ) Anal. Chem. 73, 256A-265A. Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), where typically the position of the signals in two-dimensional NMR spectra of the free antigen and the antigen complexed with the antigen binding peptide, such as an antibody, are compared. The antigen typically is selectively isotopically labeled with 15N so that only signals corresponding to the antigen and no signals from the antigen binding peptide are seen in the NMR-spectrum. Antigen signals originating from amino acids involved in the interaction with the antigen binding peptide typically will shift position in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids involved in the binding can be identified that way. See, e. g., Ernst Schering Res Found Workshop. 2004; (44): 149-67; Huang et al., Journal of Molecular Biology, Vol. 281 (1 ) pp. 61 -67 (1998); and Saito and Patterson, Methods. 1996 Jun; 9 (3): 516-24.

Epitope mapping/characterization also can be performed using mass spectrometry methods. See, e.g., Downard, J Mass Spectrom. 2000 Apr; 35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1 ; 71 (9): 1792-1801 . Protease digestion techniques also can be useful in the context of epitope mapping and identification. Antigenic determinant- relevant regions/sequences can be determined by protease digestion, e.g., by using trypsin in a ratio of about 1 :50 to CD39 or o/n digestion at and pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. The peptides protected from trypsin cleavage by the anti-CD39 binder can subsequently be identified by comparison of samples subjected to trypsin digestion and samples incubated with antibody and then subjected to digestion by e.g., trypsin (thereby revealing a footprint for the binder). Other enzymes like chymotrypsin, pepsin, etc., also or alternatively can be used in similar epitope characterization methods. Moreover, enzymatic digestion can provide a quick method for analyzing whether a potential antigenic determinant sequence is within a region of the CD39 polypeptide that is not surface exposed and, accordingly, most likely not relevant in terms of immunogenicity/antigenicity.

Site-directed mutagenesis is another technique useful for elucidation of a binding epitope. For example, in "alanine-scanning", each residue within a protein segment is replaced with an alanine residue, and the consequences for binding affinity measured. If the mutation leads to a significant reduction in binding affinity, it is most likely involved in binding. Monoclonal antibodies specific for structural epitopes (i.e., antibodies which do not bind the unfolded protein) can be used to verify that the alanine-replacement does not influence over- all fold of the protein. See, e.g., Clackson and Wells, Science 1995; 267:383-386; and Wells, Proc Natl Acad Sci USA 1996; 93:1-6. Electron microscopy can also be used for epitope "foot-printing". For example, Wang et al., Nature 1992; 355:275-278 used coordinated application of cryoelectron micros-copy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of a Fab-fragment on the capsid surface of native cowpea mosaic virus.

Other forms of "label-free" assay for epitope evaluation include surface plasmon resonance (SPR, BIACORE) and reflectometric interference spectroscopy (RifS). See, e.g., Fagerstam et al., Journal Of Molecular Recognition 1990;3:208-14; Nice et al., J. Chroma- togr. 1993; 646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37:3308-331 1 ; Kroger et al., Biosensors and Bioelectronics 2002; 17:937-944.

Anti-CD39 binding proteins can advantageously comprise an Fc domain, e.g., that is bound by human FcRn polypeptides and thereby provides stability in vivo. The Fc domain can be used in a form that can mediate ADCC and/or CDC (e.g. an Fc domain such as human lgG1 that has effector function, binds to human Fey receptors), and/or the antibodies can be conjugated to a moiety of interest (e.g. a cytotoxic moiety, a detectable moiety). In other embodiments, the Fc domain can be prepared such that it does not have substantial specific binding to human Fey receptors, e.g., any one or more of CD16A, CD16B, CD32A, CD32B and/or CD64). Proteins and antibodies having such Fc domains 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 as cell based assays, to assess Fc receptor binding are well known in the art, and are described in, e.g., WO 03/101485.

In one embodiment, the antibody can comprise one or more specific mutations in the Fc region that result in "Fc silent" antibodies that have 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 (EU numbering). In one embodiment, the antibody is an lgG4 and comprises one, two or three substitutions at residues 327, 330 and/or 331 (EU numbering). Examples of silent Fc lgG1 antibodies are the LALA mutant comprising L234A and L235A mutation in the lgG1 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 lgG1 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 (H268QA 309L/A330S/A331 S); 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/P331 S); 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 WO201 1/066501 ).

In one embodiment, the antibody can comprise one or more specific mutations in the Fc region that result in improved stability of an antibody of the disclosure, e.g. comprising multiple aromatic amino acid residues and/or having high hydrophobicity. 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/P331 S or (L234F/L235E/P331 S). Another example of such an Fc domain comprises substitutions at Kabat residues L234, L235, G237 and P331 (e.g., L234A/L235E/G237A/P331 S). 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 one embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234X-I substitution, a L235X ₂ substitution, and a P331 X ₃ substitution, wherein X-i is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, and X ₃ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X ₂ is glutamic acid or a conservative substitution thereof; optionally wherein X ₃ is a serine or a conservative substitution thereof. In another embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234X-I substitution, a L235X ₂ substitution, a G237X ₄ substitution and a P331X ₄ substitution, wherein X-i is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, X ₃ is any amino acid residue other than glycine, and X ₄ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X ₂ is glutamic acid or a conservative substitution thereof; optionally, X ₃ is alanine or a conservative substitution thereof; optionally X ₄ is a serine or a conservative substitution thereof. In another embodiment, the antibody comprises an Fc domain, optionally of human lgG1 isotype, comprising: a L234Xi substitution, a L235X ₂ substitution, a G237X ₄ substitution, G330X ₄ substitution, and a P331X ₅ substitution, wherein X-i is any amino acid residue other than leucine, X ₂ is any amino acid residue other than leucine, X ₃ is any amino acid residue other than glycine, X ₄ is any amino acid residue other than alanine, and X ₅ is any amino acid residue other than proline; optionally wherein X-i is an alanine or phenylalanine or a conservative substitution thereof; optionally wherein X ₂ is glutamic acid or a conservative substitution thereof; optionally, X ₃ is alanine or a conservative substitution thereof; optionally, X ₄ is serine or a conservative substitution thereof; optionally X ₅ is a serine or a conservative substitution thereof. In the shorthand notation used here, the format is: Wild type residue: Position in polypeptide: Mutant residue, wherein residue positions are indicated according to EU numbering according to Kabat.

In one embodiment, an 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):

A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K V D K R V E P K S C D K T H T C P P C P A P E A E G G P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A S I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO : 62)

In one embodiment, an 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):

A S T K G P S V F P L A P S s K S T S G G T A A L G c L V K D Y F P

E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L s S V

V T V P S S s L G T Q T Y I C N V N H K P s N T K V D K R V E P K S

C D K T H T c P P C P A P E F E G G P S V F L F P P K P K D T L M I

S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A

K T K P R E E Q Y N s T Y R V V S V L T V L H Q D W L N G K E Y K C

K V s N K A L P A S I E K T I S K A K G Q P R E P Q V Y T L P P S R

E E M T K N Q V S L T C L V K G F Y P S D I A V E w E S N G Q P E N

N Y K T T P P V L D s D G S F F L Y S K L T V D K s R W Q Q G N V F

S C S V M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO : 63)

In one embodiment, an 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):

A S T K G P S V F P L A P S s K S T S G G T A A L G c L V K D Y F P

E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L s S V

V T V P S S s L G T Q T Y I C N V N H K P s N T K V D K R V E P K S

C D K T H T c P P C P A P E A E G A P S V F L F P P K P K D T L M I

S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A

K T K P R E E Q Y N s T Y R V V S V L T V L H Q D w L N G K E Y K C

K V s N K A L P s S I E K T I S K A K G Q P R E P Q V Y T L P P S R

E E M T K N Q V s L T C L V K G F Y P S D I A V E w E S N G Q P E N

N Y K T T P P V L D s D G S F F L Y S K L T V D K s R W Q Q G N V F

S C S V M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO : 64)

In one embodiment, an 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):

A S T K G P S V F P L A P S S K S T S G G T A A L G C L V K D Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q T Y I C N V N H K P S N T K V D K R V E P K S C D K T H T C P P C P A P E A E G A P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A S I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K (SEQ ID NO : 65)

In one embodiment, the antibody comprises an Fc domain comprising an amino acid substitution that increases binding to human FcRn polypeptides in order to increase the in vivo half-life of the antibody. Exemplary mutations are described in Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691, the disclosure of which is incorporated herein by reference. Examples of substitutions used in antibodies of human lgG1 isotype are substitutions at residues M252, S254 and T256; substitutions at residues T250 and M428; substitutions at residue N434; substitutions at residues H433 and N434; substitutions at residues T307, E380 and N434; substitutions at residues T307, E380, and N434; substitutions at residues M252, S254, T256, H433, N434 and 436; substitutions at residue I253; substitutions at residues P257, N434, D376 and N434.

In one embodiment, the antibody comprises an Fc domain comprising an amino acid substitution that confers decreased sensitivity to cleavage by proteases. Matrix metalloproteinases (MMPs) represent the most prominent family of proteinases associated with tumorigenesis. While cancer cells can express MMPs, the bulk of the extracellular MMP is provided by different types of stromal cells that infiltrate the tumor and each produce a specific set of proteinases and proteinase inhibitors, which are released into the extracellular space and specifically alter the milieu around the tumor. The MMPs present in the tumor microenvironment can cleave antibodies within the hinge region and may thus lead to the inactivation of therapeutic antibodies that are designed to function within the tumor site. In one embodiment, the Fc domain comprising an amino acid substitution has decreased sensitivity to cleavage by any one, two, three or more (or all of) of the proteases selected from the group consisting of: GluV8, IdeS, gelatinase A (MMP2), gelatinase B (MMP-9), matrix metalloproteinase-7 (MMP-7), stromelysin (MMP-3), and macrophage elastase (MMP- 12). In one embodiment, the antibody decreased sensitivity to cleavage comprises an Fc domain comprising an amino acid substitution at residues E233-L234 and/or L235. In one embodiment, the antibody comprises an Fc domain comprising an amino acid substitution at residues E233, L234, L235 and G236. In one embodiment, the antibody comprises an Fc domain comprising an amino acid substitution at one or more residues 233-238, e.g., such that E233-L234-L235-G236 sequence is replaced by P233-V234-A235 (G236 is deleted). See, e.g., W099/58572 and WO2012087746, the disclosures of which are incorporated herein by reference.

An antigen-binding compound can at any desired stage be assessed for its ability to inhibit the enzymatic activity of CD39, notably to block the ATPase activity of CD39 and to reduce the production of ADP, AMP and/or adenosine by a CD39-expressing cell, and in turn restore the activity of and/or relieve the adenosine-mediated inhibition of lymphocytes.

The inhibitory activity (e.g., immune enhancing potential) of an antibody can be assessed for example, in an assay to detect the disappearance (hydrolysis) of ATP and/or the generation of AMP. An example of such an assay is assessing generation of AMP by detection AMP after incubating CD39-expressing cells (e.g., Ramos cells) with a test antibody, and measuring in supernatants the generation of AMP.

A decrease in hydrolysis of ATP into AMP, and/or a decrease in generation of AMP, in the presence of antibody indicate the antibody inhibits CD39. In one embodiment, an antibody preparation is capable of causing at least a 60% decrease in the enzymatic activity of a CD39 polypeptide, preferably at least a 70%, 80% or 90% decrease in the enzymatic activity of a CD39 polypeptide.

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

The antibody can be tested for its ability to internalize or to induce down-modulation of CD39, e.g., whether by internalization or induction of CD39 shedding from the cell surface. Whether an anti-CD39 antibody internalizes upon binding CD39 on a mammalian cell, or whether a CD39 polypeptide undergoes intracellular internalization (e.g., upon being bound by an antibody) can be determined. In other examples, to test internalization in vivo, the test antibody is labeled and introduced into an animal known to have CD39 expressed on the surface of certain cells. The antibody can be radiolabeled or labeled with fluorescent or gold particles, for instance. Animals suitable for this assay include a mammal such as a nude mouse that contains a human CD39-expressing B cells, T cells, TReg cells, tumor transplant or xenograft, or a mouse into which cells transfected with human CD39 have been introduced, or a transgenic mouse expressing the human CD39 transgene. Appropriate controls include animals that did not receive the test antibody or that received an unrelated antibody, and animals that received an antibody to another antigen on the cells of interest, which antibody is known to be internalized upon binding to the antigen. The antibody can be administered to the animal, e.g., by intravenous injection. At suitable time intervals, tissue sections of the animal can be prepared using known methods or as described in the experimental examples below, and analyzed by light microscopy or electron microscopy, for internalization as well as the location of the internalized antibody in the cell. For internalization in vitro, the cells can be incubated in tissue culture dishes in the presence or absence of the relevant antibodies added to the culture media and processed for microscopic analysis at desired time points. The presence of an internalized, labeled antibody in the cells can be directly visualized by microscopy or by autoradiography if radiolabeled antibody is used. Optionally, in microscopy, co-localization with a known polypeptide or other cellular component can be assessed; for example co-localization with endosomal/lysosomal marker LAMP-1 (CD107a) can provide information about the subcellular localization of the internalized antibody. Alternatively, in a quantitative biochemical assay, a population of cells comprising CD39-expressing cells are contacted in vitro or in vivo with a radiolabeled test antibody and the cells (if contacted in vivo, cells are then isolated after a suitable amount of time) are treated with a protease or subjected to an acid wash to remove un-internalized antibody on the cell surface. The cells are ground up and the amount of protease resistant, radioactive counts per minute (cpm) associated with each batch of cells is measured by passing the homogenate through a scintillation counter. Based on the known specific activity of the radiolabeled antibody, the number of antibody molecules internalized per cell can be deduced from the scintillation counts of the ground- up cells. Cells are "contacted" with antibody in vitro preferably in solution form such as by adding the cells to the cell culture media in the culture dish or flask and mixing the antibody well with the media to ensure uniform exposure of the cells to the antibody.

Fragments and derivatives of antibodies (which are encompassed by the term

"antibody" or "antibodies" as used in this application, unless otherwise stated or clearly contradicted by context) can be produced by techniques that are known in the art. "Fragments" comprise a portion of the intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F (ab') 2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide").

An anti-CD39 compound can be incorporated in a pharmaceutical formulation comprising in a concentration from 1 mg/ml to 500 mg/ml, wherein said formulation has a pH from 2.0 to 10.0. 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.

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

In another embodiment, 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.

In a another embodiment, 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.

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 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 peptide pharmaceutical formulation. 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.

Pharmaceutical compositions containing an antibody may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen. Administration of pharmaceutical compositions may be through several routes of administration, for example, subcutaneous, intramuscular, intraperitoneal, intravenous, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment.

Diagnosis and treatment of malignancies

Methods of treating an individual, notably a human patient, using a CD39 binding compound (e.g., anti-CD39 antibody) as described herein are also provided for. In one embodiment, the disclosure provides for the use of a CD39 binding compound (e.g., antibody) as described herein in the preparation of a pharmaceutical composition for administration to a human patient. Typically, the patient suffers from, or is at risk for, cancer or an infectious disease (e.g., a viral infection, bacterial infection).

For example, in one aspect, provided is a method of restoring or potentiating the activity of lymphocytes in a patient in need thereof, comprising the step of administering a neutralizing CD39 binding compound (e.g., anti-CD39 antibody) of the disclosure to said patient.

In one embodiment, the method directed at increasing the activity of lymphocytes (e.g., T cells) in patients having a disease in which increased lymphocyte activity is beneficial or which is caused or characterized by immunosuppression, immunosuppressive cells, or, e.g., adenosine generated by CD4 T cells, CD8 T cells, B cells). The methods will be particularly useful for example patients having a solid tumor in which it is suspected the tumor microenvironment (and CD39-mediated adenosine production therein) may contribute to lack of recognition by the immune system (immune escape). The tumor may, for example, be characterized by CD39-expressing immune cells, e.g., CD4 T cells, CD8 T cells, B cells.

More specifically, the methods and compositions are utilized for the treatment of a variety of cancers and other proliferative diseases, and infectious diseases. Because these methods operate by reducing adenosine that inhibits the anti-target cell (e.g., anti-tumor) activity of lymphocytes and possibly additionally by increasing ATP that can increase the anti-tumor activity of lymphocytes, they are applicable to a very broad range of cancers and infectious disease. Representative examples of cancers that can be treated include in particular solid tumors in which adenosine in the tumor microenvironment may play a strong role in suppressing the anti-tumor immune response. In one embodiment, a human patient treated with an anti-CD39 antibody has liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, breast cancer, lung cancer, non- small cell lung cancer (NSCLC), castrate resistant prostate cancer (CRPC), melanoma, uterine cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies including, for example, multiple myeloma, B- cell lymphoma, Hodgkin lymphoma/primary mediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers. The present disclosure is also applicable to treatment of metastatic cancers. Patients can be tested or selected for one or more of the above described clinical attributes prior to, during or after treatment.

In one embodiment, the anti-CD39 antibody is administered in an amount effective to achieve and/or maintain in an individual (e.g., for 1 , 2, 3, 4 weeks, and/or until the subsequent administration of antigen binding compound) a blood concentration of at least the EC ₅ o, optionally the EC70, optionally substantially the EC100, for neutralization of the enzymatic activity of CD39. In one embodiment, the active amount of anti-CD39 antibody is an amount effective to achieve the EC ₅₀ , optionally the EC ₇₀ , optionally substantially the EC-ioo, for neutralization of the enzymatic activity of CD39 in an extravascular tissue of an individual. In one embodiment, the active amount of anti-CD39 antibody is an amount effective to achieve (or maintain) in an individual the EC ₅₀ , optionally the EC ₇₀ , optionally substantially the EC100, for inhibition of neutralize the enzymatic activity of CD39.

Optionally, in one embodiment, in contrast to some antibodies that are directed to the depletion of CD39-expressing tumor cells by ADCC (which, e.g., can provide full efficacy at concentrations equal or substantially lower than that which provides receptor saturation), the anti-CD39 antibody does not exhibit substantial Fey receptor-mediated activity and is administered in an amount effective to neutralize the enzymatic activity of, optionally further CD39, without substantially causing down-modulation of CD39 expression, for a desired period of time, e.g., 1 week, 2 weeks, a month, until the next successive administration of anti-CD39 antibody.

In one embodiment, the anti-CD39 antibody is administered in an amount effective to achieve and/or maintain (e.g., for 1 , 2, 3, 4 weeks, and/or until the subsequent administration of anti-CD39 antibody) in an individual a blood concentration of at least the EC ₅₀ , optionally the EC ₇₀ , optionally substantially the EC1 ₀₀ , for inhibition of CD39-mediated catabolism of ATP to AMP (e.g., by assessing neutralization of ATPase activity in B cells, optionally Ramos lymphoma cells, by quantifying hydrolysis of ATP to AMP). In one embodiment, the amount of anti-CD39 antibody is an amount effective to achieve (or maintain), in an extravascular tissue of an individual, the EC ₅ o, optionally the EC70, optionally substantially the EC100, for inhibition of CD39-mediated catabolism of ATP to AMP.

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

In one embodiment, provided is a method for treating or preventing cancer in an individual, the method comprising administering to the individual an anti-CD39 antibody in an amount that achieves or maintains for a specified period of time a concentration in circulation, optionally in an extravascular tissue of interest (e.g., the tumor or tumor environment), that is higher than the EC ₅₀ , optionally EC ₇₀ or optionally Edoo, for binding to CD39-expressing cells (e.g., as assessed by titrating anti-CD39 antibody on CD39- expressing cells, for example Ramos cells). Optionally the concentration achieved is at least 20%, 50% or 100% higher than the EC ₅ o, optionally EC70 or optionally EC100, for binding to CD39-expressing cells.

The EC50, 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 Ramos cells by quantifying hydrolysis of ATP to AMP (or ATP to downstream adenosine). "EC ₅₀ " 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. "EC ₇₀ " 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. "EC1 ₀₀ " 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 some embodiments, particularly for the treatment of solid tumors, the concentration achieved is designed to lead to a concentration in tissues (outside of the vasculature, e.g., in the tumor or tumor environment) that corresponds to at least the EC ₅₀ or EC ₇₀ for neutralization of the enzymatic activity, optionally at about, or at least about, the ECioo-

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

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

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

In one embodiment, provided is a method for treating or preventing a disease (e.g., a cancer, a solid tumor, a hematological tumor) in an individual, the method comprising administering to an individual having disease (e.g., a cancer, a solid tumor) an anti-CD39 antibody that neutralizes the enzymatic activity of CD39 for at least one administration cycle, the administration cycle comprising at least a first and second (and optionally a 3 ^rd , 4 ^th , 5 ^th , 6 ^th , 7 ^th and/or 8 ^th or further) administration of the anti-CD39 antibody, wherein the anti-CD39 antibody is administered in an amount effective to achieve, or to maintain between two successive administrations, a blood (serum) concentration of anti-CD39 antibody of at least 0.1 μg/ml, optionally at least 0.2 μg/ml, optionally at least 1 μg/ml, or optionally at least 2 μg/ml (e.g., for treatment of a hematological tumor), or optionally at least about 1 μg/ml, 2 μg/ml, 10 μg/ml, or 20 μg/ml, e.g., between 1 -100 μg/ml, 1 -50 μg/ml, 1 -20 μg/ml, or 1 -10 μg/ml (e.g., for treatment of a solid tumor, for treatment of a hematological tumor). In one embodiment, a specified continuous blood concentration is maintained, wherein the blood concentration does not drop substantially below the specified blood concentration for the duration of the specified time period (e.g., between two administrations of antibody, number of weeks, 1 week, 2 weeks, 3 weeks, 4 weeks), i.e. although the blood concentration can vary during the specified time period, the specified blood concentration maintained represents a minimum or "trough" concentration. In one embodiment, a therapeutically active amount of an anti-CD39 antibody is an amount of such antibody capable of providing (at least) the EC ₅₀ concentration, optionally the EC ₇₀ concentration optionally the Edoo concentration, in blood and/or in a tissue for neutralization of the enzymatic activity of CD39 for a period of at least about 1 week, about 2 weeks, or about one month, following administration of the antibody.

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

Prior to or during a course of treatment with an anti-CD39 antibody of the disclosure, adenosine, ADP and/or AMP levels can also be assessed within and/or adjacent to a patient's tumor to assess whether the patient is benefitting from treatment with an anti-CD39 antibody. Decreased levels of adenosine, ATP, ADP and/or AMP compared following an administration (or dosing of antibody) compared to levels prior to treatment (or dosing of antibody) may indicate an individual is benefitting from treatment with an anti-CD39 antibody of the disclosure (including but not limited to an antibody that inhibits substrate-bound CD39). Optionally, if a patient is benefitting from treatment with the anti-CD39 antibody, methods can further comprise administering a further dose of the anti-CD39 antibody to the patient (e.g., continuing treatment).

In one embodiment, assessing adenosine, ADP and/or AMP levels within and/or adjacent to a patient's tumor the tissue sample comprises obtaining from the patient a biological sample of a human tissue selected from the group consisting of tissue from a cancer patient, e.g., cancer tissue, tissue proximal to or at the periphery of a cancer, cancer adjacent tissue, adjacent non-tumorous tissue or normal adjacent tissue, and detecting adenosine, ATP, ADP and/or AMP levels within the tissue. The levels from the patient can be comparing the level to a reference level, e.g., corresponding to a healthy individual. 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-expressing cells (or adenosine, ATP, ADP and/or AMP) in the tumor environment, optionally within the tumor and/or within adjacent tissue, and

b) upon a determination that tumor environment comprises CD39-expressing cells (or adenosine, ATP, ADP and/or AMP), optionally at a level that is increased compared to a reference level (e.g., corresponding to a healthy individual or an individual not deriving substantial benefit from an anti-CD39 antibody), administering to the individual an anti-CD39 antibody.

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 cells in circulation that express vascular CD39 (e.g., from a blood sample), and

b) upon a detection of cells in circulation that express vascular CD39, optionally at a level that is increased compared to a reference level (e.g., corresponding to a healthy individual or an individual not deriving substantial benefit from an anti-CD39 antibody), administering to the individual an anti-CD39 antibody.

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 cells that express vascular CD39 in the tumor environment, optionally within the tumor and/or within adjacent tissue, and

b) upon a detection of cells in the tumor environment that express vascular CD39, optionally at a level that is increased compared to a reference level (e.g., corresponding to a healthy individual or an individual not deriving substantial benefit from an anti-CD39 antibody), administering to the individual an anti-CD39 antibody.

Optionally, in any of the methods, detecting CD39-expressing cells (or adenosine,

ATP, ADP and/or AMP) within the tumor environment comprises obtaining from the individual a biological sample that comprises cancer tissue and/or tissue proximal to or at the periphery of a cancer (e.g., cancer adjacent tissue, adjacent non-tumorous tissue or normal adjacent tissue), and detecting levels of CD39-expressing cells (or adenosine, ATP, ADP and/or

AMP). CD39-expressing cells may comprise, for example, tumor cells, CD4 T cells, CD8 T cells, TReg cells, B cells.

A patient having a cancer can be treated with the anti-CD39 antibody with our 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, TReg cells, B cells). Optionally, the treatment methods can comprises a step of detecting a CD39 nucleic acid or polypeptide in a biological sample of a tumor from an individual (e.g., in cancer tissue, tissue proximal to or at the periphery of a cancer, cancer adjacent tissue, adjacent non-tumorous tissue or normal adjacent tissue). A determination that a biological sample comprises cells expressing CD39 (e.g., prominently expressing; expressing CD39 at a high level, high intensity of staining with an anti-CD39 antibody, compared to a reference) indicates that the patient has a cancer that may have a strong benefit from treatment with an agent that inhibits CD39. In one embodiment, the method comprises determining the level of expression of a CD39 nucleic acid or polypeptide in a biological sample and comparing the level to a reference level corresponding to a healthy individual. A determination that a biological sample comprises cells expressing CD39 nucleic acid or polypeptide at a level that is increased compared to the reference level indicates that the patient has a cancer that can be treated with an anti- CD39 antibody of the disclosure. Optionally, detecting a CD39 polypeptide in a biological sample comprises detecting CD39 polypeptide expressed on the surface of a malignant cell, a CD4 T cell, CD8 T cell, TReg cell, B cell. In one embodiment, a determination that a biological sample comprises cells that prominently expresses CD39 nucleic acid or polypeptide indicates that the patients has a cancer that can be treated with an anti-CD39 antibody of the disclosure. "Prominently expressed", when referring to a CD39 polypeptide, means that the CD39 polypeptide is expressed in a substantial number of cells taken from a given patient. While the definition of the term "prominently expressed" is not bound by a precise percentage value, in some examples a receptor said to be "prominently expressed" will be present on at least 10%, 20% 30%, 40%, 50°%, 60%, 70%, 80%, or more of the tumor cells taken from a patient.

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 performing a biopsy) from the individual that comprises cells from the cancer environment (e.g., 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) determining the CD39 polypeptide status of cells within the tumor environment, optionally within the tumor and/or within adjacent tissue, and

b) upon a determination that tumor environment comprises cells that express CD39 polypeptide, optionally at a level that is increased compared to a reference level, administering to the individual an anti-CD39 antibody. In one embodiment, the cells are tumor cells. In another embodiment, the cells within the tumor environment, tumor and/or adjacent tissue are non-malignant immune cells, e.g., B cells, T cells, TReg cells. Optionally, determining the CD39 polypeptide status within the tumor environment comprises obtaining from the individual a biological sample that comprises cancer tissue and/or tissue proximal to or at the periphery of a cancer (e.g., cancer adjacent tissue, adjacent non-tumorous tissue or normal adjacent tissue), bringing said cells into contact with an antibody that binds a CD39 polypeptide, and detecting cells that express CD39.

The anti-CD39 agent can advantageously be used to cancer in an individual whose cancer is non-inflammatory, or not eliciting a sufficient anti-tumor immune response (e.g. whose tumor environment is not characterized by substantial inflammation or immune effector cells). For example, a method for the treatment or prevention of a cancer in an individual in need thereof can comprise: a) determining whether the tumor environment, optionally within the tumor and/or within adjacent tissue, is characterized by inflammation and/or pro-inflammatory cells, and b) upon a determination that tumor environment is not sufficiently characterized by inflammation and/or pro-inflammatory cells, optionally compared to a reference level, administering to the individual an antigen binding protein of the disclosure.

The antibody compositions may be used in as monotherapy or combined treatments with one or more other therapeutic agents, including agents normally utilized for the particular therapeutic purpose for which the antibody is being administered. The additional therapeutic agent will normally be administered in amounts and treatment regimens typically used for that agent in a monotherapy for the particular disease or condition being treated. Such therapeutic agents include, but are not limited to anti-cancer agents and chemotherapeutic agents.

In one embodiment, the second or additional second therapeutic agent is an antibody or other Fc domain-containing protein capable of inducing ADCC toward a cell to which it is bound, e.g., via CD16 expressed by an NK cell. Typically, such antibody or other protein will comprise a domain that binds to an antigen of interest, e.g., an antigen present on a tumor cell (tumor antigen), and an Fc domain or portion thereof, and will exhibit binding to the antigen via the antigen binding domain and to Fey receptors (e.g., CD16) via the Fc domain. In one embodiment, its ADCC activity will be mediated at least in part by CD16. In one embodiment, the additional therapeutic agent is an antibody having a native or modified human Fc domain, for example a Fc domain from a human lgG1 or lgG3 antibody. The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well understood in the art, and refers to a cell-mediated reaction in which non-specific cytotoxic cells that express Fc receptors (FcRs) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils. The term "ADCC-inducing antibody" refers to an antibody that demonstrates ADCC as measured by assay(s) known to those of skill in the art. Such activity is typically characterized by the binding of the Fc region with various FcRs. Without being limited by any particular mechanism, those of skill in the art will recognize that the ability of an antibody to demonstrate ADCC can be, for example, by virtue of it subclass (such as lgG1 or lgG3), by mutations introduced into the Fc region, or by virtue of modifications to the carbohydrate patterns in the Fc region of the antibody.

In the treatment methods, the CD39-binding compound and the second therapeutic agent can be administered separately, together or sequentially, or in a cocktail. In some embodiments, the antigen-binding compound is administered prior to the administration of the second therapeutic agent. For example, the CD39-binding compound can be administered approximately 0 to 30 days prior to the administration of the second therapeutic agent. In some embodiments, an CD39-binding compound 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 second therapeutic agent. In some embodiments, a CD39- binding compound is administered concurrently with the administration of the therapeutic agents. In some embodiments, a CD39-binding compound is administered after the administration of the second therapeutic agent. For example, a CD39-binding compound can be administered approximately 0 to 30 days after the administration of the second therapeutic agent. In some embodiments, a CD39-binding compound 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 second therapeutic agent.

Examples

Example 1 : Study of anti-CD39/CD39 complexes by X-ray diffraction

Purification and cystallogenesis

Protein production: Anti-CD39 antibody having the VH and VL CDRs of the parental VH and VL of SEQ ID NOS: 2 and 3 was modified by introduction of human VH and VL acceptor frameworks, and found to retain CD39 binding and neutralization of ATPase activity, moreover without induction of intracellular internalization of CD39. The humanized VH and VL are shown in SEQ ID NOS: 4 and 5, respectively. The VH and Vk sequences of each antibody were cloned into vectors containing the hulgGI CH1 constant domain and the huCk constant domain respectively. The two obtained vectors were co-transfected into the CHO cell line. The established pool of cell was used to produce the Fab antibody in the CHO medium. CD39 protein was produced in CHO cells using standard methods.

Protein purification:

Antibody Fab fragments were purified in two steps, by affinity chromatography on Nickel-beads (Ni-NTA) followed by Size Exclusion Chromatography (SEC).

CD39/Fab complexes were purified in five steps. First, purified Fab were added to CD39 recombinant protein culture supernatant in order to form the complexes directly in culture medium. Complexes were purified from culture supernatant by affinity chromatography on Nickel-beads thanks to the Fab his tag. Ni-NTA purified complexes were then separated from free Fabs by ion exchange chromatography (I EC). CD39/Fab complexes were treated with PNGaseF in order to reduce the complexity of the antigen glycosylations. Finally, deglycosylated complexes were separated from PNGaseF by a second SEC and concentrated to about 15 mg/mL for crystallogenesis. Crystallogenesis was performed separately on ab Fab alone and CD39/fab complexes by an automated process using standard crystallogneneis kit, Wizard, MDL and Morpheus. Anti-CD39 Fab were crystallized in 0.1 M Mes pH 6.5, 1.8M ammonium sulfate buffer; Crystals were frozen in 30% glycerol cryoprotectant and analysed at the Soleil synchrotron in Saclay using the Proxima 1 beamline. Fab/CD39 complexes were crystallized in 0.1 M citrate pH5.5, 2M ammonium sulfate buffer. Crystals were frozen in 20 % glycerol cryoprotectant and analysed at the IBS synchrotron in Grenoble.

Crystals of Fab and of Ag/Ab complexes diffracted at 2.14 and 2.26 A respectively. Structures were solved by molecular isomorphous replacement (MIR) using pdb templates of Fabs and pdb templates of the rat CD39 molecule.

Results

The 3-dimensional structure showed that binding of the neutralizing anti-CD39 to the target antigen CD39 entirely relies on the heavy chain variable domain (Summary table 2; Table 3; Figure 1 ). The anti-CD39 antibody light chain does not contact the antigen directly (Figure 1 and Figure 2).

A total of 37 heavy chain residues are interfacing with CD39. Of these, 1 1 (-30%) are Kabat framework (FR) residues and 26 (~ 70%) are Kabat complementarity determining region (CDR) residues. 7 of the interfacing FR residues contact the glycan at the asparagine residue at position 292 of CD39 and 4 are interfacing with the CD39 protein but all have a minor contribution to the interface. Among the 1 1 FR residues facing the antigen, three were not conserved from the parental murine antibody (see Table 4). The parental residues were substituted by human residues showing very conservative physicochemical properties and structures: A68 replaced by V, E72 replaced by D and A72a replaced by T. Substitution of FR parental interfacing residues with human ones had no impact on antigen binding or antibody blocking activity.

Although the anti-CD39 light chain does not contact CD39 directly it appears to play an important role in the spatial organization of the heavy chain paratope. Indeed, the anti- CD39 heavy chain has a long CDR_H3 which strongly interacts with the light chain CDRs (Figure 2 ; Table 5). The CDR_H3 is positioned just above the light chain CDRs, between the antigen and the VL domain. Light chain CDRs form numerous interactions with CDR_H3. The light chain CDRs drive the orientation and the positioning of the CDR_H3 loop, they restrain the flexibility and motion of the CDR_H3 loop and they contribute indirectly to the binding to CD39. Interestingly, the CDR_H3 has a particularly high number of aromatic residues (principally tyrosines), and these aromatic residues permit a light chain/CDR_H3/CD39 matrix where the CDR_H3 is trapped between the VL CDRs that, together with some FR residues, form a paratope directed to the CDR_H3, and CD39. The matrix is stabilized by several pi-interactions between aromatic residues in CDR_H3 and respective contact residues in the VL CDRs and CD39.

The anti-CD39 heavy chain binds to both the CD39 N-terminal domain 1 and C- terminal domain 2 of CD39). The anti-CD39 binding site is located at the apex of the two CD39 domains and at the entry of the catalytic cleft (Figure 3; Table 2; Table 6). The N- terminal domain 1 of CD39 has a major contribution to the epitope (Table 6), and half (13) of the 26 heavy chain paratope residues form a direct bond with CD39-N-terminal domain 1 . In contrast, only two heavy chain paratope residues form a direct bond with the C-terminal domain 2 of CD39. Instead, the C-terminal domain 2 of CD39 interacts with anti-CD39 antibody by the N292-sugar moiety and 8 AA residues located at the domain apex and cleft entry (Table 7). The 8 amino acid residues include both CDR (in Kabat CDR2) and framework residues (in both Kabat FR1 and FR3).

The fact that anti-CD39 antibody binds simultaneously to both CD39 domains 1 and 2, the latter optimized by binding via the N292-sugar moiety, likely explains how the antibody blocks the enzymatic activity. Indeed, based on structural data available in the literature (PDB database, reference 3ZX3) domain motion may be a key parameter required for the ability of CD39 to hydrolyse substrate. Moreover, the human CD39 structure obtained from the anti-CD39/CD39 co-crystal complex shows only one unique enzyme conformation corresponding to one fixed relative positioning of the two domains. On the contrary, rat CD39 when crystalized alone (pdb entry 3ZX3) exists under different conformations (i.e. with slightly different positions of the domains 1 and 2). The human CD39/anti-CD39 frozen conformation perfectly superimposes with rat CD39 form A of the pdb crystal 3ZX3 (Figure 4). Binding of the antibody to both domains at the same time thus likely inhibits domain motion and block the enzyme in a given frozen status.

Table 2: VH residues interfacing with CD39

Table 3: Buried surface at the antigen/antibody interface.

Table 4: VH FR residues of humanized and parental antibody interfacing with

Table 5: VL residues interfacing with Heavy chain CDR H3

Table 6: CD39 interfacing residues

Table 7: Residues in VH that bind the CD39 N292 glycan

Example 2: Production of CDR- and/or FR- modified anti-CD39 antibodies based on crystals of Fab and of Ag/Ab complexes

Based on the study of anti-CD39/CD39 complexes by X-ray diffraction described in Example 1 , CDRs and FR residues in the anti-CD39 antibodies were considered for potential substitutions and produced.

Analysis of heavy chain residues interfacing with CD39 and/or the antibody light chain The threonine of the heavy chain at Kabat position 30 (T30) is in the Kabat FR1 and contacts a glutamine at position 96 (reference to SEQ ID NO: 1 of the CD39 polypeptide (CD39-Q96). The peptide bond O of T30 interacts with CD39-Q96. A possible substitution at position 30 is a serine. The histidine of the heavy chain at Kabat position 31 (in the Kabat CDR1 ) (H31 ) is exposed at the molecular surface, oriented toward CD39 and it forms a salt bridge with CD39-E140. It may also contact L144 of CD39 by Pi-alkyl interaction. The tyrosine of the heavy chain at Kabat position 32 (Y32) is in the Kabat CDR1 ; while it contacts CD39 it is not oriented toward CD39. Only the Calpha and peptide bond part of Y32 is interfacing with CD39. Examples for possible substitutions of Y32 include all residues except possibly P and G, as these two residues may constrain the φ and φ angles. The glycine of the heavy chain at Kabat position 33 (G33) is in the Kabat CDR1 and may form a h-bond with CD39-Q96. G33 occupies a key position at the interface and is in very close contact with CD39. Substitution with A33 impairs antibody binding to CD39, indicating that steric hindrance is key parameter for that position in the paratope. Any G33 substitution should avoid residues that cause steric hindrance. The tryptophan of the heavy chain at Kabat position 50 (W50) is in the Kabat CDR2 and occupies a key position at center of the VH paratope. It may form Pi-alkyl bond with CD39-K97 and influences the positioning of residue R99 of the heavy chain CDR3 (CDR_H3-R99). The asparagine of the heavy chain at Kabat position 52 (N52) is in the Kabat CDR2 and is totally buried at the Ab/Ag interface, forming an h-bond with valine at position 95 of CD39 (CD39-V95). Residue 52 is an important position. An exemplary potential substitution at position 52 is glutamine. The threonine of the heavy chain at Kabat position 52a (T52a) is in the Kabat CDR2 and also occupies a key position. Although it is oriented toward the C-terminal domain 2 of CD39 (referred to also as D2 or CTD2), it forms an h-bond with CD39-Q96, a residue located in N-terminal domain (domain 1 ) of CD39 (referred to also as D1 or NTD1 ). The bond may be critical for the global positioning of the CDR_H2 loop. An exemplary potential substitution at position 52a is serine. The tyrosine of the heavy chain at Kabat position 53 (Y53) is in the Kabat CDR2 and also occupies a key position. It is oriented inside the CD39 groove and may form different interactions with residues V95 and L137 of CD39. An exemplary potential substitution at position 53 is phenylalanine. A substitution with glutamic acid (E) is expected to reduce binding to CD39.

The Kabat CDR2 of the VH binds both the N- and the C-terminal domain of CD39, providing an important contribution to the mechanism of action of the antibody. The CDR2 contains a first series of residues that bind to the N-terminal domain (Kabat positions 50, 52, 52a and 53), and a second series of residues that bind the C-terminal domain of CD39 (Kabat positions 54, 57, 59 and 65). Binding to the C-terminal domain is to a large extent via the glycan at residue N292 of CD39, and involves not only CDR2 but also numerous FR3 residues. The residues of the V _H that are involved in binding to the glycosylation at residue N292 of CD39 are in the CDR2 (residues at Kabat positions 57, 59 and 65) and FR3 (residues at Kabat positions 67, 68, 69, 70, 71 ). Residues at Kabat positions 72, 72a and 72b are further present at the interface with CD39, and K19 (FR1 ) and Q78 interact with each other but may make a minor contribution to the glycan binding surface.

The threonine at Kabat position 54 (T54) of the heavy chain is in the Kabat CDR2 and is fully buried at the interface and positioned inside the enzymatic active site groove of CD39. The peptide bond O of T54 forms an h-bond with K297. However, as the radical part of T54 is not involved in CD39 contact, exemplary potential substitutions include other residues with comparable steric hindrance and physicochemical properties; serine for instance, may occupy the same position without any impact on Ag binding. Other possible substitutions include N, or optionally A or G, or optionally residues other than large residues (e.g. K or R) or hydrophobic residues (e.g., M, I, L, F, W, V or Y). The glycine at Kabat position 55 (G55) of the heavy chain is in the Kabat CDR2 and is partially buried at the interface. G55 is unlikely to be a critical residue. The local environment is not very crowded and slightly bigger residues may occupy the same place without impacting binding to the antigen. Examples of substitutions at this positions include A, S, T, N, Q (some of those could make new contacts with CD39 and reinforce the binding), generally any resides other than D and E (potential conflict with CD39-D295) or large residues (e.g., K or R). The glutamic acid at Kabat position 56 (E56) of the heavy chain is in the Kabat CDR2 and is a key residue of the paratope. It forms two bonds with K97 of CD39. An exemplary potential substitution at position 56 is aspartic acid. The threonine at Kabat position 58 (T58) of the heavy chain is in the Kabat CDR2 and is partially buried at the interface. It has a minor contribution to the paratope, if any. Exemplary potential substitutions at position 58 include hydrophilic residues that may conserve the beta-sheet organization (e.g., S, N, Q, H, E, D, R, K, A, Y), or generally residues other than hydrophobic residues (e.g., V, I, L, M, F, W) and or other other than P.

Residues at Kabat positions 57, 59 and 65 are involved in binding to the N292-linked glycan on CD39. The proline at Kabat position 57 (P57) of the heavy chain (in the Kabat CDR2) is partially buried below the NAG31 of N292. The tyrosine at Kabat position 59 (Y59) of the heavy chain (in the Kabat CDR2) forms a h-bond with NAG32 of N292 and may be oriented by a Pi-alkyl interaction with P57. This residue is preferably not substituted. The glycine at Kabat position 65 (G65) of the heavy chain (in the Kabat CDR2) makes a minor contribution to the interface with NAG32 of N292. Potential substitutions at G65 include residues with that do not introduce sufficient steric hindrance to impact binding to the antigen.

In FR3, the phenylalanine at Kabat position 67 (F67) of the heavy chain (in the Kabat FR3) is completely buried inside the VH domain hydrophobic cavity and only the Calpha part of the residue contributes to the interface with the N292 sugar (at the NAG32 moiety). Example of possible substitutions at position 67 include any hydrophobic FR residue usually found at that position which would maintain the beta-strand position and VH domain structure integrity, for example alanine, leucine, valine or isoleucine. The valine at Kabat position 68 (V68) of the heavy chain (in the Kabat FR3) is almost completely buried below the sugar moiety (interaction with NAG31 , NAG32 and/or FUC33). Example of possible substitutions at position 68 include hydrophobic residue, for example alanine (A is present in the parental antibody FR3), and the substitution of A with V does not impair the binding to the N292- sugar, or threonine. Other examples for possible substitutions include leucine. The phenylalanine at Kabat position 69 (F69) of the heavy chain (in the Kabat FR3) is completely buried inside the VH domain hydrophobic cavity and only the Calpha part of the residue contributes to the interface with the sugar (at the NAG31 and/or NAG32 moiety). Example of possible substitutions at position 69 include any hydrophobic FR residue usually found at that position which would maintain the beta-strand position and VH domain structure integrity. The serine at Kabat position 70 (S70) of the heavy chain (in the Kabat FR3) is almost completely buried below the sugar and forms an h-bond with FUC33 (and NAG31 ). Particularly in the context of a humanized VH (from a parental VH), S70 is advantageously not substituted thereby minimizing potential immunogenicity. The leucine at Kabat position 71 (L71 ) of the heavy chain is in the Kabat framework (FR3) and the peptide bond O of L71 likely forms a h-bond with S294 of CD39 and makes a minor contribution of the Calpha part to the interface with N292 (FUC33 moiety). L71 is buried inside the hydrophobic cavity of the VH domain and it is located just below the CDR_H2. Examples of possible substitutions at position 71 are threonine, valine or alanine; preferably acceptor frameworks having R, K or P are avoided or substituted to have a L, T, V or A at residue 71. In one embodiment, the segment of residues at Kabat position 67-71 has the amino acid sequence: FVFSL. Finally, the lysine at Kabat position 19 (K19) of the heavy chain (in the Kabat FR1 ) makes a minor contribution to the interface with the NAG31 moiety on the N292 glycan (as well as Q78) Similarly, the glutamine at Kabat position 78 (Q78) also makes a minor contribution to the interface with the FUC33 moiety on the N292 glycan and may form an h-bond with K19.

The three residues D72, T72a and S72b of the heavy chain are in the Kabat framework (FR3) and are partially buried at the Ab/Ag interface. These residues contribute to the overall folding of the VH domain and to the CDR_H2 positioning. Therefore they can be replaced by residues with equivalent physicochemical properties that will not impact domain structure and potential immunogenicity. For example, in the anti-CD39 antibody in the crystallized CD39 complex, D72 and T72a have been replaced parental residues E72 and A72a respectively. When substitutions are envisioned, it is to be noted that the D72 and S72b are bound by three H-bonds, which in turn positions the D72 in an orientation that shapes the CD39 binding surface, as shown in Figure 5 (panel A) and Figure 6. If S72b is substituted with a residue that results in the loss of the 72-72b interaction, the side chain of aspartic acid at position 72 may be affected such that CD39 binding is diminished as shown in Figure 5 (panel B). Thus, is S72b is substituted, e.g., with an alanine, then S72 should be substituted with a small residue, e.g. alanine. If S72b remains unsubstituted, D72 can be substituted, for example, with glutamic acid or alanine so as to maintain the 72-72b interaction. Thus, possible residue pairs at positions 72 and 72b can be: A/A, D/S, E/S and A/S. The threonine at Kabat position 72a can optionally be substituted by an alanine. Thus, the residues at positions 72, 72a, 72b can for example have any of the following sequences: ATA, DTS, ETS and ATS, AAA, DAS, EAS or AAS.

The arginine at Kabat position 95 (R95) of the heavy chain is in the Kabat CDR3 and is exposed at the molecular surface within the paratope and oriented toward CD39. The residue may form a H-bond with CD39-Q96. An example of a potential substitution at this position is lysine. The arginine at Kabat position 96 (R96) of the heavy chain is in the Kabat CDR3 and is not exposed on the paratope surface. It is oriented toward the VL domain and may form a salt bridge with Kabat residue E56 (VL-E56) within the Kabat CDR-L2. The salt bridge is not visible in the electron density map. An example of a potential substitution at this position is lysine. The glutamic acid at Kabat position 98 (E98) of the heavy chain (in the Kabat CDR3) does not appear to play an important role in binding CD39 or the VL. Examples of substitutions at this position are amino acids other than P and G. The glycine at Kabat position 99 (G99) of the heavy chain is in the Kabat CDR3, located at the top of the CDR_H3 loop, and forms a h-bond with CD39-R154. The tyrosine at Kabat position 100a (Y100a) of the heavy chain (in the Kabat CDR3) is exposed at the molecular surface. The phenol radical does not interact with any residues, however the peptide bond O of the residue forms an h- bond with VL-Y32(CDR_L1 ). Examples of potential substitutions include residues other than P, optionally further other than G). (Y100a is one of the residues that will be mutated). The valine at Kabat position 100b (V100b) of the heavy chain (in the Kabat CDR3) is oriented toward CD39 and likely forms hydrophobic interaction with CD39-V151. The Calpha part of residue is facing the VL paratope. Examples of potential substitutions include A, I and L. The phenylalanine at Kabat position 100c (F100c) of the heavy chain (in the Kabat CDR3) is oriented toward the VL domain. F100c may participate to the hydrophobic CDR_H3A L interface environment. An example of a potential substitution is tyrosine. The tyrosine at Kabat position 100d (Y100d) of the heavy chain (in the Kabat CDR3) is a key residue of the CD39A H interface. The radical contacts CD39 and Calpha part forms a h-bond with VL CDR_L3. The residue is likely to play a major role in CDR_H3 positioning and binding to CD39. The tyrosine at Kabat position 100e (Y100e) of the heavy chain (in the Kabat CDR3) is a key residue of the CDR_H3A L interaction. Examples of potential substitutions include phenylalanine or tryptophan, and residues other than P, G, E and D, or residues other than small hydrophilic residues such T, S. The phenylalanine at Kabat position 10Of (F100f) of the heavy chain (in the Kabat CDR3) is completely buried at the CDR_H3A L interface. The aspartic acid at Kabat position 101 (D101 ) of the heavy chain (in the Kabat CDR3) is partially buried at the CDR_H3A L interface. Examples of potential substitutions include S, T, E, N or other residues that are not large and hydrophilic, or residues other than R or K because of a risk of creating a salt bridge with VL-E56. The tyrosine at Kabat position 102 (Y102) of the heavy chain (in the Kabat CDR3) is not critical regarding interaction with VL. Y102 is stacked to VH-V2 and may play a role in CDR_H3 positioning.

Analysis of light chain residues interfacing with Heavy chain CDR_H3

The serine in the light chain at Kabat position 31 (S31 ) is almost completely buried at the interface with CDR_H3. It is facing CDR_H3-Y105. Examples for possible substitutions of S31 include small residues such as T, A or N, or generally residues other than large residues (e.g., K, R, I, L) or residues which may stack with CDRJH3-Y105 (e.g., F, Y, W). The tyrosine in the light chain at Kabat position 32 (Y32) is oriented toward the Ab/Ag interface and forms a h-bond with Y105 O-Calpha part on the inner side of the CDR_H3 loop. Y32 seems to occupy an important position in the VL paratope. The phenylalanine in the light chain at Kabat position 33 (F33) is completely buried inside the VL domain. Only the Calpha part of F33 is facing the CD_H3. Examples for possible substitutions of F33 include all that may occupy the same position inside the hydrophobic cavity of the VL domain (e.g., L, I, M, V, optionally further W), or optionally residues other than D, E, N and K, optionally further residues other than Q and R. The serine in the light chain at Kabat position 34 (S34) is 100 % buried below the CDR_H3. Examples for possible substitutions of S34 include small residues (A, T, G, N or H), or residues other than large residues (e.g., K, R) or hydrophobic residues (e.g., M, I, L, F, W and Y). The tyrosine in the light chain at Kabat position 49 (Y49) is an important residue for the positioning of CDR_H3 onto the VL paratope. It is stacked to CDR_H3-Y109. Examples for possible substitutions of Y49 include phenyalanine. The glutamine in the light chain at Kabat position 89 (Q89) is 100% buried below the CDR_H3 and forms a h-bond with F1 10. The histidine in the light chain at Kabat position 91 (H91 ) is almost totally buried below the CDR_H3 loop. It forms a h-bond with peptide bond O of Y108. Examples for possible substitutions of H91 include asparagine, or residues other than large (e.g. K, R) or hydrophobic residues (e.g. M, I, L, F, W, V). The four residues at Kabat positions 94 to 97 (T94, P95, Y96 and T97) in the light chain CDR_L3 contact the VH domain framework, and may indirectly influence CDR_H2 positioning and flexibility.

Rational design and production of CDR-mutated VH/VL to improve pharmacological properties

Parental humanized (CDR-grafted) antibody and the CDR-mutated antibodies mAbl to mAb39 having the VH and Vk variable regions shown below were designed to integrate substitutions that could enhance stability by reduction of hydrophobicity (removal of aromatic residues), reduction of immunogenicity (substitution with residues present in human germline sequence at the particular position) and/or increase in affinity. Figure 7 shows the mutations made in the CDRs at the antibody surface.

Exemplary anti-CD39 V _H and V _L amino acid sequences shown in the table below comprising amino acid modifications in CDRs to improve their pharmacological properties were prepared.

Table: CDR-mutated VH Domains

SEQ VH domain amino acid sequence CDR mutation Designation ID NO for heavy chain

6 QVQLVQSGSELKKPGASVKVSCKASGYTFTHYGMNWVRQA D61 Q HH1 PGQGLKWMGWINTYTGEPTYAQDFKGRFVFSLDTSVSTAY LQISSLKAEDTAVYYCARRRYEGNYVFYYFDYWGQGTTVT VSS

7 QVQLVQSGSELKKPGASVKVSCKASGYTFTHYGMNWVRQA Y(100a)S HH2 PGQGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAY LQISSLKAEDTAVYYCARRRYEGNSVFYYFDYWGQGTTVT VSS

8 QVQLVQSGSELKKPGASVKVSCKASGYTFTHYGMNWVRQA D61 Q / HH3 PGQGLKWMGWINTYTGEPTYAQDFKGRFVFSLDTSVSTAY Y(100a)S

LQISSLKAEDTAVYYCARRRYEGNSVFYYFDYWGQGTTVT VSS

9 QVQLVQSGSELKKPGASVKVSCKASGYTFTHYGMNWVRQA Y53E HH4 PGQGLKWMGWINTETGEPTYADDFKGRFVFSLDTSVSTAY

LQISSLKAEDTAVYYCARRRYEGNYVFYYFDYWGQGTTVT VSS

Table: CDR-mutated VL Domains

Antibodies were produced as Fc silent recombinant chimeric human lgG1 antibodies with a heavy chain N297Q (Kabat EU numbering) mutation which results in lack of N-linked glycosylation and loss of binding to human Fey receptors CD16A, CD16B, CD32A, CD32B and CD64. The CDR-mutated V _H and V _L were incorporated in all different combinations, including with a parental humanized VH and a VL of SEQ ID NOS 4 and 5, respectively, into antibodies mAbl to mAb39 whose sequence (by reference to the respective SEQ ID NOS) is provided below in table below. Briefly, the VH and VL sequences of each antibody as shown below were cloned into vectors containing the hulgGI constant domains (harbouring a N297Q mutation) and the huCk constant domain respectively. The two obtained vectors were co-transfected into the CHO cell line. The established pool of cell was used to produce the antibody in the CHO medium. The antibody was then purified using protein A.

Table

Antibody VH SEQ ID VL SEQ ID

NO NO

mAbl 4 10

mAb2 4 1 1

mAb3 4 12

mAb4 4 13

mAb5 4 14

mAb6 4 15

mAb7 4 16

mAb8 6 10

mAb9 6 1 1

mAb10 6 12

mAb1 1 6 13

mAb12 6 14

mAb13 6 15

mAb14 6 16

mAb15 7 10

mAb16 7 1 1

mAb17 7 12

mAb18 7 13

mAb19 7 14

mAb20 7 15

mAb21 7 16

mAb22 8 10

mAb23 8 1 1

mAb24 8 12 mAb25 8 13

mAb26 8 14

mAb27 8 15

mAb28 8 16

mAb29 9 10

mAb30 9 1 1

mAb31 9 12

mAb32 9 13

mAb33 9 14

mAb34 9 15

mAb35 9 16

mAb36 6 5

mAb37 7 5

mAb38 8 5

mAb39 9 5

Example 3: Ab titration on Ramos tumor cells by flow cytometry

Ramos human lymphoma cells that endogenously express CD39 were used to evaluate ability of different anti-CD39 antibodies to bind human CD39. Briefly, 10 ⁵ Ramos cells were resuspended in PBS/0.2% BSA/0.02% NaN3 (named "staining buffer") are distributed into round bottom 96W-microplat.es. Dose-range of anti-CD39 antibodies of Example 2 were added to the plates and cells are incubated for 45 min at 4°C. Cells were washed three times in staining buffer by spinning plates at 400 g for 3 min at 4°C. PE- coupled goat anti-mouse or goat anti-human IgG Fc fragment secondary antibodies (Beckman Coulter) diluted in staining buffer were added to the cells and plates are incubated for 30 additional minutes at 4°C. Cells were washed three times as described above and analyzed on an Accury C6 flow cytometer equipped with an HTFC plate reader. Median of fluorescence vs. antibodies concentration was plotted on graphs and EC50 is calculated using GraphPad Prism program.

Antibodies having heavy chains H1 (parental, VL of SEQ ID NO: 4), HH1 , HH2, HH3 and HH4 of Example 2, in each case combined with each of light chains of L0 (parental, VL of SEQ ID NO: 5), LH1 , LH2, LH4, LH5, LH6 and LH7 were tested for binding to Ramos cells. While the antibodies having the heavy/light chain combinations H0/LH1 , H0/LH2, H0/LH4, H0/LH5, H0/LH6, H0/LH7 HH1/LH1 , HH1/LH2, HH1/LH4, HH1/LH5, HH1/LH6, HH1/LH7, HH2/LH1 , HH2/LH2, HH2/LH4, HH2/LH5, HH2/LH6, HH2/LH7, HH3/LH1 , HH3/LH2, HH3/LH4, HH3/LH5, HH3/LH6 and HH3/LH7 were all expected to retain binding to CD39 based on the analysis of the crystal structure, HH4 antibodies having a glutamic acid substitution at Kabat residue 53 (i.e. antibodies HH4/L0, HH4/LH 1 , HH4/LH2, HH4/LH4, HH4/LH5, HH4/LH6 and HH4/LH7) were all expected to result in a loss of binding, as the aromatic residue (tyrosine) of the heavy chain at Kabat position 53 (Y53) is oriented inside the CD39 groove and may form different interactions with residues V95 and L137 of CD39.

Results showed that each antibody having the H1 , HH 1 , HH2 or HH3 heavy chain retained binding affinity to Ramos cells, irrespective of the light chain with which it was paired. However, all antibodies having the HH4 heavy chain lost binding affinity to Ramos cells.

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.