Novel anti-CD39 antibodies

Abstract

The present disclosure provides anti-CD39 antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding them, pharmaceutical compositions comprising them, and uses thereof.

Description

[Technical Field]

[0001]

[0001] The present disclosure relates generally to novel anti-CD39 antibodies. [Background technology]

[0002]

[0002] CD39, also known as ecto-nucleoside triphosphate diphosphohydrolase 1 (ENTPDase 1), is an integral membrane protein that converts ATP or ADP to AMP, which is then dephosphorylated by CD73 to convert it to adenosine. Adenosine is a potent immunosuppressant that binds to adenosine receptors (e.g., A2A receptors) on the surface of CD4, CD8 T cells, and natural killer (NK) cells, inhibiting T cell and NK cell responses and thereby suppressing the immune system. Adenosine also binds to A2A and A2B receptors on macrophages and dendritic cells, inhibiting phagocytosis and antigen presentation and increasing the secretion of tumor-promoting factors such as VEGF, TGFβ, and IL-6. The enzymatic activities of CD39 and CD73 play a key role in regulating the duration, magnitude, and chemical nature of purinergic signals delivered to immune cells through the conversion of ADP and ATP to AMP, and AMP to adenosine, respectively (Luca Antonioli et al., Trends Mol Med. 2013 Jun;19(6):355-367). Increased adenosine levels mediated by CD39 and CD73 create an immunosuppressive environment, thereby promoting cancer development and progression. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0003] There remains a need for new anti-CD39 antibodies. [Means for solving the problem]

[0004] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or more than one antibody.

[0005] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to human CD39, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and / or a light chain variable region comprising LCDR1, LCDR2, and LCDR3; a) HCDR1 comprises an amino acid sequence selected from the group consisting of NYGMN (SEQ ID NO: 1), KYWMN (SEQ ID NO: 2), NYWMN (SEQ ID NO: 3), DTFLH (SEQ ID NO: 4), DYNMY (SEQ ID NO: 5), DTYVH (SEQ ID NO: 6); b) HCDR2 is LINTYTGEPTYADDFKD (SEQ ID NO: 7), EIRLKSNKYGTHYAESVKG (SEQ ID NO: 8), QIRLNPDNYATHX1AESVKG (SEQ ID NO: 9), X 58 IDPAX 59 X 60 NIKYDPKFQG (SEQ ID NO: 151), FIDPYNGYTSYNQKFKG (SEQ ID NO: 11), RIDPAIDNSKYDPKFQG (SEQ ID NO: 12), c) HCDR3 comprises an amino acid sequence selected from the group consisting of KGIYYDYVWFFDV (SEQ ID NO: 13), QLDLYWFFDV (SEQ ID NO: 14), HGX2RGFAY (SEQ ID NO: 15), SPYYYGSGYRIFDV (SEQ ID NO: 16), IYGYDDAYYFDY (SEQ ID NO: 17), YYCALYDGYNVYAMDY (SEQ ID NO: 18); d) LCDR1 comprises an amino acid sequence selected from the group consisting of KASQDINRYIA (SEQ ID NO: 19), RASQSISDYLH (SEQ ID NO: 20), KSSQSLLDSDGRTHLN (SEQ ID NO: 21), SAFSSVNYMH (SEQ ID NO: 22), SATSSVSYMH (SEQ ID NO: 23), RSSKNLLHSNGITYLY (SEQ ID NO: 24); e) LCDR2 comprises an amino acid sequence selected from the group consisting of YTSTLLP (SEQ ID NO: 25), YASQSIS (SEQ ID NO: 26), LVSKLDS (SEQ ID NO: 27), TTSNLAS (SEQ ID NO: 28), STSNLAS (SEQ ID NO: 29), RASTLAS (SEQ ID NO: 30); f) LCDR3 comprises an amino acid sequence selected from the group consisting of LQYSNLLT (SEQ ID NO: 31), QNGHSLPLT (SEQ ID NO: 32), WQGTLFPWT (SEQ ID NO: 33), QQRSTYPFT (SEQ ID NO: 34), QQRITYPFT (SEQ ID NO: 35), AQLLELPHT (SEQ ID NO: 36); X1 is Y or F, X2 is S or T, and X 58 is R or K, and X 59 is N, G, S, or Q, and X 60 is G, A, or D.

[0006]

[0006] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, and / or HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and / or HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, and / or LCDR1 comprises the amino acid sequence of SEQ ID NO: 21, and / or LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and / or LCDR3 comprises the amino acid sequence of SEQ ID NO: 33, and X1 and X2 are as defined above.

[0007]

[0007] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, and / or HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 38, and / or HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40 and SEQ ID NO: 41, and / or LCDR1 comprises the amino acid sequence of SEQ ID NO: 21, and / or LCDR2 comprises the amino acid sequence of SEQ ID NO: 27, and / or LCDR3 comprises the amino acid sequence of SEQ ID NO: 33.

[0008] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, and / or HCDR2 comprises the amino acid sequence of SEQ ID NO: 151, and / or HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, and / or LCDR1 comprises the amino acid sequence of SEQ ID NO: 22, and / or LCDR2 comprises the amino acid sequence of SEQ ID NO: 28, and / or LCDR3 comprises the amino acid sequence of SEQ ID NO: 34, and X 58 , X 59 , and X 60 is as defined above.

[0009] In some embodiments, the heavy chain variable region of an antibody or antigen-binding fragment thereof provided herein comprises: a) an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 7, and an HCDR3 comprising the sequence of SEQ ID NO: 13, or b) an HCDR1 comprising the sequence of SEQ ID NO: 2, an HCDR2 comprising the sequence of SEQ ID NO: 8, and an HCDR3 comprising the sequence of SEQ ID NO: 14, or c) an HCDR1 comprising the sequence of SEQ ID NO: 3, an HCDR2 comprising the sequence of SEQ ID NO: 37, and an HCDR3 comprising the sequence of SEQ ID NO: 40, or d) an HCDR1 comprising the sequence of SEQ ID NO: 3, an HCDR2 comprising the sequence of SEQ ID NO: 38, and an HCDR3 comprising the sequence of SEQ ID NO: 41, or e) an HCDR1 comprising the sequence of SEQ ID NO: 4, an HCDR2 comprising the sequence of SEQ ID NO: 10, and an HCDR3 comprising the sequence of SEQ ID NO: 16, or f) an HCDR1 comprising the sequence of SEQ ID NO: 4, an HCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, and an HCDR3 comprising the sequence of SEQ ID NO: 16, or g) an HCDR1 comprising the sequence of SEQ ID NO: 5, an HCDR2 comprising the sequence of SEQ ID NO: 11, and an HCDR3 comprising the sequence of SEQ ID NO: 17, or h) HCDR1 comprising the sequence of SEQ ID NO: 6, HCDR2 comprising the sequence of SEQ ID NO: 12, and HCDR3 comprising the sequence of SEQ ID NO: 18 Includes.

[0010] In some embodiments, the light chain variable region of an antibody or antigen-binding fragment thereof provided herein comprises: a) an LCDR1 comprising the sequence of SEQ ID NO: 19, an LCDR2 comprising the sequence of SEQ ID NO: 25, and an LCDR3 comprising the sequence of SEQ ID NO: 31, or b) an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 32, or c) an LCDR1 comprising the sequence of SEQ ID NO: 21, an LCDR2 comprising the sequence of SEQ ID NO: 27, and an LCDR3 comprising the sequence of SEQ ID NO: 33, or d) LCDR1 comprising the sequence of SEQ ID NO: 22, LCDR2 comprising the sequence of SEQ ID NO: 28, and LCDR3 comprising the sequence of SEQ ID NO: 34, or e) an LCDR1 comprising the sequence of SEQ ID NO: 23, an LCDR2 comprising the sequence of SEQ ID NO: 29, and an LCDR3 comprising the sequence of SEQ ID NO: 35, or f) LCDR1 comprising the sequence of SEQ ID NO: 24, LCDR2 comprising the sequence of SEQ ID NO: 30, and LCDR3 comprising the sequence of SEQ ID NO: 36 Includes.

[0011] In some embodiments, in the antibody or antigen-binding fragment thereof provided herein, (a) HCDR1 comprises the sequence of SEQ ID NO: 1, HCDR2 comprises the sequence of SEQ ID NO: 7, HCDR3 comprises the sequence of SEQ ID NO: 13, LCDR1 comprises the sequence of SEQ ID NO: 19, LCDR2 comprises the sequence of SEQ ID NO: 25, and LCDR3 comprises the sequence of SEQ ID NO: 31, or (b) HCDR1 comprises the sequence of SEQ ID NO: 2, HCDR2 comprises the sequence of SEQ ID NO: 8, HCDR3 comprises the sequence of SEQ ID NO: 14, LCDR1 comprises the sequence of SEQ ID NO: 20, LCDR2 comprises the sequence of SEQ ID NO: 26, and LCDR3 comprises the sequence of SEQ ID NO: 32, or (c) HCDR1 comprises the sequence of SEQ ID NO: 3, HCDR2 comprises the sequence of SEQ ID NO: 37, HCDR3 comprises the sequence of SEQ ID NO: 40, LCDR1 comprises the sequence of SEQ ID NO: 21, LCDR2 comprises the sequence of SEQ ID NO: 27, and LCDR3 comprises the sequence of SEQ ID NO: 33, or (d) HCDR1 comprises the sequence of SEQ ID NO: 3, HCDR2 comprises the sequence of SEQ ID NO: 38, HCDR3 comprises the sequence of SEQ ID NO: 41, LCDR1 comprises the sequence of SEQ ID NO: 21, LCDR2 comprises the sequence of SEQ ID NO: 27, and LCDR3 comprises the sequence of SEQ ID NO: 33, or (e) HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises the sequence of SEQ ID NO: 10, HCDR3 comprises the sequence of SEQ ID NO: 16, LCDR1 comprises the sequence of SEQ ID NO: 22, LCDR2 comprises the sequence of SEQ ID NO: 28, and LCDR3 comprises the sequence of SEQ ID NO: 34, or (f) HCDR1 comprises the sequence of SEQ ID NO: 4, HCDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, HCDR3 comprises the sequence of SEQ ID NO: 16, LCDR1 comprises the sequence of SEQ ID NO: 22, LCDR2 comprises the sequence of SEQ ID NO: 28, and LCDR3 comprises the sequence of SEQ ID NO: 34, or (g) HCDR1 comprises the sequence of SEQ ID NO:5, HCDR2 comprises the sequence of SEQ ID NO:11, HCDR3 comprises the sequence of SEQ ID NO:17, LCDR1 comprises the sequence of SEQ ID NO:23, LCDR2 comprises the sequence of SEQ ID NO:29, and LCDR3 comprises the sequence of SEQ ID NO:35, or (h) HCDR1 comprises the sequence of SEQ ID NO: 6, HCDR2 comprises the sequence of SEQ ID NO: 12, HCDR3 comprises the sequence of SEQ ID NO: 18, LCDR1 comprises the sequence of SEQ ID NO: 24, LCDR2 comprises the sequence of SEQ ID NO: 30, and LCDR3 comprises the sequence of SEQ ID NO: 36.

[0012] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more of heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of light chains LFR1, LFR2, LFR3, and LFR4; (a) HFR1 is X 19 VQLVX 20 SGX 21 X 22 X 23 X 24 KPGX 25 SX 26 X27 X 28 SCX 29 ASGX 30 X 31 X 32 X 33 (SEQ ID NO: 76) or a homologous sequence having at least 80% sequence identity thereto; (b) HFR2 is WVX 34 QX 35 PGX 36 X 37 LEWX 38 X 39 (SEQ ID NO: 77) or a homologous sequence having at least 80% sequence identity thereto; (c) HFR3 sequence is X 40 X 41 TX 42 X 43 X 44 DX 45 SX 46 X 47 TX 48 YX 49 X 50 X 51 X 52 SLX 53 X 54 EDTAVYYCX 55 X 56 (SEQ ID NO: 78) or a homologous sequence having at least 80% sequence identity thereto; (d) HFR4 is WGQGTX 57 VTVSS (SEQ ID NO: 126) or a homologous sequence having at least 80% sequence identity thereto; (e) LFR1 comprises the sequence X3IVX4TQSPATLX5X6SPGERX7TX8X9C (SEQ ID NO: 80), or a homologous sequence having at least 80% sequence identity thereto; (f)LFR2 is WYQQKPGQX 10 PX 11 LLIY (SEQ ID NO: 81) or a homologous sequence having at least 80% sequence identity thereto; (g)LFR3 is GX 12 PX 13 RFSGSGSGTX 14 X 15 TLTISSX16 EPEDFAVYX 17 C (SEQ ID NO: 82), or a homologous sequence having at least 80% sequence identity thereto; (h) LFR4 is FGX 18 GTKLEIK (SEQ ID NO: 152) or a homologous sequence thereof having at least 80% sequence identity; X3 is E or Q, X4 is L or M, X5 is S or T, X6 is L, V, or A, X7 is A or V, X8 is L or I, X9 is S or T, and X 10 is A or S, and X 11 is R or K, and X 12 is I or V, and X 13 is A or T, and X 14 is D or S, and X 15 is F or Y and X 16 is L, M, or V, and X 17 is Y or F, and X 18 is G or Q, and X 19 is Q or E, and X 20 is E or Q, and X 21 is G or A, and X 22 is G or E, and X 23 is L or V, and X 24 is V or K, and X 25 is G or A, and X 26 is L, M, or V, and X 27 is R or K, and X 28 is V or L, and X 29 is A or K, and X 30 is F or Y and X 31 is N or T, and X 32 is F or L, and X 33 is S or K, and X 34 is R or K, and X 35 is A or S, and X 36 is K or Q, and X 37 is R or G, and X 38 is M, I, or V, and X 39 is G or A, and X40 is R or K, and X 41 is V, A, or F, and X 42 is I or L, and X 43 is S or T, and X 44 is R or A, and X 45 is D or T, and X 46 is K, A, or S, and X 47 is S or N, and X 48 is L, V, or A, and X 49 is M or L, and X 50 is Q or E, and X 51 is M or L, and X 52 is S, I, or N, and X 53 is R or K, and X 54 is S or T, and X 55 is A or T, and X 56 is R, N, or T, and X 57 is T or L.

[0013] In some embodiments, HFR1 is EVQLVESGGGLVKPGGSX 61 RLSCAASGFTFS (SEQ ID NO: 154), or a homologous sequence having at least 80% sequence identity thereto, and HFR2 is WVRQX 62 PGKGLEWVX 63 (SEQ ID NO: 155), or a homologous sequence having at least 80% sequence identity thereto, and HFR3 is RFTISRDDSKNTX 64 YLQMNSLKTEDTAVYYCTT (SEQ ID NO: 156), or a homologous sequence having at least 80% sequence identity thereto; HFR4 comprises the sequence WGQGTTVTVSS (SEQ ID NO: 79), or a homologous sequence having at least 80% sequence identity thereto; and LFR1 comprises EIVX 65 TQSPATLSX 66 SPGERX 67 TLSC (SEQ ID NO: 157) or a homologous sequence having at least 80% sequence identity thereto, and LFR2 is WYQQKPGQX 68PRLLIY (SEQ ID NO: 158) or a homologous sequence having at least 80% sequence identity thereto, and LFR3 is GIPARFSGSGSGTDFTLTISSX 69 EPEDFAVYX 70 C (SEQ ID NO: 159), or a homologous sequence having at least 80% sequence identity thereto; LFR4 comprises the sequence FGGGTKLEIK (SEQ ID NO: 153), or a homologous sequence having at least 80% sequence identity thereto; X 61 is L or M, and X 62 is A or S, and X 63 is G or A, and X 64 is L or V, and X 65 is L or M, and X 66 is L or V, and X 67 is A or V, and X 68 is A or S, and X 69 is L or V, and X 70 is Y or F.

[0014] In some embodiments, HFR1 is X 71 VQLVQSGAEVKKPGASVKX 72 SCKASGYX 73 LK (SEQ ID NO: 160), or a homologous sequence having at least 80% sequence identity thereto, and HFR2 is WVX 74 QAPGQX 75 LEWX 76 G (SEQ ID NO: 161), or a homologous sequence having at least 80% sequence identity thereto, and HFR3 comprises X 77 X 78 TX 79 TX 80 DTSX 81 X 82 TAYX 83 ELX 84 SLRSEDTAVYYCAX 85 (SEQ ID NO: 149), or a homologous sequence having at least 80% sequence identity thereto, and HFR4 is WGQGTX 57 VTVSS (SEQ ID NO: 126) or a homologous sequence having at least 80% sequence identity thereto, and LFR1 is X86 IVLTQSPATLX 87 X 88 SPGERX 89 TX 90 X 91 C (SEQ ID NO: 150), or a homologous sequence having at least 80% sequence identity thereto, and LFR2 is WYQQKPGQX 10 PX 11 LLIY (SEQ ID NO: 81), or a homologous sequence having at least 80% sequence identity thereto, and LFR3 is GX 92 PX 93 RFSGSGSGTX 94 X 95 TLTISSX 96 LFR4 comprises the sequence of FGQGTKLEIK (SEQ ID NO: 83) or a homologous sequence having at least 80% sequence identity thereto; X 10 , X 11 , and X 57 is as defined above, and X 71 is Q or E, and X 72 is V or L, and X 73 is N or T, and X 74 is R or K, and X 75 is R or G, and X 76 is M or I, and X 77 is R or K, and X 78 is V or A, and X 79 is I or L, and X 80 is R or A, and X 81 is A or S, and X 82 is S or N, and X 83 is M or L, and X 84 is S or I, and X 85 is R or N, and X 86 is E or Q, and X 87 is S or T, and X 88 is L or A, and X 89 is A or V, and X 90 is L or I, and X 91 is S or T, and X92 is I or X 93 is A or T, and X 94 is D or S, and X 95 is F or Y and X 96 is L or M.

[0015]

[0015] In some embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 84-86, 115, 119-120, and 131, HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 87-90 and 121-123, HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 91-97, 116-117, and 124-125, HFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 79 and 118, LFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 98-103 and 127-129, LFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 104, 105, and 130, LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 106-110 and 132-133, and LFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 83 and 153.

[0016]

[0016] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 60, 62, 64, 66, 140, 141, 142, 146, 147, 39, and homologous sequences thereof having at least 80% sequence identity and retaining specific binding affinity to human CD39, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 61, 63, 65, 67, 143, 144, 145, 111, 112, 63, and homologous sequences thereof having at least 80% sequence identity and retaining specific binding affinity to human CD39.

[0017]

[0017] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 68, 70, 72, 74, and homologous sequences thereof having at least 80% sequence identity and retaining specific binding affinity to human CD39, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, 75, and homologous sequences thereof having at least 80% sequence identity and retaining specific binding affinity to human CD39.

[0018] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are (1) a heavy chain variable region comprising the sequence of SEQ ID NO: 42 and a light chain variable region comprising the sequence of SEQ ID NO: 51; or (2) a heavy chain variable region comprising the sequence of SEQ ID NO: 43 and a light chain variable region comprising the sequence of SEQ ID NO: 52; or (3) a heavy chain variable region comprising the sequence of SEQ ID NO: 44 and a light chain variable region comprising the sequence of SEQ ID NO: 53; or (4) a heavy chain variable region comprising the sequence of SEQ ID NO: 45 and a light chain variable region comprising the sequence of SEQ ID NO: 54; or (5) a heavy chain variable region comprising the sequence of SEQ ID NO: 47 and a light chain variable region comprising the sequence of SEQ ID NO: 56; or (6) a heavy chain variable region comprising the sequence of SEQ ID NO: 49 and a light chain variable region comprising the sequence of SEQ ID NO: 58; or (7) a heavy chain variable region comprising the sequence of SEQ ID NO: 50 and a light chain variable region comprising the sequence of SEQ ID NO: 59; or (8) a heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 61; or (9) A heavy chain variable region comprising the sequence of SEQ ID NO: 62 and a light chain variable region comprising the sequence of SEQ ID NO: 61; or (10) A heavy chain variable region comprising the sequence of SEQ ID NO: 64 and a light chain variable region comprising the sequence of SEQ ID NO: 61; or (11) A heavy chain variable region comprising the sequence of SEQ ID NO: 66 and a light chain variable region comprising the sequence of SEQ ID NO: 61, or (12) A heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 63; or (13) A heavy chain variable region comprising the sequence of SEQ ID NO: 62 and a light chain variable region comprising the sequence of SEQ ID NO: 63; or (14) A heavy chain variable region comprising the sequence of SEQ ID NO: 64 and a light chain variable region comprising the sequence of SEQ ID NO: 63; or (15) A heavy chain variable region comprising the sequence of SEQ ID NO: 66 and a light chain variable region comprising the sequence of SEQ ID NO: 63; or (16) A heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 65; or (17) A heavy chain variable region comprising the sequence of SEQ ID NO: 62 and a light chain variable region comprising the sequence of SEQ ID NO: 65; or (18) A heavy chain variable region comprising the sequence of SEQ ID NO: 64 and a light chain variable region comprising the sequence of SEQ ID NO: 65; or (19) A heavy chain variable region comprising the sequence of SEQ ID NO: 66 and a light chain variable region comprising the sequence of SEQ ID NO: 65; or (20) A heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 67; or (21) A heavy chain variable region comprising the sequence of SEQ ID NO: 62 and a light chain variable region comprising the sequence of SEQ ID NO: 67; or (22) A heavy chain variable region comprising the sequence of SEQ ID NO: 64 and a light chain variable region comprising the sequence of SEQ ID NO: 67; or (23) A heavy chain variable region comprising the sequence of SEQ ID NO: 66 and a light chain variable region comprising the sequence of SEQ ID NO: 67; or (24) A heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 61; or (25) A heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 61; or (26) A heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 61; or (27) A heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 143; or (28) A heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 143; or (29) A heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 143; or (30) A heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 143; or (31) A heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 144; or (32) A heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 144; or (33) A heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 144; or (34) A heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 144; or (35) A heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 145; or (36) A heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 145; or (37) A heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 145; or (38) A heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 145; or (39) A heavy chain variable region comprising the sequence of SEQ ID NO: 146 and a light chain variable region comprising the sequence of SEQ ID NO: 111; or (40) A heavy chain variable region comprising the sequence of SEQ ID NO: 146 and a light chain variable region comprising the sequence of SEQ ID NO: 112; or (41) A heavy chain variable region comprising the sequence of SEQ ID NO: 147 and a light chain variable region comprising the sequence of SEQ ID NO: 111; or (42) A heavy chain variable region comprising the sequence of SEQ ID NO: 39 and a light chain variable region comprising the sequence of SEQ ID NO: 63; or (43) A heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 69; or (44) A heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 69; or (45) A heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 69; or (46) A heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 69; or (47) A heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 71; or (48) A heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 71; or (49) A heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 71; or (50) A heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 71; or (51) A heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 73; or (52) A heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 73; or (53) A heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 73; or (54) A heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 73; or (55) A heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 75; or (56) A heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 75; or (57) A heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 75; or (58) A heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 75. Includes.

[0019]

[0019] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more amino acid residue substitutions or modifications and still retain specific binding affinity for human CD39. In some embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences and / or in one or more of the non-CDR sequences of the heavy chain variable region or light chain variable region. In some embodiments, at least one of the substitutions is a conservative substitution.

[0020]

[0020] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM. In some embodiments, the Fc region is derived from human IgG1 or IgG4. In some embodiments, the Fc region derived from human IgG1 or IgG4 has reduced effector function. In some embodiments, the Fc region derived from human IgG1 comprises the L234A and / or L235A mutations. In some embodiments, the Fc region derived from human IgG4 optionally has reduced effector function. In some embodiments, the Fc region derived from human IgG4 comprises the S228P mutation, and / or the L235E mutation, and / or the F234A and L235A mutations.

[0021] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are humanized. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, chimeric antibodies, labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, or fusion proteins.

[0022]

[0022] In some embodiments, the antibody or antigen-binding fragment thereof provided herein is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single-chain domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0023] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have a cytotoxicity of 10 or more as measured by a FACS (fluorescence-activated cell sorting) assay. -8 EC below M 50 It can specifically bind to human CD39 at 1000 kJ / s.

[0024] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are a) specifically binds to human CD39 but does not specifically bind to mouse CD39 as measured by a FACS assay; b) 10 as measured by FACS assay -8 EC below M 50 specifically binds to cynomolgus monkey CD39. c) 10 as measured by Biacore assay -7 M or less (for example, 5 × 10 -8 M or less, 3×10 -8 M or less, 2×10 -8 M or less, 1×10 -8 M or less, or 8 x 10 -9 M or less) K D specifically binds to human CD39 at d) 10 as measured by Octet assay -8 M or less (for example, 8 × 10 -9 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 1×10 -9 M or less, or 9 x 10-10 M or less) K D specifically binds to human CD39 at e) an IC of 50 nM or less (e.g., 1 nM or less, 5 nM or less, 10 nM or less, or 30 nM or less) as measured by an ATPase activity assay 50 inhibits ATPase activity in CD39-expressing cells, f) capable of enhancing ATP-mediated monocyte activation at a concentration of 50 nM or less (e.g., 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, or 0.2 nM or less) as measured by analysis of CD80, CD86, and / or CD40 expression by FACS assay; g) capable of enhancing ATP-mediated T cell activation in peripheral blood mononuclear cells (PBMCs) at concentrations of 25 nM or less as measured by IL-2 secretion, IFN-γ secretion, and CD4+ or CD8+ T cell proliferation; h) is capable of enhancing ATP-mediated DC activation at a concentration of 25 nM or less (or 10 nM or less, or 5 nM or less, or 1 nM or less, or 0.5 nM or less, or 0.2 nM or less), as measured by analysis of CD83 expression by FACS assay, or by the ability of activated dendritic cells (DCs) to promote T cell proliferation, or by the ability of activated DCs to promote IFN-γ production in a mixed lymphocyte reaction (MLR) assay; i) CD4 induced by adenosine (hydrolyzed from ATP) at concentrations of 1 nM or less (e.g., 0.1 nM or less, 0.01 nM or less) as measured by FACS assay + Can block the inhibition of T cell proliferation, j) can inhibit tumor growth in a NK cell or macrophage cell dependent manner; k) T cell proliferation, CD25 + Human CD8 inhibited by eATP as measured by cell and viable cell populations + can reverse T cell proliferation, and l) capable of enhancing the release of IL1β from human macrophages induced by LPS stimulation at a concentration of 50 nM or less (or 12.5 nM or less, or 3.13 nM or less, or 0.78 nM or less, or 0.2 nM or less, or 0.049 nM or less, or 0.012 nM or less, or 0.003 nM or less, or 0.0008 nM or less) as measured by ELISA assay; The compound has one or more properties selected from the group consisting of:

[0025] In another aspect, the present disclosure provides an anti-CD39 antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment thereof provided herein for binding to human CD39. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 43 and a light chain variable region comprising the sequence of SEQ ID NO: 52 for binding to human CD39. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 44 and a light chain variable region comprising the sequence of SEQ ID NO: 53 for binding to human CD39, or competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 45 and a light chain variable region comprising the sequence of SEQ ID NO: 54. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 47 and a light chain variable region comprising the sequence of SEQ ID NO: 56 for binding to human CD39.

[0026]

[0026] In another aspect, the present disclosure provides an anti-CD39 antibody or antigen-binding fragment thereof that specifically binds to an epitope of CD39, wherein the epitope includes one or more residues selected from the group consisting of Q96, N99, E143, R147, R138, M139, E142, K5, E100, D107, V81, E82, R111, and V115.

[0027] In some embodiments, the epitope comprises one or more residues selected from the group consisting of Q96, N99, E143, and R147. In some embodiments, the epitope comprises one or more residues selected from the group consisting of R138, M139, and E142. In some embodiments, the epitope comprises one or more residues selected from the group consisting of K5, E100, and D107. In some embodiments, the epitope comprises one or more residues selected from the group consisting of V81, E82, R111, and V115. In some embodiments, the CD39 is human CD39. In some embodiments, the CD39 is human CD39 comprising the amino acid sequence of SEQ ID NO: 162.

[0028]

[0028] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is none of antibody 9-8B, antibody T895, and antibody I394, where antibody 9-8B comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 46 and a light chain variable region comprising the sequence of SEQ ID NO: 48, antibody T895 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 55 and a light chain variable region comprising the sequence of SEQ ID NO: 57, and antibody I394 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 113 and a light chain variable region comprising the sequence of SEQ ID NO: 114.

[0029]

[0029] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are bispecific. In some embodiments, the antibodies or antigen-binding fragments provided herein can specifically bind to a second antigen other than CD39 or a second epitope on CD39. In certain embodiments, the second antigen is selected from the group consisting of TGF-beta, CD73, PD1, PDL1, 4-1BB, CTLA4, TIGIT, GITA, VISTA, TIGIT, B7-H3, B7-H4, B7-H5, CD112R, Siglec-15, LAG3, and TIM-3.

[0030] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are linked to one or more conjugate moieties, which in some embodiments comprise a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, or other anti-cancer drug.

[0031]

[0031] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present disclosure and one or more pharmaceutically acceptable carriers.

[0032] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof of the present disclosure.

[0032]

[0033] In another aspect, the present disclosure provides a vector comprising an isolated polynucleotide of the present disclosure.

[0034] In another aspect, the present disclosure provides a host cell comprising a vector of the present disclosure.

[0033]

[0035] In another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof and / or pharmaceutical composition of the present disclosure and a second therapeutic agent.

[0036] In another aspect, the present disclosure provides a method of expressing an antibody or antigen-binding fragment thereof of the present disclosure, comprising culturing a host cell of the present disclosure under conditions in which a vector of the present disclosure is expressed.

[0034]

[0037] In another aspect, the present disclosure provides a method of treating, preventing, or alleviating a CD39-related disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, and / or a pharmaceutical composition of the present disclosure.

[0035]

[0038] In another aspect, the present disclosure provides a method of treating, preventing, or alleviating a disease treatable by reducing the ATPase activity of CD39 in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, and / or a pharmaceutical composition of the present disclosure.

[0036]

[0039] In another aspect, the present disclosure provides a method of treating, preventing, or alleviating a disease associated with adenosine-mediated inhibition of T cell, monocyte, macrophage, dendritic cell, antigen-presenting cell, NK, and / or B cell activity in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, and / or a pharmaceutical composition of the present disclosure.

[0037]

[0040] In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the cancer is selected from the group consisting of anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, and the like. The cancers include skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, throat cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T- or B-cell lymphoma, GI organ stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma. In some embodiments, the cancer is leukemia, lymphoma, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, or breast cancer. In some embodiments, the subject has been identified as having cancer cells or tumor-infiltrating immune cells or immunosuppressive cells that express CD39, optionally at a level significantly higher than that normally found in non-cancer cells or non-immunosuppressive cells. Examples of immune suppressive cells expressing CD39 include myeloid-derived suppressor cells (MDSCs) or regulatory T cells (Tregs).

[0038]

[0041] In some embodiments, the disease, disorder, or condition is an autoimmune disease or infection. In some embodiments, the autoimmune disease is immune thrombocytopenia, systemic sclerosis, sclerosis, adult respiratory distress syndrome, eczema, asthma, Sjögren's syndrome, Addison's disease, giant cell arteritis, immune complex nephritis, immune thrombocytopenic purpura, autoimmune thrombocytopenia, celiac disease, psoriasis, dermatitis, colitis, or systemic lupus erythematosus. In some embodiments, the infection is a viral infection or a bacterial infection. In some embodiments, the infection is HIV infection, HBV infection, HCV infection, inflammatory bowel disease, or Crohn's disease.

[0039]

[0042] In some embodiments, the subject is a human. In some embodiments, the administration is oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular. In some embodiments, the method further comprises administering a therapeutically effective amount of a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an anti-cancer drug, a radiation therapy agent, an immunotherapy agent, an anti-angiogenesis agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, and a cytokine.

[0040]

[0043] In another aspect, the present disclosure provides a method for modulating the activity of CD39 in a CD39-positive cell, comprising exposing the CD39-positive cell to an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure. In some embodiments, the cell is an immune cell.

[0041]

[0044] In another aspect, the present disclosure provides a method for detecting the presence or amount of CD39 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure, and determining the presence or amount of CD39 in the sample.

[0042]

[0045] In another aspect, the present disclosure provides a method of diagnosing a CD39-related disease, disorder, or condition in a subject, comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure; b) determining the presence or amount of CD39 in the sample; and c) correlating the presence or amount of CD39 with the presence or status of the CD39-related disease, disorder, or status in the subject.

[0043]

[0046] In another aspect, the present disclosure provides use of an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating, preventing, or ameliorating a CD39-related disease, disorder, or condition in a subject.

[0044]

[0047] In another aspect, the present disclosure provides use of an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure in the manufacture of a diagnostic reagent for diagnosing a CD39-related disease, disorder, or condition in a subject.

[0045]

[0048] In another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof of the present disclosure and / or a pharmaceutical composition of the present disclosure, useful for detecting CD39. [Brief explanation of the drawings]

[0046] [Figure 1]

[0049] FIG. 1 shows ATP-mediated enhanced T cell proliferation by anti-CD39 monoclonal antibodies mAB21 and mAb23. [Figure 2]

[0050] Figure 2 shows blockade of ATP-mediated inhibition of T cell proliferation by anti-CD39 chimeric antibodies c14, c19, c21, and c23. hIgG4 refers to the human IgG4 isotype control antibody. [Figure 3]

[0051] FIG. 3 shows the expression level of CD39 on dendritic cells (DCs). [Figure 4A]

[0052] FIG. 4A shows ATP-mediated DC activation by anti-CD39 chimeric antibodies c14, c19, c21, and c23 as measured by CD86 expression using FACS. [Figure 4B] FIG. 4B shows ATP-mediated DC activation by anti-CD39 chimeric antibodies c14, c19, c21, and c23 as measured by CD83 expression using FACS. [Figure 4C] FIG. 4C shows ATP-mediated DC activation by anti-CD39 chimeric antibodies c14, c19, c21, and c23 as measured by HLA-DR expression using FACS. [Figure 5]

[0053] FIG. 5 shows tumor growth after treatment with anti-CD39 chimeric antibodies c23-hIgG4 and c23-hIgG1 in mice inoculated with MOLP-8 cells (a human multiple myeloma cell line). [Figure 6]

[0054] Figure 6A shows the binding characteristics of humanized antibody hu23.H5L5 to ENTPD1 (i.e., CD39), ENTPD2 (i.e., CD39L1), ENTPD3 (i.e., CD39L3), ENTPD5 (i.e., CD39L4), and ENTPD6 (i.e., CD39L2) proteins, respectively. Figure 6B shows the binding of negative control hIgG4 to ENTPD1 (i.e., CD39), ENTPD2 (i.e., CD39L1), ENTPD3 (i.e., CD39L3), ENTPD5 (i.e., CD39L4), and ENTPD6 (i.e., CD39L2) proteins, respectively. [Figure 7]

[0055] 7A and 7B show the binding activity of the c23 humanized antibody to MOLP-8 cells by FACS. [Figure 8]

[0056] FIG. 8 shows the binding activity of the c23 humanized antibody (obtained by yeast display) to MOLP-8 cells. [Figure 9]

[0057] 9A and 9B show ATPase inhibition of c23 humanized antibody in SK-MEL-28 cells by FACS. [Figure 10A]

[0058] 10A to 10C show the binding activity of the c14 humanized antibody to MOLP-8 cells as determined by FACS. [Figure 10B] 10A to 10C show the binding activity of the c14 humanized antibody to MOLP-8 cells as determined by FACS. [Figure 10C] 10A to 10C show the binding activity of the c14 humanized antibody to MOLP-8 cells as determined by FACS. [Figure 11A]

[0059] FIG. 11A shows ATP-mediated T cell activation in PMBC by humanized antibody hu23.H5L5 as measured by IL-2. [Figure 11B] FIG. 11B shows ATP-mediated T cell activation in PMBCs by humanized antibody hu23.H5L5 as measured by IFN-γ. [Figure 11C]FIG. 11C shows ATP-mediated T cell activation in PMBCs by humanized antibody hu23.H5L5 as measured by CD4+ T cell proliferation. [Figure 11D] FIG. 11D shows ATP-mediated T cell activation in PMBCs by humanized antibody hu23.H5L5 as measured by CD8+ T cell proliferation. [Figure 12A]

[0060] FIG. 12A shows the binding activity of humanized antibodies hu23.H5L5 and hu14.H1L1 to SK-MEL-5 cells by FACS. [Figure 12B] FIG. 12B shows the binding activity of humanized antibodies hu23.H5L5 and hu14.H1L1 to SK-MEL-28 cells by FACS. [Figure 12C] FIG. 12C shows the binding activity of humanized antibodies hu23.H5L5 and hu14.H1L1 to MOLP-8 cells by FACS. [Figure 12D] FIG. 12D shows the binding activity of humanized antibodies hu23.H5L5 and hu14.H1L1 to CHOK1-cynoCD39 cells by FACS. [Figure 12E] FIG. 12E shows the binding activity of humanized antibodies hu23.H5L5 and hu14.H1L1 to CHOK1-mCD39 cells by FACS. [Figure 13]

[0061] Figure 13A shows the ATPase inhibitory activity of humanized antibodies hu23.H5L5 and hu14.H1L1 on SK-MEL-5, and Figure 13B shows the ATPase inhibitory activity of humanized antibodies hu23.H5L5 and hu14.H1L1 on MOLP-8. [Figure 14A]

[0062] Figure 14A shows ATP-mediated monocyte activation by the anti-CD39 humanized antibody hu23.H5L5 as measured by CD80 expression. [Figure 14B] Figure 14B shows ATP-mediated monocyte activation by the anti-CD39 humanized antibody hu23.H5L5 as measured by CD86 expression. [Figure 14C]Figure 14C shows ATP-mediated monocyte activation by the anti-CD39 humanized antibody hu23.H5L5 as measured by CD40 expression. [Figure 15A]

[0063] FIG. 15A shows that humanized antibody hu23.H5L5 increases ATP-mediated DC activation as measured by CD83 expression. [Figure 15B] FIG. 15B shows that humanized antibody hu23.H5L5 increases ATP-mediated DC activation as measured by enhanced T cell proliferation. [Figure 15C] FIG. 15C shows that humanized antibody hu23.H5L5 increases ATP-mediated DC activation as measured by T cell activation. [Figure 16]

[0064] FIG. 16 shows tumor growth inhibition by humanized antibodies hu23.H5L5 and hu14.H1L1 in MOLP-8 xenografted mice. [Figure 17]

[0065] Figure 17 shows tumor growth inhibition of anti-CD39 humanized antibody hu23.H5L5 in NK-depleted MOLP-8 xenografted mice. [Figure 18]

[0066] Figure 18 shows tumor growth inhibition of anti-CD39 humanized antibody hu23.H5L5 in macrophage-depleted MOLP-8 xenografted mice. [Figure 19A]

[0067] FIG. 19A shows the results of epitope binning of humanized antibodies hu23.H5L5 and hu14.H1L1 with reference antibodies. [Figure 19B] FIG. 19B shows the epitope classification of the antibodies tested. [Figure 20]

[0068] FIG. 20 shows the effect of anti-CD39 humanized antibody hu23.H5L5 on IL1β release from human macrophages induced by LPS stimulation. [Figure 21]

[0069] Figure 21 shows tumor growth inhibition of different doses (0.03 mg / kg, 0.3 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg) of humanized antibody hu23.H5L5 in PBMC-fed mice. [Figure 22]

[0070] FIG. 22 shows the results of epitope mapping of humanized antibody hu23.H5L5, chimeric antibodies c34 and c35, and reference antibodies T895, I394, and 9-8B. [Figure 23A]

[0071] Figure 23A shows extracellular ATP-inhibited CD8+ T cell proliferation reversed by humanized antibody hu23.H5L5 as measured by T cell proliferation. [Figure 23B] Figure 23B shows extracellular ATP-inhibited CD8+ T cell proliferation as measured by CD25+ cells, reversed by humanized antibody hu23.H5L5. [Figure 23C] Figure 23C shows the proliferation of extracellular ATP-inhibited CD8+ T cells reversed by humanized antibody hu23.H5L5 as measured by viable cell population. DETAILED DESCRIPTION OF THE INVENTION

[0047]

[0072] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of the present disclosure. It is understood that various equivalents, changes, and modifications can be made to those skilled in the art without departing from the scope of the present disclosure, and that such equivalent embodiments are intended to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0048]

[0073] definition

[0074] As used herein, the term "antibody" includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. Naturally occurring, intact antibodies contain two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each consisting of a variable region (VH) and a first, second, third, and optionally, fourth constant region (CH1, CH2, CH3, and CH4, respectively). Mammalian light chains are classified as lambda or kappa, each consisting of a variable region (VL) and a constant region. Antibodies are "Y" shaped, with the stem of the Y consisting of the second and third constant regions of two heavy chains linked together via disulfide bonds. Each arm of the Y contains the variable region and first constant region of one heavy chain bound to the variable and constant regions of one light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, and HCDR3).The boundaries of the CDRs of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the rules of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J. Mol. Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). The three CDRs are inserted between adjacent sections known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3, and LFR4, and heavy chain FRs including HFR1, HFR2, HFR3, and HFR4), which are more highly conserved than the CDRs and form a scaffold supporting highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains.The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0049]

[0075] In certain embodiments, the antibodies provided herein encompass any antigen-binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise the intact native antibody structure. Examples of antigen-binding fragments include, without limitation, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies. An antigen-binding fragment can bind to the same antigen as the parent antibody.

[0050]

[0076] "Fab," with respect to an antibody, means the portion of the antibody that consists of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0051]

[0077] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0078] "F(ab')2" means a dimer of Fab'.

[0079] "Fc," with respect to antibodies (e.g., of an IgG, IgA, or IgD isotype), refers to the portion of an antibody consisting of the second and third constant domains of a first heavy chain linked via disulfide bonds to the second and third constant domains of a second heavy chain. For antibodies of the IgM and IgE isotypes, the Fc further comprises a fourth constant domain. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0052]

[0080] "Fv," with respect to an antibody, refers to the minimum fragment of the antibody that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0081] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to each other either directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)).

[0053]

[0082] By "single chain Fv-Fc antibody" or "scFv-Fc" is meant an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0083] "Camelized single domain antibodies," "heavy chain antibodies," or "HCAbs" are antibodies that contain two V HHeavy chain antibodies refer to antibodies containing heavy chain domains and not containing light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38(1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302(2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies originally came from the Camelidae family (camels, dromedaries, and llamas). Camelized antibodies lack light chains but retain genuine antigen-binding capacity (Hamers-Casterman C. et al., Nature. June 3; 363(6428): 446-8(1993); Nguyen VK. et al., Immunogenetics. April; 54(1): 39-47(2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101(2003)). The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November; 21(13): 3490-8. Epub 2007 Jun 15(2007)).

[0054]

[0084] "Nanobody" refers to an antibody fragment consisting of a VHH domain of a heavy chain antibody and two constant domains, CH2 and CH3.

[0085] A "diabody" or "dAb" includes small antibody fragments with two antigen-binding sites, V L V linked to domain H Domain Included (V H -V L or V L -V H) (See, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8(1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0055]

[0086] "Domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. H The domains are covalently joined with peptide linkers to create bivalent or multivalent domain antibodies. H The domains may target the same or different antigens.

[0056]

[0087] As used herein, the term "valent" refers to the presence of a specific number of antigen-binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment having only one single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment having multiple antigen-binding sites. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0057]

[0088] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes, which may be on the same antigen or on two different antigens.

[0058]

[0089] In certain embodiments, an "scFv dimer" is a dimer of another V H -V L V dimerized with the moiety (dimerized via a peptide linker) H -V L and a bivalent diabody or bispecific scFv (BsFv) comprising one part V H V in other parts L In other embodiments, an "scFv dimer" comprises two V (linked by a peptide linker) and two V (linked by a peptide linker) that cooperate to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). L1 -V H2 V associated with (also linked by a peptide linker) H1 -V L2 and a bispecific diabody comprising V H1 and V L1 cooperated, V H2 and V L2 cooperate, with each cooperated pair having a different antigen specificity.

[0059]

[0090] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" is a fragment of three peptide chains, i.e., linked by peptide linkers (e.g., long flexible linkers), connecting each of the two Vs via disulfide bridges. L Two V's bonded to the moiety H In some embodiments, dsFv-dsFv' are bispecific, in which the disulfide-paired heavy and light chains have different antigen specificities.

[0060]

[0091] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of its heavy and / or light chain derived from one species and the remainder of its heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0061]

[0092] As used herein, the term "humanized" means that the antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human.

[0062]

[0093] As used herein, the term "affinity" refers to the strength of the non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen.

[0094] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. Specific binding is defined as the K D value, i.e., the ratio of the dissociation rate to the association rate (k off / k on The binding affinity can be characterized by the binding affinity expressed by K D may be determined using any conventional method known in the art, including, but not limited to, surface plasmon resonance, Octet, microscale thermophoresis, HPLC-MS, and FACS assays. -6 M or less (for example, 5 × 10 -7 M or less, 2×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10-9 M or less, or 10 -9 M or less) K D The value can indicate the specific binding between the antibody or antigen-binding fragment thereof and CD39 (e.g., human CD39).

[0063]

[0095] As used herein, the ability to "compete for binding to human CD39" refers to the ability of a first antibody or antigen-binding fragment to inhibit, to any detectable extent, the binding interaction between human CD39 and a second anti-CD39 antibody. In certain embodiments, an antibody or antigen-binding fragment that competes for binding to human CD39 inhibits the binding interaction between human CD39 and the second anti-human CD39 antibody by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 95%, or greater than 99%.

[0064]

[0077] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes within the antigen. Epitopes may be linear or conformational (i.e., involving separated amino acid residues). For example, if an antibody or antigen-binding fragment blocks at least 85%, or at least 90%, or at least 95% of the binding of a reference antibody to the antigen, the antibody or antigen-binding fragment may be considered to bind to the same / closely related epitope as the reference antibody.

[0065] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group along with a side chain characteristic of each amino acid. In this disclosure, the names of amino acids may also be represented as standard one-letter or three-letter codes, which are summarized below.

[0066] [Table 1]

[0067]

[0079] A "conservative substitution" with respect to an amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not result in significant changes in the conformational structure of a protein and thus can retain the biological activity of the protein.

[0068]

[0080] As used herein, the term "homologous" means a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0069]

[0081] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to maximize the number of identical amino acids (or nucleic acids). In other words, the percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) with respect to the reference sequence being compared by the total number of amino acid residues (or bases) in either the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for calculating percent amino acid (or nucleic acid) sequence identity can be achieved using publicly available tools such as BLASTN, BLASTp (available from the US National Center for Biotechnology Information (NCBI) website; see also Altschul S F et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the European Bioinformatics Institute website; see also Higgins D Get al., Methods in Enzymology, 266:383-402 (1996); Larkin M A et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize appropriate parameters for the alignment, for example by selecting a suitable algorithm.

[0070] As used herein, "effector function" refers to the biological activity attributable to the binding of the Fc region of an antibody to its effector, such as the C1 complex and Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is mediated by the interaction of an antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC), which is mediated by the binding of the Fc region of an antibody to an Fc receptor on an effector cell, and phagocytosis. Effector function can be assessed using various assays, such as Fc receptor binding assays, C1q binding assays, and cytolytic assays.

[0071] An "isolated" material has been altered by the hand of man from its natural state. If an "isolated" composition or material occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it exists in a substantially pure state sufficiently separated from the coexisting materials in its natural state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (ion exchange chromatography or reverse-phase HPLC).

[0072] As used herein, the term "vector" refers to a vehicle into which a genetic element is operably inserted, resulting in expression of the genetic element, thereby producing the protein, RNA, or DNA encoded by the genetic element, or in replication of the genetic element. A vector can be used to transform, transduce, or transfect a host cell to result in expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector may contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, a vector may contain an origin of replication. A vector may also contain materials that aid in the vector's entry into a cell, including, but not limited to, a viral particle, a liposome, or a protein coating. The vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0073]

[0085] As used herein, the phrase "host cell" means a cell into which exogenous polynucleotides and / or vectors can be or have been introduced. The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0074] The term "anti-tumor activity" refers to a reduction in tumor cell proliferation, viability, or metastatic activity. For example, anti-tumor activity can be demonstrated by a decrease in the rate of abnormal cell proliferation that occurs during treatment, or a stable or reduced tumor size, or prolonged survival with treatment, compared to untreated controls. Such activity can be assessed using accepted in vitro or in vivo tumor models, including, but not limited to, xenograft models, allograft models, mouse mammary tumor virus (MMTV) models, and other models known in the art for studying anti-tumor activity.

[0075]

[0088] As used herein, "treating" or "treatment" of a disease, disorder, or condition includes preventing or alleviating the disease, disorder, or condition, slowing the onset or rate of occurrence of the disease, disorder, or condition, reducing the risk of developing the disease, disorder, or condition, preventing or delaying the onset of symptoms associated with the disease, disorder, or condition, reducing or terminating symptoms associated with the disease, disorder, or condition, completely or partially ameliorating the disease, disorder, or condition, curing the disease, disorder, or condition, or some combination thereof.

[0076] The terms "diagnosis," "diagnose," or "diagnosing" refer to the identification of a pathological state, disease, or condition, such as the identification of a CD39-associated disease, or the identification of a subject having a CD39-associated disease that may benefit from a particular treatment regimen. In some embodiments, diagnosis includes the identification of an abnormal amount or activity of CD39. In some embodiments, diagnosis refers to the identification of cancer or an autoimmune disease in a subject.

[0077] As used herein, the term "biological sample" or "sample" refers to a biological composition containing cells and / or other molecular entities obtained or derived from a subject of interest and characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. Biological samples include, but are not limited to, cells, tissues, organs, and / or biological fluids of a subject obtained by any method known to those of skill in the art. In some embodiments, the biological sample is a fluid sample. In some embodiments, the fluid sample is whole blood, plasma, serum, mucus (including nasal discharge and sputum), peritoneal fluid, pleural fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, abdominal fluid, ascites, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lungs, kidneys, skin, or blood vessels of a subject.

[0078] As used herein, "CD39" refers to an integral membrane protein, also known as ENTPD1 or ENTPDase 1, that converts ATP to AMP. Structurally, it is characterized by two transmembrane domains, a small cytoplasmic domain, and a large extracellular hydrophobic domain. In certain embodiments, CD39 is human CD39. As used herein, CD39 may be from mouse and other animal species, such as cynomolgus monkeys, among others. An exemplary sequence of the human CD39 protein is disclosed in NCBI Ref Seq No. NP_001767.3. An exemplary sequence of the Mus musculus (mouse) CD39 protein is disclosed in NCBI Ref Seq No. NP_033978.1. An exemplary sequence of the cynomolgus monkey (monkey) CD39 protein is disclosed in NCBI Ref Seq No. XP_015311945.1.

[0079] In addition to CD39, the ENTPDase family also includes several other members, including ENTPDases 2, 3, 4, 5, 6, 7, and 8 (also known as ENTPD2, 3, 4, 5, 6, 7, and 8, which are used interchangeably in this disclosure). Four of the ENTPDases are typical cell surface-localized enzymes (ENTPDases 1, 2, 3, and 8) with catalytic sites facing the extracellular side. ENTPDases 5 and 6 exhibit intracellular localization and are secreted after heterologous expression. ENTPDases 4 and 7 are entirely intracellular and face the lumen of cytoplasmic organelles. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein specifically bind to CD39 (i.e., ENTPDase 1) but do not bind to other family members, such as ENTPDases 2, 3, 5, or 6.

[0080] The term "anti-CD39 antibody" means an antibody capable of specifically binding to CD39 (e.g., human or monkey CD39). The term "anti-human CD39 antibody" means an antibody capable of specifically binding to human CD39.

[0081] As used herein, a "CD39-associated" disease, disorder, or condition refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of CD39. In some embodiments, the CD39-associated disease, disorder, or condition is an immune-related disorder, such as, for example, an autoimmune disease. In some embodiments, the CD39-associated disease, disorder, or condition is a disorder associated with excessive cell proliferation, such as, for example, cancer. In certain embodiments, the CD39-associated disease or condition is characterized by expression or overexpression of CD39 and / or a CD39-associated gene, such as the ENTPD1, ​​2, 3, 4, 5, 6, 7, or 8 gene.

[0082]

[0095] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation and physiologically compatible with the recipient thereof.

[0083]

[0096] As used herein, the term "CD39-positive cells" refers to cells (eg, phagocytes) that express CD39 on their surface.

[0097] Anti-CD39 antibody

[0098] The present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof. The anti-CD39 antibodies and antigen-binding fragments provided herein are capable of specifically binding to CD39.

[0084]

[0099] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have a serotonin activity of 10 or higher by Biacore assay. -7 M or less, 8×10 -8 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 8×10 -9 M or less, 5×10 -9 M or less, 2×10 -9 M or less, 10 -9 M or less, 8×10 -10 M or less, 7×10 -10 M or less, or 6 x 10 -10 K below M D The Biacore assay specifically binds to human CD39 at a K value. The Biacore assay is based on surface plasmon resonance technology. See, for example, Murphy, M. et al., Current protocols in protein science, Chapter 19, unit 19.14, 2006. In certain embodiments, D The K value is measured by the method described in Example 5.1 of this disclosure. D Values ​​are measured at about 25°C or about 37°C. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have a K measured at 25°C and a K measured at 37°C. D Similar values, e.g., K measured at 37°C D Approximately 80% to 150%, approximately 90% to 130%, approximately 90% to 120%, or approximately 90% to 110% of the value D It has a value.

[0085]

[0100] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have a 10 -8 M or less, 8×10 -9 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, 1×10 -9 M or less, 9×10 -10 M or less, 8×10 -10 M or less, 7×10 -10 M or less, or 6 x 10 -10 K below M D The Octet assay specifically binds to human CD39 at a value of 0.05%. The Octet assay is based on biolayer interferometry technology. See, for example, Abdiche, Yasmina N., et al. Analytical biochemistry 386.2(2009):172-180 and Sun Y S., Instrumentation Science & Technology, 2014, 42(2):109-127. In certain embodiments, K D Values ​​are measured by the method described in Example 5.1 of this disclosure.

[0086]

[0101] The binding of the antibodies or antigen-binding fragments thereof provided herein to human CD39 is measured at a "50% effective concentration" (EC 50 ) value. 50 The values ​​refer to the concentration of antibody at which 50% of its maximal binding is observed. EC 50 Values ​​can be measured by binding assays known in the art, for example, direct or indirect binding assays such as enzyme-linked immunosorbent assays (ELISAs), FACS assays, and other binding assays. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein exhibit a binding activity of 10 or more, as measured by FACS (fluorescence-activated cell sorting) assay. -7 M or less, 8×10 -8 M or less, 5×10 -8M or less, 2×10 -8 M or less, 10 -8 M or less, 8×10 -9 M or less, 5×10 -9 M or less, 2×10 -9 M or less, 10 -9 M or less, 8×10 -10 M or less, 7×10 -10 M or less, or 6 x 10 -10 EC below M 50 (i.e., 50% binding concentration) specifically binds to human CD39. In certain embodiments, binding is measured by ELISA or FACS assay.

[0087]

[0102] In some embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human CD39 (i.e., ENTPDase 1). In some embodiments, the antibodies or antigen-binding fragments provided herein do not bind to other members of the ENTPDase family. In some embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human CD39 but do not specifically bind to ENTPDase 2, 3, 5, or 6, as measured, for example, by an ELISA assay.

[0088]

[0103] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein specifically bind to human CD39 but do not specifically bind to mouse CD39, as measured, for example, by a FACS assay.

[0089]

[0104] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein exhibit a cytotoxicity of 10 or more by FACS assay. -7 M or less, 8×10 -8 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 10 -8 M or less, 8×10 -9 M or less, 5×10 -9 M or less, 2×10 -9 M or less, 10 -9 M or less, 8×10 -10 M or less, 7×10-10 M or less, or 6 x 10 -10 EC below M 50 It specifically binds to cynomolgus monkey CD39.

[0090]

[0105] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein have an IC of 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 8 nM or less, 5 nM or less, 3 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07 nM or less, 0.06 nM or less, or 0.05 nM or less as measured by an ATPase activity assay. 50 inhibits ATPase activity in CD39-expressing cells. ATPase activity assays can be determined using any method known in the art, for example, by colorimetric detection of phosphate released as a result of ATPase activity. In certain embodiments, ATPase activity is determined by the method described in Example 3.3 of the present disclosure.

[0091]

[0106] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can enhance ATP-mediated monocyte activation at concentrations of 50 nM or less (e.g., 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, or 0.2 nM or less), as measured by analysis of CD80, CD86, and / or CD40 expression by FACS assay, where upregulation of CD80, CD86, and / or CD40 is indicative of monocyte activation. ATP-mediated monocyte activation can be determined using methods known in the art, for example, by the method described in Example 5.5 of the present disclosure.

[0092]

[0107] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein inhibit the secretion of IL-2, or the secretion of IFN-γ, or the secretion of CD4 + or CD8+ ATP-mediated T cell activation in PBMCs can be enhanced at concentrations of 25 nM or less, 20 nM or less, 15 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less, as measured by proliferation of T cells, for example, by the methods described in Example 5.5 of the present disclosure.

[0093]

[0108] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can enhance ATP-mediated dendritic cell (DC) activation at concentrations of 25 nM or less (or 10 nM or less, or 5 nM or less, or 1 nM or less, or 0.5 nM or less), as measured by analysis of CD83 expression by FACS assay.

[0094]

[0109] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can enhance ATP-mediated DC activation at concentrations of 25 nM or less (or 10 nM or less, or 5 nM or less, or 1 nM or less, or 0.5 nM or less), as measured by the ability of activated DCs to promote T cell proliferation.

[0095]

[0110] The antibodies and antigen-binding fragments thereof provided herein can enhance ATP-mediated DC activation at concentrations of 25 nM or less (or 10 nM or less, or 5 nM or less, or 1 nM or less, or 0.5 nM or less), as measured by the ability of activated DCs to promote IFN-γ production in a mixed lymphocyte reaction (MLR) assay.

[0096]

[0111] The activity of ATP-mediated DC maturation can be determined using methods known in the art, for example, the methods described in Example 5.5 of the present disclosure.

[0112] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein inhibit adenosine (hydrolyzed from ATP)-induced CD4 T cell proliferation at a concentration of 1 nM or less (e.g., 0.1 nM or less, 0.01 nM or less) as measured by a FACS assay. +Inhibition of T cell proliferation can be blocked. T cell proliferation can be determined using methods known in the art, for example, the methods described in Example 3.4 of this disclosure.

[0097]

[0113] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can inhibit tumor growth in a mammal in an NK cell or macrophage cell-dependent manner.

[0098]

[0114] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein inhibit T cell proliferation, CD25 + Human CD8 inhibited by eATP as measured by cell and viable cell populations + Can reverse T cell proliferation. % of T cell proliferation, CD25 + The % of cells, and % of viable cells, can be determined using methods known in the art, such as those described in Example 3.4 of this disclosure.

[0099]

[0115] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can enhance IL1β release from human macrophages induced by LPS stimulation at a concentration of 50 nM or less (or 12.5 nM or less, or 3.13 nM or less, or 0.78 nM or less, or 0.2 nM or less, or 0.049 nM or less, or 0.012 nM or less, or 0.003 nM or less, or 0.0008 nM or less), as measured by ELISA assay. IL1β release from macrophages can be determined using methods known in the art, for example, the methods described in Example 5.5.4 of the present disclosure.

[0100]

[0116] Illustrative anti-CD39 antibodies

[0117] In certain embodiments, the present disclosure provides a method for the detection of NYGMN (SEQ ID NO: 1), KYWMN (SEQ ID NO: 2), NYWMN (SEQ ID NO: 3), DTFLH (SEQ ID NO: 4), DYNMY (SEQ ID NO: 5), DTYVH (SEQ ID NO: 6), LINTYTGEPTYADDFKD (SEQ ID NO: 7), EIRLKSNKYGTHYAESVKG (SEQ ID NO: 8), QIRLNPDNYATHX1AESVKG (SEQ ID NO: 9), X 58 IDPAX 59 X 60 NIKYDPKFQG (SEQ ID NO: 151), FIDPYNGYTSYNQKFKG (SEQ ID NO: 11), RIDPAIDNSKYDPKFQG (SEQ ID NO: 12), KGIYYDYVWFFDV (SEQ ID NO: 13), QLDLYWFFDV (SEQ ID NO: 14), HGX2RGFAY (SEQ ID NO: 15), SPYYYGSGYRIFDV (SEQ ID NO: 16), IYGYDDAYYFDY (SEQ ID NO: 17), YYCALYDGYNVYAMDY (SEQ ID NO: 18), KASQDINRYIA (SEQ ID NO: 19), RASQSISDYLH (SEQ ID NO: 20), KSSQSLLDSDGRTHLN (SEQ ID NO: 21), SAFSSVNYMH (SEQ ID NO: 22), SATSSVSYMH (SEQ ID NO: 23), RSSKNLLHSNGITYLY (SEQ ID NO: 24) X1 is Y or F, X2 is S or T, and X3 is S or T; and X4 is Y or F; and X5 is S or T; and X6 is S or T; and X7 is Y or F; and X8 is S or T; and X9 is S or T; and X1 is Y or F; and X1 is S or T; and X2 is S or T; and X5 is S or T; and X6 is S or T; and X7 is S or T; and X8 is S or T; and X9 is S or T; and X1 is Y or F; and X2 is S or T; and X5 is S or T; and X6 is S or T; and X7 is S or T; and X8 is S or T; and X9 is S or T; and X9 is S or T; and X1 is Y or F; and X2 is S or T; and X1 is Y or F; and X2 is S or T; and X5 is S or T; and X1 is Y or F; and X2 is S or T; and X5 is S or T; and X6 is S or T; and X7 is S or T; and X8 is S or T; and X9 is S or T; and X1 is Y or F; and X2 is S or T; and X1 is S or T ...2 is S or T; and X2 is S or T; and X2 is S or T; and X2 is S or T; and 58 is R or K, and X 59 is N, G, S, or Q, and X 60is G, A, or D. In certain embodiments, the present disclosure further encompasses antibodies and antigen-binding fragments thereof having substitutions of one, two, or no more than three amino acid residues in any of SEQ ID NOs: 1-9, 11-36, and 151.

[0101]

[0118] As used herein, the antibody "mAb13" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:42 and a light chain variable region having the sequence of SEQ ID NO:51.

[0102]

[0119] As used herein, the antibody "mAb14" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:43 and a light chain variable region having the sequence of SEQ ID NO:52.

[0103]

[0120] As used herein, the antibody "mAb19" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:44 and a light chain variable region having the sequence of SEQ ID NO:53.

[0104]

[0121] As used herein, the antibody "mAb21" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:45 and a light chain variable region having the sequence of SEQ ID NO:54.

[0105]

[0122] As used herein, the antibody "mAb23" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:47 and a light chain variable region having the sequence of SEQ ID NO:56.

[0106]

[0123] As used herein, the antibody "mAb34" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:49 and a light chain variable region having the sequence of SEQ ID NO:58.

[0107]

[0124] As used herein, the antibody "mAb35" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:50 and a light chain variable region having the sequence of SEQ ID NO:59.

[0108]

[0125] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof that comprise one or more (e.g., one, two, three, four, five, or six) CDR sequences of antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34, or mAb35.

[0109]

[0126] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof comprising: HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6; HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, 11 to 12, and 151; and HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18; and / or LCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 24; LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 to 30; and LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 36.

[0110]

[0127] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 7, an HCDR3 comprising the sequence of SEQ ID NO: 13, and / or an LCDR1 comprising the sequence of SEQ ID NO: 19, an LCDR2 comprising the sequence of SEQ ID NO: 25, and an LCDR3 comprising the sequence of SEQ ID NO: 31.

[0111]

[0128] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 2, an HCDR2 comprising the sequence of SEQ ID NO: 8, an HCDR3 comprising the sequence of SEQ ID NO: 14, and / or an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 32.

[0112]

[0129] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 3, an HCDR2 comprising the sequence of SEQ ID NO: 37, an HCDR3 comprising the sequence of SEQ ID NO: 40, and / or an LCDR1 comprising the sequence of SEQ ID NO: 21, an LCDR2 comprising the sequence of SEQ ID NO: 27, and an LCDR3 comprising the sequence of SEQ ID NO: 33.

[0113]

[0130] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 3, an HCDR2 comprising the sequence of SEQ ID NO: 38, an HCDR3 comprising the sequence of SEQ ID NO: 41, and / or an LCDR1 comprising the sequence of SEQ ID NO: 21, an LCDR2 comprising the sequence of SEQ ID NO: 27, and an LCDR3 comprising the sequence of SEQ ID NO: 33.

[0114]

[0131] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 4, an HCDR2 comprising the sequence of SEQ ID NO: 10, an HCDR3 comprising the sequence of SEQ ID NO: 16, and / or an LCDR1 comprising the sequence of SEQ ID NO: 22, an LCDR2 comprising the sequence of SEQ ID NO: 28, and an LCDR3 comprising the sequence of SEQ ID NO: 34.

[0115]

[0132] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 5, an HCDR2 comprising the sequence of SEQ ID NO: 11, an HCDR3 comprising the sequence of SEQ ID NO: 17, and / or an LCDR1 comprising the sequence of SEQ ID NO: 23, an LCDR2 comprising the sequence of SEQ ID NO: 29, and an LCDR3 comprising the sequence of SEQ ID NO: 35.

[0116]

[0133] In certain embodiments, the present disclosure provides anti-CD39 antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO: 6, an HCDR2 comprising the sequence of SEQ ID NO: 12, an HCDR3 comprising the sequence of SEQ ID NO: 18, and / or an LCDR1 comprising the sequence of SEQ ID NO: 24, an LCDR2 comprising the sequence of SEQ ID NO: 30, and an LCDR3 comprising the sequence of SEQ ID NO: 36.

[0117]

[0134] Table 1 below shows the CDR amino acid sequences of antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34, and mAb35. The boundaries of the CDRs were defined or identified according to Kabat's rules. Table 2 below shows the amino acid sequences of the heavy and light chain variable regions of antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34, and mAb35.

[0118] [Table 2]

[0119] [Table 3-1]

[0120] [Table 3-2]

[0121]

[0135] Given that antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34, and mAb35 can each bind to CD39 and that antigen-binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, the HCDR1, HCDR2, and HCDR3 sequences, and LCDR1, LCDR2, and LCDR3 sequences of antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34, and mAb35 can be "mixed and matched" (i.e., the CDRs of different antibodies are mixed and matched, but each antibody must contain an HCDR1, HCDR2, and HCDR3, and an LCDR1, LCDR2, and LCDR3) to create anti-CD39 binding molecules of the disclosure. The CD39 binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples. Preferably, when VH CDR sequences are mixed and matched, the HCDR1, HCDR2, and / or HCDR3 sequences from a particular VH sequence are replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, the LCDR1, LCDR2, and / or LCDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequences. For example, the HCDR1s of antibodies mAb13 and mAb19 share some structural similarity and are therefore amenable to mixing and matching. It will be readily apparent to those skilled in the art that novel VH and VL sequences can be created by replacing one or more VH and / or VL CDR sequences for monoclonal antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34, and mAb35 with structurally similar sequences from the CDR sequences disclosed herein.

[0122]

[0136] It is known that CDR is involved in antigen binding.However, it has been found that not all six CDRs are essential or invariant.In other words, it is possible to replace, change or modify one or more CDRs in anti-CD39 antibody mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 and mAb35 while substantially maintaining specific binding affinity to CD39.

[0123]

[0137] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof are capable of specifically binding to CD39. The CDR sequences provided in Table 1 above are derived from mouse antibodies, but they can be grafted into any suitable FR sequences of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant techniques.

[0124]

[0138] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or antigen-binding fragments thereof are desirable for their reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can essentially be performed by substituting non-human (e.g., mouse) CDR genes with the corresponding human CDR genes in human immunoglobulin genes (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0125]

[0139] To this end, suitable human heavy and light chain variable domains can be selected using methods known in the art. In an illustrative example, a "best-fit" approach can be used, in which the variable domain sequence of a non-human (e.g., rodent) antibody is screened or BLASTed against known human variable domain sequences, and the human sequence that is closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al., (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, frameworks derived from the consensus sequence of all human antibodies may be used for grafting non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0126]

[0140] In some embodiments, the disclosure provides 16 humanized antibodies of c14, designated hu14.H1L1, hu14.H2L1, hu14.H3L1, hu14.H4L1, hu14.H1L2, hu14.H2L2, hu14.H3L2, hu14.H4L2, hu14.H1L3, hu14.H2L3, hu14.H3L3, hu14.H4L3, hu14.H1L4, hu14.H2L4, hu14.H3L4, and hu14.H4L4, respectively. The SEQ ID NOs for the heavy and light chain variable regions of each humanized antibody of c14 are set forth in Table 16 in Example 5.1. Each of the 16 humanized antibodies of c14 comprises an HCDR1 comprising the sequence of SEQ ID NO: 2, an HCDR2 comprising the sequence of SEQ ID NO: 8, an HCDR3 comprising the sequence of SEQ ID NO: 14, an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 32. The boundaries of the CDRs were defined or identified according to the rules of Kabat.

[0127]

[0141] In some embodiments, the present disclosure provides hu23.H1L1, hu23.H2L1, hu23.H3L1, hu23.H4L1, hu23.H1L2, hu23.H2L2, hu23.H3L2, hu23.H4L2, hu23.H1L3, hu23.H2L3, hu23.H3L3, hu23.H4L3, hu23.H1L4, hu23.H2L4, hu23.H3L4, and hu23.H4L4, respectively. Thirty-one humanized antibodies of c23 are provided, designated hu23.H5L1, hu23.H6L1, hu23.H7L1, hu23.H1L5, hu23.H5L5, hu23.H6L5, hu23.H7L5, hu23.H1L6, hu23.H5L6, hu23.H6L6, hu23.H7L6, hu23.H1L7, hu23.H5L7, hu23.H6L7, and hu23.H7L7. The SEQ ID NOs for the heavy and light chain variable regions of each humanized antibody of c23 are set forth in Tables 13 and 14 in Example 5.1. Each of the 31 humanized antibodies for antibody c23 above comprises an HCDR1 comprising the sequence of SEQ ID NO: 4, an HCDR2 comprising the sequence of SEQ ID NO: 10, an HCDR3 comprising the sequence of SEQ ID NO: 16, an LCDR1 comprising the sequence of SEQ ID NO: 22, an LCDR2 comprising the sequence of SEQ ID NO: 28, and an LCDR3 comprising the sequence of SEQ ID NO: 34. The boundaries of the CDRs were defined or identified according to the rules of Kabat.

[0128]

[0142] In some embodiments, the present disclosure also provides six humanized antibodies that have the same CDRs as c23 except for different amino acid sequences in HCDR2. In some embodiments, the amino acid sequences in HCDR2 of these humanized antibodies of variants of c23 (c23') are: X 58 IDPAX 59 X 60 NIKYDPKFQG (SEQ ID NO: 151), wherein X 58 is R or K, and X 59 is N, G, S, or Q, and X 60is G, A, or D. In some embodiments, the amino acid sequence of HCDR2 of these c23 variant (c23') humanized antibodies comprises a sequence selected from the group consisting of RIDPAGGNIKYDPKFQG (SEQ ID NO: 134), RIDPASGNIKYDPKFQG (SEQ ID NO: 135), RIDPAQGNIKYDPKFQG (SEQ ID NO: 136), RIDPANANIKYDPKFQG (SEQ ID NO: 137), RIDPANDNIKYDPKFQG (SEQ ID NO: 138), and KIDPANGNIKYDPKFQG (SEQ ID NO: 139). The boundaries of the CDRs are defined or identified by the Kabat rules.

[0129]

[0143] In some embodiments, the present disclosure also provides four humanized antibodies against the c23 variants by yeast display, designated hu23.201, hu23.203, hu23.207, and hu23.211. The heavy and light chain variable regions of humanized antibodies hu23.201, hu23.203, hu23.207, and hu23.211 are shown in Table 15 in Example 5.1. Each of the four humanized antibodies, hu23.201, hu23.203, hu23.207, and hu23.211, comprises an HCDR1 comprising the sequence of SEQ ID NO:4, an HCDR2 comprising the sequence of SEQ ID NO:10, an HCDR3 comprising the sequence of SEQ ID NO:16, an LCDR1 comprising the sequence of SEQ ID NO:22, an LCDR2 comprising the sequence of SEQ ID NO:28, and an LCDR3 comprising the sequence of SEQ ID NO:34. The boundaries of the CDRs were defined or identified according to the rules of Kabat.

[0130]

[0144] Table 3 below shows four variants of the humanized c14 heavy chain variable region (i.e., hu14.VH_1, hu14.VH_2, hu14.VH_3, and hu14.VH_4) and four variants of the humanized c14 light chain variable region (i.e., hu14.VL_1, hu14.VL_2, hu14.VL_3, and hu14.VL_4). Table 4 below shows the amino acid sequences of the FRs for the humanized c14 heavy and light chain variable regions. Table 5 below shows the heavy and light chain FR amino acid sequences of each of the 16 humanized antibodies for chimeric antibody c14, designated hu14.H1L1, hu14.H2L1, hu14.H3L1, hu14.H4L1, hu14.H1L2, hu14.H2L2, hu14.H3L2, hu14.H4L2, hu14.H1L3, hu14.H2L3, hu14.H3L3, hu14.H4L3, hu14.H1L4, hu14.H2L4, hu14.H3L4, and hu14.H4L4, respectively. The heavy and light chain variable regions of these 16 humanized antibodies are shown in Table 16 in Example 5.1.

[0131] [Table 4]

[0132] [Table 5]

[0133] [Table 6]

[0134]

[0145] Table 6 below shows seven variants of the humanized c23 heavy chain variable region (i.e., hu23.VH_1, hu23.VH_2, hu23.VH_3, hu23.VH_4, hu23.VH_5, hu23.VH_6, and hu23.VH_7) and seven variants of the humanized c23 light chain variable region (i.e., hu23.VL_1, hu23.VL_2, hu23.VL_3, hu23.VL_4, hu23.VL_5, hu23.VL_6, and hu23.VL_7). Table 7 below shows the amino acid sequences of the heavy and light chain variable regions of four humanized antibodies for the chimeric antibody c23 obtained by yeast display. Table 8 below shows the FR amino acid sequences of 35 humanized antibodies for c23. Table 9 below shows the FR amino acid sequences for the heavy and light chains of each of the 35 humanized antibodies of c23.

[0135] [Table 7-1]

[0136] [Table 7-2]

[0137] [Table 8]

[0138] [Table 9]

[0139] [Table 9-2]

[0140] [Table 10]

[0141]

[0146] In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein consist essentially of all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FRs and, if present, the constant region are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be derived from different human immunoglobulin genes, e.g., the FR sequences are derived from one human antibody and the constant region is derived from another human antibody. In some embodiments, the humanized antibodies or antigen-binding fragments thereof comprise human heavy chains HFR1-4 and / or light chains LFR1-4.

[0142]

[0147] In some embodiments, a human-derived FR region may comprise the same amino acid sequence as the original human immunoglobulin from which it was derived. In some embodiments, one or more amino acid residues in the human FR are substituted with the corresponding residue from the parent non-human antibody. This is desirable in certain embodiments to allow the humanized antibody or fragment thereof to closely resemble the structure of the non-human parent antibody, thereby optimizing binding characteristics (e.g., increasing binding affinity). In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises the substitution of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in each of the human FR sequences, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue in all of the FR sequences of the heavy or light chain variable domains. In some embodiments, such changes in amino acid residues can occur only in the heavy chain FR regions, only in the light chain FR regions, or in both chains. In certain embodiments, one or more amino acids in the human FR sequences are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequence are backmutated to the corresponding amino acid of the parent non-human antibody to increase binding affinity.

[0143]

[0148] In certain embodiments, the present disclosure provides X 19 VQLVX 20 SGX 21 X 22 X 23X 24 KPGX 25 SX 26 X 27 X 28 SCX 29 ASGX 30 X 31 X 32 X 33 Heavy chain HFR1, WVX comprising the sequence of (SEQ ID NO: 76) or a homologous sequence having at least 80% sequence identity 34 QX 35 PGX 36 X 37 LEWX 38 X 39 (SEQ ID NO: 77) or a homologous sequence having at least 80% sequence identity thereto 40 X 41 TX 42 X 43 X 44 DX 45 SX 46 X 47 TX 48 YX 49 X 50 X 51 X 52 SLX 53 X 54 EDTAVYYCX 55 X 56 (SEQ ID NO: 78) or a homologous sequence having at least 80% sequence identity thereto, and 57 Also provided are humanized anti-CD39 antibodies and antigen-binding fragments thereof, comprising a heavy chain HFR4 comprising the sequence of VTVSS (SEQ ID NO: 126) or a homologous sequence having at least 80% sequence identity thereto, wherein X 19 is Q or E, and X 20 is E or Q, and X 21 is G or A, and X 22 is G or E, and X 23 is L or V, and X 24 is V or K, and X 25 is G or A, and X 26 is L, M, or V, and X 27 is R or K, and X 28 is V or L, and X 29is A or K, and X 30 is F or Y and X 31 is N or T, and X 32 is F or L, and X 33 is S or K, and X 34 is R or K, and X 35 is A or S, and X 36 is K or Q, and X 37 is R or G, and X 38 is M, I, or V, and X 39 is G or A, and X 40 is R or K, and X 41 is V, A, or F, and X 42 is I or L, and X 43 is S or T, and X 44 is R or A, and X 45 is D or T, and X 46 is K, A, or S, and X 47 is S or N, and X 48 is L, V, or A, and X 49 is M or L, and X 50 is Q or E, and X 51 is M or L, and X 52 is S, I, or N, and X 53 is R or K, and X 54 is S or T, and X 55 is A or T, and X 56 is R, N, or T, and X 57 is T or L.

[0144]

[0149] In certain embodiments, the present disclosure provides a light chain LFR1 comprising the sequence of X3IVX4TQSPATLX5X6SPGERX7TX8X9C (SEQ ID NO: 80), or a homologous sequence having at least 80% sequence identity thereto, WYQQKPGQX 10 PX 11 A light chain LFR2, GX comprising the sequence of LLIY (SEQ ID NO: 81) or a homologous sequence having at least 80% sequence identity thereto 12 PX 13 RFSGSGSGTX 14 X 15 TLTISSX16 EPEDFAVYX 17 C (SEQ ID NO: 82) or a homologous sequence having at least 80% sequence identity thereto, and 18 Also provided are humanized anti-CD39 antibodies and antigen-binding fragments thereof, comprising a light chain LFR4 comprising the sequence of GTKLEIK (SEQ ID NO: 152) or at least 80% homologous sequence thereof, wherein X3 is E or Q, X4 is L or M, X5 is S or T, X6 is L, V, or A, X7 is A or V, X8 is L or I, X9 is S or T, and X 10 is A or S, and X 11 is R or K, and X 12 is I or V, and X 13 is A or T, and X 14 is D or S, and X 15 is F or Y and X 16 is L, M, or V, and X 17 is Y or F, and X 18 is G or Q.

[0145]

[0150] In certain embodiments, the present disclosure provides 61 Heavy chain HFR1, WVRQX, comprising the sequence RLSCAASGFTFS (SEQ ID NO: 154) or a homologous sequence having at least 80% sequence identity thereto 62 PGKGLEWVX 63 Heavy chain HFR2, RFTISRDDSKNTX comprising the sequence of (SEQ ID NO: 155) or a homologous sequence having at least 80% sequence identity 64 Also provided are humanized anti-CD39 antibodies and antigen-binding fragments thereof, comprising a heavy chain HFR3 comprising the sequence of YLQMNSLKTEDTAVYYCTT (SEQ ID NO: 156) or a homologous sequence having at least 80% sequence identity thereto, and a heavy chain HFR4 comprising the sequence of WGQGTTVTVSS (SEQ ID NO: 79) or a homologous sequence having at least 80% sequence identity thereto, wherein X 61 is L or M, and X 62 is A or S, and X 63 is G or A, and X 64is L or V.

[0146]

[0151] In certain embodiments, the present disclosure provides EIVX 65 TQSPATLSX 66 SPGERX 67 Light chain LFR1, WYQQKPGQX, comprising TLSC (SEQ ID NO: 157) or a homologous sequence having at least 80% sequence identity thereto 68 Light chain LFR2, GIPARFSGSGSGTDFTLTISSX, comprising the sequence of PRLLIY (SEQ ID NO: 158) or a homologous sequence having at least 80% sequence identity thereto 69 EPEDFAVYX 70 Also provided is a humanized anti-CD39 antibody and antigen-binding fragment thereof, comprising a light chain LFR3 comprising the sequence of X C (SEQ ID NO: 159) or a homologous sequence having at least 80% sequence identity thereto, and a light chain LFR4 comprising FGGGTKLEIK (SEQ ID NO: 153) or a homologous sequence having at least 80% sequence identity thereto, wherein X 65 is L or M, and X 66 is L or V, and X 67 is A or V, and X 68 is A or S, and X 69 is L or V, and X 70 is Y or F.

[0147]

[0152] In certain embodiments, the present disclosure provides X 71 VQLVQSGAEVKKPGASVKX 72 SCKASGYX 73 Heavy chain HFR1, WVX comprising the sequence of LK (SEQ ID NO: 160) or a homologous sequence having at least 80% sequence identity thereto 74 QAPGQX 75 LEWX 76 heavy chain HFR2, X comprising the sequence of G (SEQ ID NO: 161) or a homologous sequence having at least 80% sequence identity thereto; 77 X 78 TX 79 TX 80 DTSX 81 X 82 TAYX 83 ELX 84SLRSEDTAVYYCAX 85 Heavy chain HFR3, WGQGTX comprising the sequence of (SEQ ID NO: 149) or a homologous sequence having at least 80% sequence identity 57 Also provided are humanized anti-CD39 antibodies and antigen-binding fragments thereof, comprising a heavy chain HFR4 comprising VTVSS (SEQ ID NO: 126) or a homologous sequence having at least 80% sequence identity thereto, wherein X 57 is as defined above, and X 71 is Q or E, and X 72 is V or L, and X 73 is N or T, and X 74 is R or K, and X 75 is R or G, and X 76 is M or I, and X 77 is R or K, and X 78 is V or A, and X 79 is I or L, and X 80 is R or A, and X 81 is A or S, and X 82 is S or N, and X 83 is M or L, and X 84 is S or I, and X 85 is R or N.

[0148]

[0153] In certain embodiments, the present disclosure provides X 86 IVLTQSPATLX 87 X 88 SPGERX 89 TX 90 X 91 Light chain LFR1, WYQQKPGQX, comprising the sequence of C (SEQ ID NO: 150) or a homologous sequence having at least 80% sequence identity thereto 10 PX 11 A light chain LFR2, GX comprising the sequence of LLIY (SEQ ID NO: 81) or a homologous sequence having at least 80% sequence identity thereto 92 PX 93 RFSGSGSGTX 94 X 95 TLTISSX 96Also provided are humanized anti-CD39 antibodies and antigen-binding fragments thereof, comprising a light chain LFR3 comprising the sequence of EPEDFAVYYC (SEQ ID NO: 148) or a homologous sequence having at least 80% sequence identity thereto, and a light chain LFR4 comprising the sequence of FGQGTKLEIK (SEQ ID NO: 83) or a homologous sequence having at least 80% sequence identity thereto, wherein X 10 and X 11 is as defined above, and X 86 is E or Q, and X 87 is S or T, and X 88 is L or A, and X 89 is A or V, and X 90 is L or I, and X 91 is S or T, and X 92 is I or V, and X 93 is A or T, and X 94 is D or S, and X 95 is F or Y and X 96 is L or M.

[0149]

[0154] In certain embodiments, the present disclosure also provides humanized anti-CD39 antibodies and antigen-binding fragments thereof, comprising a heavy chain HFR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 84-86, 115, 119-120, and 131; a heavy chain HFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 87-90 and 121-123; a heavy chain HFR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 91-97, 116-117, and 124-125; and a heavy chain HFR4 comprising a sequence selected from the group consisting of SEQ ID NOs: 79 and 118; and / or a light chain LFR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 98-103 and 127-129; a light chain LFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 104, 105, and 130; a light chain LFR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 106-110 and 132-133; and a light chain LFR4 comprising a sequence selected from the group consisting of SEQ ID NOs: 83 and 153.

[0150]

[0155] In certain embodiments, the present disclosure provides hu14.VH_1 (SEQ ID NO: 68), hu14.VH_2 (SEQ ID NO: 70), hu14.VH_3 (SEQ ID NO: 72), hu14.VH_4 (SEQ ID NO: 74), hu23.VH_1 (SEQ ID NO: 60), hu23.VH_2 (SEQ ID NO: 62), hu23.VH_3 (SEQ ID NO: 64), hu23.VH_4 (SEQ ID NO: 66), hu23.VH_5 (SEQ ID NO: 140). Also provided are humanized anti-CD39 and antigen-binding fragments thereof, comprising HFR1, HFR2, HFR3, and / or HFR4 sequences contained in a heavy chain variable region selected from the group consisting of hu23.VH_6 (SEQ ID NO: 141), hu23.VH_7 (SEQ ID NO: 142), hu23.201H (SEQ ID NO: 146), hu23.207H (SEQ ID NO: 147), and hu23.211H (SEQ ID NO: 39).

[0151]

[0156] In certain embodiments, the present disclosure provides hu14.VL_1 (SEQ ID NO: 69), hu14.VL_2 (SEQ ID NO: 71), hu14.VL_3 (SEQ ID NO: 73), hu14.VL_4 (SEQ ID NO: 75), hu23.VL_1 (SEQ ID NO: 61), hu23.VL_2 (SEQ ID NO: 63), hu23.VL_3 (SEQ ID NO: 65), hu23.VL_4 (SEQ ID NO: 67), hu23.VL_5 (SEQ ID NO: 143). Also provided are humanized anti-CD39 and antigen-binding fragments thereof, comprising LFR1, LFR2, LFR3, and / or LFR4 sequences contained in a light chain variable region selected from the group consisting of hu23.VL_6 (SEQ ID NO: 144), hu23.VL_7 (SEQ ID NO: 145), hu23.201L (SEQ ID NO: 111), hu23.203L (SEQ ID NO: 112), and hu23.211L (SEQ ID NO: 63).

[0152]

[0157] In certain embodiments, the humanized anti-CD39 antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NOs: 39, 60, 62, 64, 66, 68, 70, 72, 74, 140, 141, 142, 146, 147, and / or a light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 111, 112, 143, 144, and 145.

[0153]

[0158] The present disclosure provides: 1) "hu14.H1L1", which comprises the heavy chain variable region of hu14.VH_1 (SEQ ID NO: 68) and the light chain variable region of hu14.VL_1 (SEQ ID NO: 69); 2) "hu14.H2L1," comprising the heavy chain variable region of hu14.VH_2 (SEQ ID NO: 70) and the light chain variable region of hu14.VL_1 (SEQ ID NO: 69); 3) "hu14.H3L1," comprising the heavy chain variable region of hu14.VH_3 (SEQ ID NO: 72) and the light chain variable region of hu14.VL_1 (SEQ ID NO: 69); 4) "hu14.H4L1," comprising the heavy chain variable region of hu14.VH_4 (SEQ ID NO: 74) and the light chain variable region of hu14.VL_1 (SEQ ID NO: 69); 5) "hu14.H1L2", comprising the heavy chain variable region of hu14.VH_1 (SEQ ID NO: 68) and the light chain variable region of hu14.VL_2 (SEQ ID NO: 71); 6) "hu14.H2L2," comprising the heavy chain variable region of hu14.VH_2 (SEQ ID NO: 70) and the light chain variable region of hu14.VL_2 (SEQ ID NO: 71); 7) "hu14.H3L2," comprising the heavy chain variable region of hu14.VH_3 (SEQ ID NO: 72) and the light chain variable region of hu14.VL_2 (SEQ ID NO: 71); 8) "hu14.H4L2," comprising the heavy chain variable region of hu14.VH_4 (SEQ ID NO: 74) and the light chain variable region of hu14.VL_2 (SEQ ID NO: 71); 9) "hu14.H1L3," comprising the heavy chain variable region of hu14.VH_1 (SEQ ID NO: 68) and the light chain variable region of hu14.VL_3 (SEQ ID NO: 73); 10) "hu14.H2L3," comprising the heavy chain variable region of hu14.VH_2 (SEQ ID NO: 70) and the light chain variable region of hu14.VL_3 (SEQ ID NO: 73); 11) "hu14.H3L3," comprising the heavy chain variable region of hu14.VH_3 (SEQ ID NO: 72) and the light chain variable region of hu14.VL_3 (SEQ ID NO: 73); 12) "hu14.H4L3," comprising the heavy chain variable region of hu14.VH_4 (SEQ ID NO: 74) and the light chain variable region of hu14.VL_3 (SEQ ID NO: 73); 13) "hu14.H1L4," comprising the heavy chain variable region of hu14.VH_1 (SEQ ID NO: 68) and the light chain variable region of hu14.VL_4 (SEQ ID NO: 75); 14) "hu14.H2L4," comprising the heavy chain variable region of hu14.VH_2 (SEQ ID NO: 70) and the light chain variable region of hu14.VL_4 (SEQ ID NO: 75); 15) "hu14.H3L4," comprising the heavy chain variable region of hu14.VH_3 (SEQ ID NO: 72) and the light chain variable region of hu14.VL_4 (SEQ ID NO: 75); and 16) "hu14.H4L4," comprising the heavy chain variable region of hu14.VH_4 (SEQ ID NO: 74) and the light chain variable region of hu14.VL_4 (SEQ ID NO: 75) Also provided is an exemplary humanized antibody of chimeric antibody c14 comprising:

[0154]

[0159] The present disclosure provides: 1) "hu23.H1L1", which comprises the heavy chain variable region of hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region of hu23.VL_1 (SEQ ID NO: 61); 2) "hu23.H2L1," comprising the heavy chain variable region of hu23.VH_2 (SEQ ID NO: 62) and the light chain variable region of hu23.VL_1 (SEQ ID NO: 61); 3) "hu23.H3L1," comprising the heavy chain variable region of hu23.VH_3 (SEQ ID NO: 64) and the light chain variable region of hu23.VL_1 (SEQ ID NO: 61); 4) "hu23.H4L1," comprising the heavy chain variable region of hu23.VH_4 (SEQ ID NO: 66) and the light chain variable region of hu23.VL_1 (SEQ ID NO: 61); 5) "hu23.H1L2," comprising the heavy chain variable region of hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region of hu23.VL_2 (SEQ ID NO: 63); 6) "hu23.H2L2," comprising the heavy chain variable region of hu23.VH_2 (SEQ ID NO: 62) and the light chain variable region of hu23.VL_2 (SEQ ID NO: 63); 7) "hu23.H3L2," comprising the heavy chain variable region of hu23.VH_3 (SEQ ID NO: 64) and the light chain variable region of hu23.VL_2 (SEQ ID NO: 63); 8) "hu23.H4L2," comprising the heavy chain variable region of hu23.VH_4 (SEQ ID NO: 66) and the light chain variable region of hu23.VL_2 (SEQ ID NO: 63); 9) "hu23.H1L3," comprising the heavy chain variable region of hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region of hu23.VL_3 (SEQ ID NO: 65); 10) "hu23.H2L3," comprising the heavy chain variable region of hu23.VH_2 (SEQ ID NO: 62) and the light chain variable region of hu23.VL_3 (SEQ ID NO: 65); 11) "hu23.H3L3," comprising the heavy chain variable region of hu23.VH_3 (SEQ ID NO: 64) and the light chain variable region of hu23.VL_3 (SEQ ID NO: 65); 12) "hu23.H4L3," comprising the heavy chain variable region of hu23.VH_4 (SEQ ID NO: 66) and the light chain variable region of hu23.VL_3 (SEQ ID NO: 65); 13) "hu23.H1L4," comprising the heavy chain variable region of hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region of hu23.VL_4 (SEQ ID NO: 67); 14) "hu23.H2L4," comprising the heavy chain variable region of hu23.VH_2 (SEQ ID NO: 62) and the light chain variable region of hu23.VL_4 (SEQ ID NO: 67); 15) "hu23.H3L4," comprising the heavy chain variable region of hu23.VH_3 (SEQ ID NO: 64) and the light chain variable region of hu23.VL_4 (SEQ ID NO: 67); 16) "hu23.H4L4," comprising the heavy chain variable region of hu23.VH_4 (SEQ ID NO: 66) and the light chain variable region of hu23.VL_4 (SEQ ID NO: 67); 17) "hu23.H5L1," comprising the heavy chain variable region of hu23.VH_5 (SEQ ID NO: 140) and the light chain variable region of hu23.VL_1 (SEQ ID NO: 61); 18) "hu23.H6L1," comprising the heavy chain variable region of hu23.VH_6 (SEQ ID NO: 141) and the light chain variable region of hu23.VL_1 (SEQ ID NO: 61); 19) "hu23.H7L1," comprising the heavy chain variable region of hu23.VH_7 (SEQ ID NO: 142) and the light chain variable region of hu23.VL_1 (SEQ ID NO: 61); 20) "hu23.H1L5," comprising the heavy chain variable region of hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region of hu23.VL_5 (SEQ ID NO: 143); 21) "hu23.H5L5," comprising the heavy chain variable region of hu23.VH_5 (SEQ ID NO: 140) and the light chain variable region of hu23.VL_5 (SEQ ID NO: 143); 22) "hu23.H6L5," comprising the heavy chain variable region of hu23.VH_6 (SEQ ID NO: 141) and the light chain variable region of hu23.VL_5 (SEQ ID NO: 143); 23) "hu23.H7L5," comprising the heavy chain variable region of hu23.VH_7 (SEQ ID NO: 142) and the light chain variable region of hu23.VL_5 (SEQ ID NO: 143); 24) "hu23.H1L6," comprising the heavy chain variable region of hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region of hu23.VL_6 (SEQ ID NO: 144); 25) "hu23.H5L6," comprising the heavy chain variable region of hu23.VH_5 (SEQ ID NO: 140) and the light chain variable region of hu23.VL_6 (SEQ ID NO: 144); 26) "hu23.H6L6," comprising the heavy chain variable region of hu23.VH_6 (SEQ ID NO: 141) and the light chain variable region of hu23.VL_6 (SEQ ID NO: 144); 27) "hu23.H7L6," comprising the heavy chain variable region of hu23.VH_7 (SEQ ID NO: 142) and the light chain variable region of hu23.VL_6 (SEQ ID NO: 144); 28) "hu23.H1L7," comprising the heavy chain variable region of hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region of hu23.VL_7 (SEQ ID NO: 145); 29) "hu23.H5L7," comprising the heavy chain variable region of hu23.VH_5 (SEQ ID NO: 140) and the light chain variable region of hu23.VL_7 (SEQ ID NO: 145); 30) "hu23.H6L7," comprising the heavy chain variable region of hu23.VH_6 (SEQ ID NO: 141) and the light chain variable region of hu23.VL_7 (SEQ ID NO: 145); 31) "hu23.H7L7," comprising the heavy chain variable region of hu23.VH_7 (SEQ ID NO: 142) and the light chain variable region of hu23.VL_7 (SEQ ID NO: 145); 32) "hu23.201," comprising the heavy chain variable region of hu23.201H (SEQ ID NO: 146) and the light chain variable region of hu23.201L (SEQ ID NO: 111); 33) "hu23.203," comprising the heavy chain variable region of hu23.201H (SEQ ID NO: 146) and the light chain variable region of hu23.203L (SEQ ID NO: 112); 34) "hu23.207," comprising the heavy chain variable region of hu23.207H (SEQ ID NO: 147) and the light chain variable region of hu23.201L (SEQ ID NO: 111); and 35) "hu23.211," comprising the heavy chain variable region of hu23.211H (SEQ ID NO: 39) and the light chain variable region of hu23.211L (SEQ ID NO: 63) Also provided is an exemplary humanized antibody of chimeric antibody c23, comprising:

[0155]

[0160] These exemplary humanized anti-CD39 antibodies retained specific binding ability or affinity for CD39 and were at least as good as or better than the parental murine antibodies mAb14 or mAb23 in that respect.

[0156]

[0161] In some embodiments, the anti-CD39 antibodies and antigen-binding fragments provided herein comprise all or a portion of a heavy chain variable domain and / or all or a portion of a light chain variable domain. In one embodiment, the anti-CD39 antibodies or antigen-binding fragments provided herein are single-domain antibodies consisting of all or a portion of a heavy chain variable domain provided herein. Further information on such single-domain antibodies is available in the art (see, e.g., U.S. Patent No. 6,248,516).

[0157]

[0162] In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, optionally including heavy and / or light chain constant regions. In certain embodiments, the heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM. In certain embodiments, the light chain constant region comprises Cκ or Cλ. The constant region of the anti-CD39 antibodies or antigen-binding fragments thereof provided herein may be identical to the sequence of the wild-type constant region or may differ in one or more mutations.

[0158]

[0163] In certain embodiments, the heavy chain constant region comprises an Fc region. The Fc region is known to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody complement-dependent cytotoxicity (CDC). The Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, it is recognized that the Fc regions of IgG1 and IgG3 induce both ADCC and CDC more effectively than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided herein comprise an Fc region of an IgG1 or IgG3 isotype capable of inducing ADCC or CDC, or comprise a constant region of an IgG4 or IgG2 isotype with reduced or depleted effector function. In some embodiments, the Fc region derived from human IgG1 has reduced effector function. In some embodiments, the Fc region derived from human IgG1 comprises the mutations L234A and / or L235A. In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein comprise a wild-type human IgG4 Fc region or other wild-type human IgG4 allele. In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein comprise a human IgG4 Fc region comprising the S228P mutation and / or the L235E mutation, and / or the F234A and L235A mutations. In some embodiments, the Fc region derived from human IgG4 comprises the S228P mutation and / or the F234A and L235A mutations.

[0159]

[0164] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have sufficient specific binding affinity for human CD39 to provide for diagnostic and / or therapeutic uses.

[0160]

[0165] The antibodies or antigen-binding fragments thereof provided herein can be monoclonal, polyclonal, humanized, chimeric, recombinant, bispecific, multispecific, labeled, bivalent, anti-idiotypic, or fusion proteins. Recombinant antibodies are antibodies prepared using recombinant methods in vitro rather than in an animal.

[0161]

[0166] In certain embodiments, the present disclosure provides an anti-CD39 antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment thereof provided herein for binding to CD39. In certain embodiments, the present disclosure provides an anti-CD39 antibody or antigen-binding fragment thereof that competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 43 and a light chain variable region comprising the sequence of SEQ ID NO: 52 for binding to human CD39. In certain embodiments, the present disclosure provides an anti-CD39 antibody or antigen-binding fragment thereof that competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 44 and a light chain variable region comprising the sequence of SEQ ID NO: 53 for binding to human CD39. In certain embodiments, the present disclosure provides an anti-CD39 antibody or antigen-binding fragment thereof that competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 45 and a light chain variable region comprising the sequence of SEQ ID NO: 54 for binding to human CD39, or that competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 47 and a light chain variable region comprising the sequence of SEQ ID NO: 56 for binding to human CD39.

[0162]

[0096] In some embodiments, the present disclosure provides an anti-CD39 antibody or antigen-binding fragment thereof that specifically binds to an epitope of CD39, wherein the epitope includes one or more residues selected from the group consisting of Q96, N99, E143, R147, R138, M139, E142, K5, E100, D107, V81, E82, R111, and V115.

[0163]

[0167] In some embodiments, the epitope comprises one or more residues selected from the group consisting of Q96, N99, E143, and R147, hi some embodiments, the epitope comprises all of residues Q96, N99, E143, and R147.

[0164]

[0168] In some embodiments, the epitope comprises one or more residues selected from the group consisting of R138, M139, and E142. In some embodiments, the epitope comprises all of residues R138, M139, and E142.

[0165]

[0169] In some embodiments, the epitope comprises one or more residues selected from the group consisting of K5, E100, and D107, hi some embodiments, the epitope comprises all of residues K5, E100, and D107.

[0166]

[0170] In some embodiments, the epitope comprises one or more residues selected from the group consisting of V81, E82, R111, and V115. In some embodiments, the epitope comprises all of residues V81, E82, R111, and V115.

[0167]

[0171] In some embodiments, the CD39 is human CD39. In some embodiments, the CD39 is human CD39 comprising the amino acid sequence of SEQ ID NO: 162.

[0172] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof provided herein is not any of antibody 9-8B, antibody T895, and antibody I394.

[0168]

[0173] As used herein, "9-8B" refers to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:46 and a light chain variable region having the amino acid sequence of SEQ ID NO:48.

[0169]

[0174] As used herein, "T895" refers to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 55 and a light chain variable region having the amino acid sequence of SEQ ID NO: 57.

[0170]

[0175] As used herein, "I394" refers to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:113 and a light chain variable region having the amino acid sequence of SEQ ID NO:114.

[0171]

[0176] Antibody variants

[0177] The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0172]

[0178] In certain embodiments, antibody variants comprise one or more of the CDR sequences provided in Table 1 above, one or more of the non-CDR sequences of the heavy or light chain variable regions provided in Tables 4, 5, 8, and 9 above, and / or one or more modifications or substitutions in the constant region (e.g., Fc region). Such variants retain the binding specificity of their parent antibodies for CD39 but have one or more desirable properties conferred by the modification or substitution. For example, antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or eliminated effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., introduced one or more cysteine ​​residues).

[0173]

[0179] Methods known in the art, such as "alanine scanning mutagenesis," may be used to screen parent antibody sequences to identify suitable or preferred residues for modification or substitution (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and the modified antibodies are produced and screened for the desired properties. If substitution at a particular amino acid position demonstrates a desired change in function, that position can be identified as a potential residue for modification or substitution. Potential residues may be further evaluated by substituting them with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0174]

[0180] Affinity variants

[0181] Affinity variants of antibodies may comprise modifications or substitutions in one or more CDR sequences provided in Table 1 above, one or more FR sequences provided in Tables 4, 5, 8, and 9 above, or the heavy or light chain variable region sequences provided in Tables 2, 3, 6, and 7 above. Because it is known in the art that a CDR region is flanked by two FR regions in the variable region, FR sequences can be easily identified by one skilled in the art based on the CDR sequences in Table 1 above and the variable region sequences in Tables 2, 3, 6, and 7 above. Affinity variants retain the specific binding affinity of the parent antibody to CD39, or even have improved CD39 specific binding affinity over the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR sequence, FR sequence, or variable region sequence comprises a conservative substitution.

[0175]

[0182] Those skilled in the art will understand that one or more amino acid residues can be substituted in the CDR sequences provided in Table 1 above, and the variable region sequences provided in Tables 2, 3, 6, and 7 above, and the resulting antibody or antigen-binding fragment can still retain, or even have improved, binding affinity or capacity for CD39. To achieve this goal, various methods known in the art can be used. For example, phage display technology can be used to generate and express libraries of antibody variants (such as Fab or scFv variants), which can then be screened for binding affinity to human CD39. As another example, computer software can be used to virtually simulate the binding of an antibody to human CD39 and identify amino acid residues on the antibody that form the binding interface. Such residues can be avoided for substitution to prevent a reduction in binding affinity, or targeted for substitution to provide stronger binding.

[0176]

[0183] In certain embodiments, the humanized antibodies or antigen-binding fragments thereof provided herein contain substitutions of one or more amino acid residues in one or more of the CDR sequences and / or in one or more of the FR sequences. In certain embodiments, the affinity variants contain a total of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less substitutions in the CDR and / or FR sequences.

[0177]

[0184] In certain embodiments, an anti-CD39 antibody or antigen-binding fragment thereof comprises one, two, or three CDR sequences that share at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the CDRs listed in Table 1 above, yet retains specific binding affinity for CD39 at a similar or even higher level than its parent antibody.

[0178]

[0185] In certain embodiments, an anti-CD39 antibody or antigen-binding fragment thereof comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to a variable region sequence listed in Tables 2, 3, 6, and 7 above, while retaining specific binding affinity for CD39 at a similar or even higher level than its parent antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, or deleted in the variable region sequences listed in Tables 2, 3, 6, and 7 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (e.g., within the FRs).

[0179]

[0186] Glycosylation variants

[0187] The anti-CD39 antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants obtained by increasing or decreasing the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[0180]

[0188] An antibody or antigen-binding fragment thereof may contain one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, in tripeptide sequences such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine. Removal of native glycosylation sites can be conveniently achieved, for example, by altering the amino acid sequence so that one of the above tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted. New glycosylation sites can likewise be created by introducing such a tripeptide sequence or a serine or threonine residue.

[0181]

[0189] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments provided herein comprise one or more mutations at positions selected from the group consisting of N55, G56, and N239 to remove one or more deamidation sites. In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments provided herein comprise a mutation at N55 (e.g., N55G, N55S, or N55Q), and / or a mutation at G56 (e.g., G56A, G56D), and / or a mutation at N297 (e.g., N297A, N297Q, or N297G). These mutations have been tested and are not believed to adversely affect the binding affinity of the antibodies provided herein.

[0182]

[0190] Cysteine ​​engineered variants

[0191] The anti-CD39 antibodies or antigen-binding fragments thereof provided herein also include cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0183]

[0192] A free cysteine ​​residue is a residue that is not part of a disulfide bridge. Cysteine-engineered variants are useful for conjugating, for example, cytotoxic and / or imaging compounds, labels, or especially radioisotopes at the engineered cysteine ​​site, for example, by maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0184]

[0193] Fc variants

[0194] The anti-CD39 antibodies or antigen-binding fragments thereof provided herein also encompass Fc variants comprising modification or substitution of one or more amino acid residues in the Fc region and / or hinge region to provide altered effector functions, e.g., ADCC and CDC. Methods for modifying ADCC activity by antibody engineering have been described in the art, e.g., Shields RL et al., J Biol Chem. 2001, 276(9):6591-604; Idusogie EE et al., J Immunol. 2000, 164(8):4178-84; Steurer W et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE et al., J Immunol. 2001, 166(4):2571-5; Lazar GA et al., PNAS, 2006, 103(11):4005-4010; Ryan MC et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards JO et al., Mol. Cancer See Ther. 2008, 7(8):2517-27; Shields R. Let al., J. Biol. Chem. 2002, 277:26733-26740; Shinkawa T. et al., J. Biol. Chem. 2003, 278:3466-3473.

[0185]

[0195] The CDC activity of the antibodies or antigen-binding fragments provided herein can also be modified, for example, by improving or diminishing C1q binding and / or CDC (see, e.g., WO 99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fe domain variants). One or more amino acids selected from amino acid residues 329, 331, and 322 in the Fc region can be replaced with different amino acid residues to modify C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see, e.g., U.S. Pat. No. 6,194,551 by Idusogie et al.). One or more amino acid substitutions can also be introduced to modify the antibody's ability to fix complement (see PCT Publication WO 94 / 29351 by Bodmer et al.).

[0186]

[0196] In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein have reduced effector function and comprise one or more amino acid substitutions in IgG1 at positions selected from the group consisting of 234, 235, 237, 238, 268, 297, 309, 330, and 331. In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and comprise one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S, or any combination thereof. In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and comprise the mutations L234A and L235A. In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein are of the IgG2 isotype and contain one or more amino acid substitutions selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, or any combination thereof (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein are of the IgG4 isotype and contain one or more amino acid substitutions selected from the group consisting of S228P, N297A, N297Q, N297G, L235E, F234A, L235A, or any combination thereof. In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein are of the IgG2 / IgG4 cross-isotype. Examples of IgG2 / IgG4 cross-isotypes are described in Rother RP et al., Nat Biotechnol 25:1256-1264 (2007).

[0187]

[0197] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and comprise one or more amino acid substitutions at one or more of 228, 234, and 235. In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and comprise the S228P and / or L235E and / or F234A and L235A mutations in the Fc region.

[0188]

[0198] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants bind to FcRn at acidic pH, allowing it to escape degradation in lysosomes and then translocate and be released extracellularly, and therefore can have an extended pharmacokinetic half-life. Methods for engineering antibodies or antigen-binding fragments thereof to improve their binding affinity to FcRn are known in the art; see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277(2010); and Hinton, P. et al., J. Immunology, 176:346-356(2006).

[0189]

[0199] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions at the interface of the Fc region that facilitate and / or promote heterodimerization. These modifications include the introduction of a protrusion into a first Fc polypeptide and a cavity into a second Fc polypeptide, with the protrusion positioned within the cavity to promote interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0190]

[0200] antigen-binding fragment

[0201] Anti-CD39 antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-CD39 antibodies provided herein, including, for example, the exemplary antibodies whose CDRs are shown in Table 1 above and whose variable sequences are shown in Tables 2, 3, 6, and 7, as well as various variants thereof (affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.).

[0191]

[0202] In certain embodiments, the anti-CD39 antigen-binding fragments provided herein are diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies.

[0192]

[0203] Various techniques can be used to produce such antigen-binding fragments. Illustrative methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form an F(ab')2 fragment (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0193]

[0204] In certain embodiments, the antigen-binding fragment is an scFv. The production of scFvs is described, for example, in WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. ScFvs can be fused at the amino or carboxyl terminus to an effector protein to provide a fusion protein (see, for example, Antibody Engineering, edited by Borrebaeck).

[0194]

[0205] In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule with more than two valencies is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0195]

[0206] A bivalent molecule is monospecific if both binding sites are specific for binding to the same antigen or epitope. In certain embodiments, this provides stronger binding to the antigen or epitope than its monovalent counterpart. Similarly, multivalent molecules can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valence of the binding site and the second valence of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have the same specificity but different sequences).

[0196]

[0207] A bivalent molecule can also be bispecific if the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific if two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0197]

[0208] bispecific antibody

[0209] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is bispecific. In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is further linked to a second functional moiety having a different binding specificity than the CD39 antibody or antigen-binding fragment thereof.

[0198]

[0210] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein can specifically bind to a second antigen other than CD39 or a second epitope on CD39. In certain embodiments, the second antigen is selected from the group consisting of TGFbeta, CD73, PD1, PDL1, 4-1BB, CTLA4, TIGIT, GITA, VISTA, TIGIT, B7-H3, B7-H4, B7-H5, CD112R, Siglec-15, LAG3, and TIM-3.

[0199]

[0211] Conjugates

[0212] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof further comprises one or more conjugate moieties. The conjugate moiety can be linked to the antibody or antigen-binding fragment thereof. The conjugate moiety is a moiety that can bind to the antibody or antigen-binding fragment thereof. It is contemplated that various conjugate moieties can be linked to the antibodies or antigen-binding fragments thereof provided herein (see, for example, "Conjugate Vaccines," Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be linked to the antibody or antigen-binding fragment thereof by covalent bonding, affinity bonding, intercalation, coordinate bonding, complexation, association, blending, or addition, among others. In some embodiments, the antibody or antigen-binding fragment thereof can be linked to one or more conjugates via a linker.

[0200]

[0213] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein may be engineered to contain specific sites in addition to the epitope-binding moiety that are available for attachment to one or more conjugate moieties. For example, such sites may contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, that facilitate covalent linkage to the conjugate moiety.

[0201]

[0214] In certain embodiments, an antibody or antigen-binding fragment thereof may be linked to a conjugate moiety indirectly or through another conjugate moiety. For example, an antibody or antigen-binding fragment thereof provided herein may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. In some embodiments, the conjugate moiety comprises a clearance modifier (e.g., a polymer such as PEG that extends half-life), a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label, an enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, a purification moiety, or other anti-cancer drug.

[0202]

[0215] A "toxin" can be any agent that is harmful to or damages or kills cells. Examples of toxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethoxysilane, methylparaben), methylparaben, ... These include, without limitation, cisplatin, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin binders.

[0203]

[0216] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, lucerifers, glucoamylase, lysozyme, saccharide oxidases, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P, other lanthanides), luminescent labels, plastid moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0204]

[0217] In certain embodiments, the conjugate moiety may be a clearance modifier that helps extend the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and others. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody varies, and when two or more polymers are attached, they may be the same or different molecules.

[0205]

[0218] In certain embodiments, the conjugated moiety can be a purification moiety such as a magnetic bead.

[0219] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as the basis for a conjugate.

[0206]

[0220] Polynucleotides and Recombinant Methods

[0221] The present disclosure provides isolated polynucleotides encoding the anti-CD39 antibodies or antigen-binding fragments thereof provided herein. As used herein, the terms "nucleic acid" or "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0207]

[0222] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains). The encoding DNA may also be obtained by synthetic methods.

[0208]

[0223] The isolated polynucleotide encoding an anti-CD39 antibody or antigen-binding fragment thereof can be inserted into a vector for further cloning (amplification of the DNA) or expression using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0209]

[0224] The present disclosure provides vectors comprising the isolated polynucleotides provided herein. In certain embodiments, the polynucleotides provided herein encode an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phage, and M13 phage, as well as the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, and p GEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, p Includes LexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0210]

[0225] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or DNA expression in the vectors herein are prokaryotic cells, yeast, or the higher eukaryotic cells described above. Prokaryotic cells suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Escherichia, Enterobacteriaceae, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillus, e.g., B. subtilis and B. licheniformis, Pseudomonas, e.g., P. aeruginosa, and Streptomyces.

[0211]

[0226] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-CD39 antibody-encoding vectors. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used. However, Schizosaccharomyces pombe is also effective against Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus, Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanniomyces Many other genera, species, and strains of Schwanniomycetes, such as A. occidentalis, and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger, are commonly available and useful herein.

[0212]

[0227] Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as viruses herein in accordance with the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0213]

[0228] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and the human hepatocellular carcinoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, or 293, and their derivatives.

[0214]

[0229] Host cells are transformed with the above-described expression or cloning vectors for production of anti-CD39 antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, antibodies may be produced by homologous recombination, as known in the art. In certain embodiments, host cells are capable of producing the antibodies or antigen-binding fragments thereof provided herein.

[0215]

[0230] The present disclosure also provides a method for expressing an antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions in which a vector of the present disclosure is expressed. The host cells used to produce the antibody or antigen-binding fragment thereof provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Re. 30,985 may be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those of skill in the art may also be included at appropriate concentrations. Temperature, pH, and other culture conditions will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0216]

[0231] When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris from host cells or lysed fragments is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, a Pellicon ultrafiltration unit from Amicon or Millipore. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and an antibiotic may be included to prevent the growth of adventitious contaminants.

[0217]

[0232] The anti-CD39 antibody or antigen-binding fragment thereof prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0218]

[0233] In certain embodiments, solid-phase immobilized protein A is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are also available. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0219]

[0234] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, preferably performed at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0220]

[0235] Pharmaceutical Composition

[0236] The present disclosure further provides pharmaceutical compositions comprising an anti-CD39 antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.

[0221]

[0237] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestrants or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other ingredients known in the art, or various combinations thereof.

[0222]

[0238] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments thereof and conjugates provided herein reduces oxidation of the antibodies or antigen-binding fragments thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Accordingly, in certain embodiments, pharmaceutical compositions are provided comprising one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine. Further provided are methods of preventing oxidation, extending shelf life, and / or improving efficacy of the antibodies or antigen-binding fragments provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0223]

[0239] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride for injection, Ringer's injection, isotonic dextrose injection, sterile water for injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphates or citrates; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifiers such as Polysorbate 80 (TWEEN-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Pharmaceutical compositions in multi-dose containers may contain antimicrobial agents used as carriers, including phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0224]

[0240] The pharmaceutical compositions can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0225]

[0241] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or solid form suitable for producing a liquid solution, suspension, emulsion, etc. The injectable preparation may include a sterile and / or non-pyrogenic solution ready for injection, a sterile dry soluble product such as a lyophilized powder ready for mixing with a solvent immediately before use, including hypodermic tablets, a sterile suspension ready for injection, a sterile dry insoluble product ready for mixing with a vehicle immediately before use, and a sterile and / or non-pyrogenic emulsion. The solution may be aqueous or non-aqueous.

[0226]

[0242] In certain embodiments, unit-dose preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration should be sterile and non-pyrogenic, as known and practiced in the art.

[0227]

[0243] In certain embodiments, sterile, lyophilized powders are prepared by dissolving the antibodies or antigen-binding fragments disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological properties of the powder or a reconstituted solution prepared from the powder. Possible excipients include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent, in one embodiment, has a near-neutral pH and may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-CD39 antibody or antigen-binding fragment thereof, or a composition thereof. Overfilling the vial by a small amount (e.g., about 10%) more than needed for the dose or set of doses is permissible to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0228]

[0244] The lyophilized powder is reconstituted with water for injection to provide a formulation for parenteral administration. In one embodiment, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the selected therapy and can be determined empirically.

[0229]

[0245] kit

[0246] In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein. In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an anti-cancer drug, radiation therapy, an immunotherapeutic agent, an anti-angiogenic agent, targeted therapy, cell therapy, gene therapy, hormone therapy, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, and a cytokine.

[0230]

[0247] Such kits may further include, if desired, one or more of a variety of conventional pharmaceutical kit components, such as a container containing one or more pharmaceutically acceptable carriers, additional containers, and the like, as will be readily apparent to one of skill in the art. Instructions, such as an insert or label, indicating the amounts of the components to be administered, guidelines for administration, and / or guidelines for mixing the components, may also be included in the kit.

[0231]

[0248] How to use

[0249] The present disclosure also provides methods for treating a CD39-related disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein. In certain embodiments, the subject is a human.

[0232]

[0250] In some embodiments, the CD39-associated disease, disorder, or condition is characterized by expression or overexpression of CD39.

[0251] In certain embodiments, the CD39-related disease, disorder, or condition is cancer. In certain embodiments, the cancer is a CD39-expressing cancer. As used herein, a "CD39-expressing" cancer refers to a cancer characterized by expression of CD39 protein in cancer cells, tumor-infiltrating immune cells, or immunosuppressive cells, or by expression of CD39 at a level significantly higher than that expected in normal cells in cancer cells, tumor-infiltrating immune cells, or immunosuppressive cells. Various methods can be used to determine the presence and / or amount of CD39 in a test biological sample from a subject. For example, the test biological sample can be exposed to an anti-CD39 antibody or antigen-binding fragment thereof that binds to and detects expressed CD39 protein. Alternatively, CD39 can be detected at the nucleic acid expression level using qPCR, reverse transcriptase PCR, microarray, SAGE, FISH, or other methods. In some embodiments, the test sample is derived from cancer cells or tissue, or tumor-infiltrating immune cells. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample from the same individual from which the test sample was obtained. For example, the reference sample can be a non-disease sample adjacent or near the test sample (eg, a tumor).

[0233]

[0252] In certain embodiments, the cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal tract cancer, skin cancer , prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, throat cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T- or B-cell lymphoma, GI organ stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma. In certain embodiments, the cancer is leukemia, lymphoma, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, or breast cancer.

[0234]

[0253] In some embodiments, the subject has been identified as having cancer cells or tumor-infiltrating immune or immunosuppressive cells that express CD39, optionally at levels significantly higher than those normally found in non-cancerous or non-immunosuppressive cells.

[0235]

[0254] In some embodiments, the disease, disorder, or condition is an autoimmune disease or infection. In some embodiments, the autoimmune disease is immune thrombocytopenia, systemic sclerosis, sclerosis, adult respiratory distress syndrome, eczema, asthma, Sjögren's syndrome, Addison's disease, giant cell arteritis, immune complex nephritis, immune thrombocytopenic purpura, autoimmune thrombocytopenia, celiac disease, psoriasis, dermatitis, colitis, or systemic lupus erythematosus. In some embodiments, the infection is a viral infection or a bacterial infection. In some embodiments, the infection is HIV infection, HBV infection, HCV infection, inflammatory bowel disease, or Crohn's disease.

[0236]

[0255] In another aspect, provided is a method of treating a disease, disorder, or condition in a subject that would benefit from modulating the activity of CD39, comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein. In certain embodiments, the disease, disorder, or condition is a CD39-associated disease, disorder, or condition as defined above.

[0237]

[0256] The therapeutically effective amount of an antibody or antigen-binding fragment provided herein will depend on various factors known in the art, such as weight, age, past medical history, current medications, the subject's health status and the potential for cross-reactivity, allergies, sensitivities, and adverse side reactions, as well as the route of administration and the extent of disease occurrence. Doses may be proportionately reduced or increased by the practitioner (e.g., physician or veterinarian) as dictated by these and other circumstances or requirements.

[0238]

[0257] In some embodiments, the antibodies or antigen-binding fragments provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose may vary over the course of treatment. For example, in certain embodiments, initial administered doses may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response.

[0239]

[0258] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) and may, for example, be administered as a single dose or as several divided doses administered over time.

[0240]

[0259] The antibodies or antigen-binding fragments thereof provided herein may be administered by any route known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0241]

[0260] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibodies or antigen-binding fragments thereof disclosed herein may be administered in combination with a second therapeutic agent, such as a chemotherapeutic agent, an anti-cancer drug, a radiotherapy agent, an immunotherapy agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, and a cytokine.

[0242]

[0261] As used herein, the term "immunotherapy" refers to a type of therapy that stimulates the immune system to fight diseases such as cancer or generally strengthens the immune system. Examples of immunotherapies include, without limitation, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0243]

[0262] "Targeted therapy" is a type of therapy that acts on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not in normal cells or that are more abundant in cancer cells, or target molecules in the cancer microenvironment that contribute to cancer growth and survival. Targeted therapy targets therapeutic agents to tumors, thereby sparing normal tissues from the effects of the therapeutic agents.

[0244]

[0263] In some of these embodiments, antibodies or antigen-binding fragments thereof provided herein that are administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in some of these embodiments, the antibody or antigen-binding fragment thereof and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof that is administered "in combination with" another therapeutic agent need not be administered simultaneously with or in the same composition as that agent. An antibody or antigen-binding fragment thereof that is administered before or after another agent is considered to be administered "in combination with" that agent, as that term is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with an antibody or antigen-binding fragment thereof disclosed herein are administered according to the schedule listed in the product information sheet for that additional therapeutic agent, or according to protocols known in the art, such as those described in the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)).

[0245]

[0264] The present disclosure further provides a method for modulating the activity of CD39 in a CD39-positive cell, comprising exposing the CD39-positive cell to an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the CD39-positive cell is an immune cell.

[0246]

[0265] In another aspect, the present disclosure provides a method of detecting the presence or amount of CD39 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein, and / or a pharmaceutical composition provided herein, and determining the presence or amount of CD39 in the sample.

[0247]

[0266] In another aspect, the present disclosure provides a method of diagnosing a CD39-related disease, disorder, or condition in a subject, comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein; b) determining the presence or amount of CD39 in the sample; and c) correlating the presence or amount of CD39 with the presence or status of the CD39-related disease, disorder, or status in the subject.

[0248]

[0267] In another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety, and / or a pharmaceutical composition provided herein, useful for detecting a CD39-related disease, disorder, or condition. The kit may further comprise instructions for use.

[0249]

[0268] In another aspect, the present disclosure also provides the use of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein in the manufacture of a medicament for treating, preventing, or ameliorating a CD39-related disease, disorder, or condition in a subject, and in the manufacture of a diagnostic reagent for diagnosing a CD39-related disease, disorder, or condition.

[0250]

[0269] In another aspect, the present disclosure provides a method for treating, preventing, or alleviating a disease treatable by reducing the ATPase activity of CD39 in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein. For example, the antibody or antigen-binding fragment thereof provided herein may be administered to reduce the ATPase activity of CD39-expressing cancer cells, tumor-infiltrating immune cells, or immunosuppressive cells. In some embodiments, the subject is a human. In some embodiments, the subject has a disease, disorder, or condition selected from the group consisting of cancer, autoimmune disease, and infectious disease.

[0251]

[0270] In another aspect, the present disclosure provides a method of treating, preventing, or alleviating a disease associated with adenosine-mediated inhibition of T cell, monocyte, macrophage, DC, APC, NK, and / or B cell activity in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, and / or a pharmaceutical composition provided herein.

[0252]

[0271] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below are within the scope of the present invention, in whole or in part. These specific compositions, materials, and methods are not intended to limit the invention, but merely illustrate specific embodiments within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein while remaining within the boundaries of the invention. It is the intention of the inventors that such variations be included within the scope of the invention. [Example]

[0253] Example 1. Preparation of materials

[0272] 1.1. Generation of reference antibodies

[0273] Anti-CD39 reference antibodies were generated based on published sequences. Antibody 9-8B is disclosed in patent application WO 2016 / 073845A1, and the sequences of its heavy and light chain variable regions are included herein as SEQ ID NOs: 46 and 48, respectively. Antibody T895 is disclosed in patent application WO 2019 / 027935A1 as antibody 31895, and the sequences of its heavy and light chain variable regions are included herein as SEQ ID NOs: 55 and 57, respectively. Antibody I394 is disclosed in patent application WO 2018 / 167267A1, and the sequences of its heavy and light chain variable regions are included herein as SEQ ID NOs: 113 and 114, respectively. The heavy and light chain variable regions of antibodies 9-8B, T895, and I394 are shown in Table 10 below. DNA encoding the reference antibodies was cloned and expressed in Expi293 cells (Invitrogen). The cell culture medium was collected and centrifuged to remove the cell pellet, and the collected supernatant was purified by Protein A affinity chromatography (MabselectSure, GE Healthcare) to obtain the reference antibody preparation.

[0254] [Table 11]

[0255]

[0274] 1.2. Generation of human, cynomolgus monkey, and mouse CD39 stable-expressing cell lines

[0275] DNA sequences encoding full-length human CD39 (NP_001767.3), cynomolgus monkey CD39 (XP_015311944.1), and mouse CD39 (NP_033978.1) were cloned into expression vectors and subsequently transfected and expressed in HEK293 cells. Transfected cells expressing human CD39, cynomolgus monkey CD39, and mouse CD39, respectively, were cultured in selective medium. Single-cell clones stably expressing human CD39, cynomolgus monkey CD39, or mouse CD39 were isolated by limiting dilution. The cells were then screened by FACS using anti-human CD39 antibodies (BD, Cat#555464), anti-cynomolgus monkey CD39 (9-8B), and anti-mouse CD39 (Biolegend, Cat#143810).

[0256]

[0276] Similarly, CHOK1 cells (Invitrogen) transfected with human CD39, cynomolgus monkey CD39, and mouse CD39 expression plasmids were cultured in selective medium. Single-cell clones stably expressing human CD39, cynomolgus monkey CD39, or mouse CD39 were isolated by limiting dilution and subsequently screened by FACS using anti-human CD39, anti-cynomolgus monkey CD39, or anti-mouse CD39 antibodies.

[0257]

[0277] The stable cell lines were designated HEK293-hCD39, HEK293-cynoCD39, HEK293-mCD39, CHOK1-hCD39, CHOK1-cynoCD39, and CHOK1-mCD39, respectively, and all of them showed high expression and ATPase activity.

[0258]

[0278] 1.3. Production of Recombinant Proteins

[0279] A DNA sequence encoding the extracellular domain (ECD) of human CD39 was cloned into an expression vector and transfected into HEK293 cells to express the recombinant ECD protein.

[0259] Example 2. Antibody generation

[0280] 2.1. Immunization and Hybridoma Generation and Screening

[0281] To generate antibodies against CD39, Balb / c and SJL / J mice (SLAC) were immunized with recombinantly expressed human CD39 antigen or fragments thereof, DNA encoding full-length human CD39, and / or cells expressing human CD39. Over the course of the immunization protocol, immune responses were monitored by plasma or serum samples obtained by tail vein or retroorbital bleeding. Mice with sufficient titers of anti-CD39 antibodies were used for fusions. Splenocytes and / or lymph node cells from immunized mice were isolated and fused to a mouse myeloma cell line (SP2 / 0). The resulting hybridomas were screened for the production of CD39-specific antibodies by ELISA assay using human CD39 ECD recombinant protein or by the Acumen assay (TTP Labtech) using CHOK1-hCD39 cells stably expressing human CD39. hCD39-specific hybridoma clones were confirmed by FACS and enzyme activity blocking assays and subcloned to obtain stable hybridoma clones. After one or two rounds of subcloning, hybridoma monoclones were expanded for antibody production and frozen as stocks.

[0260]

[0282] Antibody-secreting hybridomas were subcloned by limiting dilution. Stable subclones were cultured in vitro to generate antibody in tissue culture medium for characterization. After one or two rounds of subcloning, hybridoma monoclones were expanded for antibody production.

[0261]

[0283] After approximately 14 days of culture, the hybridoma cell culture medium was collected and purified by protein A affinity chromatography (GE). The hybridoma antibody clones were designated mAb13, mAb14, Ab19, mAb21, mAb23, mAb34, and mAb35, respectively.

[0262] Example 3. Antibody Characterization

[0284] 3.1. Antibodies

[0285] Hybridoma antibody clones mAb13, mAb14, Ab19, mAb21, mAb23, mAb34, and mAb35 were characterized in a series of binding and functional assays as described below.

[0263]

[0286] 3.2. Binding affinity to human CD39, cynomolgus monkey CD39, and mouse CD39

[0287] FACS was used to determine antibody binding to cell lines expressing CD39 naturally (SK-MEL-28) or recombinantly (CHOK1-hCD39, CHOK1-cynoCD39, and CHOK1-mCD39), or to cells lacking CD39 expression (CHOK1-blank) as a negative control.

[0264]

[0288] CHOK1-hCD39, CHOK1-cCD39, CHOK1-mCD39, and CHOK1-blank cells were maintained in culture medium according to ATCC protocols. Cells were harvested and grown at a cell density of 3 × 10 6 The cells were resuspended in blocking buffer at 3 × 10 cells / ml. The cells were added to 100 μl / well (3 × 10 5The cells were transferred to a 96-well FACS plate at 100 μL / well, the plate was centrifuged, and washed twice with FACS buffer (PBS, 1% FBS, 0.05% Tween-20). Serial four-fold dilutions of anti-CD39 antibodies were prepared in FACS buffer, starting at 30 μg / ml. Reference antibody 9-8B and mouse / human control IgG were used as positive and negative controls, respectively. Cells were resuspended in 100 μL / well of diluted antibody, and the plate was incubated at 4°C for 60 minutes. The plate was washed with FACS buffer, and Alexa-Fluor® 488-labeled secondary antibody (1:1000 in FACS buffer) was added to each well and incubated at 4°C for 30 minutes. The plate was washed with FACS buffer, and the cells were resuspended in 100 μL / well of PBS. The cells were then analyzed using a FACSVerse™, and the mean fluorescence intensity was determined. A full binding curve for CD39-expressing cells was generated by testing a range of antibody concentrations. Apparent affinities were determined for each antibody using Prism software.

[0265]

[0289] Similarly, human CD39-expressing cells SK-MEL-5, SK-MEL-28, or MOLP-8 were incubated with gradient concentrations of anti-CD39 antibody for 30 minutes at 4°C. Cells were washed three times with FACS buffer and then incubated with fluorescently labeled secondary antibodies (goat anti-mouse IgG or goat anti-human IgG) for 30 minutes at 4°C. Cells were washed three times, then resuspended in FACS buffer and analyzed by flow cytometry on a BD Celesta. Data were plotted and analyzed using GraphPad Prism 8.02.

[0266]

[0290] The binding affinities of the seven purified hybridoma antibodies are summarized in Table 11 in comparison with the known anti-CD39 antibody 9-8B. All hybridoma antibodies bound to human and cynomolgus monkey CD39 in a dose-dependent manner, but none recognized mouse CD39 in FACS studies.

[0267] [Table 12]

[0268]

[0291] 3.3. Detection of ATPase inhibition

[0292] CD39-expressing cells, SK-MEL-5 and MOLP-8, were washed with PBS buffer and incubated with the antibody gradient at 37°C for 30 minutes. 50 mM ATP was added to each well and incubated with the cells for 16 hours. The supernatant was collected, and orthophosphate production from ATP degradation was measured using a Malachite Green Phosphate Detection Kit (R&D systems, catalog #DY996) according to the manufacturer's instructions. Isotype and / or 9-8B were used as controls. Data were plotted and analyzed using GraphPad Prism 8.02. EC 50 is the concentration of the indicated antibody that achieves 50% of the signal in this assay.

[0269]

[0293] As summarized in Table 11, all of the seven purified hybridoma antibodies had better ATPase inhibitory activity compared to the reference antibody 9-8B.

[0294] 3.4. ATP-mediated T cell proliferation inhibition assay

[0295] Human T cells labeled with CSFE and stimulated with anti-CD3 and anti-CD28 were incubated with anti-CD39 antibody or an isotype control in the presence of ATP. T cell proliferation was analyzed by FACS using CSFE dilution. mIgG2a was used as an isotype control.

[0270]

[0296] The T cell proliferation activities of selected anti-CD39 antibodies mAb21 and mAb23 are shown in Figure 1 and summarized in Table 11. EC 50 is the concentration of the indicated antibody that achieves 50% of the signal in this assay. Both antibodies enhanced T cell proliferation in a dose-dependent manner; i.e., both antibodies blocked ATP-mediated inhibition of T cell proliferation.

[0271]

[0297] 3.5. Epitope Binning

[0298] Anti-CD39 antibodies were labeled using an Alex488 labeling kit, diluted at a series of concentrations, mixed with CHOK1-hCD39 cells, and bound ECs were analyzed using FACS. 50 The blocking efficiency of unlabeled antibodies against labeled antibodies was tested. Briefly, mononuclear CHOK1-hCD39 cells were cultured at 2 × 10 6 The antibody gradient was mixed to a final volume of 100 μl, and an equal amount of Alex488-labeled antibody was added to the EC 80 The 96-well plate was incubated at 4°C for 1 hour, spun down, and washed three times with 200 μl of FACS buffer. FACS analysis was performed using a FACScelesta machine, and data were analyzed using Flowjo software. Blocking rates were calculated, and antibodies with a competition rate of over 80% compared to non-competing wells (Alex488-labeled antibody alone) were assigned to one epitope group.

[0272]

[0299] The competition results are shown in Table 12. Based on the competition results, the four anti-CD39 hybridoma antibodies (mAb14, mAb19, mAb21, and mAb23) can be classified into four different epitope groups, as shown in Table 11. Specifically, the anti-CD39 antibodies mAb19 and mAb21 compete for highly similar epitopes and are classified into epitope group I, as shown in Table 11. mAb14 did not compete with any of the other antibodies tested and was classified into epitope group IV, as shown in Table 11. mAb23 showed cross-competition with mAb19 and mAb21 and was classified into epitope group II in Table 11.

[0273] [Table 13]

[0274]

[0300] 3.6. Hybridoma sequencing

[0301] RNA was isolated from the monoclonal hybridoma cells and reverse transcribed into cDNA using a commercially available kit. The cDNA was then used as a template to amplify the heavy and light chain variable regions with primers from the Mouse Ig-Primer Set (Novagen). PCR products of the correct size were collected, purified, and ligated with the appropriate plasmid vector. The ligation product was transformed into DH5α competent cells. Clones were selected, and the inserted fragments were analyzed by DNA sequencing.

[0275]

[0302] The variable region sequences of the hybridoma antibodies are provided in Table 2 herein. Example 4. Generation and characterization of chimeric antibodies

[0303] 4.1. Generation and Production of Chimeric Antibodies

[0304] DNA encoding the variable regions of four selected hybridoma antibodies (mAb14, mAb19, mAb21, and mAb23) was synthesized and subcloned into expression vectors previously containing human IgG constant genes. The vectors were transfected into mammalian cells for recombinant protein expression, and the expressed antibodies were purified on a protein A affinity column. The resulting chimeric antibodies are referred to herein as c14, c19, c21, and c23, where the prefix "c" indicates "chimeric" and the numbers indicate the clone of the hybridoma antibody. For example, the number "14" indicates that the antibody is from hybridoma antibody mAb14.

[0276]

[0305] 4.2. Characterization of Chimeric Antibodies

[0306] The four purified chimeric antibodies were tested for their ability to block ATP-mediated inhibition of T cell proliferation (similar to the method described in Example 3.4). As shown in Figure 2, the anti-CD39 chimeric antibodies c14, c19, c21, and c23 dose-dependently inhibited CD4 T cell proliferation (at concentrations ranging from 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM). + Blocked the suppression of T cell proliferation. CFSE-CD4 +T and hIgG4 were used as positive and negative controls for ATP-mediated T cell proliferation, respectively.

[0277]

[0307] The four purified chimeric antibodies were further tested for their ability to enhance ATP-induced dendritic cell (DC) activation and maturation in the presence of ATP, which induces DC maturation through stimulation of the P2Y11 receptor on monocyte-derived dendritic cells.

[0278]

[0308] Briefly, human monocytes were isolated from healthy human blood and differentiated into moDCs in the presence of GM-CSF and IL-4 for 6 days. The differentiated moDCs were then treated with different doses of four anti-CD39 chimeric antibodies in the presence of ATP for an additional 24 hours. DC maturation was then assessed by analyzing the expression of CD86, CD83, and HLA-DR by FACS assay.

[0279]

[0309] Figure 3 shows the levels of CD39 on the DC surface by FACS. Figures 4A-4C show the expression of CD86 (Figure 4A), CD83 (Figure 4B), and HLA-DR (Figure 4C) after antibody treatment, respectively. ATP-induced DC maturation was indicated by increased expression of CD86, CD83, and HLA-DR compared with vehicle treatment. All four anti-CD39 antibodies, c14, c19, c21, and c23, showed significant effects on enhancing ATP-induced DC maturation.

[0280]

[0310] The chimeric antibody was also tested in vivo for antitumor activity. NOD-SCID mice were inoculated in the right hind flank region with tumor cells (10 × 10 ) in 0.1 ml of PBS mixed with Matrigel (1:1) for tumor development. 6 The average tumor size was approximately 80 mm 3 Mice were randomized when they reached 100 mg / kg. Treatment began on the same day as randomization at 30 mg / kg and was administered twice weekly. Tumor volumes were measured in two dimensions using calipers twice weekly after randomization, and the volume was calculated in mm using the formula: V = (L × W × W) / 2. 3where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L). Dosing and tumor and body weight measurements were performed in a laminar flow cabinet. Data were analyzed using two-way ANOVA with Graphpad Prism.

[0281]

[0311] The tumor growth results for the chimeric anti-CD39 antibody c23 are shown in Figure 5. Both human IgG1 and IgG4 isotypes of c23 were obtained and tested. Both chimeric antibodies, c23-hIgG4 and c23-hIgG1, demonstrated antitumor efficacy compared to the vehicle group, with no significant difference identified between c23-hIgG4 and c23-hIgG1.

[0282] Example 5. Antibody humanization and affinity maturation

[0312] 5.1. Humanization

[0313] The chimeric antibodies c23 and c14 were selected as clones for humanization. The antibody sequences were aligned with human germline sequences to identify the best-fit model. Based on their homology with the original mouse antibody sequence, the best-matched human germline sequence was selected as the template for humanization. CDRs from the mouse antibody sequence were then grafted onto the template, along with residues to maintain the core structure of the top and center of the antibody. Optimized mutations were introduced into the framework regions to generate humanized heavy chain variable region variants and humanized light chain variable region variants, which were then mixed and matched to provide multiple humanized antibody clones. After grafting and mutation, the humanized antibodies retained similar binding affinities to human CD39-expressing cells. The humanized antibodies were further evaluated by CD39 ATPase inhibition assays and in vitro immune cell activation assays. In vivo studies were also performed on some of the humanized antibodies.

[0283]

[0314] A total of 31 humanized antibody clones were obtained for c23, consisting of seven humanized c23 heavy chain variable region variants (i.e., hu23.VH_1, hu23.VH_2, hu23.VH_3, hu23.VH_4, hu23.VH_5, hu23.VH_6, and hu23.VH_7) and seven humanized c23 light chain variable region variants (i.e., hu23.VL_1, hu23.VL_2, hu23.VL_3, hu23.VL_4, hu23.VL_5, hu23.VL_6, and hu23.VL_7), mixed and matched. The 31 humanized antibody clones are designated as hu23.H1L1, hu23.H1L2, etc., as shown in Table 9 above and Tables 13, 14, and 15 below. Here, the prefix "hu" indicates "humanized" and the suffix, eg, "H1L1," represents the sequence number of the c23 humanized antibody clone having variable regions of the hu23.VH_1 and hu23.VL_1 variants.

[0284] [Table 14]

[0285] [Table 15]

[0286] [Table 16]

[0287]

[0315] Similarly, a total of 16 humanized antibodies were obtained for c14 by mixing and matching four variants of the humanized c14 heavy chain variable region (i.e., hu14.VH_1, hu14.VH_2, hu14.VH_3, and hu14.VH_4) and four variants of the humanized c14 light chain variable region (i.e., hu14.VL_1, hu14.VL_2, hu14.VL_3, and hu14.VL_4). The 16 humanized antibody clones are designated by the same notation, hu14.H1L1, hu14.H1L2, etc., as shown in Table 16 below.

[0288] [Table 17]

[0289]

[0316] Several humanized antibody clones were also obtained by yeast display of c23. Briefly, the mouse heavy and light chain sequences were aligned with an in-house database of human antibody sequences. IGHV1-3, which is the template with the highest homology for CDR grafting of the heavy and light chains, respectively, was used. * 01 and IGKV3-11 * 01 was selected. Backmutations were identified using a high-throughput method using yeast display. Specifically, positions contributing to the CDR conformation (Vernier zone residues) were identified, and a library of backmutations was created by incorporating both template and mouse residues at each position during DNA synthesis. Final candidates were identified by sequencing the top binders to the human CD39 protein. The humanized antibodies for c23 obtained via yeast display are designated hu23.201 (having VH / VL of SEQ ID NOs: 146 / 111), hu23.203 (having VH / VL of SEQ ID NOs: 146 / 112), hu23.207 (having VH / VL of SEQ ID NOs: 147 / 111), and hu23.211 (having VH / VL of SEQ ID NOs: 39 / 63).

[0290]

[0317] The humanized antibodies in Tables 13, 14, 15, and 16 were recombinantly produced and tested for binding affinity, demonstrating that they can retain specific binding to human CD39. Humanized antibodies with relatively high affinity were further evaluated in functional assays, including a CD39 blocking assay and an in vitro immune cell activation assay.

[0291]

[0318] In particular, the humanized antibodies hu23.H5L5, hu23.201, and hu14.H1L1, as well as the reference antibodies I394 and T895, were characterized for their binding affinity to human CD39 using Biacore (GE). Briefly, the antibodies to be tested were captured onto a CM5 chip (GE) using a Human Antibody Capture Kit (GE). 6xHis-tagged human CD39 antigen was serially diluted for multiple doses and injected at 30 μl / min for 180 seconds. The buffer flow rate was maintained for 400 seconds for dissociation. 3M MgCl2 was used for chip regeneration. Association and dissociation curves were fitted to a 1:1 binding model, with Ka / Kd / K for each antibody. D Values ​​were calculated. The affinity data for the antibodies tested are summarized in Table 17 below.

[0292] [Table 18]

[0293]

[0319] Furthermore, the humanized antibodies hu23.H5L5 and hu14.H1L2, as well as the reference antibodies I394, T895, and 9-8B, were characterized for their binding affinity to human CD39 using the Octet assay (Creative Biolabs) according to the manufacturer's manual. Briefly, the antibodies were coupled to the sensor, and then the sensor was immersed in a gradient of CD39 (starting at 200 nM, with 2-fold dilutions for a total of 8 doses). The binding responses were measured in real time. The results were generally consistent. The affinity data for the tested antibodies are summarized in Table 18 below.

[0294] [Table 19]

[0295]

[0320] Furthermore, a single NG motif (N55G56) that is susceptible to deamidation was identified in the HCDR2 of a humanized antibody clone of the c23 antibody (e.g., hu23.H5L5). To remove the deamidation site, various mutations were introduced into N55 or G56. It was found that N55 and G56 could be mutated to various residues while still retaining specific binding to human CD39. For example, single-point replacement of N55 with G, S, or Q maintained antibody binding affinity and had no adverse effect on binding to human CD39. Similarly, replacement of G56 with A or D ensured that the mutant antibody retained specific binding and binding affinity to human CD39. Other mutations were expected to be similarly effective.

[0296]

[0321] 5.2. Detection of binding specificity

[0322] The binding specificity of purified humanized antibody hu23.H5L5 to ENTPDase family members was detected by ELISA assay. Briefly, ENTPD1 (i.e., CD39) and ENTPD2 / 3 / 5 / 6 proteins were coated onto a 96-well ELISA plate overnight at 4°C. The following day, the ELISA plate was washed and blocked with 200 μl / well of blocking buffer (1% BSA and 0.05% Tween 20 in PBS) for 2 hours. A gradient of hu23.H5L5 was then applied in duplicate to wells and stained with anti-hIgG-HRP. After washing, the plate was developed with TMB substrate and stopped with 2N HCl. OD450 was recorded using a plate reader and plotted using Graphpad Prism. The binding specificity of hu23.H5L5 is shown in Figure 6. Figure 6A shows that humanized antibody hu23.H5L5 specifically binds to human CD39 but does not bind to any of the ENTPD2 / 3 / 5 / 6 proteins, and Figure 6B shows that the negative control hIgG4 does not bind to any of the ENTPD1 / 2 / 3 / 5 / 6 proteins.

[0297]

[0323] 5.3. Characterization of Humanized Antibodies

[0324] The binding affinity of the c23 humanized antibody was determined by FACS using a method similar to that described in Example 3.2. The c23 humanized antibody clones showing good binding affinity are listed in Tables 19 and 20 below and are also shown in Figures 7A, 7B, and 8. EC 50 is the concentration of the indicated antibody that achieves 50% of the signal in this assay.

[0298] [Table 20]

[0299] [Table 21]

[0300]

[0325] Selected c23 humanized antibodies were tested in an ATPase inhibition assay (as described in Example 3.3) on SK-MEL-28 cells. Inhibition plots for the indicated antibodies are shown in Figures 9A and 9B and summarized in Table 21. hu23.H5L5 and hu23.201 were selected for further validation.

[0301] [Table 22]

[0302]

[0326] The binding affinity of the c14 humanized antibody was determined by FACS using MOLP-8 cells expressing human CD39 using methods similar to those described in Example 3.2.

[0327] The humanized antibody clones of c14 that showed good binding affinity are shown in Figures 10A, 10B, and 10C. EC 50 are summarized in Table 22.

[0303] [Table 23]

[0304]

[0328] 5.4. Epitope Binning

[0329] Selected humanized antibodies were tested for competitive binding (method described in Example 3.5). The results of epitope binning of humanized antibodies hu23.H5L5 and hu14.H1L1 with the reference antibodies are shown in Figure 19A.

[0305]

[0330] Based on the competition results (shown in Figure 19A), the two humanized anti-CD39 antibodies, hu23.H5L5 and hu14.H1L1, could be classified into two distinct epitope groups (see Figure 19B). Specifically, the anti-CD39 antibody hu23.H5L5 competed with reference antibodies I394, T895, and 9-8B for a highly similar epitope and was classified into epitope group I. The anti-CD39 antibody hu23.H5L5, c34, and c35 did not compete with any other antibodies tested except for partial competition with T895 and were classified into epitope group II.

[0306]

[0331] 5.5. Characterization of Optimized Humanized Antibodies

[0332] 5.5.1 Blockade of CD39 with hu23.H5L5 improved human T cell proliferation in the presence of extracellular ATP (eATP).

[0307]

[0333] Human PBMCs stimulated with anti-CD3 and anti-CD28 antibodies were incubated with 25 nM of the humanized anti-CD39 antibody hu23.H5L5 or vehicle in the presence of ATP. Cell culture supernatants were collected for detection of IL-2 and IFN-γ secretion, respectively. + T cells and CD8 + T cell proliferation was analyzed on day 5 by FACS using Cell Trace Violet dye dilution.

[0308]

[0334] As shown in Figures 11A-11D, hu23.H5L5 inhibited CD4 + and CD8 +It significantly enhanced T cell proliferation and stimulated their production of IL-2 and IFN-γ. As shown in Figures 11A, 11B, and 11D, hu23.H5L5 exhibited significantly greater activity than I394 in enhancing T cell activation in PBMCs.

[0309]

[0335] Human CD8 + T cells were also isolated from PBMCs of healthy donors, labeled with a cell proliferation dye, activated with anti-CD3 and anti-CD28 antibodies, and treated with different doses of the humanized anti-CD39 antibody hu23.H5L5 or the reference antibody I394 for a total treatment period of 5 days. 200 μM ATP was added to the cells on day 3 after the start of the CD39 blockade treatment. On day 5, CD8 T cells were analyzed by flow cytometry. + T cell proliferation %, CD25 + The % of cells and % of live cells were analyzed.

[0310]

[0336] As shown in Figures 23A-23C, hu23.H5L5 inhibited the activity of human CD8 + It significantly reversed T cell proliferation.

[0337] The binding affinity of humanized antibodies hu23.H5L5 and hu14.H1L1 was tested by FACS on a variety of cells according to methods similar to those described in Example 3.2.

[0311]

[0338] Figures 12A-12E show the binding affinity of antibodies hu23.H5L5 and hu14.H1L1 to SK-MEL-5 (Figure 12A), SK-MEL-28 (Figure 12B), MOLP-8 (Figure 12C), CHOK1-cynoCD39 (Figure 12D), and CHOK1-mCD39 (Figure 12E), respectively. Reference antibodies T895 and I394 were tested in parallel as control antibodies. As shown in Figure 12 and summarized in Table 23, both antibodies hu23.H5L5 and hu14.H1L1 bind to SK-MEL-5 in a dose-dependent manner and with subnanomolar or nanomolar EC 50These antibodies bound to human and cynomolgus monkey CD39-expressing cells with similar affinity by FACS. Neither of these antibodies recognized mouse CD39 in FACS studies. The different maximum signals (mean fluorescence intensity, MFI) between cells for each antibody are likely due to differences in their expression levels.

[0312] [Table 24]

[0313]

[0339] Figure 13 shows that hu23.H5L5 blocked CD39 ATPase activity in SK-MEL-5 (Figure 13A) or MOLP-8 (Figure 13B) cells similarly to the reference antibodies T895 and I394 (methods described in Example 3.3). The results are summarized in Table 24.

[0314]

[0340] hu23.H5L5 has an enzyme-blocking IC of 70 pM in SK-MEL-5 cells and 330 pM in MOLP-8 cells 50 , similar to or slightly better than the reference antibodies T895 and I394. However, hu14.H1L1 failed to reach saturation blocking in both cells, and 9-8B was identified as a non-blocker in this assay.

[0315] [Table 25]

[0316]

[0341] 5.5.2 Blockade of CD39 with hu23.H5L5 enhanced ATP-mediated monocyte activation.

[0342] The humanized antibody hu23.H5L5 was also tested in an ATP-mediated monocyte activation assay. ATP-mediated pro-inflammatory activity plays an important role in regulating the function of various immune cell types, including monocytes. To assess whether CD39 blockade can enhance ATP-mediated monocyte activation, human monocytes were purified from healthy human blood and then incubated with anti-CD39 antibodies at various concentrations ranging from 0.2 nM to 100 nM in the presence of ATP. hu23.H5L5 was shown to be effective in inducing monocyte activation at 0.2 nM, the lowest concentration tested. Monocyte activation was assessed by analyzing the expression of CD80 (Figure 14A), CD86 (Figure 14B), and CD40 (Figure 14C) by FACS assay (hu23.H5L5 concentration was 50 nM). Reference anti-CD39 antibodies I394 and T895 were used as controls, and hIgG4 was used as an isotype control.

[0317]

[0343] The results are shown in Figure 14. Stimulation with ATP alone showed upregulated expression of CD80 and CD86, indicating monocyte activation. The anti-CD39 humanized antibody hu23.H5L5 further enhanced ATP-mediated monocyte activation, as demonstrated by upregulation of CD80, CD86, and CD40 at levels comparable to those enhanced by the reference antibody I394. The reference antibody T895 had no significant effect on ATP-induced activated monocytes.

[0318]

[0344] 5.5.3 Blockade of CD39 with hu23.H5L5 enhanced ATP-mediated DC activation.

[0345] The selected humanized antibody, hu23.H5L5, was also tested in an ATP-mediated DC activation assay (following a similar method described in Example 4.2). Briefly, DC maturation was assessed by analyzing CD83 expression by FACS assay. ATP induced DC maturation, as indicated by increased expression of CD83 (Figure 15A). hu23.H5L5 increased CD83 expression in a dose-dependent manner starting at a low level of 0.2 nM, and significantly increased CD83 expression at an antibody level of 0.6 nM, which is more potent than both the reference antibodies T895 and I394.

[0319]

[0346] To further evaluate the resulting effect of ATP-mediated DC activation on T cell activation, ATP-activated DCs were washed and then incubated with allogeneic T cells for a mixed lymphocyte reaction (MLR). T cell proliferation (Figure 15B) and IFN-γ production from activated T cells were analyzed (Figure 15C).

[0320]

[0347] Compared with the reference antibodies I394 and T895, the anti-CD39 antibody hu23.H5L5 exhibited a dose-dependent and significant effect on enhancing ATP-induced DC maturation. Reference I394 exhibited similar but slightly weaker activity, whereas the effect of T895 was significantly milder. Consistently, as shown in Figures 15B and 15C, enhanced ATP-mediated moDC maturation by the anti-CD39 blocking antibody hu23.H5L5 resulted in higher T cell proliferation and IFN-γ production in MLR assays.

[0321]

[0348] 5.5.4 Blockade of CD39 by hu23.H5L5 enhanced IL-1β release from human macrophages induced by LPS stimulation.

[0349] Human CD14 from healthy human PBMCs + T cells were isolated and then enriched for CD14 + 2 × 10 monocytes 6 M1-like macrophages were generated by seeding cells at a density of 100 cells / well into 6-well plates and culturing them with 100 ng / mL human GM-CSF for 6 days. In vitro differentiated macrophages were treated with increasing doses of hu23.H5L5 or reference antibody I394 for 1 hour, followed by stimulation with 10 ng / mL LPS for 3 hours and 800 μM ATP for 2 hours. IL-1β was quantified in the cell culture supernatants by ELISA.

[0322]

[0350] The results are shown in Figure 20. Asterisks indicate significant differences between the respective conditions. As shown in Figure 20, hu23.H5L5 significantly promoted IL1β release from human macrophages induced by LPS stimulation, and hu23.H5L5 exhibited significantly higher activity than the reference antibody I394 in promoting IL1β release from human macrophages induced by LPS stimulation.

[0323]

[0351] 5.6. In vivo studies

[0352] The effect of humanized antibodies hu23.H5L5 and hu14.H1L1 was determined in MOLP-8 xenografted mice according to the methods described in Example 4.2.

[0324]

[0353] The results are shown in Figure 16. All of the anti-CD39 antibodies inhibited tumor growth compared to the vehicle group. The efficacy observed for I394 was somewhat weaker than the other antibodies, including hu23.H5L5 and hu14.H1L1.

[0325]

[0354] The antitumor efficacy of the humanized antibody hu23.H5L5 was also tested in vivo in a PBMC adoptive animal model (NCG mice, inoculated with 5 M MOLP-8 cells / mouse) by testing a range of different doses (0.03 mg / kg, 0.3 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg, ip, BIW x 6 doses) according to the method described in Example 4.2.

[0326]

[0355] The results are shown in Figure 21. As shown in Figure 21, the humanized antibody hu23.H5L5 potently inhibited tumor growth at all doses tested.

[0356] We also determined whether the anti-CD39 antibody's antitumor efficacy was dependent on NK cells or macrophage cells. NK-depleting treatment with anti-asialo-GM1 was administered intraperitoneally at 20 μl / mouse starting on day 7 and once every 5 days. Macrophage-depleting treatment with clodronate liposomes was also administered intravenously at 200 μl / mouse starting on days 7 and 9 and once weekly. Blood sample analysis data demonstrated that the reagents significantly depleted mononuclear phagocytes or NK cells.

[0327]

[0357] The tumor growth inhibitory effect of hu23.H5L5 was abolished in models depleted of NK (Figure 17) or macrophage (Figure 18) cells, suggesting that the antitumor effect of anti-CD39 antibodies is dependent on NK cells and macrophages.

[0328]

[0358] Specifically, as shown in Figure 17, anti-asialo-GM1 slightly enhanced tumor growth at a late stage compared to vehicle. Compared with the group treated with hu23.H5L5, the tumor growth inhibitory effect of hu23.H5L5 was completely abolished by combining it with anti-asialo-GM1. As shown in Figure 18, clodronate liposomes had no effect on tumor growth compared to vehicle. However, clodronate liposome treatment completely abolished the tumor growth inhibitory effect of hu23.H5L5.

[0329] Example 6. Epitope Mapping

[0359] To define the epitope of anti-CD39 antibodies, CD39 mutants were designed and defined by substitution of amino acids exposed on the surface of human CD39. As shown in Table 25 below, the mutants were cloned into expression vectors fused with a C-terminal EGFP sequence and transfected into HEK-293F cells. The targeted amino acid mutations are shown using the numbering of UniProtKB-P49961 (ENTP1_HUMAN). This is the wild-type amino acid sequence of human CD39 and is represented herein as SEQ ID NO: 162. For example, V77G means that the valine at position 77 of SEQ ID NO: 162 has been replaced with glycine.

[0330] [Table 26-1] [Table 26-2]

[0331]

[0360] Briefly, human CD39 mutants were generated by gene synthesis and then cloned into the expression vector pCMV3-GFPSpark. Vectors containing the validated mutant sequences were prepared and transfected into HEK293F cells. Three days after transfection, cells were harvested to test for EGFP expression of the transgene. A dose range of antibodies (starting at 100 nM, 11 three-fold dilutions) was tested against the 20 mutants generated and stained with AlexFluor647-labeled anti-hIgG by FACS. Antibody binding was described as relative binding, calculated by dividing the AlexFluor647 intensity by the GFP intensity. The results are shown in Figure 22.

[0332]

[0361] As shown in Figure 22, humanized antibody hu23.H5L5 lost binding to mutants KW27-6 and KW27-20, but not to the other mutants. Mutant KW27-6 contains amino acid substitutions at residues Q96, N99, E143, and R147, indicating that one or more, or all, of the mutant residues are important for the core epitope of hu23.H5L5. Mutant KW27-20 contains amino acid substitutions at residues R138, M139, and E142, indicating that one or more, or all, of the mutant residues are also important for the core epitope of hu23.H5L5.

[0333]

[0362] As shown in Figure 22, chimeric antibody c34 lost binding to mutant KW27-16 but not to any of the other mutants. Mutant KW27-16 contains amino acid substitutions at residues K5, E100, and D107, indicating that one or more, or all, of the mutant residues are important for the core epitope of c34.

[0334]

[0363] As shown in Figure 22, chimeric antibody c35 lost binding to mutant KW27-2 but not to any of the other mutants. Mutant KW27-2 contains amino acid substitutions at residues V81, E82, R111, and V115, indicating that one or more, or all, of the mutant residues are important for the core epitope of c35.

[0335]

[0364] As shown in Figure 22, the reference antibody T895 lost binding to mutant KW27-20 but not to any of the other mutants. Mutant KW27-20 contains amino acid substitutions at residues R138, M139, and E142, indicating that one or more, or all, of the mutant residues are important for the core epitope of T895.

[0336]

[0365] As shown in Figure 22, the reference antibody I394 lost binding to mutants KW27-6 and KW27-20, but not to the other mutants. Mutant KW27-6 contains amino acid substitutions at residues Q96, N99, E143, and R147, indicating that one or more, or all, of the mutant residues are important for the core epitope of I394. Mutant KW27-20 contains amino acid substitutions at residues R138, M139, and E142, indicating that one or more, or all, of the mutant residues are important for the core epitope of I394.

[0337]

[0366] As shown in Figure 22, the reference antibody 9-8B lost binding to mutant KW27-6 but not to any of the other mutants. Mutant KW27-6 contains amino acid substitutions at residues Q96, N99, E143, and R147, indicating that one or more, or all, of the mutant residues are important for the core epitope of 9-8B. Aspects of the invention [Aspect 1] An antibody or antigen-binding fragment thereof capable of specifically binding to human CD39, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 and / or a light chain variable region comprising LCDR1, LCDR2, and LCDR3; a) the HCDR1 comprises an amino acid sequence selected from the group consisting of NYGMN (SEQ ID NO: 1), KYWMN (SEQ ID NO: 2), NYWMN (SEQ ID NO: 3), DTFLH (SEQ ID NO: 4), DYNMY (SEQ ID NO: 5), and DTYVH (SEQ ID NO: 6); b) the HCDR2 is selected from the group consisting of LINTYTGEPTYADDFKD (SEQ ID NO: 7), EIRLKSNKYGTHYAESVKG (SEQ ID NO: 8), QIRLNPDNYATHX1AESVKG (SEQ ID NO: 9), X 58 IDPAX 59 X 60 NIKYDPKFQG (SEQ ID NO: 151), FIDPYNGYTSYNQKFKG (SEQ ID NO: 11), RIDPAIDNSKYDPKFQG (SEQ ID NO: 12), c) the HCDR3 comprises an amino acid sequence selected from the group consisting of KGIYYDYVWFFDV (SEQ ID NO: 13), QLDLYWFFDV (SEQ ID NO: 14), HGX2RGFAY (SEQ ID NO: 15), SPYYYGSGYRIFDV (SEQ ID NO: 16), IYGYDDAYYFDY (SEQ ID NO: 17), YYCALYDGYNVYAMDY (SEQ ID NO: 18); d) the LCDR1 comprises an amino acid sequence selected from the group consisting of KASQDINRYIA (SEQ ID NO: 19), RASQSISDYLH (SEQ ID NO: 20), KSSQSLLDSDGRTHLN (SEQ ID NO: 21), SAFSSVNYMH (SEQ ID NO: 22), SATSSVSYMH (SEQ ID NO: 23), RSSKNLLHSNGITYLY (SEQ ID NO: 24); e) the LCDR2 comprises an amino acid sequence selected from the group consisting of YTSTLLP (SEQ ID NO: 25), YASQSIS (SEQ ID NO: 26), LVSKLDS (SEQ ID NO: 27), TTSNLAS (SEQ ID NO: 28), STSNLAS (SEQ ID NO: 29), and RASTLAS (SEQ ID NO: 30); f) the LCDR3 comprises an amino acid sequence selected from the group consisting of LQYSNLLT (SEQ ID NO: 31), QNGHSLPLT (SEQ ID NO: 32), WQGTLFPWT (SEQ ID NO: 33), QQRSTYPFT (SEQ ID NO: 34), QQRITYPFT (SEQ ID NO: 35), AQLLELPHT (SEQ ID NO: 36); X1 is Y or F, X2 is S or T, and X 58 is R or K, and X 59 is N, G, S, or Q, and X 60 is G, A, or D. [Aspect 2] a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 3, and / or b) said HCDR2 comprises the amino acid sequence of SEQ ID NO: 9, and / or c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 15, and / or d) said LCDR1 comprises the amino acid sequence of SEQ ID NO: 21; and / or e) said LCDR2 comprises the amino acid sequence of SEQ ID NO: 27; and / or f) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 33; 2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein X1 and X2 are as defined in embodiment 1. [Aspect 3] The HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 38, and / or 3. The antibody or antigen-binding fragment thereof according to embodiment 2, wherein the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40 and SEQ ID NO: 41. [Aspect 4] a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, and / or b) said HCDR2 comprises the amino acid sequence of SEQ ID NO: 151; and / or c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, and / or d) said LCDR1 comprises the amino acid sequence of SEQ ID NO: 22; and / or e) said LCDR2 comprises the amino acid sequence of SEQ ID NO: 28; and / or f) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 34; X 58 , X 59 and X 60 2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein: [Aspect 5] The heavy chain variable region a) an HCDR1 comprising the sequence of SEQ ID NO: 1, an HCDR2 comprising the sequence of SEQ ID NO: 7, and an HCDR3 comprising the sequence of SEQ ID NO: 13, or b) an HCDR1 comprising the sequence of SEQ ID NO: 2, an HCDR2 comprising the sequence of SEQ ID NO: 8, and an HCDR3 comprising the sequence of SEQ ID NO: 14, or c) an HCDR1 comprising the sequence of SEQ ID NO: 3, an HCDR2 comprising the sequence of SEQ ID NO: 37, and an HCDR3 comprising the sequence of SEQ ID NO: 40, or d) an HCDR1 comprising the sequence of SEQ ID NO: 3, an HCDR2 comprising the sequence of SEQ ID NO: 38, and an HCDR3 comprising the sequence of SEQ ID NO: 41, or e) an HCDR1 comprising the sequence of SEQ ID NO: 4, an HCDR2 comprising the sequence of SEQ ID NO: 10, and an HCDR3 comprising the sequence of SEQ ID NO: 16, or f) an HCDR1 comprising the sequence of SEQ ID NO: 4, an HCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, and an HCDR3 comprising the sequence of SEQ ID NO: 16, or g) an HCDR1 comprising the sequence of SEQ ID NO: 5, an HCDR2 comprising the sequence of SEQ ID NO: 11, and an HCDR3 comprising the sequence of SEQ ID NO: 17, or h) HCDR1 comprising the sequence of SEQ ID NO: 6, HCDR2 comprising the sequence of SEQ ID NO: 12, and HCDR3 comprising the sequence of SEQ ID NO: 18 5. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, comprising: [Aspect 6] The light chain variable region is a) an LCDR1 comprising the sequence of SEQ ID NO: 19, an LCDR2 comprising the sequence of SEQ ID NO: 25, and an LCDR3 comprising the sequence of SEQ ID NO: 31, or b) an LCDR1 comprising the sequence of SEQ ID NO: 20, an LCDR2 comprising the sequence of SEQ ID NO: 26, and an LCDR3 comprising the sequence of SEQ ID NO: 32, or c) an LCDR1 comprising the sequence of SEQ ID NO: 21, an LCDR2 comprising the sequence of SEQ ID NO: 27, and an LCDR3 comprising the sequence of SEQ ID NO: 33, or d) LCDR1 comprising the sequence of SEQ ID NO: 22, LCDR2 comprising the sequence of SEQ ID NO: 28, and LCDR3 comprising the sequence of SEQ ID NO: 34, or e) an LCDR1 comprising the sequence of SEQ ID NO: 23, an LCDR2 comprising the sequence of SEQ ID NO: 29, and an LCDR3 comprising the sequence of SEQ ID NO: 35, or f) LCDR1 comprising the sequence of SEQ ID NO: 24, LCDR2 comprising the sequence of SEQ ID NO: 30, and LCDR3 comprising the sequence of SEQ ID NO: 36 6. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, comprising: [Aspect 7] a) the HCDR1 comprises the sequence of SEQ ID NO: 1, the HCDR2 comprises the sequence of SEQ ID NO: 7, the HCDR3 comprises the sequence of SEQ ID NO: 13, the LCDR1 comprises the sequence of SEQ ID NO: 19, the LCDR2 comprises the sequence of SEQ ID NO: 25, and the LCDR3 comprises the sequence of SEQ ID NO: 31, or b) the HCDR1 comprises the sequence of SEQ ID NO: 2, the HCDR2 comprises the sequence of SEQ ID NO: 8, the HCDR3 comprises the sequence of SEQ ID NO: 14, the LCDR1 comprises the sequence of SEQ ID NO: 20, the LCDR2 comprises the sequence of SEQ ID NO: 26, and the LCDR3 comprises the sequence of SEQ ID NO: 32, or c) the HCDR1 comprises the sequence of SEQ ID NO: 3, the HCDR2 comprises the sequence of SEQ ID NO: 37, the HCDR3 comprises the sequence of SEQ ID NO: 40, the LCDR1 comprises the sequence of SEQ ID NO: 21, the LCDR2 comprises the sequence of SEQ ID NO: 27, and the LCDR3 comprises the sequence of SEQ ID NO: 33, or d) the HCDR1 comprises the sequence of SEQ ID NO: 3, the HCDR2 comprises the sequence of SEQ ID NO: 38, the HCDR3 comprises the sequence of SEQ ID NO: 41, the LCDR1 comprises the sequence of SEQ ID NO: 21, the LCDR2 comprises the sequence of SEQ ID NO: 27, and the LCDR3 comprises the sequence of SEQ ID NO: 33, or e) the HCDR1 comprises the sequence of SEQ ID NO: 4, the HCDR2 comprises the sequence of SEQ ID NO: 10, the HCDR3 comprises the sequence of SEQ ID NO: 16, the LCDR1 comprises the sequence of SEQ ID NO: 22, the LCDR2 comprises the sequence of SEQ ID NO: 28, and the LCDR3 comprises the sequence of SEQ ID NO: 34, or f) the HCDR1 comprises the sequence of SEQ ID NO: 4, the HCDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, the HCDR3 comprises the sequence of SEQ ID NO: 16, the LCDR1 comprises the sequence of SEQ ID NO: 22, the LCDR2 comprises the sequence of SEQ ID NO: 28, and the LCDR3 comprises the sequence of SEQ ID NO: 34, or g) the HCDR1 comprises the sequence of SEQ ID NO: 5, the HCDR2 comprises the sequence of SEQ ID NO: 11, the HCDR3 comprises the sequence of SEQ ID NO: 17, the LCDR1 comprises the sequence of SEQ ID NO: 23, the LCDR2 comprises the sequence of SEQ ID NO: 29, and the LCDR3 comprises the sequence of SEQ ID NO: 35, or h) An antibody or antigen-binding fragment thereof according to any one of aspects 1 to 6, wherein the HCDR1 comprises the sequence of SEQ ID NO: 6, the HCDR2 comprises the sequence of SEQ ID NO: 12, the HCDR3 comprises the sequence of SEQ ID NO: 18, the LCDR1 comprises the sequence of SEQ ID NO: 24, the LCDR2 comprises the sequence of SEQ ID NO: 30, and the LCDR3 comprises the sequence of SEQ ID NO: 36. [Embodiment 8] Further comprising one or more of heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of light chains LFR1, LFR2, LFR3, and LFR4; a) The HFR1 is X 19 VQLVX 20 SGX 21 X 22 X 23 X 24 KPGX 25 SX 26 X 27 X 28 SCX 29 ASGX 30 X 31 X 32 X 33(SEQ ID NO: 76) or a homologous sequence having at least 80% sequence identity thereto; b) The HFR2 is WVX 34 QX 35 PGX 36 X 37 LEWX 38 X 39 (SEQ ID NO: 77) or a homologous sequence having at least 80% sequence identity thereto; c) the HFR3 sequence is X 40 X 41 TX 42 X 43 X 44 DX 45 SX 46 X 47 TX 48 YX 49 X 50 X 51 X 52 SLX 53 X 54 EDTAVYYCX 55 X 56 (SEQ ID NO: 78) or a homologous sequence having at least 80% sequence identity thereto; d) The HFR4 is WGQGTX 57 VTVSS (SEQ ID NO: 126) or a homologous sequence having at least 80% sequence identity thereto; e) the LFR1 comprises the sequence X3IVX4TQSPATLX5X6SPGERX7TX8X9C (SEQ ID NO: 80) or a homologous sequence having at least 80% sequence identity thereto; f) The LFR2 is WYQQKPGQX 10 PX 11 LLIY (SEQ ID NO: 81) or a homologous sequence having at least 80% sequence identity thereto; g) The LFR3 is GX 12 PX 13 RFSGSGSGTX 14 X 15 TLTISSX 16 EPEDFAVYX 17 C (SEQ ID NO: 82), or a homologous sequence having at least 80% sequence identity thereto; h) The LFR4 is FGX 18 GTKLEIK (SEQ ID NO: 152) or a homologous sequence thereof having at least 80% sequence identity; X3 is E or Q, X4 is L or M, X5 is S or T, X6 is L, V, or A, X7 is A or V, X8 is L or I, X9 is S or T, and X 10 is A or S and X 11 is R or K, and X 12 is I or V, and X 13 is A or T and X 14 is D or S, and X 15 is F or Y and X 16 is L, M, or V, and X 17 is Y or F and X 18 is G or Q and X 19 is Q or E and X 20 is E or Q and X 21 is G or A and X 22 is G or E and X 23 is L or V, and X 24 is V or K, and X 25 is G or A and X 26 is L, M, or V, and X 27 is R or K, and X 28 is V or L, and X 29 is A or K and X 30 is F or Y and X 31 is N or T and X 32 is F or L, and X 33 is S or K, and X 34 is R or K, and X 35 is A or S and X 36 is K or Q and X 37 is R or G and X 38 is M, I, or V, and X 39 is G or A and X 40 is R or K, and X 41 is V, A, or F and X 42 is I or L, and X 43is S or T and X 44 is R or A and X 45 is D or T and X 46 is K, A, or S, and X 47 is S or N and X 48 is L, V, or A, and X 49 is M or L, and X 50 is Q or E and X 51 is M or L, and X 52 is S, I, or N and X 53 is R or K, and X 54 is S or T and X 55 is A or T and X 56 is R, N, or T and X 57 is T or L. [Aspect 9] a) The HFR1 is EVQLVESGGGLVKPGGSX 61 RLSCAASGFTFS (SEQ ID NO: 154) or a homologous sequence having at least 80% sequence identity thereto; b) The HFR2 is WVRQX 62 PGKGLEWVX 63 (SEQ ID NO: 155) or a homologous sequence having at least 80% sequence identity thereto; c) the HFR3 is RFTISRDDSKNTX 64 YLQMNSLKTEDTAVYYCTT (SEQ ID NO: 156) or a homologous sequence having at least 80% sequence identity thereto; d) the HFR4 comprises the sequence WGQGTTVTVSS (SEQ ID NO: 79) or a homologous sequence having at least 80% sequence identity thereto; e) the LFR1 is EIVX 65 TQSPATLSX 66 SPGERX 67 TLSC (SEQ ID NO: 157) or a homologous sequence having at least 80% sequence identity thereto; f) The LFR2 is WYQQKPGQX 68PRLLIY (SEQ ID NO: 158) or a homologous sequence having at least 80% sequence identity thereto; g) the LFR3 is GIPARFSGSGSGTDFTLTISSX 69 EPEDFAVYX 70 C (SEQ ID NO: 159), or a homologous sequence having at least 80% sequence identity thereto; h) the LFR4 comprises the sequence FGGGTKLEIK (SEQ ID NO: 153) or a homologous sequence having at least 80% sequence identity thereto; X 61 is L or M, and X 62 is A or S and X 63 is G or A and X 64 is L or V, and X 65 is L or M, and X 66 is L or V, and X 67 is A or V and X 68 is A or S and X 69 is L or V, and X 70 is Y or F. [Aspect 10] a) The HFR1 is X 71 VQLVQSGAEVKKPGASVKX 72 SCKASGYX 73 LK (SEQ ID NO: 160) or a homologous sequence having at least 80% sequence identity thereto; b) The HFR2 is WVX 74 QAPGQX 75 LEWX 76 G (SEQ ID NO: 161), or a homologous sequence having at least 80% sequence identity thereto; c) The HFR3 is X 77 X 78 TX 79 TX 80 DTSX 81 X 82 TAYX 83 ELX 84 SLRSEDTAVYYCAX 85 (SEQ ID NO: 149) or a homologous sequence having at least 80% sequence identity thereto; d) The HFR4 is WGQGTX 57 VTVSS (SEQ ID NO: 126) or a homologous sequence having at least 80% sequence identity thereto; e) The LFR1 is X 86 IVLTQSPATLX 87 X 88 SPGERX 89 TX 90 X 91 C (SEQ ID NO: 150), or a homologous sequence having at least 80% sequence identity thereto; f) said LFR2 is WYQQKPGQX 10 PX 11 LLIY (SEQ ID NO: 81) or a homologous sequence having at least 80% sequence identity thereto; g) The LFR3 is GX 92 PX 93 RFSGSGSGTX 94 X 95 TLTISSX 96 EPEDFAVYYC (SEQ ID NO: 148) or a homologous sequence having at least 80% sequence identity thereto; h) the LFR4 comprises the sequence FGQGTKLEIK (SEQ ID NO: 83) or a homologous sequence having at least 80% sequence identity thereto; X 10 , X 11 , and X 57 is as defined in embodiment 8, and X 71 is Q or E and X 72 is V or L, and X 73 is N or T and X 74 is R or K, and X 75 is R or G and X 76 is M or I, and X 77 is R or K, and X 78 is V or A and X 79 is I or L, and X 80 is R or A and X 81 is A or S and X 82 is S or N and X 83 is M or L, and X 84is S or I, and X 85 is R or N, and X 86 is E or Q and X 87 is S or T and X 88 is L or A, and X 89 is A or V and X 90 is L or I, and X 91 is S or T and X 92 is I or X 93 is A or T and X 94 is D or S, and X 95 is F or Y and X 96 is L or M. [Embodiment 11] The HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 84 to 86, 115, 119 to 120, and 131; the HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 87 to 90, and 121 to 123; the HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 91 to 97, 116 to 117, and 124 to 125; the HFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 79 and 118; the LFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 98 to 103, and 127 to 129; said LFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 104, 105, and 130; the LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 106 to 110, and 132 to 133; 9. The antibody or antigen-binding fragment thereof according to embodiment 7 or 8, wherein the LFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 83 and 153. [Embodiment 12] A heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 60, 62, 64, 66, 140, 141, 142, 146, 147, and 39, and homologous sequences thereof that have at least 80% sequence identity and retain specific binding affinity to human CD39; and A light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 61, 63, 65, 67, 143, 144, 145, 111, 112, 63, and homologous sequences thereof that have at least 80% sequence identity and retain specific binding affinity to human CD39. 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, comprising: [Embodiment 13] A heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 68, 70, 72, and 74, and homologous sequences thereof that have at least 80% sequence identity and retain specific binding affinity to human CD39; and A light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, and 75, and homologous sequences thereof that have at least 80% sequence identity and retain specific binding affinity to human CD39. 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, comprising: [Aspect 14] a) a heavy chain variable region comprising the sequence of SEQ ID NO: 42 and a light chain variable region comprising the sequence of SEQ ID NO: 51, or b) a heavy chain variable region comprising the sequence of SEQ ID NO: 43 and a light chain variable region comprising the sequence of SEQ ID NO: 52, or c) a heavy chain variable region comprising the sequence of SEQ ID NO: 44 and a light chain variable region comprising the sequence of SEQ ID NO: 53, or d) a heavy chain variable region comprising the sequence of SEQ ID NO: 45 and a light chain variable region comprising the sequence of SEQ ID NO: 54, or e) a heavy chain variable region comprising the sequence of SEQ ID NO: 47 and a light chain variable region comprising the sequence of SEQ ID NO: 56, or f) a heavy chain variable region comprising the sequence of SEQ ID NO: 49 and a light chain variable region comprising the sequence of SEQ ID NO: 58, or g) a heavy chain variable region comprising the sequence of SEQ ID NO: 50 and a light chain variable region comprising the sequence of SEQ ID NO: 59, or h) a heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 63, or i) a heavy chain variable region comprising the sequence of SEQ ID NO: 62 and a light chain variable region comprising the sequence of SEQ ID NO: 63, or j) a heavy chain variable region comprising the sequence of SEQ ID NO: 64 and a light chain variable region comprising the sequence of SEQ ID NO: 63, or k) a heavy chain variable region comprising the sequence of SEQ ID NO: 66 and a light chain variable region comprising the sequence of SEQ ID NO: 63, or l) a heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 65, or m) a heavy chain variable region comprising the sequence of SEQ ID NO: 62 and a light chain variable region comprising the sequence of SEQ ID NO: 65, or n) a heavy chain variable region comprising the sequence of SEQ ID NO: 64 and a light chain variable region comprising the sequence of SEQ ID NO: 65, or o) a heavy chain variable region comprising the sequence of SEQ ID NO: 66 and a light chain variable region comprising the sequence of SEQ ID NO: 65, or p) a heavy chain variable region comprising the sequence of SEQ ID NO: 60 and a light chain variable region comprising the sequence of SEQ ID NO: 67, or q) a heavy chain variable region comprising the sequence of SEQ ID NO: 62 and a light chain variable region comprising the sequence of SEQ ID NO: 67, or r) a heavy chain variable region comprising the sequence of SEQ ID NO: 64 and a light chain variable region comprising the sequence of SEQ ID NO: 67, or s) a heavy chain variable region comprising the sequence of SEQ ID NO: 66 and a light chain variable region comprising the sequence of SEQ ID NO: 67, or t) a heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 61, or u) a heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 61, or v) a heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 61, or w) a heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 143, or x) a heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 143, or y) a heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 143, or z) a heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 144, or aa) a heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 144, or bb) a heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 144, or cc) a heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 145, or dd) a heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 145, or ee) a heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 145, or ff) a heavy chain variable region comprising the sequence of SEQ ID NO: 146 and a light chain variable region comprising the sequence of SEQ ID NO: 111, or gg) a heavy chain variable region comprising the sequence of SEQ ID NO: 146 and a light chain variable region comprising the sequence of SEQ ID NO: 112, or hh) a heavy chain variable region comprising the sequence of SEQ ID NO: 147 and a light chain variable region comprising the sequence of SEQ ID NO: 111, or ii) a heavy chain variable region comprising the sequence of SEQ ID NO: 39 and a light chain variable region comprising the sequence of SEQ ID NO: 63, or jj) a heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 69, or kk) a heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 69, or ll) a heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 69, or mm) a heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 69, or nn) a heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 71, or oo) a heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 71, or pp) a heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 71, or qq) a heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 71, or rr) a heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 73, or ss) a heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 73, or tt) a heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 73, or uu) a heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 73, or vv) a heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 75, or ww) a heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 75, or xx) a heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 75, or yy) a heavy chain variable region comprising the sequence of SEQ ID NO: 74, and a light chain variable region comprising the sequence of SEQ ID NO: 75 14. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 13, comprising: [Aspect 15] An antibody or antigen-binding fragment thereof according to any one of aspects 1 to 14, which further comprises substitution or modification of one or more amino acid residues and yet retains specific binding affinity for human CD39. [Aspect 16] An antibody or antigen-binding fragment thereof according to Aspect 15, wherein at least one of the substitutions or modifications is in one or more of the CDR sequences of the heavy chain variable region or light chain variable region and / or in one or more of the non-CDR sequences. [Embodiment 17] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 16, further comprising an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG. [Aspect 18] An antibody or antigen-binding fragment thereof according to Aspect 17, wherein the Fc region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM. [Aspect 19] An antibody or antigen-binding fragment thereof according to Aspect 18, wherein the Fc region derived from human IgG1 comprises the mutations L234A and L235A. [Aspect 20] An antibody or antigen-binding fragment thereof according to any one of aspects 1 to 19, which is humanized. [Aspect 21] An antibody or antigen-binding fragment thereof according to any one of aspects 1 to 20, which is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotype antibody, or a fusion protein. [Aspect 22] An antibody or antigen-binding fragment thereof according to any one of aspects 1 to 21, which is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single-domain antibody, nanobody, domain antibody, or bivalent domain antibody. [Aspect 23] 10 as measured by FACS assay -8 EC below M 50 23. The antibody or antigen-binding fragment thereof according to any of embodiments 1 to 22, which is capable of specifically binding to human CD39 at [Embodiment 24] a) specifically binds to human CD39 but does not specifically bind to mouse CD39 as measured by a FACS assay; b) 10 as measured by FACS assay -8 EC below M 50 specifically binds to cynomolgus monkey CD39. c) 10 as measured by Biacore assay -7 M or less (for example, 5 × 10 -8 M or less, 3×10 -8 M or less, 2×10 -8 M or less, 1×10 -8M or less, or 8 x 10 -9 M or less) K D specifically binds to human CD39 at d) 10 as measured by Octet assay -8 M or less (for example, 8 × 10 -9 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 1×10 -9 M or less, or 9 x 10 -10 M or less) K D specifically binds to human CD39 at e) an IC of 50 nM or less (e.g., 1 nM or less, 5 nM or less, 10 nM or less, or 30 nM or less) as measured by an ATPase activity assay 50 inhibits ATPase activity in CD39-expressing cells, f) capable of enhancing ATP-mediated monocyte activation at a concentration of 10 nM or less (e.g., 5 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, or 0.2 nM or less) as measured by analysis of CD80, CD86, and / or CD40 expression by FACS assay; g) capable of enhancing ATP-mediated T cell activation in PBMCs at concentrations of 25 nM or less as measured by IL-2 secretion, IFN-γ secretion, and CD4+ or CD8+ T cell proliferation; h) is capable of enhancing ATP-mediated DC activation at a concentration of 25 nM or less (or 10 nM or less, or 5 nM or less, or 1 nM or less, or 0.5 nM or less), as measured by analysis of CD83 expression by FACS assay, or by the ability of activated dendritic cells (DCs) to promote T cell proliferation, or by the ability of activated DCs to promote IFN-γ production in a mixed lymphocyte reaction (MLR) assay; i) CD4 induced by adenosine (hydrolyzed from ATP) at concentrations of 1 nM or less (e.g., 0.1 nM or less, 0.01 nM or less) as measured by FACS assay + Can block the inhibition of T cell proliferation, j) capable of inhibiting tumor growth in a mammalian NK cell or macrophage cell dependent manner; k) T cell proliferation, CD25 + Human CD8 inhibited by eATP as measured by cell and viable cell populations + can reverse T cell proliferation, and l) capable of enhancing the release of IL1β from human macrophages induced by LPS stimulation at a concentration of 50 nM or less (or 12.5 nM or less, or 3.13 nM or less, or 0.78 nM or less, or 0.2 nM or less, or 0.049 nM or less, or 0.012 nM or less, or 0.003 nM or less, or 0.0008 nM or less) as measured by ELISA assay; 24. The antibody or antigen-binding fragment thereof according to any of embodiments 1 to 23, wherein the antibody or antigen-binding fragment has one or more properties selected from the group consisting of: [Aspect 25] An anti-CD39 antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof described in any one of aspects 1 to 24 for binding to human CD39. [Aspect 26] An antibody or antigen-binding fragment thereof according to aspect 25, which competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 43 and a light chain variable region comprising the sequence of SEQ ID NO: 52 for binding to human CD39. [Aspect 27] An antibody or antigen-binding fragment thereof according to aspect 25, which competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 44 and a light chain variable region comprising the sequence of SEQ ID NO: 53 for binding to human CD39, or competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 45 and a light chain variable region comprising the sequence of SEQ ID NO: 54. [Aspect 28] An antibody or antigen-binding fragment thereof according to aspect 25, which competes with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 47 and a light chain variable region comprising the sequence of SEQ ID NO: 56 for binding to human CD39. [Aspect 29] An anti-CD39 antibody or antigen-binding fragment thereof that specifically binds to an epitope of CD39, wherein the epitope comprises one or more residues selected from the group consisting of Q96, N99, E143, R147, R138, M139, E142, K5, E100, D107, V81, E82, R111, and V115. [Aspect 30] An anti-CD39 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 29, wherein the epitope comprises one or more residues selected from the group consisting of Q96, N99, E143, and R147. [Aspect 31] An anti-CD39 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 29, wherein the epitope comprises one or more residues selected from the group consisting of R138, M139, and E142. [Aspect 32] An anti-CD39 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 29, wherein the epitope comprises one or more residues selected from the group consisting of K5, E100, and D107. [Aspect 33] An anti-CD39 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 29, wherein the epitope comprises one or more residues selected from the group consisting of V81, E82, R111, and V115. [Aspect 34] An anti-CD39 antibody or antigen-binding fragment thereof according to any one of aspects 1 to 33, wherein the CD39 is human CD39 comprising the amino acid sequence of SEQ ID NO: 162. [Embodiment 35] The antibody is not any of antibody 9-8B, antibody T895, and antibody I394; antibody 9-8B comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 46 and a light chain variable region comprising the sequence of SEQ ID NO: 48; antibody T895 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 55 and a light chain variable region comprising the sequence of SEQ ID NO: 57; 35. The antibody or antigen-binding fragment thereof according to any of embodiments 28 to 34, wherein the antibody I394 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 113 and a light chain variable region comprising the sequence of SEQ ID NO: 114. [Embodiment 36] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 35, which is bispecific. [Aspect 37] An antibody or antigen-binding fragment thereof according to aspect 36, which is capable of specifically binding to a second antigen other than CD39 or a second epitope on CD39. [Embodiment 38] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 37, linked to one or more conjugate moieties. [Aspect 39] The antibody or antigen-binding fragment thereof described in Aspect 38, wherein the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, a purification moiety, or other anti-cancer drug. [Aspect 40] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of aspects 1 to 39 and one or more pharmaceutically acceptable carriers. [Aspect 41] An isolated polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 39. [Aspect 42] A vector comprising the isolated polynucleotide described in Aspect 41. [Aspect 43] A host cell comprising the vector described in Aspect 42. [Aspect 44] A kit comprising an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 39, and / or a pharmaceutical composition according to aspect 40, and a second therapeutic agent. [Aspect 45] A method for expressing an antibody or antigen-binding fragment thereof described in any one of aspects 1 to 39, comprising the step of culturing a host cell described in aspect 43 under conditions in which the vector described in aspect 42 is expressed. [Aspect 46] A method for treating, preventing, or alleviating a CD39-related disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of aspects 1 to 39, and / or a pharmaceutical composition described in aspect 40. [Aspect 47] A method for treating, preventing, or alleviating a disease treatable by reducing the ATPase activity of CD39 in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of aspects 1 to 39, and / or a pharmaceutical composition described in aspect 40. [Aspect 48] A method for treating, preventing, or alleviating a disease associated with adenosine-mediated inhibition of T, monocyte, macrophage, DC, APC, NK, and / or B cell activity in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of aspects 1 to 39, and / or a pharmaceutical composition described in aspect 40. [Aspect 49] A method according to any one of aspects 46 to 48, wherein the disease, disorder, or condition is cancer. [Aspect 50] The cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal tract cancer, skin cancer, or prostate cancer. 50. The method of embodiment 49, wherein the cancer is selected from the group consisting of thyroid cancer, pituitary cancer, vaginal cancer, thyroid cancer, throat cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T- or B-cell lymphoma, GI organ stromal tumor, soft tissue tumor, hepatocellular carcinoma, and adenocarcinoma. [Aspect 51] The method described in aspect 49, wherein the cancer is leukemia, lymphoma, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, or breast cancer. [Aspect 52] A method described in any one of aspects 46 to 51, wherein the subject has been identified as having cancer cells or tumor-infiltrating immune cells or immunosuppressive cells that express CD39, optionally at a level significantly higher than the level normally found in non-cancer cells or non-immunosuppressive cells. [Aspect 53] A method according to any one of aspects 46 to 48, wherein the disease, disorder, or condition is an autoimmune disease or infection. [Aspect 54] The method of aspect 53, wherein the autoimmune disease is immune thrombocytopenia, systemic sclerosis, sclerosis, adult respiratory distress syndrome, eczema, asthma, Sjogren's syndrome, Addison's disease, giant cell arteritis, immune complex nephritis, immune thrombocytopenic purpura, autoimmune thrombocytopenia, celiac disease, psoriasis, dermatitis, colitis, or systemic lupus erythematosus. [Aspect 55] The method of aspect 53, wherein the infection is HIV infection, HBV infection, HCV infection, inflammatory bowel disease, or Crohn's disease. [Aspect 56] A method according to any one of aspects 46 to 55, wherein the subject is a human. [Aspect 57] The method of any one of aspects 46 to 56, wherein the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration. [Aspect 58] The method of any one of aspects 46 to 57, further comprising administering a therapeutically effective amount of a second therapeutic agent. [Aspect 59] The method described in Aspect 58, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anticancer drugs, radiation therapy agents, immunotherapy agents, antiangiogenic agents, targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines. [Aspect 60] A method for regulating the activity of CD39 in CD39-positive cells, comprising the step of exposing the CD39-positive cells to an antibody or antigen-binding fragment thereof described in any one of aspects 1 to 39 and / or a pharmaceutical composition described in aspect 40. [Aspect 61] The method described in aspect 60, wherein the cell is an immune cell. [Aspect 62] A method for detecting the presence or amount of CD39 in a sample, comprising the steps of contacting the sample with an antibody or antigen-binding fragment thereof described in any one of aspects 1 to 39 and / or a pharmaceutical composition described in aspect 40, and determining the presence or amount of CD39 in the sample. [Aspect 63] A method for diagnosing a CD39-related disease, disorder, or condition in a subject, comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof described in any of aspects 1 to 39 and / or a pharmaceutical composition described in aspect 40; b) determining the presence or amount of CD39 in the sample; and c) correlating the presence or amount of CD39 with the presence or status of the CD39-related disease, disorder, or condition in the subject. [Aspect 64] Use of an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 39 and / or a pharmaceutical composition according to aspect 40 in the manufacture of a medicament for treating, preventing, or alleviating a CD39-related disease, disorder, or condition in a subject. [Aspect 65] Use of an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 39 and / or a pharmaceutical composition according to aspect 40 in the manufacture of a diagnostic reagent for diagnosing a CD39-related disease, disorder, or condition in a subject. [Aspect 66] A kit useful for detecting CD39, comprising an antibody or antigen-binding fragment thereof according to any one of aspects 1 to 39 and / or a pharmaceutical composition according to aspect 40.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to human CD39, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3; a) the HCDR1 comprises the sequence of SEQ ID NO: 1, the HCDR2 comprises the sequence of SEQ ID NO: 7, the HCDR3 comprises the sequence of SEQ ID NO: 13, the LCDR1 comprises the sequence of SEQ ID NO: 19, the LCDR2 comprises the sequence of SEQ ID NO: 25, and the LCDR3 comprises the sequence of SEQ ID NO: 31; or b) the HCDR1 comprises the sequence of SEQ ID NO: 2, the HCDR2 comprises the sequence of SEQ ID NO: 8, the HCDR3 comprises the sequence of SEQ ID NO: 14, the LCDR1 comprises the sequence of SEQ ID NO: 20, the LCDR2 comprises the sequence of SEQ ID NO: 26, and the LCDR3 comprises the sequence of SEQ ID NO: 32; or c) the HCDR1 comprises the sequence of SEQ ID NO: 3, the HCDR2 comprises the sequence of SEQ ID NO: 37, the HCDR3 comprises the sequence of SEQ ID NO: 40, the LCDR1 comprises the sequence of SEQ ID NO: 21, the LCDR2 comprises the sequence of SEQ ID NO: 27, and the LCDR3 comprises the sequence of SEQ ID NO: 33; or d) the HCDR1 comprises the sequence of SEQ ID NO: 3, the HCDR2 comprises the sequence of SEQ ID NO: 38, the HCDR3 comprises the sequence of SEQ ID NO: 41, the LCDR1 comprises the sequence of SEQ ID NO: 21, the LCDR2 comprises the sequence of SEQ ID NO: 27, and the LCDR3 comprises the sequence of SEQ ID NO: 33; or f) the HCDR1 comprises the sequence of SEQ ID NO: 4, the HCDR2 comprises the sequence of SEQ ID NO: 10, the HCDR3 comprises the sequence of SEQ ID NO: 16, the LCDR1 comprises the sequence of SEQ ID NO: 22, the LCDR2 comprises the sequence of SEQ ID NO: 28, and the LCDR3 comprises the sequence of SEQ ID NO: 34; or g) the HCDR1 comprises the sequence of SEQ ID NO: 4, the HCDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, the HCDR3 comprises the sequence of SEQ ID NO: 16, the LCDR1 comprises the sequence of SEQ ID NO: 22, the LCDR2 comprises the sequence of SEQ ID NO: 28, and the LCDR3 comprises the sequence of SEQ ID NO: 34; or h) the HCDR1 comprises the sequence of SEQ ID NO:5, the HCDR2 comprises the sequence of SEQ ID NO:11, the HCDR3 comprises the sequence of SEQ ID NO:17, the LCDR1 comprises the sequence of SEQ ID NO:23, the LCDR2 comprises the sequence of SEQ ID NO:29, and the LCDR3 comprises the sequence of SEQ ID NO:35; or i) An antibody or antigen-binding fragment thereof, wherein the HCDR1 comprises the sequence of SEQ ID NO: 6, the HCDR2 comprises the sequence of SEQ ID NO: 12, the HCDR3 comprises the sequence of SEQ ID NO: 18, the LCDR1 comprises the sequence of SEQ ID NO: 24, the LCDR2 comprises the sequence of SEQ ID NO: 30, and the LCDR3 comprises the sequence of SEQ ID NO:

36.

2. further comprising one or more of heavy chains HFR1, HFR2, HFR3, and HFR4, and / or one or more of light chains LFR1, LFR2, LFR3, and LFR4; a) the HFR1 is X １９ VQLVX ２０ SGX ２１ X ２２ X ２３ X ２４ KPGX ２５ SX ２６ X ２７ X ２８ SCX ２９ ASGX ３０ X ３１ X ３２ X ３３ (SEQ ID NO: 76), b) the HFR2 is WVX ３４ QX ３５ PGX ３６ X ３７ LEWX ３８ X ３９ (SEQ ID NO: 77), c) the HFR3 sequence is X ４０ X ４１ TX ４２ X ４３ X ４４ DX ４５ SX ４６ X ４７ TX ４８ YX ４９ X ５０ X ５１ X ５２ SLX ５３ X ５４ EDTAVYYCX ５５ X ５６ (SEQ ID NO: 78), d) The HFR4 is WGQGTX ５７ VTVSS (SEQ ID NO: 126), e) the LFR1 is X ３ IVX ４ TQSPATLX ５ X ６ SPGERX ７ TX ８ X ９ C (SEQ ID NO: 80), f) The LFR2 is WYQQKPGQX １０ PX １１ LLIY (SEQ ID NO: 81), g) The LFR3 is GX １２ PX １３ RFSGSGSGTX １４ X １５ TLTISSX １６ EPEDFAVYX １７ C (SEQ ID NO: 82), h) The LFR4 is FGX １８ GTKLEIK (SEQ ID NO: 152), X ３ is E or Q, and X ４ is L or M, and X ５ is S or T, and X ６ is L, V, or A, and X ７ is A or V, and X ８ is L or I, and X ９ is S or T, and X １０ is A or S, and X １１ is R or K, and X １２ is I or V, and X １３ is A or T, and X １４ is D or S, and X １５ is F or Y, and X １６ is L, M, or V, and X １７ is Y or F, and X １８ is G or Q, and X １９ is Q or E, and X ２０ is E or Q, and X ２１ is G or A, and X ２２ is G or E, and X ２３ is L or V, and X ２４ is V or K, and X ２５ is G or A, and X ２６ is L, M, or V, and X ２７ is R or K, and X ２８ is V or L, and X ２９ is A or K, and X ３０ is F or Y, and X ３１ is N or T, and X ３２ is F or L, and X ３３ is S or K, and X ３４ is R or K, and X ３５ is A or S, and X ３６ is K or Q, and X ３７ is R or G, and X ３８ is M, I, or V, and X ３９ is G or A, and X ４０ is R or K, and X ４１ is V, A, or F, and X ４２ is I or L, and X ４３ is S or T, and X ４４ is R or A, and X ４５ is D or T, and X ４６ is K, A, or S, and X ４７ is S or N, and X ４８ is L, V, or A, and X ４９ is M or L, and X ５０ is Q or E, and X ５１ is M or L, and X ５２ is S, I, or N, and X ５３ is R or K, and X ５４ is S or T, and X ５５ is A or T, and X ５６ is R, N, or T, and X ５７ The antibody or antigen-binding fragment thereof according to claim 1, wherein is T or L.

3. a) The HFR1 is EVQLVESGGGLVKPGGSX ６１ comprising the sequence RLSCAASGFTFS (SEQ ID NO: 154), b) the HFR2 is WVRQX ６２ PGKGLEWVX ６３ (SEQ ID NO: 155), c) the HFR3 is RFTISRDDSKNTX ６４ YLQMNSLKTEDTAVYYCTT (SEQ ID NO: 156), d) said HFR4 comprises the sequence WGQGTTVTVSS (SEQ ID NO: 79); e) the LFR1 is EIVX ６５ TQSPATLSX ６６ SPGERX ６７ TLSC (SEQ ID NO: 157), f) The LFR2 is WYQQKPGQX ６８ comprising the sequence of PRLLIY (SEQ ID NO: 158), g) the LFR3 is GIPARFSGSGSGTDFTLTISSX ６９ EPEDFAVYX ７０ C (SEQ ID NO: 159), and h) said LFR4 comprises the sequence FGGGTKLEIK (SEQ ID NO: 153); X ６１ is L or M, and X ６２ is A or S, and X ６３ is G or A, and X ６４ is L or V, and X ６５ is L or M, and X ６６ is L or V, and X ６７ is A or V, and X ６８ is A or S, and X ６９ is L or V, and X ７０ The antibody or antigen-binding fragment thereof according to claim 2, wherein is Y or F.

4. a) the HFR1 is X ７１ VQLVQSGAEVKKPGASVKX ７２ SCKASGYX ７３ LK (SEQ ID NO: 160), b) the HFR2 is WVX ７４ QAPGQX ７５ LEWX ７６ G (SEQ ID NO: 161), c) said HFR3 is X ７７ X ７８ TX ７９ TX ８０ DTSX ８１ X ８２ TAYX ８３ ELX ８４ SLRSEDTAVYYCAX ８５ (SEQ ID NO: 149), d) The HFR4 is WGQGTX ５７ VTVSS (SEQ ID NO: 126), e) the LFR1 is X ８６ IVLTQSPATLX ８７ X ８８ SPGERX ８９ TX ９０ X ９１ C (SEQ ID NO: 150), f) The LFR2 is WYQQKPGQX １０ PX １１ LLIY (SEQ ID NO: 81), g) The LFR3 is GX ９２ PX ９３ RFSGSGSGTX ９４ X ９５ TLTISSX ９６ EPEDFAVYYC (SEQ ID NO: 148), and h) said LFR4 comprises the sequence FGQGTKLEIK (SEQ ID NO: 83); X １０ , X １１ , and X ５７ is as defined in claim 2, and X ７１ is Q or E, and X ７２ is V or L, and X ７３ is N or T, and X ７４ is R or K, and X ７５ is R or G, and X ７６ is M or I, and X ７７ is R or K, and X ７８ is V or A, and X ７９ is I or L, and X ８０ is R or A, and X ８１ is A or S, and X ８２ is S or N, and X ８３ is M or L, and X ８４ is S or I, and X ８５ is R or N, and X ８６ is E or Q, and X ８７ is S or T, and X ８８ is L or A, and X ８９ is A or V, and X ９０ is L or I, and X ９１ is S or T, and X ９２ is I or X ９３ is A or T, and X ９４ is D or S, and X ９５ is F or Y, and X ９６ The antibody or antigen-binding fragment thereof according to claim 2, wherein is L or M.

5. the HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 84-86, 115, 119-120, and 131; the HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 87-90, and 121-123; the HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 91-97, 116-117, and 124-125; said HFR4 comprising a sequence selected from the group consisting of SEQ ID NOs: 79 and 118; the LFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 98-103, and 127-129; said LFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 104, 105, and 130; the LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 106-110, and 132-133; The antibody or antigen-binding fragment thereof according to claim 2, wherein the LFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 83 and 153.

6. a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 60, 62, 64, 66, 140, 141, 142, 146, 147, 39; and A light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 61, 63, 65, 67, 143, 144, 145, 111, 112, and 63. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising:

7. a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 68, 70, 72, and 74; and A light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, and 75. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising:

8. a) a heavy chain variable region comprising the sequence of SEQ ID NO: 42 and a light chain variable region comprising the sequence of SEQ ID NO: 51, or b) a heavy chain variable region comprising the sequence of SEQ ID NO: 43 and a light chain variable region comprising the sequence of SEQ ID NO: 52, or c) a heavy chain variable region comprising the sequence of SEQ ID NO: 44 and a light chain variable region comprising the sequence of SEQ ID NO: 53; or d) a heavy chain variable region comprising the sequence of SEQ ID NO: 45 and a light chain variable region comprising the sequence of SEQ ID NO: 54; or e) a heavy chain variable region comprising the sequence of SEQ ID NO: 47 and a light chain variable region comprising the sequence of SEQ ID NO: 56; or f) a heavy chain variable region comprising the sequence of SEQ ID NO: 49 and a light chain variable region comprising the sequence of SEQ ID NO: 58; or g) a heavy chain variable region comprising the sequence of SEQ ID NO: 50 and a light chain variable region comprising the sequence of SEQ ID NO: 59; or h) a heavy chain variable region comprising the sequence of SEQ ID NO: 60, and a light chain variable region comprising the sequence of SEQ ID NO: 63, or i) a heavy chain variable region comprising the sequence of SEQ ID NO: 62, and a light chain variable region comprising the sequence of SEQ ID NO: 63, or j) a heavy chain variable region comprising the sequence of SEQ ID NO: 64 and a light chain variable region comprising the sequence of SEQ ID NO: 63; or k) a heavy chain variable region comprising the sequence of SEQ ID NO: 66 and a light chain variable region comprising the sequence of SEQ ID NO: 63; or l) a heavy chain variable region comprising the sequence of SEQ ID NO: 60, and a light chain variable region comprising the sequence of SEQ ID NO: 65, or m) a heavy chain variable region comprising the sequence of SEQ ID NO: 62, and a light chain variable region comprising the sequence of SEQ ID NO: 65; or n) a heavy chain variable region comprising the sequence of SEQ ID NO: 64, and a light chain variable region comprising the sequence of SEQ ID NO: 65; or o) a heavy chain variable region comprising the sequence of SEQ ID NO: 66, and a light chain variable region comprising the sequence of SEQ ID NO: 65, or p) a heavy chain variable region comprising the sequence of SEQ ID NO: 60, and a light chain variable region comprising the sequence of SEQ ID NO: 67, or q) a heavy chain variable region comprising the sequence of SEQ ID NO: 62 and a light chain variable region comprising the sequence of SEQ ID NO: 67; or r) a heavy chain variable region comprising the sequence of SEQ ID NO: 64, and a light chain variable region comprising the sequence of SEQ ID NO: 67, or s) a heavy chain variable region comprising the sequence of SEQ ID NO: 66, and a light chain variable region comprising the sequence of SEQ ID NO: 67, or t) a heavy chain variable region comprising the sequence of SEQ ID NO: 140, and a light chain variable region comprising the sequence of SEQ ID NO: 61, or u) a heavy chain variable region comprising the sequence of SEQ ID NO: 141 and a light chain variable region comprising the sequence of SEQ ID NO: 61; or v) a heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 61; or w) a heavy chain variable region comprising the sequence of SEQ ID NO: 140, and a light chain variable region comprising the sequence of SEQ ID NO: 143; or x) a heavy chain variable region comprising the sequence of SEQ ID NO: 141, and a light chain variable region comprising the sequence of SEQ ID NO: 143, or y) a heavy chain variable region comprising the sequence of SEQ ID NO: 142, and a light chain variable region comprising the sequence of SEQ ID NO: 143, or z) a heavy chain variable region comprising the sequence of SEQ ID NO: 140, and a light chain variable region comprising the sequence of SEQ ID NO: 144, or aa) a heavy chain variable region comprising the sequence of SEQ ID NO: 141, and a light chain variable region comprising the sequence of SEQ ID NO: 144, or bb) a heavy chain variable region comprising the sequence of SEQ ID NO: 142 and a light chain variable region comprising the sequence of SEQ ID NO: 144; or cc) a heavy chain variable region comprising the sequence of SEQ ID NO: 140 and a light chain variable region comprising the sequence of SEQ ID NO: 145; or dd) a heavy chain variable region comprising the sequence of SEQ ID NO: 141, and a light chain variable region comprising the sequence of SEQ ID NO: 145, or ee) a heavy chain variable region comprising the sequence of SEQ ID NO: 142, and a light chain variable region comprising the sequence of SEQ ID NO: 145, or ff) a heavy chain variable region comprising the sequence of SEQ ID NO: 146 and a light chain variable region comprising the sequence of SEQ ID NO: 111, or gg) a heavy chain variable region comprising the sequence of SEQ ID NO: 146 and a light chain variable region comprising the sequence of SEQ ID NO: 112, or hh) a heavy chain variable region comprising the sequence of SEQ ID NO: 147, and a light chain variable region comprising the sequence of SEQ ID NO: 111; or ii) a heavy chain variable region comprising the sequence of SEQ ID NO: 39, and a light chain variable region comprising the sequence of SEQ ID NO: 63; or jj) a heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 69; or kk) a heavy chain variable region comprising the sequence of SEQ ID NO: 70, and a light chain variable region comprising the sequence of SEQ ID NO: 69, or ll) a heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 69, or mm) a heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 69; or nn) a heavy chain variable region comprising the sequence of SEQ ID NO: 68, and a light chain variable region comprising the sequence of SEQ ID NO: 71, or oo) a heavy chain variable region comprising the sequence of SEQ ID NO: 70 and a light chain variable region comprising the sequence of SEQ ID NO: 71, or pp) a heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 71, or qq) a heavy chain variable region comprising the sequence of SEQ ID NO: 74, and a light chain variable region comprising the sequence of SEQ ID NO: 71, or rr) a heavy chain variable region comprising the sequence of SEQ ID NO: 68, and a light chain variable region comprising the sequence of SEQ ID NO: 73, or ss) a heavy chain variable region comprising the sequence of SEQ ID NO: 70, and a light chain variable region comprising the sequence of SEQ ID NO: 73, or tt) a heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 73, or uu) a heavy chain variable region comprising the sequence of SEQ ID NO: 74 and a light chain variable region comprising the sequence of SEQ ID NO: 73; or vv) a heavy chain variable region comprising the sequence of SEQ ID NO: 68 and a light chain variable region comprising the sequence of SEQ ID NO: 75; or ww) a heavy chain variable region comprising the sequence of SEQ ID NO: 70, and a light chain variable region comprising the sequence of SEQ ID NO: 75, or xx) a heavy chain variable region comprising the sequence of SEQ ID NO: 72 and a light chain variable region comprising the sequence of SEQ ID NO: 75; or yy) a heavy chain variable region comprising the sequence of SEQ ID NO: 74, and a light chain variable region comprising the sequence of SEQ ID NO: 75 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising:

9. 9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, further comprising an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the Fc region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the Fc region derived from human IgG1 comprises the mutations L234A and L235A.

12. 12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, which is humanized.

13. 13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotype antibody, or a fusion protein.

14. Diabody, Fab, Fab', F(ab') ２ , Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv) ２ , a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody, a camelized single-domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody.

15. 10 as measured by FACS assay －８ EC below M ５０ 15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, which is capable of specifically binding to human CD39 at 1500 ng / mL.

16. a) specifically binds to human CD39 but not to mouse CD39 as measured by a FACS assay; b) 10 as measured by FACS assay －８ EC below M ５０ specifically binds to cynomolgus monkey CD39 at c) 10 as measured by Biacore assay －７ M or less (for example, 5 x 10 －８ M or less, 3×10 －８ M or less, 2×10 －８ M or less, 1×10 －８ M or less, or 8 x 10 －９ M or less) K Ｄ specifically binds to human CD39 at a value d) 10 as measured by Octet assay －８ M or less (for example, 8 x 10 －９ M or less, 5×10 －９ M or less, 4×10 －９ M or less, 3×10 －９ M or less, 1×10 －９ M or less, or 9 x 10 －１０ M or less) K Ｄ specifically binds to human CD39 at a value e) an IC of 50 nM or less (e.g., 1 nM or less, 5 nM or less, 10 nM or less, or 30 nM or less) as measured by an ATPase activity assay ５０ inhibits ATPase activity in CD39-expressing cells, f) is capable of enhancing ATP-mediated monocyte activation at a concentration of 10 nM or less (e.g., 5 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, or 0.2 nM or less) as measured by analysis of CD80, CD86, and / or CD40 expression by FACS assay; g) capable of enhancing ATP-mediated T cell activation in PBMCs at concentrations up to 25 nM as measured by IL-2 secretion, IFN-γ secretion, and proliferation of CD4+ or CD8+ T cells; h) is capable of enhancing ATP-mediated DC activation at concentrations of 25 nM or less (or 10 nM or less, or 5 nM or less, or 1 nM or less, or 0.5 nM or less), as measured by analysis of CD83 expression by FACS assay, or by the ability of activated dendritic cells (DCs) to promote T cell proliferation, or by the ability of activated DCs to promote IFN-γ production in a mixed lymphocyte reaction (MLR) assay; i) CD4 induced by adenosine (hydrolyzed from ATP) at concentrations of 1 nM or less (e.g., 0.1 nM or less, 0.01 nM or less) as measured by FACS assay ＋ can block the inhibition of T cell proliferation; j) capable of inhibiting tumor growth in a mammalian NK cell or macrophage cell dependent manner; k) T cell proliferation, CD25 ＋ Human CD8 inhibited by eATP as measured by cell and viable cell populations ＋ can reverse T cell proliferation, and l) capable of enhancing IL1β release from human macrophages induced by LPS stimulation at a concentration of 50 nM or less (or 12.5 nM or less, or 3.13 nM or less, or 0.78 nM or less, or 0.2 nM or less, or 0.049 nM or less, or 0.012 nM or less, or 0.003 nM or less, or 0.0008 nM or less) as measured by ELISA assay; 16. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, having one or more properties selected from the group consisting of:

17. 17. An anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, which specifically binds to an epitope of CD39, wherein the epitope comprises one or more residues selected from the group consisting of Q96, N99, E143, R147, R138, M139, E142, K5, E100, D107, V81, E82, R111, and V115.

18. 18. The anti-CD39 antibody or antigen-binding fragment thereof of claim 17, wherein the epitope comprises one or more residues selected from the group consisting of Q96, N99, E143, and R147.

19. 18. The anti-CD39 antibody or antigen-binding fragment thereof of claim 17, wherein the epitope comprises one or more residues selected from the group consisting of R138, M139, and E142.

20. 18. The anti-CD39 antibody or antigen-binding fragment thereof of claim 17, wherein the epitope comprises one or more residues selected from the group consisting of K5, E100, and D107.

21. 18. The anti-CD39 antibody or antigen-binding fragment thereof of claim 17, wherein the epitope comprises one or more residues selected from the group consisting of V81, E82, R111, and V115.

22. 22. An anti-CD39 antibody or antigen-binding fragment thereof according to any one of claims 1 to 21, wherein the CD39 is human CD39 comprising the amino acid sequence of SEQ ID NO:

162.

23. None of antibody 9-8B, antibody T895, and antibody I394 antibody 9-8B comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 46 and a light chain variable region comprising the sequence of SEQ ID NO: 48; antibody T895 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 55 and a light chain variable region comprising the sequence of SEQ ID NO: 57; 23. An antibody or antigen-binding fragment thereof according to any one of claims 16 to 22, wherein the antibody I394 comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 113 and a light chain variable region comprising the sequence of SEQ ID NO:

114.

24. 24. The antibody or antigen-binding fragment thereof of any one of claims 1 to 23, which is bispecific.

25. 25. The antibody or antigen-binding fragment thereof of claim 24, which is capable of specifically binding to a second antigen other than CD39 or a second epitope on CD39.

26. 26. The antibody or antigen-binding fragment thereof of any one of claims 1 to 25, linked to one or more conjugate moieties.

27. 27. The antibody or antigen-binding fragment thereof of claim 26, wherein the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, a purification moiety, or other anti-cancer drug.

28. 28. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and one or more pharmaceutically acceptable carriers.

29. 28. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 27.

30. 30. A vector comprising the isolated polynucleotide of claim 29.

31. A host cell comprising the vector of claim 30.

32. 29. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 27, and / or a pharmaceutical composition according to claim 28, and a second therapeutic agent.

33. 38. A method for expressing an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27, comprising culturing a host cell described in claim 31 under conditions in which the vector described in claim 30 is expressed.

34. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28 in the manufacture of a medicament for use in a method for treating, preventing, or alleviating a CD39-related disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28.

35. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28 in the manufacture of a medicament for use in a method for treating, preventing, or alleviating a disease treatable by reducing the ATPase activity of CD39 in a subject, the method comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28.

36. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28 in the manufacture of a medicament for use in a method for treating, preventing, or alleviating a disease associated with adenosine-mediated inhibition of T, monocyte, macrophage, DC, APC, NK, and / or B cell activity in a subject, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28 to a subject.

37. 37. The use of any one of claims 34 to 36, wherein the disease, disorder, or condition is cancer.

38. The cancers include anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatic bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal tract cancer, skin cancer, prostate cancer, and lower intestinal cancer.

38. The use of claim 37, which is for pituitary cancer, vaginal cancer, thyroid cancer, throat cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T- or B-cell lymphoma, GI organ stromal tumor, soft tissue tumor, hepatocellular carcinoma, adenocarcinoma.

39. 38. The use of claim 37, wherein the cancer is leukemia, lymphoma, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, or breast cancer.

40. 40. The use of any one of claims 34 to 39, wherein the subject has been identified as having cancer cells or tumor-infiltrating immune or immunosuppressive cells that express CD39, optionally at levels significantly higher than levels normally found in non-cancerous or non-immunosuppressive cells.

41. 37. The use of any one of claims 34 to 36, wherein the disease, disorder, or condition is an autoimmune disease or an infection.

42. 42. The use of claim 41, wherein the autoimmune disease is immune thrombocytopenia, systemic sclerosis, sclerosis, adult respiratory distress syndrome, eczema, asthma, Sjogren's syndrome, Addison's disease, giant cell arteritis, immune complex nephritis, immune thrombocytopenic purpura, autoimmune thrombocytopenia, celiac disease, psoriasis, dermatitis, colitis, or systemic lupus erythematosus.

43. 42. The use of claim 41, wherein the infection is an HIV infection, an HBV infection, an HCV infection, an inflammatory bowel disease, or Crohn's disease.

44. 44. The use according to any one of claims 34 to 43, wherein the subject is a human.

45. 45. The use of any one of claims 34 to 44, wherein the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

46. 46. The use of any one of claims 34 to 45, wherein the method further comprises administering a therapeutically effective amount of a second therapeutic agent.

47. 47. The use of claim 46, wherein the second therapeutic agent is selected from the group consisting of a chemotherapeutic agent, an anti-cancer drug, a radiotherapy agent, an immunotherapy agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, and a cytokine.

48. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28 in the manufacture of a medicament for use in a method for modulating the activity of CD39 in CD39-positive cells, the method comprising a step of exposing the CD39-positive cells to an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28.

49. 49. The use of claim 48, wherein the cell is an immune cell.

50. A method for detecting the presence or amount of CD39 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28, and determining the presence or amount of CD39 in the sample.

51. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28 in the manufacture of a kit for use in a method for diagnosing a CD39-related disease, disorder, or condition in a subject, the method comprising the steps of: a) contacting a sample obtained from the subject with the antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or the pharmaceutical composition described in claim 28; b) determining the presence or amount of CD39 in the sample; and c) correlating the presence or amount of CD39 with the presence or status of a CD39-related disease, disorder, or condition in the subject.

52. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28 in the manufacture of a medicament for treating, preventing, or alleviating a CD39-related disease, disorder, or condition in a subject.

53. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28 in the manufacture of a diagnostic reagent for diagnosing a CD39-related disease, disorder, or condition in a subject.

54. A kit comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 27 and / or a pharmaceutical composition described in claim 28, which is useful for detecting CD39.

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