Novel anti-CD39 antibodies

Abstract

The present invention provides an anti-CD39 antibody or an antigen-binding fragment thereof, an isolated polynucleotide encoding the antibody or the antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and uses thereof.

Description

Technical Field

[0001] The present invention generally relates to novel anti-CD39 antibodies. Background Art

[0002] CD39, also known as ectonucleoside triphosphate diphosphohydrolase 1 (ENTPDase1), is an integral membrane protein that converts ATP or ADP into AMP, which is then dephosphorylated to adenosine by CD73. Adenosine is a potent immunosuppressant and binds to adenosine receptors (e.g., A2A receptors) located on the surface of CD4, CD8 T cells, and natural killer (NK) cells, and inhibits T cell and NK cell responses, thereby suppressing the immune system. Adenosine also binds to A2A or A2B receptors on macrophages and dendritic cells, inhibiting phagocytosis and antigen presentation, and increasing the secretion of tumor-promoting factors (e.g., VEGF, TGFb, and IL-6). The enzymatic activities of CD39 and CD73 play a strategic role in calibrating the duration, intensity, and chemical nature of purinergic signals transmitted to immune cells by converting ADP and ATP into AMP and AMP into 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 that promotes cancer initiation and progression.

[0003] There remains a need for novel anti-CD39 antibodies. Summary of the invention

[0004] Throughout this application, the articles "a", "an", and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an antibody" refers to 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, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein:

[0006] (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); and

[0007] (b) the HCDR2 comprises an amino acid sequence 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); and

[0008] (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), and YYCALYDGYNVYAMDY (SEQ ID NO: 18); and

[0009] (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); and

[0010] (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); and

[0011] (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), and AQLLELPHT (SEQ ID NO: 36);

[0012] Where X1 is Y or F, X2 is S or T, X 58 is R or K, X 59 is N, G, S or Q, X 60 It is G, A or D.

[0013] In some embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, and / or the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9, and / or the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15, and / or the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 21, and / or the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 27, and / or the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 33, wherein X1 and X2 are as defined above.

[0014] In some embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, and / or the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:37 and SEQ ID NO:38, and / or the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:40 and SEQ ID NO:41, and / or the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:21, and / or the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:27, and / or the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:33.

[0015] In some embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, and / or the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 151, and / or the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 16, and / or the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, and / or the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, and / or the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34, wherein X 58 , X 59 and X 60 As defined above.

[0016] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof provided herein comprises:

[0017] (a) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 1, the HCDR2 comprises the sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the sequence shown in SEQ ID NO: 13; or

[0018] (b) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 2, the HCDR2 comprises the sequence shown in SEQ ID NO: 8, and the HCDR3 comprises the sequence shown in SEQ ID NO: 14; or

[0019] (c) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 3, the HCDR2 comprises the sequence shown in SEQ ID NO: 37, and the HCDR3 comprises the sequence shown in SEQ ID NO: 40; or

[0020] (d) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 3, the HCDR2 comprises the sequence shown in SEQ ID NO: 38, and the HCDR3 comprises the sequence shown in SEQ ID NO: 41; or

[0021] (e) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO:4, the HCDR2 comprises the sequence shown in SEQ ID NO:10, and the HCDR3 comprises the sequence shown in SEQ ID NO:16; or

[0022] (f) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO:4, the HCDR2 comprises a sequence selected from the group consisting of SEQ ID NO:134, 135, 136, 137, 138 and 139, and the HCDR3 comprises the sequence shown in SEQ ID NO:16; or

[0023] (g) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 5, the HCDR2 comprises the sequence shown in SEQ ID NO: 11, and the HCDR3 comprises the sequence shown in SEQ ID NO: 17; or

[0024] (h) HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO:6, the HCDR2 comprises the sequence shown in SEQ ID NO:12, and the HCDR3 comprises the sequence shown in SEQ ID NO:18.

[0025] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof provided herein comprises:

[0026] (a) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO: 19, the LCDR2 comprises the sequence shown in SEQ ID NO: 25, and the LCDR3 comprises the sequence shown in SEQ ID NO: 31; or

[0027] (b) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO: 20, the LCDR2 comprises the sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the sequence shown in SEQ ID NO: 32; or

[0028] (c) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO:21, the LCDR2 comprises the sequence shown in SEQ ID NO:27, and the LCDR3 comprises the sequence shown in SEQ ID NO:33; or

[0029] (d) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO: 22, the LCDR2 comprises the sequence shown in SEQ ID NO: 28, and the LCDR3 comprises the sequence shown in SEQ ID NO: 34; or

[0030] (e) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO:23, the LCDR2 comprises the sequence shown in SEQ ID NO:29, and the LCDR3 comprises the sequence shown in SEQ ID NO:35; or

[0031] (f) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the sequence shown in SEQ ID NO:24, the LCDR2 comprises the sequence shown in SEQ ID NO:30, and the LCDR3 comprises the sequence shown in SEQ ID NO:36.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises:

[0033] (a) the HCDR1 comprises the sequence shown in SEQ ID NO: 1, the HCDR2 comprises the sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the sequence shown in SEQ ID NO: 13; the LCDR1 comprises the sequence shown in SEQ ID NO: 19, the LCDR2 comprises the sequence shown in SEQ ID NO: 25, and the LCDR3 comprises the sequence shown in SEQ ID NO: 31; or

[0034] (b) the HCDR1 comprises the sequence shown in SEQ ID NO: 2, the HCDR2 comprises the sequence shown in SEQ ID NO: 8, and the HCDR3 comprises the sequence shown in SEQ ID NO: 14; the LCDR1 comprises the sequence shown in SEQ ID NO: 20, the LCDR2 comprises the sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the sequence shown in SEQ ID NO: 32; or

[0035] (c) the HCDR1 comprises the sequence shown in SEQ ID NO: 3, the HCDR2 comprises the sequence shown in SEQ ID NO: 37, and the HCDR3 comprises the sequence shown in SEQ ID NO: 40; the LCDR1 comprises the sequence shown in SEQ ID NO: 21, the LCDR2 comprises the sequence shown in SEQ ID NO: 27, and the LCDR3 comprises the sequence shown in SEQ ID NO: 33; or

[0036] (d) the HCDR1 comprises the sequence shown in SEQ ID NO: 3, the HCDR2 comprises the sequence shown in SEQ ID NO: 38, and the HCDR3 comprises the sequence shown in SEQ ID NO: 41; the LCDR1 comprises the sequence shown in SEQ ID NO: 21, the LCDR2 comprises the sequence shown in SEQ ID NO: 27, and the LCDR3 comprises the sequence shown in SEQ ID NO: 33; or

[0037] (e) the HCDR1 comprises the sequence shown in SEQ ID NO: 4, the HCDR2 comprises the sequence shown in SEQ ID NO: 10, and the HCDR3 comprises the sequence shown in SEQ ID NO: 16; the LCDR1 comprises the sequence shown in SEQ ID NO: 22, the LCDR2 comprises the sequence shown in SEQ ID NO: 28, and the LCDR3 comprises the sequence shown in SEQ ID NO: 34; or

[0038] (f) the HCDR1 comprises the sequence shown in 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, and the HCDR3 comprises the sequence shown in SEQ ID NO:16; the LCDR1 comprises the sequence shown in SEQ ID NO:22, the LCDR2 comprises the sequence shown in SEQ ID NO:28, and the LCDR3 comprises the sequence shown in SEQ ID NO:34; or

[0039] (g) the HCDR1 comprises the sequence shown in SEQ ID NO: 5, the HCDR2 comprises the sequence shown in SEQ ID NO: 11, and the HCDR3 comprises the sequence shown in SEQ ID NO: 17; the LCDR1 comprises the sequence shown in SEQ ID NO: 23, the LCDR2 comprises the sequence shown in SEQ ID NO: 29, and the LCDR3 comprises the sequence shown in SEQ ID NO: 35; or

[0040] (h) the HCDR1 comprises the sequence shown in SEQ ID NO: 6, the HCDR2 comprises the sequence shown in SEQ ID NO: 12, and the HCDR3 comprises the sequence shown in SEQ ID NO: 18; the LCDR1 comprises the sequence shown in SEQ ID NO: 24, the LCDR2 comprises the sequence shown in SEQ ID NO: 30, and the LCDR3 comprises the sequence shown in SEQ ID NO: 36.

[0041] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further include one or more of the heavy chain HFR1, HFR2, HFR3 and HFR4, and / or one or more of the light chain LFR1, LFR2, LFR3 and LFR4, wherein:

[0042] (a) The HFR1 comprises X 19 VxDV 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 thereof having at least 80% sequence identity,

[0043] (b) The HFR2 includes WVX 34 QX 35 PGX 36 X 37 LEWX 38 X 39 (SEQ ID NO: 77) or a homologous sequence thereof having at least 80% sequence identity,

[0044] (c) the HFR3 sequence comprises X 40 X 41 TX 42 X 43 X 44 DX 45 SX 46 X 47 TX 48 YX 49 X 50 X 51 X52 SLX 53 X 54 EDTAVYYCX 55 X 56 (SEQ ID NO: 78) or a homologous sequence thereof having at least 80% sequence identity,

[0045] (d) The HFR4 includes WGQGTX 57 The sequence shown in VTVSS (SEQ ID NO: 126) or a homologous sequence thereof having at least 80% sequence identity,

[0046] (e) the LFR1 comprises the sequence shown as X3IVX4TQSPATLX5X6SPGERX7TX8X9C (SEQ ID NO: 80) or a homologous sequence thereof having at least 80% sequence identity,

[0047] (f) The LFR2 includes WYQQKPGQX 10 PX 11 The sequence shown in LLIY (SEQ ID NO: 81) or a homologous sequence thereof having at least 80% sequence identity,

[0048] (g) The LFR3 includes GX 12 PX 13 RFSGSGSGTX 14 X 15 TLTISSX 16 EPEDFAVYX 17 C (SEQ ID NO: 82) or a homologous sequence thereof having at least 80% sequence identity, and

[0049] (h) The LFR4 includes FGX 18 The sequence shown as GTKLEIK (SEQ ID NO: 152) or a homologous sequence thereof having at least 80% sequence identity,

[0050] Where 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; X 10 A or S; X 11 is R or K; X 12 is I or V; X 13 A or T; X 14 Is D or S; X 15 is F or Y; X 16 L, M or V; X 17 is Y or F; X 18 is G or Q; X19 It is Q or E; X 20 is E or Q; X 21 is G or A; X 22 is G or E; X 23 L or V; X 24 is V or K; X 25 is G or A; X 26 L, M or V; X 27 is R or K; X 28 is V or L; X 29 A or K; X 30 is F or Y; X 31 is N or T; X 32 Is F or L; X 33 is S or K; X 34 is R or K; X 35 A or S; X 36 It is K or Q; X 37 is R or G; X 38 is M, I or V; X 39 is G or A; X 40 is R or K; X 41 is V, A or F; X 42 Is I or L; X 43 is S or T; X 44 is R or A; X 45 is D or T; X 46 is K, A or S; X 47 is S or N; X 48 is L, V or A; X 49 M or L; X 50 It is Q or E; X 51 M or L; X 52 is S, I or N; X 53 is R or K; X 54 is S or T; X 55 A or T; X 56 is R, N or T; and X 57 It is T or L.

[0051] In some embodiments, the HFR1 comprises EVQLVESGGGLVKPGGSX 61 The sequence shown in RLSCAASGFTFS (SEQ ID NO: 154) or a homologous sequence thereof having at least 80% sequence identity, wherein the HFR2 comprises WVRQX 62 PGKGLEWVX 63 (SEQ ID NO: 155) or a homologous sequence thereof having at least 80% sequence identity, wherein the HFR3 comprises RFTISRDDSKNTX64 YLQMNSLKTEDTAVYYCTT (SEQ ID NO: 156) or a homologous sequence thereof having at least 80% sequence identity, the HFR4 comprises a sequence as shown in WGQGTTVTVSS (SEQ ID NO: 79) or a homologous sequence thereof having at least 80% sequence identity, the LFR1 comprises a sequence as shown in EIVX 65 TQSPATLSX 66 SPGERX 67 TLSC (SEQ ID NO: 157) or a homologous sequence thereof having at least 80% sequence identity, wherein the LFR2 comprises WYQQKPGQX 68 PRLLIY (SEQ ID NO: 158) or a homologous sequence thereof having at least 80% sequence identity, wherein the LFR3 comprises GIPARFSGSGSGTDFTLTISSX 69 EPEDFAVYX 70 C (SEQ ID NO: 159) or a homologous sequence thereof having at least 80% sequence identity, and the LFR4 comprises a sequence as shown in FGGGTKLEIK (SEQ ID NO: 153) or a homologous sequence thereof having at least 80% sequence identity, wherein X 61 L or M; X 62 A or S; X 63 is G or A; X 64 L or V; X 65 It is L or M; X 66 L or V; X 67 A or V; X 68 A or S; X 69 L or V; X 70 It is Y or F.

[0052] In some embodiments, the HFR1 comprises X 71 VQLVQSGAEVKKPGASVKX 72 SCKASGYX 73 LK (SEQ ID NO: 160) or a homologous sequence thereof having at least 80% sequence identity, wherein the HFR2 comprises WVX 74 QX 75 LEWX 76 G (SEQ ID NO: 161) or a homologous sequence thereof having at least 80% sequence identity, wherein the HFR3 comprises 77 X 78 TX 79 TX80 DTSX 81 X 82 TAYX 83 ELX 84 SLRSEDTAVYYCAX 85 (SEQ ID NO: 149) or a homologous sequence thereof having at least 80% sequence identity, wherein the HFR4 comprises WGQGTX 57 The sequence shown in VTVSS (SEQ ID NO: 126) or a homologous sequence thereof having at least 80% sequence identity, the LFR1 comprises X 86 IVLTQSPATLX 87 X 88 SPGERX 89 TX 90 X 91 C (SEQ ID NO: 150) or a homologous sequence thereof having at least 80% sequence identity, wherein the LFR2 comprises WYQQKPGQX 10 PX 11 The sequence shown in LLIY (SEQ ID NO: 81) or a homologous sequence thereof having at least 80% sequence identity, wherein the LFR3 comprises GX 92 PX 93 RFSGSGSGTX 94 X 95 TLTISSX 96 EPEDFAVYYC (SEQ ID NO: 148) or a homologous sequence thereof having at least 80% sequence identity, and the LFR4 comprises a sequence as shown in FGQGTKLEIK (SEQ ID NO: 83) or a homologous sequence thereof having at least 80% sequence identity, wherein X 10 , X 11 and X 57 As defined above, X 71 It is Q or E; X 72 is V or L; X 73 N or T; X 74 is R or K; X 75 is R or G; X 76 is M or I; X 77 is R or K; X 78 is V or A; X 79 Is I or L; X 80 is R or A; X 81 A or S; X 82 is S or N; X 83 M or L; X 84 is S or I; X 85is R or N; X 86 is E or Q; X 87 is S or T; X 88 L or A; X 89 A or V; X 90 is L or I; X 91 is S or T; X 92 is I or V; X 93 A or T; X 94 Is D or S; X 95 is F or Y; X 96 It is L or M.

[0053] In some embodiments, the HFR1 includes a sequence selected from the group consisting of SEQ ID NOs: 84-86, 115, 119-120 and 131; the HFR2 includes a sequence selected from the group consisting of SEQ ID NOs: 87-90 and 121-123; the HFR3 includes a sequence selected from the group consisting of SEQ ID NOs: 91-97, 116-117 and 124-125; the HFR4 includes a sequence selected from the group consisting of SEQ ID NOs: 79 and 118; the LFR1 includes a sequence selected from the group consisting of SEQ ID NOs: 98-103 and 127-129; the LFR2 includes a sequence selected from the group consisting of SEQ ID NOs: 104, 105 and 130; the LFR3 includes a sequence selected from the group consisting of SEQ ID NOs: 106-110 and 132-133, and the LFR4 includes a sequence selected from the group consisting of SEQ ID NOs: 83 and 153.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 60, 62, 64, 66, 140, 141, 142, 146, 147, 39, and homologous sequences having at least 80% sequence identity thereto but still retaining specific binding affinity to human CD39; the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 61, 63, 65, 67, 143, 144, 145, 111, 112, 63, and homologous sequences having at least 80% sequence identity thereto but still retaining specific binding affinity to human CD39.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 68, 70, 72, 74, and homologous sequences having at least 80% sequence identity thereto but still retaining specific binding affinity to human CD39; the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 69, 71, 73, 75, and homologous sequences having at least 80% sequence identity thereto but still retaining specific binding affinity to human CD39.

[0056] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein include:

[0057] (1) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 42, and the light chain variable region comprises the sequence shown in SEQ ID NO: 51, or

[0058] (2) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 43, and the light chain variable region comprises the sequence shown in SEQ ID NO: 52, or

[0059] (3) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 44, and the light chain variable region comprises the sequence shown in SEQ ID NO: 53, or

[0060] (4) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 45, and the light chain variable region comprises the sequence shown in SEQ ID NO: 54, or

[0061] (5) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the sequence shown in SEQ ID NO: 56, or

[0062] (6) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 49, and the light chain variable region comprises the sequence shown in SEQ ID NO: 58, or

[0063] (7) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 50, and the light chain variable region comprises the sequence shown in SEQ ID NO: 59, or

[0064] (8) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence shown in SEQ ID NO: 61, or

[0065] (9) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 62, and the light chain variable region comprises the sequence shown in SEQ ID NO: 61, or

[0066] (10) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 64, and the light chain variable region comprises the sequence shown in SEQ ID NO: 61, or

[0067] (11) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 66, and the light chain variable region comprises the sequence shown in SEQ ID NO: 61, or

[0068] (12) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence shown in SEQ ID NO: 63, or

[0069] (13) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 62, and the light chain variable region comprises the sequence shown in SEQ ID NO: 63, or

[0070] (14) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 64, and the light chain variable region comprises the sequence shown in SEQ ID NO: 63, or

[0071] (15) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 66, and the light chain variable region comprises the sequence shown in SEQ ID NO: 63, or

[0072] (16) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence shown in SEQ ID NO: 65, or

[0073] (17) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 62, and the light chain variable region comprises the sequence shown in SEQ ID NO: 65, or

[0074] (18) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 64, and the light chain variable region comprises the sequence shown in SEQ ID NO: 65, or

[0075] (19) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 66, and the light chain variable region comprises the sequence shown in SEQ ID NO: 65, or

[0076] (20) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence shown in SEQ ID NO: 67, or

[0077] (21) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 62, and the light chain variable region comprises the sequence shown in SEQ ID NO: 67, or

[0078] (22) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 64, and the light chain variable region comprises the sequence shown in SEQ ID NO: 67, or

[0079] (23) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 66, and the light chain variable region comprises the sequence shown in SEQ ID NO: 67, or

[0080] (24) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 140, and the light chain variable region comprises the sequence shown in SEQ ID NO: 61, or

[0081] (25) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 141, and the light chain variable region comprises the sequence shown in SEQ ID NO: 61, or

[0082] (26) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 142, and the light chain variable region comprises the sequence shown in SEQ ID NO: 61, or

[0083] (27) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence shown in SEQ ID NO: 143, or

[0084] (28) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 140, and the light chain variable region comprises the sequence shown in SEQ ID NO: 143, or

[0085] (29) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 141, and the light chain variable region comprises the sequence shown in SEQ ID NO: 143, or

[0086] (30) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 142, and the light chain variable region comprises the sequence shown in SEQ ID NO: 143, or

[0087] (31) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence shown in SEQ ID NO: 144, or

[0088] (32) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 140, and the light chain variable region comprises the sequence shown in SEQ ID NO: 144, or

[0089] (33) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 141, and the light chain variable region comprises the sequence shown in SEQ ID NO: 144, or

[0090] (34) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 142, and the light chain variable region comprises the sequence shown in SEQ ID NO: 144, or

[0091] (35) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 60, and the light chain variable region comprises the sequence shown in SEQ ID NO: 145, or

[0092] (36) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 140, and the light chain variable region comprises the sequence shown in SEQ ID NO: 145, or

[0093] (37) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 141, and the light chain variable region comprises the sequence shown in SEQ ID NO: 145, or

[0094] (38) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 142, and the light chain variable region comprises the sequence shown in SEQ ID NO: 145, or

[0095] (39) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 146, and the light chain variable region comprises the sequence shown in SEQ ID NO: 111, or

[0096] (40) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 146, and the light chain variable region comprises the sequence shown in SEQ ID NO: 112, or

[0097] (41) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 147, and the light chain variable region comprises the sequence shown in SEQ ID NO: 111, or

[0098] (42) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 39, and the light chain variable region comprises the sequence shown in SEQ ID NO: 63, or

[0099] (43) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 68, and the light chain variable region comprises the sequence shown in SEQ ID NO: 69, or

[0100] (44) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 70, and the light chain variable region comprises the sequence shown in SEQ ID NO: 69, or

[0101] (45) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 72, and the light chain variable region comprises the sequence shown in SEQ ID NO: 69, or

[0102] (46) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 74, and the light chain variable region comprises the sequence shown in SEQ ID NO: 69, or

[0103] (47) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 68, and the light chain variable region comprises the sequence shown in SEQ ID NO: 71, or

[0104] (48) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 70, and the light chain variable region comprises the sequence shown in SEQ ID NO: 71, or

[0105] (49) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 72, and the light chain variable region comprises the sequence shown in SEQ ID NO: 71, or

[0106] (50) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 74, and the light chain variable region comprises the sequence shown in SEQ ID NO: 71, or

[0107] (51) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 68, and the light chain variable region comprises the sequence shown in SEQ ID NO: 73, or

[0108] (52) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 70, and the light chain variable region comprises the sequence shown in SEQ ID NO: 73, or

[0109] (53) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 72, and the light chain variable region comprises the sequence shown in SEQ ID NO: 73, or

[0110] (54) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 74, and the light chain variable region comprises the sequence shown in SEQ ID NO: 73, or

[0111] (55) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 68, and the light chain variable region comprises the sequence shown in SEQ ID NO: 75, or

[0112] (56) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 70, and the light chain variable region comprises the sequence shown in SEQ ID NO: 75, or

[0113] (57) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 72, and the light chain variable region comprises the sequence shown in SEQ ID NO: 75, or

[0114] (58) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 74, and the light chain variable region comprises the sequence shown in SEQ ID NO: 75.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more amino acid residue substitutions or modifications, but still maintains specific binding affinity to human CD39. In some embodiments, at least one of the substitutions or modifications is in one or more CDR sequences and / or one or more non-CDR sequences in the heavy chain variable region or light chain variable region. In some embodiments, at least one of the substitutions or modifications is a conservative substitution.

[0116] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further includes an Fc region, optionally including an Fc region of a human immunoglobulin (Ig), or optionally including 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 a reduced effector function. In some embodiments, the Fc region derived from human IgG1 includes L234A and / or L235A mutations. In some embodiments, the Fc region derived from human IgG4 optionally has a reduced effector function. In some embodiments, the Fc region derived from human IgG4 includes S228P mutations and / or L235E mutations and / or F234A and L235A mutations.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof provided herein is humanized. In some embodiments, the antibody or antigen-binding fragment thereof provided herein is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof provided in the present application is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide bond-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide bond-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent bifunctional antibody), multispecific antibody, camelized single chain domain antibody, nanobody, domain antibody or bivalent domain antibody.

[0119] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can be expressed as no more than 10 -8 EC of M 50 Specifically binds to human CD39, the EC 50 The results were determined by FACS (flow cytometry fluorescence sorting technology).

[0120] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein have one or more properties selected from the following group:

[0121] (a) Binds specifically to human CD39 but not mouse CD39 as determined by FACS assay;

[0122] (b) not more than 10 -8 EC of M 50 Specifically binds to cynomolgus monkey CD39, the EC 50 Determined by FACS assay;

[0123] (c) not more than 10 -7 M (e.g., no more than 5x10 -8 M, no more than 3x10 -8 M, no more than 2x10 -8 M, not more than 1x10 -8 M, or no more than 8x10 -9 M) K D The K D Values ​​were determined by Biacore assay;

[0124] (d) not more than 10 -8 M (for example, no more than 8x10 -9 M, no more than 5x10 -9 M, no more than 4x10 -9M, no more than 3x10 -9 M, not more than 1x10 -9 M, or not more than 9x10 -10 M) K D The K D Values ​​were determined by Octet analysis;

[0125] (e) with an IC of no more than 50 nM (e.g., no more than 1 nM, no more than 5 nM, no more than 10 nM, or no more than 30 nM) 50 Inhibiting ATPase activity in cells expressing CD39, the IC 50 Determined by ATPase activity assay;

[0126] (f) is capable of enhancing ATP-mediated monocyte activation at a concentration of no more than 50 nM (e.g., no more than 40 nM, no more than 30 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 3 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, or no more than 0.2 nM) as determined by FACS analysis of expression of CD80, CD86 and / or CD40;

[0127] (g) is capable of enhancing ATP-mediated T cell activation in peripheral blood mononuclear cells (PBMCs) at a concentration not exceeding 25 nM, as measured by IL-2 secretion, IFN-γ secretion, CD4 + or CD8 + T cell proliferation assay;

[0128] (h) capable of enhancing ATP-mediated dendritic cell (DC) activation at a concentration of no more than 25 nM (or no more than 10 nM, or no more than 5 nM, or no more than 1 nM, or no more than 0.5 nM, or no more than 0.2 nM), as measured by the expression of CD83 as assayed by FACS, or by the ability of activated 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;

[0129] (i) capable of blocking CD4 T cells induced by adenosine (by ATP hydrolysis) at a concentration of no more than 1 nM (e.g., no more than 0.1 nM, no more than 0.01 nM) + Inhibition of T cell proliferation, the concentrations being measured by FACS assay;

[0130] (j) capable of inhibiting tumor growth in a mammal in a NK cell- or macrophage-dependent manner;

[0131] (k) Able to reverse the inhibition of human CD8 by extracellular ATP (eATP)+ T cell proliferation, through T cell proliferation, CD25 + Cell and live cell population assays; and

[0132] (l) capable of enhancing the release of IL1β from human macrophages induced by LPS stimulation at a concentration of no more than 50 nM (or no more than 12.5 nM, or no more than 3.13 nM, or no more than 0.78 nM, or no more than 0.2 nM, or no more than 0.049 nM, or no more than 0.012 nM, or no more than 0.003 nM, or no more than 0.0008 nM), as measured by ELISA detection method.

[0133] In another aspect, the present invention provides an anti-CD39 antibody or antigen-binding fragment thereof, which competes with the antibody or antigen-binding fragment thereof provided in the present application for binding to human CD39. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof competes for binding to human CD39 with an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 43, and the light chain variable region comprises a sequence as shown in SEQ ID NO: 52. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof competes for binding to human CD39 with an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 44, and the light chain variable region comprises a sequence as shown in SEQ ID NO: 53; or competes for binding to human CD39 with an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 45, and the light chain variable region comprises a sequence as shown in SEQ ID NO: 54. In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof competes for binding to human CD39 with an antibody comprising a heavy chain variable region comprising a sequence as shown in SEQ ID NO: 47 and a light chain variable region comprising a sequence as shown in SEQ ID NO: 56.

[0134] In another aspect, the present invention provides an anti-CD39 antibody or an 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.

[0135] 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 shown in SEQ ID NO: 162.

[0136] In some embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is not any of antibody 9-8B, antibody T895 and antibody I394, wherein: antibody 9-8B includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes the sequence as shown in SEQ ID NO: 46, and the light chain variable region includes the sequence as shown in SEQ ID NO: 48; antibody T895 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes the sequence as shown in SEQ ID NO: 55, and the light chain variable region includes the sequence as shown in SEQ ID NO: 57; and antibody I394 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes the sequence as shown in SEQ ID NO: 113, and the light chain variable region includes the sequence as shown in SEQ ID NO: 114.

[0137] In some embodiments, the antibody or antigen-binding fragment thereof provided herein is bispecific. In some embodiments, the antibody or antigen-binding fragment 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.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof provided herein is linked to one or more conjugate moieties. In some embodiments, the conjugate moieties include a cleanup 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 binding agent, a purification moiety, or other anticancer drugs.

[0139] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention and one or more pharmaceutically acceptable carriers.

[0140] In another aspect, the present invention provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of the present invention.

[0141] In another aspect, the present invention provides a vector comprising the isolated polynucleotide of the present invention.

[0142] In another aspect, the present invention provides a host cell comprising the vector of the present invention.

[0143] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof and / or the pharmaceutical composition described in the present invention, and a second therapeutic agent.

[0144] In another aspect, the present invention provides a method for expressing the antibody or antigen-binding fragment thereof of the present invention, comprising culturing the host cell of the present invention under conditions for expressing the vector of the present invention.

[0145] In another aspect, the present invention provides a method for treating, preventing or alleviating a disease, disorder or condition associated with CD39 in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention and / or the pharmaceutical composition of the present invention.

[0146] In another aspect, the present invention 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 the antibody or antigen-binding fragment thereof of the present invention and / or the pharmaceutical composition of the present invention.

[0147] In another aspect, the present invention provides a method for 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 the antibody or antigen-binding fragment thereof of the present invention and / or the pharmaceutical composition of the present invention.

[0148] In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the cancer is anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, In some embodiments, the subject is a cancer cell or tumor-infiltrating immune cell or immunosuppressive cell that expresses CD39, wherein the subject is a leukemia cell, a lymphoma cell, a bladder cancer cell, a glioma cell, a glioblastoma cell, an ovarian cancer cell, a melanoma cell, a prostate cancer cell, a thyroid cancer cell, a esophageal cancer cell, or a breast cancer cell. In some embodiments, the subject is a cancer cell or tumor-infiltrating immune cell or immunosuppressive cell that expresses CD39, wherein the subject is a leukemia cell, a lymphoma cell, a bladder cancer cell, a glioma cell, a glioblastoma cell, an ovarian cancer cell, a melanoma cell, a prostate cancer cell, a thyroid cancer cell, an esophageal cancer cell, or a breast cancer cell. In some embodiments, the subject is a cancer cell or tumor-infiltrating immune cell or immunosuppressive cell that expresses CD39, wherein the subject is a leukemia cell, a lymphoma cell, a bladder cancer cell, a glioma cell, a glioblastoma cell, an ovarian cancer cell, a melanoma cell, a prostate cancer cell, a thyroid cancer cell, an esophageal cancer cell, or a breast cancer cell. In some embodiments, the subject is a cancer cell or tumor-infiltrating immune cell or immunosuppressive cell that expresses CD39, wherein the subject is a leukemia cell, a lymphoma cell, a bladder cancer cell, a glioma cell, a glioblastoma cell, an ovarian cancer cell, a melanoma cell, a prostate cancer cell, a thyroid cancer cell, an esophageal cancer cell, or a breast cancer cell. Examples of immunosuppressive cells expressing CD39 include myeloid-derived suppressor cells (MDSCs) or regulatory T cells (Tregs).

[0149] In some embodiments, the disease, disorder or condition is an autoimmune disease or infection. In some embodiments, the autoimmune disease is immune thrombocytopenia, systemic scleroderma, 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.

[0150] In some embodiments, the subject is a human. The administration is via oral, intranasal, intravenous, subcutaneous, sublingual or intramuscular administration. 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 chemotherapeutic agents, anticancer agents, radiotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators and cytokines.

[0151] In another aspect, the present invention provides a method for regulating CD39 activity in CD39-positive cells, comprising exposing the CD39-positive cells to the antibody or antigen-binding fragment thereof and / or the pharmaceutical composition of the present invention. In some embodiments, the cells are immune cells.

[0152] In another aspect, the present invention provides a method for detecting the presence or content of CD39 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition described in the present invention, and determining the presence or content of CD39 in the sample.

[0153] In another aspect, the present invention provides a method for diagnosing a disease, disorder or condition associated with CD39 in a subject, comprising: a) contacting a sample obtained from the subject with the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition described in the present invention; b) determining the presence or amount of CD39 in the sample; c) correlating the presence or amount of CD39 with the presence or status of the disease, disorder or condition associated with CD39 in the subject.

[0154] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition described in the present invention in the preparation of a medicament for treating, preventing or alleviating a disease, disorder or condition associated with CD39 in a subject.

[0155] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof of the present invention and / or the pharmaceutical composition of the present invention in the preparation of a diagnostic reagent for diagnosing a disease, disorder or condition associated with CD39 in a subject.

[0156] In another aspect, the present invention provides a kit for detecting CD39, comprising the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition described in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] Figure 1 It was shown that anti-CD39 monoclonal antibodies mAb21 and mAb23 enhanced ATP-mediated T cell proliferation.

[0158] Figure 2 Blockade of ATP-mediated inhibition of T cell proliferation by anti-CD39 chimeric antibodies c14, c19, c21 and c23 is shown. hIgG4 refers to human IgG4 isotype control antibody.

[0159] Figure 3 Shown are CD39 expression levels on dendritic cells (DCs).

[0160] Figure 4 A to Figure 4 C shows ATP-mediated DC activation by anti-CD39 chimeric antibodies c14, c19, c21, and c23, as detected by FACS for CD86 ( Figure 4 A) CD83 Figure 4 B) and HLA-DR ( Figure 4 C) expression was determined.

[0161] Figure 5 Shown is tumor growth after treatment with anti-CD39 chimeric antibodies c23-hlgG4 and c23-hlgG1 in mice inoculated with MOLP-8 cells (human multiple myeloma cell line).

[0162] Figure 6 A shows the binding properties of humanized antibody hu23.H5L5 to ENTPD1 (ie, CD39), ENTPD2 (ie, CD39L1), ENTPD3 (ie, CD39L3), ENTPD5 (ie, CD39L4) and ENTPD6 (ie, CD39L2) proteins, respectively. Figure 6 B shows the binding of negative control hIgG4 to ENTPD1 (ie, CD39), ENTPD2 (ie, CD39L1), ENTPD3 (ie, CD39L3), ENTPD5 (ie, CD39L4), and ENTPD6 (ie, CD39L2) proteins, respectively.

[0163] Figure 7 A and Figure 7 B shows the binding activity of c23 humanized antibody to MOLP-8 cells determined by FACS.

[0164] Figure 8 The binding activity of c23 humanized antibody (obtained by yeast display) to MOLP-8 cells is shown.

[0165] Fig. 9 A and Fig. 9 B shows ATPase inhibition of SK-MEL-28 cells by c23 humanized antibody as determined by FACS.

[0166] Fig.10 A to Fig.10 C shows the binding activity of c14 humanized antibody to MOLP-8 cells determined by FACS.

[0167] Fig.11 A to Fig.11 D shows ATP-mediated T cell activation of humanized antibody hu23.H5L5 in PBMCs via IL-2 ( Fig.11A) IFN-γ ( Fig.11 B) CD4 + T cell proliferation ( Fig.11 C) and CD8 + T cell proliferation ( Fig.11 D) determination.

[0168] Fig.12 A to Fig.12 E shows the interaction of humanized antibodies hu23.H5L5 and hu14.H1L1 with SK-MEL-5 cells as determined by FACS ( Fig.12 A), SK-MEL-28 cells ( Fig.12 B) MOLP-8 cells ( Fig.12 C) CHOK1-cynoCD39 cells ( Fig.12 D) and CHOK1-mCD39 cells ( Fig.12 E) binding activity.

[0169] Fig.13 A to Fig.13 B shows the effects of humanized antibodies hu23.H5L5 and hu14.H1L1 on SK-MEL-5 ( Fig.13 A) and MOLP-8( Fig.13 B) ATPase inhibitory activity.

[0170] Fig.14 A to Fig.14 C shows ATP-mediated monocyte activation by anti-CD39 humanized antibody hu23.H5L5 through CD80 ( Fig.14 A) CD86( Fig.14 B) and CD40 ( Fig.14 C) expression was determined.

[0171] Fig.15 The expression of CD83 was measured ( Fig.15 A), humanized antibody hu23.H5L5 increased ATP-mediated DC activation and enhanced T cell proliferation ( Fig.15 B) and T cell activation ( Fig.15 C).

[0172] Fig.16 Humanized antibodies hu23.H5L5 and hu14.H1L1 were shown to inhibit tumor growth in MOLP-8 xenograft mice.

[0173] Fig.17 Anti-CD39 humanized antibody hu23.H5L5 was shown to inhibit tumor growth in NK-depleted MOLP-8 xenograft mice.

[0174] Fig.18 Anti-CD39 humanized antibody hu23.H5L5 is shown to inhibit tumor growth in macrophage-depleted MOLP-8 xenograft mice.

[0175] Fig.19 A shows the epitope grouping results of humanized antibodies hu23.H5L5 and hu14.H1L1 and reference antibodies. Fig.19 B shows the epitope grouping of the antibodies tested.

[0176] Fig. 20 Shown is the effect of humanized anti-CD39 antibody hu23.H5L5 on IL1β release from human macrophages induced by LPS stimulation.

[0177] Fig.21 The inhibition of tumor growth in PBMC-transplanted mice by humanized antibody hu23.H5L5 at different doses (0.03 mg / kg, 0.3 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg) is shown.

[0178] Fig. 22 The epitope mapping results of humanized antibody hu23.H5L5, chimeric antibodies c34 and c35, and reference antibodies T895, I394, and 9-8B are shown.

[0179] Fig.23 A to 23C show the results of measuring T cell proliferation ( Fig.23 A) CD25 + cell( Fig.23 B) and living cell populations ( Fig.23 C), Humanized antibody hu23.H5L5 reverses CD8 inhibition by extracellular ATP + T cell proliferation. DETAILED DESCRIPTION

[0180] The following description of the present invention is only for illustrating various embodiments of the present invention. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A variety of equivalents, variations and modifications can be easily derived by those skilled in the art without departing from the scope of the present invention, and it should be understood that such equivalent embodiments are included within the scope of the present invention. All documents cited in the present invention, including publications, patents and patent applications, are incorporated by reference in their entirety.

[0181] definition

[0182] The term "antibody" in the present invention includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that can bind to a specific antigen. A natural complete antibody contains two heavy (H) chains and two light (L) chains. The heavy chains of mammals can be divided into α, δ, ε, γ and μ, each of which consists of a variable region (VH) and the first, second, third and fourth (optional) constant regions (CH1, CH2, CH3, CH4, respectively); the light chains of mammals can be divided into λ or κ, and each light chain consists of a variable region (VL) and a constant region. The antibody is "Y"-shaped, and the stem of the "Y"-shaped structure is composed of the second and third constant regions of two heavy chains bound by disulfide bonds. Each arm of the "Y"-shaped structure includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain. The variable regions of the light chain and the heavy chain determine the binding of the antigen. The variable region of each chain usually contains three hypervariable regions, called complementarity determining regions (CDRs) (the light chain CDR includes LCDR1, LCDR2, LCDR3, and the heavy chain CDR includes HCDR1, HCDR2, HCDR3).The CDR boundaries of the antibodies and antigen-binding fragments disclosed in the present invention can be named or identified by Kabat, IMGT, Chothia or Al-Lazikani nomenclature (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, 1989). 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)). Among them, the three CDRs are separated by flanking parts called framework regions (FR) (light chain FR includes LFR1, LFR2, LFR3 and LFR4, and heavy chain FR includes HFR1, HFR2, HFR3 and HFR4). The framework region is more highly conserved than the CDR and forms a scaffold to support the highly variable loop. The constant regions of the heavy and light chains are not related to antigen binding, but have a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of their heavy chain constant regions. Antibodies can be divided into five major classes or isotypes, depending on whether they contain α, δ, ε, γ, and μ heavy chains: IgA, IgD, IgE, IgG, and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0183] In certain embodiments, the antibodies provided by the present invention include any antigen-binding fragments thereof. The term "antigen-binding fragment" in the present invention refers to an antibody fragment formed by a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not have a complete native antibody structure. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds diabody), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen-binding fragment is able to bind to the same antigen as the parent antibody.

[0184] The "Fab" of an antibody refers to a portion of an antibody composed of a single light chain (including a variable region and a constant region) and the variable region and first constant region of a single heavy chain bound together by disulfide bonds.

[0185] "Fab'" refers to the Fab fragment including part of the hinge region.

[0186] "F(ab')2" refers to a dimer of Fab'.

[0187] The "Fc" of an antibody (e.g., IgG, IgA, or IgD isotype) refers to a portion of an antibody consisting of the second and third constant regions of the first heavy chain connected to the second and third constant regions of the second heavy chain via a disulfide bond. The Fc of IgM and IgE isotype antibodies also contains a fourth constant region. The Fc portion of an antibody is responsible for a variety of different effector functions, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

[0188] The "Fv" of an antibody refers to the smallest antibody fragment that contains a complete antigen binding site. The Fv fragment consists of the variable region of a single light chain combined with the variable region of a single heavy chain.

[0189] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody in which the light chain variable region and the heavy chain variable region are directly linked to each other or connected through a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0190] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of a scFv linked to the Fc portion of an antibody.

[0191] "Camelized single domain antibody", "heavy chain antibody" or "HCAb (Heavy-chain-only antibody)" all refer to antibodies containing two V H Heavy chain antibodies are antibodies that contain heavy chain domains but not 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. Pat. No. 6,005,079). Heavy chain antibodies originally came from Camelidae (camels, dromedaries and llamas). Although they lack light chains, camelized antibodies have confirmed full antigen binding function (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al., Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable region (VHH domain) of heavy chain antibodies is the smallest known antigen binding unit produced by adaptive immunity (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0192] "Nanobody" refers to an antibody fragment that consists of the VHH domain from a heavy chain antibody and two constant regions, CH2 and CH3.

[0193] "Diabodies" or "dAbs" include small antibody fragments with two antigen-binding sites, wherein the fragments contain V and V linked on the same polypeptide chain. H Domain and V L Domain (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). The linker between the two domains is short, so that the two domains on the same chain cannot pair with each other, thereby forcing the two domains to pair with the complementary domains of another chain to form two antigen binding sites. The two antigen binding sites can target the same or different antigens (or epitopes). In some embodiments, a "bispecific ds bifunctional antibody" is a bifunctional antibody that targets two different antigens (or epitopes).

[0194] "Domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or a light chain. H The domains are covalently linked by a peptide linker to form a bivalent or multivalent domain antibody. H The domains can target the same or different antigens.

[0195] The term "valence" used in the present invention 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 antigen binding site. 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 binding sites, four binding sites and six binding sites in an antigen binding molecule, respectively. In some embodiments, the antibody or its antigen binding fragment is bivalent.

[0196] In the present invention, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and can bind to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

[0197] In some embodiments, a "scFv dimer" is a bivalent bifunctional antibody or a bispecific scFv (BsFv) comprising two dimerized V H -V L (connected by a peptide linker) such that one portion of V H With another part of V L The two binding sites can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody comprising V L1 -V H2 (connected by a peptide linker) and V H1 -VL2 (connected by a peptide linker), such that V H1 and V L1 Collaboration, V H2 and V L2 Collaborative, and each collaborating pair has a different antigenic specificity.

[0198] "dsFv" refers to a disulfide-stabilized Fv fragment in which the variable region of a single light chain is connected to the variable region of a single heavy chain by a disulfide bond. In some embodiments, "(dsFv)2" or "(dsFv-dsFv')" contains three peptide chains: two V H The parts are connected by a peptide linker (e.g., a long flexible linker) and are respectively connected to the two V L In some embodiments, dsFv-dsFv' has bispecificity, wherein each pair of heavy chain and light chain paired by disulfide bonds has a different antigen specificity.

[0199] The term "chimeric" used in the present invention refers to an antibody or antigen-binding fragment having a portion of a heavy chain and / or a light chain derived from one species, and the remainder of the heavy chain and / or the light chain derived from another different species. In an exemplary example, a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal (e.g., a mouse). In some embodiments, the non-human animal is a mammal, such as a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.

[0200] The term "humanized" used in the present invention refers to an antibody or antigen-binding fragment comprising CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable).

[0201] The term "affinity" as used in the present invention refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, antibody) or a fragment thereof and an antigen.

[0202] "Specific binding" or "specifically binds" as used herein refers to a non-random binding reaction between two molecules, for example, a reaction between an antibody and an antigen. Specific binding can be characterized by binding affinity, for example, as measured by K D The value represents the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / k on K can be determined by any conventional method known in the art. D , including but not limited to, surface plasmon resonance, Octet method, microthermophoresis, HPLC-MS method and FACS detection method. ≤10 -6 M (e.g. ≤5x10 -7 M, ≤2x10-7 M, ≤10 -7 M, ≤5x10 -8 M, ≤2x10 -8 M, ≤10 -8 M, ≤5x10 -9 M, ≤4x10 -9 M, ≤3x10 -9 M, ≤2x10 -9 M, or ≤10 -9 M) K D The value can represent specific binding between the antibody or antigen-binding fragment thereof and CD39 (eg, human CD39).

[0203] The ability of "competitive binding to human CD39" in the present invention refers to the ability of the first antibody or its antigen-binding fragment to inhibit the binding interaction between human CD39 and the second anti-CD39 antibody to any detectable extent. In some embodiments, the antibody or antigen-binding fragment that competes for binding to human CD39 can inhibit the binding interaction between human CD39 and the second anti-CD39 antibody by at least 85% or at least 90%. In some embodiments, such inhibition can be greater than 95% or greater than 99%.

[0204] The term "epitope" used in the present invention refers to a specific atomic group or amino acid on an antigen that binds to an antibody. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitope on the antigen. An epitope may be linear or conformational (i.e., including spaced amino acid residues). For example, if an antibody or its antigen-binding fragment blocks the binding of a reference antibody to an antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or its antigen-binding fragment may be considered to bind to the same / closely related epitope as the reference antibody.

[0205] The term "amino acid" as used herein refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. The amino acid names are also represented in the present invention by standard single-letter or three-letter codes, which are summarized as follows:

[0206] name Three letter code Single letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0207] In the present invention, when "conservative substitution" is used for an amino acid sequence, it means that an amino acid residue is replaced with another amino acid residue having a side chain of 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). It is known in the art that conservative substitutions usually do not cause significant changes in the conformational structure of the protein, and therefore can retain the biological activity of the protein.

[0208] The term "homologous" as used herein refers to a nucleic acid sequence (or its complementary chain) or an amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0209] When "percent (%) sequence identity" is used for an amino acid sequence (or nucleic acid sequence), it means that after the sequence is aligned and, if necessary, intervals are introduced to maximize the number of identical amino acids (or nucleic acids), in the candidate sequence, the percentage of amino acid (or nucleic acid) residues identical to those in the reference sequence in the candidate sequence. In other words, the percentage (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) identical to the reference sequence to be compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence (whichever is shorter). Conservative substitutions of the amino acid residues may or may not be considered identical residues. The sequences can be aligned to determine the percentage sequence identity of the amino acid (or nucleic acid) sequences using tools disclosed in the art, such as BLASTN, BLASTp (National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (European Bioinformatics Institute website, see Higgins D G et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters of the tool or appropriately adjust the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0210] As used in the present invention, "effector function" refers to the biological activity of the Fc region of an antibody in combination with its effector (e.g., C1 complex and Fc receptor). Exemplary effector functions include complement-dependent cytotoxicity (CDC) mediated by the interaction of an antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by the binding of the Fc region of an antibody to Fc receptors on effector cells, and phagocytosis. Effector function can be assessed using various assays (e.g., Fc receptor binding assays, C1q binding assays, and cell lysis assays).

[0211] An "isolated" substance has been artificially altered from its natural state. If an "isolated" composition or substance occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated", but it may be considered "isolated" if the substance with which it coexists in nature is sufficiently separated and present in a substantially pure state. An "isolated nucleic acid sequence" refers to the sequence of an isolated nucleic acid molecule. In some embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that is 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% pure, wherein the purity is determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ion exchange chromatography or reverse phase HPLC).

[0212] The term "vector" in the present invention refers to a vehicle into which a genetic element can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc. The vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site. The vector can also include components that assist it in entering the cell, including but not limited to, viral particles, liposomes or protein shells. The vector can be an expression vector or a cloning vector. The vector (e.g., expression vector) provided by the present invention contains the nucleic acid sequence encoding the antibody or its antigen-binding fragment described in the present invention, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0213] In the present invention, "host cell" refers to a cell into which an exogenous polynucleotide and / or vector may be or has been introduced.

[0214] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals (e.g., non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians and reptiles). Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably in the present invention.

[0215] The term "anti-tumor activity" refers to a decrease in tumor cell proliferation, viability or metastatic activity. For example, anti-tumor activity can be shown by a decrease in the growth rate of abnormal cells that occur during treatment or a stabilization or reduction in tumor size, or by a longer survival period due to treatment compared to a control without treatment. Anti-tumor activity can be assessed using an acceptable in vitro or in vivo tumor model (including but not limited to a xenograft model, an allograft model, a mouse mammary tumor virus (MMTV) model, and other known models of anti-tumor activity known in the art).

[0216] As used in the present invention, "treatment" or "therapy" for a disease, disorder or condition includes preventing or alleviating a disease, disorder or condition, reducing the rate of occurrence or development of a disease, disorder or condition, reducing the risk of developing a disease, disorder or condition, preventing or delaying the development of symptoms associated with a disease, disorder or condition, reducing or stopping symptoms associated with a disease, disorder or condition, producing complete or partial reversal of a disease, disorder or condition, curing a disease, disorder or condition, or a combination of the above.

[0217] The terms "diagnosis", "diagnose" or "diagnosing" refer to the identification of a pathological state, disease or condition, such as the identification of a CD39-related disease, or to the identification of a subject with a CD39-related disease who may benefit from a particular treatment regimen. In some embodiments, the diagnosis comprises the identification of abnormal levels or activity of CD39. In some embodiments, the diagnosis refers to the identification of cancer or an autoimmune disease in a subject.

[0218] As used in the present invention, the term "biological sample" or "sample" refers to a biological composition obtained from or derived from a target subject, which contains cells and / or other molecular entities to be characterized and / or identified, such as characterized and / or identified based on 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 skilled 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 excretions and sputum), peritoneal fluid, pleural fluid, pleural fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis fluid, abdominal fluid, ascites or pericardial effusion. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lung, kidney, skin or blood vessel of a subject.

[0219] As used in the present invention, "CD39", also known as ENTPD1 or ENTPD enzyme 1, refers to an integral membrane protein that can convert ATP into AMP. Structurally, CD39 is characterized by two transmembrane domains, a smaller cytoplasmic domain, and a larger extracellular hydrophobic domain. In certain embodiments, the CD39 is human CD39. The CD39 used in the present invention can be from other animal species, such as mice and cynomolgus monkeys. An exemplary sequence of human CD39 protein is disclosed in NCBI RefSeq No. NP_001767.3. An exemplary sequence of mouse (Mus musculus) CD39 protein is disclosed in NCBI Ref Seq No. NP_033978.1. An exemplary sequence of cynomolgus (Cynomolgus) CD39 protein is disclosed in NCBI Ref Seq No. XP_015311945.1.

[0220] In addition to CD39, the ENTPD enzyme family also includes several other members, including ENTPD enzymes 2, 3, 4, 5, 6, 7 and 8 (also referred to as ENTPD2, 3, 4, 5, 6, 7 and 8, which can be used interchangeably in the present invention). The four ENTPD enzymes are typical cell surface-localized enzymes with catalytic sites facing the extracellular space (ENTPD enzymes 1, 2, 3, 8). ENTPD enzymes 5 and 6 show intracellular localization and secretion after heterologous expression. ENTPD enzymes 4 and 7 are completely located in the cell, facing the lumen of the cytoplasmic organelles. In some embodiments, the antibodies or antigen-binding fragments thereof provided by the present invention specifically bind to CD39 (i.e., ENTPD enzyme 1), but do not bind to other family members, such as ENTPD enzymes 2, 3, 5 or 6.

[0221] The term "anti-CD39 antibody" refers to an antibody that can specifically bind to CD39 (eg, human or monkey CD39). The term "anti-human CD39 antibody" refers to an antibody that can specifically bind to human CD39.

[0222] As used herein, a "CD39-associated" disease, disorder or condition refers to any disease or condition caused by, aggravated by, or associated with an increase or decrease in the expression or activity of CD39. In some embodiments, the CD39-associated disease, disorder or condition is an immune-related disorder, such as an autoimmune disease. In some embodiments, the CD39-associated disease, disorder or condition is a disorder associated with excessive cell proliferation, such as cancer. In certain embodiments, the CD39-associated disease or condition is characterized by expression or overexpression of CD39 and or CD39-associated genes, such as ENTPD1, ​​2, 3, 4, 5, 6, 7 or 8 genes.

[0223] The term "pharmaceutically acceptable" means that the referred carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient thereof.

[0224] As used herein, the term "CD39-positive cells" refers to cells (eg, phagocytes) expressing CD39 on the cell surface.

[0225] Anti-CD39 antibody

[0226] The present invention provides an anti-CD39 antibody and an antigen-binding fragment thereof. The anti-CD39 antibody and the antigen-binding fragment provided by the present invention can specifically bind to CD39.

[0227] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are present in an amount of no more than 10 -7 M, not more than 8×10 -8 M, not more than 5×10 -8 M, not more than 2×10 -8 M, not more than 8×10 -9 M, not more than 5×10 -9 M, not more than 2×10 - 9 M. No more than 10 -9 M, not more than 8×10 -10 M, not more than 7×10 -10 M or not more than 6×10 -10 M of K D The K DThe value is determined by Biacore detection. Biacore detection 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, K is determined by the method described in Example 5.1 of the present invention. D In certain embodiments, K D The K 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 value measured at 25°C. D The K values ​​were similar to those measured at 37 °C. D The value is equivalent to, for example, K measured at 25°C D The K values ​​are measured at 37°C. D The value is from about 80% to about 150%, from about 90% to about 130%, or from about 90% to about 120%, or from about 90% to about 110%.

[0228] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are present in an amount not exceeding 10 -8 M, no more than 8x10 -9 M, no more than 5x10 -9 M, no more than 4x10 -9 M, no more than 3x10 -9 M, no more than 2x10 -9 M, not more than 1x10 -9 M, no more than 9x10 -10 M, no more than 8x10 -10 M, no more than 7x10 -10 M or no larger than 6x10 -10 M of K D The K D The value is determined by Octet detection. Octet detection is based on biolayer interferometry (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 is determined by the method described in Example 5.1 of the present invention. D value.

[0229] The binding of the antibodies or antigen-binding fragments thereof provided by the present invention to human CD39 can also be measured using the "half maximal effective concentration" (EC 50 ) value, which refers to the antibody concentration at which 50% of the maximum binding of the antibody is observed. EC can be determined by binding assays known in the art, such as direct or indirect binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), FACS assays, and other binding assays). 50 In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are present in an amount not exceeding 10 -7 M, no more than 8x10 -8 M, no more than 5x10 -8 M, no more than 2x10 -8 M. No more than 10 -8 M, no more than 8x10 -9 M, no more than 5x10 -9 M, no more than 2x10 -9 M. No more than 10 -9 M, no more than 8x10 -10 M, no more than 7x10 -10 M or no larger than 6x10 -10 EC of M 50 (i.e. 50% binding concentration) specifically binds to human CD39, the EC 50 The values ​​are determined by FACS (flow cytometry fluorescence sorting) detection. In certain embodiments, the binding is determined by ELISA or FACS detection.

[0230] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention specifically bind to human CD39 (i.e., ENTPD enzyme 1). In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention do not bind to other members of the ENTPD enzyme family. In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention specifically bind to human CD39, but do not specifically bind to ENTPD enzymes 2, 3, 5, or 6, as determined by an ELISA assay.

[0231] 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, for example, as determined by FACS assay.

[0232] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are present in an amount of no more than 10 -7 M, no more than 8x10 -8 M, no more than 5x10 -8 M, no more than 2x10 -8 M. No more than 10-8 M, no more than 8x10 -9 M, no more than 5x10 -9 M, no more than 2x10 -9 M. No more than 10 -9 M, no more than 8x10 -10 M, or no more than 7x10 -10 M or no larger than 6x10 -10 EC of M 50 Specifically binds to cynomolgus monkey CD39, the EC 50 Determined by FACS detection.

[0233] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention have an IC of no more than 50 nM, no more than 40 nM, no more than 30 nM, no more than 20 nM, no more than 10 nM, no more than 8 nM, no more than 5 nM, no more than 3 nM, no more than 1 nM, no more than 0.9 nM, no more than 0.8 nM, no more than 0.7 nM, no more than 0.6 nM, no more than 0.5 nM, no more than 0.4 nM, no more than 0.3 nM, no more than 0.2 nM, no more than 0.1 nM, no more than 0.09 nM, no more than 0.08 nM, no more than 0.07 nM, no more than 0.06 nM, no more than 0.05 nM. 50 Inhibiting ATPase activity in cells expressing CD39, the IC 50 By ATPase activity assay. ATPase activity assays can be determined using methods known in the art, such as by colorimetric detection of phosphate released as a result of ATPase activity. In certain embodiments, ATPase activity is determined by those methods described in Example 3.3 of the present invention.

[0234] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can enhance ATP-mediated monocyte activation at a concentration of no more than 50 nM (e.g., no more than 40 nM, no more than 30 nM, no more than 20 nM, no more than 10 nM, no more than 5 nM, no more than 3 nM, no more than 2 nM, no more than 1 nM, no more than 0.5 nM, or no more than 0.2 nM), as determined by FACS analysis of CD80, CD86, and / or CD40 expression, wherein upregulation of CD80, CD86, and / or CD40 indicates monocyte activation. ATP-mediated monocyte activity can be determined using methods known in the art (e.g., those described in Example 5.5 of the present invention).

[0235] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention can enhance ATP-mediated T cell activation in PBMCs at a concentration of no more than 25 nM, no more than 20 nM, no more than 15 nM, no more than 10 nM, no more than 9 nM, no more than 8 nM, no more than 7 nM, no more than 6 nM, no more than 5 nM, no more than 4 nM, no more than 3 nM, no more than 2 nM or no more than 1 nM, which is achieved by IL-2 secretion, IFN-γ secretion, CD4 + or CD8 + T cell proliferation assays, such as those described in Example 5.5 of the present invention.

[0236] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are capable of enhancing ATP-mediated dendritic cell (DC) activation at a concentration of no more than 25 nM (or no more than 10 nM, or no more than 5 nM, or no more than 1 nM, or no more than 0.5 nM), as determined by analyzing the expression of CD83 by FACS detection.

[0237] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are capable of enhancing ATP-mediated DC activation at a concentration of no more than 25 nM (or no more than 10 nM, or no more than 5 nM, or no more than 1 nM, or no more than 0.5 nM), which is determined by the ability of activated DCs to promote T cell proliferation.

[0238] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are capable of enhancing ATP-mediated DC activation at a concentration of no more than 25 nM (or no more than 10 nM, or no more than 5 nM, or no more than 1 nM, or no more than 0.5 nM), as determined by the ability of activated DCs to promote IFN-γ production in a mixed lymphocyte reaction (MLR) assay.

[0239] ATP-mediated DC maturation can be determined using methods known in the art, such as those described in Example 5.5 of the present invention.

[0240] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention can block CD4 T cells induced by adenosine (by ATP hydrolysis) at a concentration of no more than 1 nM (e.g., no more than 0.1 nM, no more than 0.01 nM). + Inhibition of T cell proliferation, said concentration being determined by FACS detection. T cell proliferation may be determined using methods known in the art, such as those described in Example 3.4 of the present invention.

[0241] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention are capable of inhibiting tumor growth in mammals in a NK cell- or macrophage-dependent manner.

[0242] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the present invention can reverse the inhibition of human CD8 + T cell proliferation, through T cell proliferation, CD25 + Cell and live cell population assays. T cell proliferation percentage (%), CD25 + Cell percentage (%) and viable cell percentage (%).

[0243] In certain embodiments, the antibodies and antigen-binding fragments thereof provided by the invention can enhance the release of human macrophage IL1β induced by LPS stimulation at a concentration of no more than 50 nM (or no more than 12.5 nM, or no more than 3.13 nM, or no more than 0.78 nM, or no more than 0.2 nM, or no more than 0.049 nM, or no more than 0.012 nM, or no more than 0.003 nM, or no more than 0.0008 nM), and the concentration is determined by ELISA detection method. Macrophage IL1β release can be determined using methods known in the art (such as those described in Example 5.5.4 of the present invention).

[0244] Example Anti-CD39 Antibodies

[0245] In certain embodiments, the anti-CD39 antibodies (e.g., anti-human CD39 antibodies) and antigen-binding fragments thereof provided herein include one or more (e.g., 1, 2, 3, 4, 5 or 6) CDRs, wherein the CDRs include a 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), LINTYTGEPTYADDFKD (SEQ ID NO: 7), EIRLKSNKYGTHYAESVKG (SEQ ID NO: 8), QIRLNPDNYATHX1AESVKG (SEQ ID NO: 9), X 58 IDPAX 59 X 60NIKYDPKFQG (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), 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), 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) and AQLLELPHT (SEQ ID NO: 36), where X1 is Y or F, X2 is S or T, X 58 is R or K, X 59 is N, G, S or Q, X 60 is G, A or D. In certain embodiments, the present invention further includes antibodies and antigen-binding fragments thereof having no more than one, two or three amino acid residue substitutions to any one of SEQ ID NOs: 1-9, 11-36 and 151.

[0246] The antibody "mAb13" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO:42, and the light chain variable region has a sequence as shown in SEQ ID NO:51.

[0247] The antibody "mAb14" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO:43 and the light chain variable region has a sequence as shown in SEQ ID NO:52.

[0248] The antibody "mAb19" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a sequence as shown in SEQ ID NO: 44 and the light chain variable region has a sequence as shown in SEQ ID NO: 53.

[0249] The antibody "mAb21" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the sequence shown in SEQ ID NO:45 and the light chain variable region has the sequence shown in SEQ ID NO:54.

[0250] The antibody "mAb23" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the sequence shown in SEQ ID NO: 47 and the light chain variable region has the sequence shown in SEQ ID NO: 56.

[0251] The antibody "mAb34" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the sequence shown in SEQ ID NO: 49 and the light chain variable region has the sequence shown in SEQ ID NO: 58.

[0252] The antibody "mAb35" used in the present invention refers to a monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the sequence shown in SEQ ID NO: 50 and the light chain variable region has the sequence shown in SEQ ID NO: 59.

[0253] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided herein comprise one or more (e.g., 1, 2, 3, 4, 5 or 6) CDR sequences of antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 or mAb35.

[0254] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided by the present invention comprise HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6; the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-9, 11-12 and 151; the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-18; the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-24; the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-30; the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-36.

[0255] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided by the present invention comprise HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 1, the HCDR2 comprises the sequence shown in SEQ ID NO: 7, the HCDR3 comprises the sequence shown in SEQ ID NO: 13, the LCDR1 comprises the sequence shown in SEQ ID NO: 19, the LCDR2 comprises the sequence shown in SEQ ID NO: 25, and the LCDR3 comprises the sequence shown in SEQ ID NO: 31.

[0256] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided by the present invention comprise HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 2, the HCDR2 comprises the sequence shown in SEQ ID NO: 8, the HCDR3 comprises the sequence shown in SEQ ID NO: 14, the LCDR1 comprises the sequence shown in SEQ ID NO: 20, the LCDR2 comprises the sequence shown in SEQ ID NO: 26, and the LCDR3 comprises the sequence shown in SEQ ID NO: 32.

[0257] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided by the present invention comprise HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 3, the HCDR2 comprises the sequence shown in SEQ ID NO: 37, the HCDR3 comprises the sequence shown in SEQ ID NO: 40, the LCDR1 comprises the sequence shown in SEQ ID NO: 21, the LCDR2 comprises the sequence shown in SEQ ID NO: 27, and the LCDR3 comprises the sequence shown in SEQ ID NO: 33.

[0258] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided by the present invention comprise HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 3, the HCDR2 comprises the sequence shown in SEQ ID NO: 38, the HCDR3 comprises the sequence shown in SEQ ID NO: 41, the LCDR1 comprises the sequence shown in SEQ ID NO: 21, the LCDR2 comprises the sequence shown in SEQ ID NO: 27, and the LCDR3 comprises the sequence shown in SEQ ID NO: 33.

[0259] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided by the present invention comprise HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 4, the HCDR2 comprises the sequence shown in SEQ ID NO: 10, the HCDR3 comprises the sequence shown in SEQ ID NO: 16, the LCDR1 comprises the sequence shown in SEQ ID NO: 22, the LCDR2 comprises the sequence shown in SEQ ID NO: 28, and the LCDR3 comprises the sequence shown in SEQ ID NO: 34.

[0260] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided by the present invention comprise HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 5, the HCDR2 comprises the sequence shown in SEQ ID NO: 11, the HCDR3 comprises the sequence shown in SEQ ID NO: 17, the LCDR1 comprises the sequence shown in SEQ ID NO: 23, the LCDR2 comprises the sequence shown in SEQ ID NO: 29, and the LCDR3 comprises the sequence shown in SEQ ID NO: 35.

[0261] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided by the present invention comprise HCDR1, HCDR2 and HCDR3, and / or LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises the sequence shown in SEQ ID NO: 6, the HCDR2 comprises the sequence shown in SEQ ID NO: 12, the HCDR3 comprises the sequence shown in SEQ ID NO: 18, the LCDR1 comprises the sequence shown in SEQ ID NO: 24, the LCDR2 comprises the sequence shown in SEQ ID NO: 30, and the LCDR3 comprises the sequence shown in SEQ ID NO: 36.

[0262] The CDR amino acid sequences of antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 and mAb35 are shown in Table 1 below. CDR boundaries are defined or identified according to the Kabat rule. The amino acid sequences of the heavy chain variable region and the light chain variable region of antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 and mAb35 are shown in Table 2 below.

[0263] Table 1: CDR amino acid sequences of 7 monoclonal antibodies

[0264]

[0265]

[0266] Table 2: Amino acid sequences of variable regions of 7 monoclonal antibodies

[0267]

[0268]

[0269] Assuming that antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 and mAb35 can all bind to CD39, and that the antigen binding specificity is primarily provided 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., CDRs from different antibodies can be mixed and matched, but each antibody must contain HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3) to generate anti-CD39 binding molecules of the present invention. Such "mixed and matched" antibodies can be tested for CD39 binding 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 by 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 by structurally similar CDR sequences. For example, the HCDR1 of antibodies mAb13 and mAb19 have some structural similarities and are therefore easily mixed and matched. It is apparent to those skilled in the art that new VH and VL sequences can be generated by replacing one or more VH and / or VL CDR sequences with structurally similar sequences in the CDR sequences of monoclonal antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 and mAb35 disclosed in the present invention.

[0270] It is known that CDR is responsible for antigen binding. However, it has been found that not all 6 CDRs are essential or unchangeable. In other words, one or more CDRs in anti-CD39 antibodies mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 and mAb35 can be replaced or changed or modified, but the specific binding affinity to CD39 is substantially retained.

[0271] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention contain appropriate framework region (FR) sequences, as long as the antibodies and antigen-binding fragments thereof can specifically bind to CD39. The CDR sequences shown in Table 1 above are obtained from mouse antibodies, but can be grafted to any suitable FR sequence of any suitable species (e.g., mouse, human, rat, rabbit, and others) using suitable methods known in the art (e.g., recombinant technology).

[0272] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention are humanized. It is expected that humanized antibodies or antigen-binding fragments thereof have reduced immunogenicity in humans. Humanized antibodies or antigen-binding fragments thereof are chimeric in their variable regions because non-human CDR sequences are grafted into human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can be accomplished by replacing corresponding human CDR genes with non-human (e.g., mouse) CDR genes on human immunoglobulin genes (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0273] Suitable human heavy and light chain variable domains can be selected using methods known in the art to achieve this purpose. In an illustrative example, a "best-fit" method can be used, wherein non-human (e.g., rodent) antibody variable domain sequences are screened, or BLAST aligned with a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human framework for grafting 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, a framework derived from the consensus sequence of all human antibodies can be used to graft 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).

[0274] In certain embodiments, the present invention provides 16 humanized antibodies of c14, which are respectively named 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. The SEQ ID NOs of the heavy chain variable region and the light chain variable region of each humanized antibody of c14 are shown in Table 16 of Example 5.1. Each of the 16 humanized antibodies of c14 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 2, the HCDR2 includes the sequence shown in SEQ ID NO: 8, the HCDR3 includes the sequence shown in SEQ ID NO: 14, the LCDR1 includes the sequence shown in SEQ ID NO: 20, the LCDR2 includes the sequence shown in SEQ ID NO: 26, and the LCDR3 includes the sequence shown in SEQ ID NO: 32. The CDR boundaries are defined or identified according to the Kabat rules.

[0275] In some embodiments, the present invention provides 31 humanized antibodies of c23, which are respectively named 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.H2L 4, hu23.H3L4, hu23.H4L4, 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 of the heavy and light chain variable regions of each humanized antibody of c23 are shown in Tables 13 and 14 of Example 5.1. Each of the 31 humanized antibodies of c23 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 4, the HCDR2 includes the sequence shown in SEQ ID NO: 10, the HCDR3 includes the sequence shown in SEQ ID NO: 16, the LCDR1 includes the sequence shown in SEQ ID NO: 22, the LCDR2 includes the sequence shown in SEQ ID NO: 28, and the LCDR3 includes the sequence shown in SEQ ID NO: 34. The CDR boundaries are defined or identified according to the Kabat rules.

[0276] In some embodiments, the present invention also provides 6 humanized antibodies, which have the same CDR as c23, except that the amino acid sequence of HCDR2 is different. In some embodiments, the amino acid sequence of HCDR2 of these c23 variants (c23') humanized antibodies includes 58 IDPAX 59 X 60 The amino acid sequence shown in NIKYDPKFQG (SEQ ID NO: 151), wherein X 58 is R or K, X 59 is N, G, S or Q, X 60is G, A or D. In some embodiments, the amino acid sequence of HCDR2 of the humanized antibody of these c23 variants (c23') includes 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). CDR boundaries are defined or identified according to the Kabat rules.

[0277] In some embodiments, the present invention also provides four humanized antibodies of c23 variants displayed by yeast, which are named hu23.201, hu23.203, hu23.207 and hu23.211. The heavy chain variable region and light chain variable region of humanized antibodies hu23.201, hu23.203, hu23.207 and hu23.211 are shown in Table 15 of Example 5.1. Each of the four humanized antibodies hu23.201, hu23.203, hu23.207 and hu23.211 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1 includes the sequence shown in SEQ ID NO: 4, the HCDR2 includes the sequence shown in SEQ ID NO: 10, the HCDR3 includes the sequence shown in SEQ ID NO: 16, the LCDR1 includes the sequence shown in SEQ ID NO: 22, the LCDR2 includes the sequence shown in SEQ ID NO: 28, and the LCDR3 includes the sequence shown in SEQ ID NO: 34. The CDR boundaries are defined or identified according to the Kabat rules.

[0278] Table 3 below shows 4 variants of humanized c14 heavy chain variable region (i.e. hu14.VH_1, hu14.VH_2, hu14.VH_3 and hu14.VH_4) and 4 variants of 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 FRs of humanized c14 heavy chain and light chain variable regions. Table 5 below shows the FR amino acid sequences of the heavy and light chains of 16 humanized antibodies of chimeric antibody c14, and the 16 humanized antibodies are named 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, hu14.H4L4. The heavy chain variable regions and light chain variable regions of these 16 humanized antibodies are shown in Table 16 of Example 5.1.

[0279] Table 3: Amino acid sequences of the humanized variable regions of the c14 humanized antibody

[0280]

[0281]

[0282] Table 4: Amino acid sequences of humanized FRs of c14 humanized antibody

[0283]

[0284]

[0285] Table 5: FR amino acid sequences of each humanized heavy chain variable region and light chain variable region of c14 humanized antibody

[0286]

[0287] Table 6 below shows 7 variants of 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 7 variants of 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 heavy chain variable region and light chain variable region of 4 humanized antibodies for chimeric antibody c23 obtained by yeast display. Table 8 below shows the FR amino acid sequences of 35 humanized antibodies of c23. Table 9 below shows the FR amino acid sequence of each heavy chain and light chain of 35 humanized antibodies of c23.

[0288] Table 6: Amino acid sequences of the variable regions of the c23 humanized antibody

[0289]

[0290]

[0291] Table 7: Amino acid sequences of the humanized variable regions of the humanized antibody c23 obtained by yeast display

[0292]

[0293]

[0294] Table 8: Amino acid sequences of humanized FRs of c23 humanized antibody

[0295]

[0296]

[0297] Table 9: FR amino acid sequences of each humanized heavy chain variable region and light chain variable region of the humanized antibody c23

[0298]

[0299]

[0300] In certain embodiments, humanized antibodies or their antigen-binding fragments provided by the invention are substantially composed of all human sequences except non-human CDR sequences. In some embodiments, variable region FR and constant region (if present) are all or substantially from human immunoglobulin sequences. People's FR sequences and people's constant region sequences can be derived from different human immunoglobulin genes, for example, derived from a FR sequence of a human antibody and derived from the constant region of another human antibody. In some embodiments, humanized antibodies or their antigen-binding fragments comprise people's heavy chain HFR1-4 and / or light chain LFR1-4.

[0301] In some embodiments, the FR region derived from people can include the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of people's FR are replaced by corresponding residues from parental non-human antibodies. This is required in certain embodiments, so that humanized antibodies or their fragments are close to non-human parent antibody structures, to optimize binding characteristics (for example, to increase binding affinity). In certain embodiments, humanized antibodies or their antigen-binding fragments of the present invention are included in each human FR sequence and are no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue replacements, or are no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue replacements in all FR sequences of heavy chain variable region or light chain variable region. In some embodiments, the change of this amino acid residue may only be present in the heavy chain FR region, only be present in the light chain FR region, or exist on both chains. In certain embodiments, one or more amino acids of people's FR sequences are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequence are back-mutated to the corresponding amino acids of the parent non-human antibody to increase binding affinity.

[0302] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises a heavy chain HFR1, a heavy chain HFR2, a heavy chain HFR3 and a heavy chain HFR4, wherein the heavy chain HFR1 comprises a heavy chain HFR2, a heavy chain HFR3 and a heavy chain HFR4. 19 VxDV 20 SGX 21 X22X 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 thereof having at least 80% sequence identity, wherein the heavy chain HFR2 comprises WVX34 QX 35 PGX 36 X 37 LEWX 38 X 39 (SEQ ID NO: 77) or a homologous sequence thereof having at least 80% sequence identity, wherein the heavy chain HFR3 comprises 40 X 41 TX 42 X43X 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 thereof having at least 80% sequence identity, wherein the heavy chain HFR4 comprises WGQGTX 57 VTVSS (SEQ ID NO: 126) or a homologous sequence thereof having at least 80% sequence identity, wherein X 19 It is Q or E, X 20 Is E or Q, X 21 It is G or A, X 22 It is G or E, X 23 It is L or V, X 24 Is V or K, X 25 It is G or A, X 26 It is L, M or V, X 27 is R or K, X 28 It is V or L, X 29 A or K, X 30 Is F or Y, X 31 is N or T, X 32 It is F or L, X 33 Is S or K, X 34 is R or K, X 35 A or S, X 36 It is K or Q, X 37 is R or G, X 38 is M, I or V, X 39 It is G or A, X 40 is R or K, X 41 is V, A or F, X 42 Is I or L, X 43 Is S or T, X 44 is R or A, X45 Is D or T, X 46 is K, A or S, X 47 Is S or N, X 48 L, V or A, X 49 It is M or L, X 50 It is Q or E, X 51 It is M or L, X 52 is S, I or N, X 53 is R or K, X 54 Is S or T, X 55 Is A or T, X 56 is R, N or T, and X 57 It is T or L.

[0303] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises a light chain LFR1, a light chain LFR2, a light chain LFR3 and a light chain LFR4, wherein the light chain LFR1 comprises a sequence as shown in X3IVX4TQSPATLX5X6SPGERX7TX8X9C (SEQ ID NO: 80) or a homologous sequence thereof having at least 80% sequence identity, and the light chain LFR2 comprises a sequence as shown in WYQQKPGQX 10 PX 11 The sequence shown in LLIY (SEQ ID NO: 81) or a homologous sequence having at least 80% sequence identity thereto, the light chain LFR3 comprises GX 12 PX 13 RFSGSGSGTX 14 X 15 TLTISSX 16 EPEDFAVYX 17 C (SEQ ID NO: 82) or a homologous sequence thereof having at least 80% sequence identity, wherein the light chain LFR4 comprises FGX 18 GTKLEIK (SEQ ID NO: 152), or a homologous sequence thereof having at least 80% sequence identity, 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, X10 is L or I, X11 is L or I, X12 is L or I, X13 is L or I, X14 is L or I, X15 is S or T, X16 is L, V or A, X17 is A or V, X18 is L or I, X19 is S or T, X20 is L or I, X21 is L or I, X22 is L or I, X23 is S or T, X24 is L or I, X25 is S or T, X26 is L, V or A, X27 is A or V, X28 is L or I, X29 is S or T, X30 is L or 10 A or S, X 11 is R or K, X 12 Is I or V, X 13 A or T, X 14 Is D or S, X 15 Is F or Y, X 16 It is L, M or V, X 17 Is Y or F, X 18 It is G or Q.

[0304] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises a heavy chain HFR1, a heavy chain HFR2, a heavy chain HFR3 and a heavy chain HFR4, wherein the heavy chain HFR1 comprises EVQLVESGGGLVKPGGSX 61 The sequence shown in RLSCAASGFTFS (SEQ ID NO: 154) or a homologous sequence thereof having at least 80% sequence identity, wherein the heavy chain HFR2 comprises WVRQX 62 PGKGLEWVX 63 (SEQ ID NO: 155) or a homologous sequence thereof having at least 80% sequence identity, wherein the heavy chain HFR3 comprises RFTISRDDSKNTX 64 YLQMNSLKTEDTAVYYCTT (SEQ ID NO: 156) or a homologous sequence thereof having at least 80% sequence identity, the heavy chain HFR4 comprises a sequence as shown in WGQGTTVTVSS (SEQ ID NO: 79) or a homologous sequence thereof having at least 80% sequence identity, wherein X 61 For L or M, X 62 A or S, X 63 It is G or A, X 64 It is L or V.

[0305] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises a light chain LFR1, a light chain LFR2, a light chain LFR3 and a light chain LFR4, wherein the light chain LFR1 comprises EIVX 65 TQSPATLSX 66 SPGERX 67 TLSC (SEQ ID NO: 157) or a homologous sequence thereof having at least 80% sequence identity, wherein the light chain LFR2 comprises WYQQKPGQX 68 PRLLIY (SEQ ID NO: 158) or a homologous sequence thereof having at least 80% sequence identity, wherein the light chain LFR3 comprises GIPARFSGSGSGTDFTLTISSX 69 EPEDFAVYX 70 C (SEQ ID NO: 159) or a homologous sequence thereof having at least 80% sequence identity, the light chain LFR4 comprises a sequence as shown in FGGGTKLEIK (SEQ ID NO: 153) or a homologous sequence thereof having at least 80% sequence identity, wherein X 65 It is L or M, X 66 It is L or V, X67 A or V, X 68 A or S, X 69 It is L or V, X 70 It is Y or F.

[0306] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises a heavy chain HFR1, a heavy chain HFR2, a heavy chain HFR3 and a heavy chain HFR4, wherein the heavy chain HFR1 comprises a heavy chain HFR2, a heavy chain HFR3 and a heavy chain HFR4. 71 VQLVQSGAEVKKPGASVKX 72 SCKASGYX 73 LK (SEQ ID NO: 160) or a homologous sequence thereof having at least 80% sequence identity, wherein the heavy chain HFR2 comprises WVX 74 QX 75 LEWX 76 G (SEQ ID NO: 161) or a homologous sequence thereof having at least 80% sequence identity, wherein the heavy chain HFR3 comprises 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 thereof having at least 80% sequence identity, wherein the heavy chain HFR4 comprises WGQGTX 57 VTVSS (SEQ ID NO: 126) or a homologous sequence thereof having at least 80% sequence identity, wherein X 57 As defined above, X 71 It is Q or E, X 72 It is V or L, X 73 N or T, X 74 is R or K, X 75 is R or G, X 76 M or I, X 77 is R or K, X 78 Is V or A, X 79 Is I or L, X 80 is R or A, X 81 A or S, X 82 Is S or N, X 83 It is M or L, X 84 Is S or I, X 85 It is R or N.

[0307] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises a light chain LFR1, a light chain LFR2, a light chain LFR3 and a light chain LFR4, wherein the light chain LFR1 comprises 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, wherein the light chain LFR2 comprises WYQQKPGQX 10 PX 11 The sequence shown in LLIY (SEQ ID NO: 81) or a homologous sequence having at least 80% sequence identity thereto, the light chain LFR3 comprises GX 92 PX 93 RFSGSGSGTX 94 X 95 TLTISSX 96 EPEDFAVYYC (SEQ ID NO: 148) or a homologous sequence thereof having at least 80% sequence identity, the light chain LFR4 comprises a sequence as shown in FGQGTKLEIK (SEQ ID NO: 83) or a homologous sequence thereof having at least 80% sequence identity, wherein X 10 and X 11 As defined above, X 86 Is E or Q, X 87 Is S or T, X 88 It is L or A, X 89 A or V, X 90 L or I, X 91 Is S or T, X 92 Is I or V, X 93 A or T, X 94 Is D or S, X 95 Is F or Y, X 96 It is L or M.

[0308] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises a heavy chain HFR1, a heavy chain HFR2, a heavy chain HFR3 and a heavy chain HFR4 and / or a light chain LFR1, a light chain LFR2, a light chain LFR3 and a light chain LFR4, wherein the heavy chain HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 84-86, 115, 119-120 and 131, the heavy chain HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 87-90 and 121-123, the heavy chain HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 91-97, 116-117 and 124-125; the heavy chain HFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 79 and 118, the light chain LFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 98-103 and 127-129, the light chain LFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: NO: 104, 105 and 130, the light chain LFR3 comprises a sequence selected from the group consisting of SEQ ID NO: 106-110 and 132-133, and the light chain LFR4 comprises a sequence selected from the group consisting of SEQ ID NO: 83 and 153.

[0309] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises an HFR1, HFR2, HFR3 and / or HFR4 sequence in the heavy chain variable region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of 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), hu23.VH_6 (SEQ ID NO: 141), hu23.VH_7 (SEQ ID NO: 142), hu23.VH_8 (SEQ ID NO: 143), hu23.VH_9 (SEQ ID NO: 144), hu23.VH_10 (SEQ ID NO: 106), hu23.VH_11 (SEQ ID NO: 117), hu23.VH_12 (SEQ ID NO: 128), hu23.VH_13 (SEQ ID NO: 139), hu23.VH_14 (SEQ ID NO: 150), hu23.VH_15 (SEQ ID NO: 151), hu23.VH_16 (SEQ ID NO: 152), hu23.VH_17 (SEQ ID NO: 153), hu23.VH_18 (SEQ ID NO: 154), hu23.VH_19 (SEQ ID NO: 155), hu NO: 146), hu23.207H (SEQ ID NO: 147) and hu23.211H (SEQ ID NO: 39).

[0310] In certain embodiments, the present invention also provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises an LFR1, LFR2, LFR3 and / or LFR4 sequence in the light chain variable region, wherein the light chain variable region comprises a sequence selected from the group consisting of 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), hu23.VL_6 (SEQ ID NO: 144), hu23.VL_7 (SEQ ID NO: 145), hu23.201L (SEQ ID NO: 1 NO: 111), hu23.203L (SEQ ID NO: 112) and hu23.211L (SEQ ID NO: 63).

[0311] In certain embodiments, the present invention provides a humanized anti-CD39 antibody and an antigen-binding fragment thereof, which comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 39, 60, 62, 64, 66, 68, 70, 72, 74, 140, 141, 142, 146, 147, and / or a light chain variable region sequence selected from the group consisting of SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 111, 112, 143, 144 and 145.

[0312] The present invention also provides an exemplary humanized antibody of the chimeric antibody c14, comprising:

[0313] 1) "hu14.H1L1", which includes the heavy chain variable region as shown in hu14.VH_1 (SEQ ID NO: 68) and the light chain variable region as shown in hu14.VL_1 (SEQ ID NO: 69);

[0314] 2) "hu14.H2L1", which includes the heavy chain variable region as shown in hu14.VH_2 (SEQ ID NO: 70) and the light chain variable region as shown in hu14.VL_1 (SEQ ID NO: 69);

[0315] 3) "hu14.H3L1", which includes the heavy chain variable region as shown in hu14.VH_3 (SEQ ID NO: 72) and the light chain variable region as shown in hu14.VL_1 (SEQ ID NO: 69);

[0316] 4) "hu14.H4L1", which includes the heavy chain variable region as shown in hu14.VH_4 (SEQ ID NO: 74) and the light chain variable region as shown in hu14.VL_1 (SEQ ID NO: 69);

[0317] 5) "hu14.H1L2", which includes the heavy chain variable region as shown in hu14.VH_1 (SEQ ID NO: 68) and the light chain variable region as shown in hu14.VL_2 (SEQ ID NO: 71);

[0318] 6) "hu14.H2L2", which includes the heavy chain variable region as shown in hu14.VH_2 (SEQ ID NO: 70) and the light chain variable region as shown in hu14.VL_2 (SEQ ID NO: 71);

[0319] 7) "hu14.H3L2", which includes the heavy chain variable region as shown in hu14.VH_3 (SEQ ID NO: 72) and the light chain variable region as shown in hu14.VL_2 (SEQ ID NO: 71);

[0320] 8) "hu14.H4L2", which includes the heavy chain variable region as shown in hu14.VH_4 (SEQ ID NO: 74) and the light chain variable region as shown in hu14.VL_2 (SEQ ID NO: 71);

[0321] 9) "hu14.H1L3", which includes the heavy chain variable region as shown in hu14.VH_1 (SEQ ID NO: 68) and the light chain variable region as shown in hu14.VL_3 (SEQ ID NO: 73);

[0322] 10) "hu14.H2L3", which includes the heavy chain variable region as shown in hu14.VH_2 (SEQ ID NO: 70) and the light chain variable region as shown in hu14.VL_3 (SEQ ID NO: 73);

[0323] 11) "hu14.H3L3", which includes the heavy chain variable region as shown in hu14.VH_3 (SEQ ID NO: 72) and the light chain variable region as shown in hu14.VL_3 (SEQ ID NO: 73);

[0324] 12) "hu14.H4L3", which includes the heavy chain variable region as shown in hu14.VH_4 (SEQ ID NO: 74) and the light chain variable region as shown in hu14.VL_3 (SEQ ID NO: 73);

[0325] 13) "hu14.H1L4", which includes the heavy chain variable region as shown in hu14.VH_1 (SEQ ID NO: 68) and the light chain variable region as shown in hu14.VL_4 (SEQ ID NO: 75);

[0326] 14) "hu14.H2L4", which includes the heavy chain variable region as shown in hu14.VH_2 (SEQ ID NO: 70) and the light chain variable region as shown in hu14.VL_4 (SEQ ID NO: 75);

[0327] 15) "hu14.H3L4", which includes the heavy chain variable region as shown in hu14.VH_3 (SEQ ID NO: 72) and the light chain variable region as shown in hu14.VL_4 (SEQ ID NO: 75);

[0328] 16) "hu14.H4L4", which includes the heavy chain variable region shown as hu14.VH_4 (SEQ ID NO: 74) and the light chain variable region shown as hu14.VL_4 (SEQ ID NO: 75).

[0329] The present invention also provides an exemplary humanized antibody of the chimeric antibody c23, which comprises:

[0330] 1) "hu23.H1L1", which includes the heavy chain variable region as shown in hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region as shown in hu23.VL_1 (SEQ ID NO: 61);

[0331] 2) "hu23.H2L1", which includes the heavy chain variable region as shown in hu23.VH_2 (SEQ ID NO: 62) and the light chain variable region as shown in hu23.VL_1 (SEQ ID NO: 61);

[0332] 3) "hu23.H3L1", which includes the heavy chain variable region as shown in hu23.VH_3 (SEQ ID NO: 64) and the light chain variable region as shown in hu23.VL_1 (SEQ ID NO: 61);

[0333] 4) "hu23.H4L1", which includes the heavy chain variable region as shown in hu23.VH_4 (SEQ ID NO: 66) and the light chain variable region as shown in hu23.VL_1 (SEQ ID NO: 61);

[0334] 5) "hu23.H1L2", which includes the heavy chain variable region as shown in hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region as shown in hu23.VL_2 (SEQ ID NO: 63);

[0335] 6) "hu23.H2L2", which includes the heavy chain variable region as shown in hu23.VH_2 (SEQ ID NO: 62) and the light chain variable region as shown in hu23.VL_2 (SEQ ID NO: 63);

[0336] 7) "hu23.H3L2", which includes the heavy chain variable region as shown in hu23.VH_3 (SEQ ID NO: 64) and the light chain variable region as shown in hu23.VL_2 (SEQ ID NO: 63);

[0337] 8) "hu23.H4L2", which includes the heavy chain variable region as shown in hu23.VH_4 (SEQ ID NO: 66) and the light chain variable region as shown in hu23.VL_2 (SEQ ID NO: 63);

[0338] 9) "hu23.H1L3", which includes the heavy chain variable region as shown in hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region as shown in hu23.VL_3 (SEQ ID NO: 65);

[0339] 10) "hu23.H2L3", which includes the heavy chain variable region as shown in hu23.VH_2 (SEQ ID NO: 62) and the light chain variable region as shown in hu23.VL_3 (SEQ ID NO: 65);

[0340] 11) "hu23.H3L3", which includes a heavy chain variable region as shown in hu23.VH_3 (SEQ ID NO: 64) and a light chain variable region as shown in hu23.VL_3 (SEQ ID NO: 65);

[0341] 12) "hu23.H4L3", which includes the heavy chain variable region as shown in hu23.VH_4 (SEQ ID NO: 66) and the light chain variable region as shown in hu23.VL_3 (SEQ ID NO: 65);

[0342] 13) "hu23.H1L4", which includes the heavy chain variable region as shown in hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region as shown in hu23.VL_4 (SEQ ID NO: 67);

[0343] 14) "hu23.H2L4", which includes the heavy chain variable region as shown in hu23.VH_2 (SEQ ID NO: 62) and the light chain variable region as shown in hu23.VL_4 (SEQ ID NO: 67);

[0344] 15) "hu23.H3L4", which includes the heavy chain variable region as shown in hu23.VH_3 (SEQ ID NO: 64) and the light chain variable region as shown in hu23.VL_4 (SEQ ID NO: 67);

[0345] 16) "hu23.H4L4", which includes the heavy chain variable region as shown in hu23.VH_4 (SEQ ID NO: 66) and the light chain variable region as shown in hu23.VL_4 (SEQ ID NO: 67);

[0346] 17) "hu23.H5L1", which includes the heavy chain variable region as shown in hu23.VH_5 (SEQ ID NO: 140) and the light chain variable region as shown in hu23.VL_1 (SEQ ID NO: 61);

[0347] 18) "hu23.H6L1", which includes the heavy chain variable region as shown in hu23.VH_6 (SEQ ID NO: 141) and the light chain variable region as shown in hu23.VL_1 (SEQ ID NO: 61);

[0348] 19) "hu23.H7L1", which includes the heavy chain variable region as shown in hu23.VH_7 (SEQ ID NO: 142) and the light chain variable region as shown in hu23.VL_1 (SEQ ID NO: 61);

[0349] 20) "hu23.H1L5", which includes the heavy chain variable region as shown in hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region as shown in hu23.VL_5 (SEQ ID NO: 143);

[0350] 21) "hu23.H5L5", which includes the heavy chain variable region as shown in hu23.VH_5 (SEQ ID NO: 140) and the light chain variable region as shown in hu23.VL_5 (SEQ ID NO: 143);

[0351] 22) “hu23.H6L5”, which comprises a heavy chain variable region as set forth in hu23.VH_6 (SEQ ID NO: 141) and a light chain variable region as set forth in hu23.VL_5 (SEQ ID NO: 143);

[0352] 23) "hu23.H7L5", which includes the heavy chain variable region as shown in hu23.VH_7 (SEQ ID NO: 142) and the light chain variable region as shown in hu23.VL_5 (SEQ ID NO: 143);

[0353] 24) "hu23.H1L6", which includes the heavy chain variable region as shown in hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region as shown in hu23.VL_6 (SEQ ID NO: 144);

[0354] 25) "hu23.H5L6", which includes the heavy chain variable region as shown in hu23.VH_5 (SEQ ID NO: 140) and the light chain variable region as shown in hu23.VL_6 (SEQ ID NO: 144);

[0355] 26) “hu23.H6L6”, which comprises a heavy chain variable region as set forth in hu23.VH_6 (SEQ ID NO: 141) and a light chain variable region as set forth in hu23.VL_6 (SEQ ID NO: 144);

[0356] 27) "hu23.H7L6", which includes the heavy chain variable region as shown in hu23.VH_7 (SEQ ID NO: 142) and the light chain variable region as shown in hu23.VL_6 (SEQ ID NO: 144);

[0357] 28) "hu23.H1L7", which includes the heavy chain variable region as shown in hu23.VH_1 (SEQ ID NO: 60) and the light chain variable region as shown in hu23.VL_7 (SEQ ID NO: 145);

[0358] 29) "hu23.H5L7", which includes the heavy chain variable region as shown in hu23.VH_5 (SEQ ID NO: 140) and the light chain variable region as shown in hu23.VL_7 (SEQ ID NO: 145);

[0359] 30) "hu23.H6L7", which includes the heavy chain variable region as shown in hu23.VH_6 (SEQ ID NO: 141) and the light chain variable region as shown in hu23.VL_7 (SEQ ID NO: 145);

[0360] 31) "hu23.H7L7", which includes the heavy chain variable region as shown in hu23.VH_7 (SEQ ID NO: 142) and the light chain variable region as shown in hu23.VL_7 (SEQ ID NO: 145);

[0361] 32) "hu23.201", which includes the heavy chain variable region as shown in hu23.201H (SEQ ID NO: 146) and the light chain variable region as shown in hu23.201L (SEQ ID NO: 111);

[0362] 33) "hu23.203", which includes the heavy chain variable region set forth as hu23.201H (SEQ ID NO: 146) and the light chain variable region set forth as hu23.203L (SEQ ID NO: 112);

[0363] 34) "hu23.207", which includes the heavy chain variable region set forth as hu23.207H (SEQ ID NO: 147) and the light chain variable region set forth as hu23.201L (SEQ ID NO: 111);

[0364] 35) "hu23.211" comprising a heavy chain variable region as shown in hu23.211H (SEQ ID NO: 39) and a light chain variable region as shown in hu23.211L (SEQ ID NO: 63).

[0365] These exemplary humanized anti-CD39 antibodies retain specific binding ability or affinity for CD39 and are at least comparable to or even better than the parental mouse antibody mAb14 or mAb23 in this respect.

[0366] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof of the present invention comprises all or a portion of the heavy chain variable region, and / or all or a portion of the light chain variable region. In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof of the present invention is a single domain antibody, which consists of all or a portion of the heavy chain variable region of the present invention. More information about single domain antibodies is known in the art (see, e.g., U.S. Patent No. 6,248,516).

[0367] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof of the present invention further comprises an immunoglobulin (Ig) constant region, which optionally further comprises a heavy chain and / or light chain constant region. In certain embodiments, the heavy chain constant region comprises CH1, hinge and / or CH2-CH3 region (or optional CH2-CH3-CH4 region). In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof of the present invention comprises a 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 anti-CD39 antibody or antigen-binding fragment thereof of the present invention may be identical to the wild-type constant region sequence or may differ in one or more mutation points.

[0368] In certain embodiments, the heavy chain constant region includes an Fc region. It is known that the Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. The ability of the Fc regions of different Ig isotypes to induce effector functions is different. For example, it has been recognized that the Fc regions of IgG1 and IgG3 are more effective in inducing ADCC and CDC than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof of the present invention includes an Fc region of an IgG1 or IgG3 isotype, which can induce ADCC or CDC; or includes a constant region of an IgG4 or IgG2 isotype, which has reduced or depleted effector functions. In some embodiments, the Fc region derived from human IgG1 has reduced effector functions. In some embodiments, the Fc region derived from human IgG1 includes L234A and / or L235A mutations. In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof of the present invention includes an Fc region of wild-type human IgG4 or other wild-type human IgG4 alleles. In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof of the present invention comprises an Fc region of human IgG4, wherein the Fc region comprises an S228P mutation and / or an L235E mutation and / or an F234A and L235A mutation. In certain embodiments, the Fc region derived from human IgG4 comprises an S228P mutation and / or an F234A and L235A mutation.

[0369] In certain embodiments, the antibodies or fragments thereof described herein have sufficient affinity to specifically bind to human CD39 for diagnostic and / or therapeutic use.

[0370] The antibody or fragment thereof described in the present invention can be a monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a recombinant antibody, a bispecific antibody, a multispecific antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein. Recombinant antibodies are antibodies prepared in vitro using a recombinant method (rather than in an animal).

[0371] In certain embodiments, the present invention provides an anti-CD39 antibody or an antigen-binding fragment thereof, which competes with the antibody or antigen-binding fragment thereof according to the present invention for binding to human CD39. In certain embodiments, the present invention provides an anti-CD39 antibody or an antigen-binding fragment thereof, which competes with an antibody comprising a heavy chain variable region and a light chain variable region for binding to human CD39, wherein the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 43, and the light chain variable region comprises a sequence as shown in SEQ ID NO: 52. In certain embodiments, the present invention provides an anti-CD39 antibody or an antigen-binding fragment thereof, which competes with an antibody comprising a heavy chain variable region and a light chain variable region for binding to human CD39, wherein the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 44, and the light chain variable region comprises a sequence as shown in SEQ ID NO: 53. In certain embodiments, the present invention provides an anti-CD39 antibody or an antigen-binding fragment thereof, which competes for binding to human CD39 with an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence as shown in SEQ ID NO: 45, and the light chain variable region comprises the sequence as shown in SEQ ID NO: 54, or competes for binding to human CD39 with an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence as shown in SEQ ID NO: 47, and the light chain variable region comprises the sequence as shown in SEQ ID NO: 56.

[0372] In certain embodiments, the present invention provides an anti-CD39 antibody or an 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.

[0373] In certain embodiments, the epitope comprises one or more residues selected from the group consisting of Q96, N99, E143, and R147. In certain embodiments, the epitope comprises residues Q96, N99, E143, and R147.

[0374] In certain embodiments, the epitope comprises one or more residues selected from the group consisting of: R138, M139, and E142. In certain embodiments, the epitope comprises residues R138, M139, and E142.

[0375] In certain embodiments, the epitope comprises one or more residues selected from the group consisting of K5, E100, and D107. In certain embodiments, the epitope comprises residues K5, E100, and D107.

[0376] In certain embodiments, the epitope comprises one or more residues selected from the group consisting of V81, E82, R111, and V115. In certain embodiments, the epitope comprises residues V81, E82, R111, and V115.

[0377] In certain embodiments, the CD39 is human CD39. In certain embodiments, the CD39 is human CD39 comprising the amino acid sequence shown in SEQ ID NO:162.

[0378] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is not any of antibody 9-8B, antibody T895, and antibody I394.

[0379] The "9-8B" used in the present invention refers to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 46, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 48.

[0380] The "T895" used in the present invention refers to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 55, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 57.

[0381] The "I394" used in the present invention refers to an antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 113, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 114.

[0382] Antibody variants

[0383] The antibodies and antigen-binding fragments thereof provided by the present invention also include various variants of the antibody sequences provided by the present invention.

[0384] In certain embodiments, the antibody variants comprise one or more modifications or substitutions in one or more CDR sequences shown in Table 1 above, one or more non-CDR sequences of the heavy chain variable region or light chain variable region sequences of the present invention shown in Tables 4, 5, 8 and 9 above, and / or in the constant region (e.g., Fc region). These antibody variants retain the affinity of their parent for specific binding to CD39, but have the desired properties brought about by one or more of the modifications or substitutions. For example, the antibody variants may have improved antigen binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or depleted effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility with conjugation (e.g., one or more introduced cysteine ​​residues).

[0385] Parent antibody sequences can be screened to identify suitable or preferred residues to be modified or substituted using methods known in the art, such as "alanine scanning mutagenesis" (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 by uncharged or negatively charged amino acids (e.g., alanine or polyalanine), resulting in modified antibodies, and screened for target properties. If a substitution at a particular amino acid position shows a change in the target functionality, the position can be identified as a potential residue for modification or substitution. The potential residue can be further evaluated by replacing it with another residue (e.g., a cysteine ​​residue, a positively charged residue, etc.).

[0386] Affinity variants

[0387] The affinity variants of the antibody may comprise modifications or substitutions in one or more CDR sequences shown in Table 1 above, one or more FR sequences shown in Tables 4, 5, 8, 9 above, or in the heavy chain or light chain variable region sequences shown in Tables 2, 3, 6 and 7 above. A person skilled in the art can easily determine the FR sequence based on the CDR sequences in Table 1 above and the variable region sequences in Tables 2, 3, 6 and 7 above, because it is well known in the art that in the variable region, the CDR region is flanked by two FR regions. The affinity variants retain the affinity of the parent antibody for specific binding to CD39, or even have an improved affinity for specific binding to CD39 relative to the parent antibody. In certain embodiments, at least one (or all) substitutions in the CDR sequence, FR sequence or variable region sequence comprise conservative substitutions.

[0388] Those skilled in the art will appreciate that one or more amino acid residues may be substituted in the CDR sequences provided in Table 1 above, in the variable region sequences provided in Tables 2, 3, 6 and 7 above, and the resulting antibody or antigen-binding fragment still maintains binding affinity or binding capacity to CD39, or even has improved binding affinity or capacity. Various methods known in the art may be used to achieve this goal. For example, a library of antibody variants (e.g., Fab or scFv variants) may be generated and expressed using phage display technology, and then screened for affinity for binding to human CD39. For another example, computer software may be used to simulate the binding of antibodies to human CD39 and identify the amino acid residues on the antibody that form the binding interface. These residues may be avoided in substitution to prevent a reduction in binding affinity, or may be targeted for substitution to obtain stronger binding.

[0389] In certain embodiments, the humanized antibody or antigen-binding fragment thereof of the present invention comprises one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variant comprises no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substitutions in total in CDR sequences and / or FR sequences.

[0390] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises 1, 2 or 3 CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with the sequences listed in Table 1 above, while maintaining a similar or higher level of specific binding affinity to CD39 relative to its parent antibody.

[0391] In certain embodiments, the 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 with the sequences listed in Tables 2, 3, 6, and 7 above, while maintaining a similar or higher level of specific binding affinity to CD39 relative to its parent antibody. In some embodiments, a total of 1 to 10 amino acids are 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 CDR (e.g., in FR).

[0392] Glycosylation variants

[0393] The anti-CD39 antibody or antigen-binding fragment thereof of the present invention further comprises a glycosylation variant, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment thereof.

[0394] The antibody or its antigen-binding fragment may include one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) may be attached. The glycosylation of an antibody is typically N-connected or O-connected. N-connected refers to the attachment of a carbohydrate moiety to the side chain of an aspartic acid residue, e.g., an aspartic acid residue in a tripeptide sequence, such as aspartic acid-X-serine and aspartic acid-X-threonine, wherein X is any amino acid except proline. O-connected glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly to serine or threonine. Natural glycosylation sites can be easily removed, e.g., by changing the amino acid sequence so that one of the above-mentioned tripeptide sequences (for N-connected glycosylation sites) or serine or threonine residues (for O-connected glycosylation sites) present in the sequence is replaced. In a similar manner, new glycosylation sites can be generated by introducing such a tripeptide sequence or a serine or threonine residue.

[0395] In certain embodiments, the present invention provides an anti-CD39 antibody or an antigen-binding fragment thereof, comprising one or more mutations at a position selected from N55, G56 and N297 to remove one or more deamidation sites. In certain embodiments, the present invention provides an anti-CD39 antibody or an antigen-binding fragment thereof, comprising a mutation of N55 (e.g., N55G, N55S or N55Q) and / or a mutation of G56 (e.g., G56A, G56D) and / or a mutation of N297 (e.g., N297A, N297Q or N297G). These mutations were tested and are not believed to have a negative impact on the binding affinity of the antibodies provided by the present invention.

[0396] Cysteine ​​engineered variants

[0397] The anti-CD39 antibodies and antigen-binding fragments of the present invention also include cysteine ​​engineered variants, which include one or more introduced free cysteine ​​amino acid residues.

[0398] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bond. Cysteine ​​engineered variants can be used to conjugate, for example, cytotoxic compounds and / or imaging compounds, labels or radioisotopes and other substances at the site of the engineered cysteine, for example, via maleimide or haloacetyl groups. Methods for modifying antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are well known in the art, see, for example, WO2006 / 034488.

[0399] Fc variants

[0400] The anti-CD39 antibodies or antigen-binding fragments thereof described herein also include Fc variants, which include one or more amino acid residue modifications or substitutions in the Fc region and / or hinge region, for example, to provide altered effector functions, such as ADCC and CDC. Methods for altering ADCC activity by antibody engineering have been described in the prior art, see, for example, 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 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.

[0401] The CDC activity of the antibodies or antigen-binding fragments provided herein can also be altered, for example, by improving or reducing C1q binding and / or CDC (see, e.g., WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94 / 29351 for other examples of Fc region variants). One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be replaced with different amino acid residues to alter C1q binding and / or reduce or eliminate complement dependent cytotoxicity (CDC) (see U.S. Pat. No. 6,194,551 to No. Idusogie et al.). One or more amino acid substitutions can also be introduced to alter the ability of the antibody to fix complement (see PCT Publication No. WO94 / 29351 to Bodmer et al.).

[0402] 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, and any combination thereof. In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided herein are of the IgG1 isotype and comprise L234A and L235A mutations. In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof provided by the present invention is of IgG2 isotype and comprises one or more amino acid substitutions selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A and any combination thereof (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof provided by the present invention is of IgG4 isotype and comprises one or more amino acid substitutions selected from the group consisting of S228P, N297A, N297Q, N297G, L235E, F234A, L235A and any combination thereof. In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof provided by the present invention is an IgG2 / IgG4 cross-isotype. Examples of IgG2 / IgG4 cross-isotypes are described in Rother RP et al., Nat Biotechnol 25:1256-1264 (2007).

[0403] In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments thereof provided herein are of IgG4 isotype and comprise one or more amino acid substitutions at one or more positions of 228, 234, and 235. In certain embodiments, the anti-CD39 antibodies and antigen-binding fragments provided herein are of IgG4 isotype and have S228P and / or L235E and / or F234A and L235A mutations in the Fc region.

[0404] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions that can improve pH-dependent binding to neonatal Fc receptor (FcRn). Such a variant can have a prolonged pharmacokinetic half-life because it binds to FcRn at acidic pH, thereby preventing it from being degraded in lysosomes and subsequently being transferred and released outside the cell. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, e.g., 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).

[0405] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment comprises one or more amino acid substitutions at the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include the introduction of protrusions into a first Fc polypeptide and the introduction of a cavity into a second Fc polypeptide, wherein the protrusions may be located within the cavity to promote interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for producing antibodies having these modifications are well known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0406] Fab

[0407] The present invention also provides anti-CD39 antigen binding fragments. Various types of antigen binding fragments are well known in the art and can be developed based on the anti-CD39 antibodies described in the present invention, including exemplary antibodies, whose CDRs are shown in Table 1 above, whose variable region sequences are shown in Tables 2, 3, 6 and 7 above, and different variants thereof (e.g., affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered antibodies, etc.).

[0408] In certain embodiments, the anti-CD39 antigen-binding fragment of the present invention 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 and bivalent domain antibody.

[0409] A variety of techniques can be used for the production of such antigen-binding fragments. Exemplary 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 by host cells (e.g., E. coli) (e.g., for Fab, Fv, and ScFv antibody fragments), screening of phage display libraries as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0410] In certain embodiments, the antigen binding fragment is a scFv. The generation of scFv is described in, for example, WO 93 / 16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. The scFv can be fused to an effector protein at the amino or carboxyl terminus to obtain a fusion protein (see, for example, Antibody Engineering, Borrebaeck, ed.).

[0411] In certain embodiments, the anti-CD39 antibodies or antigen-binding fragments thereof provided by the present invention are divalent, tetravalent, hexavalent or multivalent. Any molecule greater than divalent is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0412] A bivalent molecule can be monospecific if both binding sites can specifically bind to the same antigen or the same epitope. In certain embodiments, this provides stronger binding to the antigen or epitope than a corresponding monovalent molecule. Similarly, multivalent molecules can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen binding portion, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences but have the same specificity).

[0413] A bivalent 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 when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0414] Bispecific Antibodies

[0415] In certain embodiments, the anti-CD39 antibody or antigen-binding fragment thereof is bispecific. In certain embodiments, the antibody or antigen-binding fragment thereof is further linked to a second functional moiety having a binding specificity different from that of the CD39 antibody or antigen-binding fragment thereof.

[0416] In certain embodiments, the bispecific antibody or antigen-binding fragment thereof provided by the present invention 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.

[0417] Conjugate

[0418] In some embodiments, the anti-CD39 antibody or its antigen binding fragment further comprises one or more conjugate parts. The conjugate part can be connected to the antibody or its antigen binding fragment. The conjugate part is a part that can be attached to the antibody or its antigen binding fragment. It can be imagined that the antibody or its antigen binding fragment described in the present invention can be connected to a variety of conjugate parts (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate parts can be connected to the antibody or its antigen binding fragment by other means such as covalent binding, affinity binding, embedding, coordinate binding, complexation, association, blending or addition. In certain embodiments, the antibody or its antigen binding fragment is connected to one or more conjugates via a linker.

[0419] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein can be engineered to contain specific sites outside of the epitope binding moiety that can be used to bind to one or more conjugate moieties. For example, the site can include one or more reactive amino acid residues (e.g., cysteine ​​or histidine residues) to facilitate covalent attachment to the conjugate moiety.

[0420] In some embodiments, the antibody or its antigen binding fragment can be connected to the conjugate part indirectly or through another conjugate part. For example, the antibody or its antigen binding fragment provided by the present invention can be conjugated with biotin and then indirectly conjugated with a second conjugate, which is conjugated with avidin. In some embodiments, the conjugate part includes a scavenging modifier (e.g., a polymer (e.g., PEG) that extends the 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 binding agent, a purification part or other anticancer drugs.

[0421] A "toxin" can be any agent that is harmful to cells or can damage or kill cells. Examples of toxins include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin 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 dacarbazine), alkylating agents (e.g., nitrogen mustard, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and dichlorodiamine platinum (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-binding agents.

[0422] Examples of detectable labels can include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioactive isotopes (e.g., 123 I. 124 I. 125 I. 131 I. 35 S. 3 H. 111 In, 112 In, 14 C. 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, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection.

[0423] In some embodiments, the conjugate moiety can be a clearance modifier, which helps to increase the half-life of the antibody. Illustrative examples include water-soluble polymers (e.g., PEG, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol / propylene glycol copolymers, etc.). The polymer can have any molecular weight and can be branched or unbranched. The number of polymers connected to the antibody can vary, and if the polymer connected is more than one, they can be the same or different molecules.

[0424] In certain embodiments, the conjugate moiety may be a purification moiety, such as a magnetic bead.

[0425] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are used as the basis of a conjugate.

[0426] Polynucleotides and recombinant methods

[0427] The present invention provides isolated polynucleotides encoding the anti-CD39 antibodies or antigen-binding fragments thereof. The term "nucleic acid" or "polynucleotide" used in the present invention refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise indicated, a specific polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as sequences explicitly indicated. 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)).

[0428] DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Encoding DNA can also be obtained synthetically.

[0429] Using recombinant techniques known in the art, the isolated polynucleotide encoding the anti-CD39 antibody or its antigen-binding fragment can be inserted into a vector for further cloning (amplification of the DNA) or for expression. A variety of vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0430] The present invention provides vectors comprising isolated polynucleotides. In certain embodiments, the polynucleotides provided by the present invention encode antibodies or antigen-binding fragments thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX , pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0431] The vector comprising the polynucleotide sequence encoding the antibody or its antigen-binding fragment can be introduced into a host cell for cloning or gene expression. The host cell suitable for cloning or expressing the DNA in the vector described in the present invention is the above-mentioned prokaryotic, yeast or higher eukaryotic cell. Prokaryotic cells suitable for the purposes of the present invention include true bacteria, such as Gram-negative bacteria or Gram-positive bacteria, for example, Enterobacteriaceae, for example, Escherichia (Escherichia) (for example, Escherichia coli (E.coli)), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (for example, Salmonella typhimurium (Salmonella typhimurium)), Serratia (Serratia) (for example, Serratia marcescens (Serratia marcescans), Shigella, Bacilli (e.g., B. subtilis and B. licheniformis), Pseudomonas (e.g., P. aeruginosa), and Streptomyces.

[0432] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast can also be used as suitable cloning or expression hosts for vectors encoding anti-CD39 antibodies. Saccharomyces cerevisiae, or baker's yeast, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are commonly used and suitable for use in the present invention, for example, Schizosaccharomyces pombe; Kluyveromyces hosts, for example, 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 lipolytica (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentali; and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus (e.g., A. nidulans and A. niger).

[0433] The host cells suitable for expressing glycosylated antibodies or antigen-binding fragments thereof provided in the present invention are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculoviral strains and variants thereof and corresponding permissive insect host cells have been found from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori. A variety of viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV, and the Bm-5 variant of Bombyx mori NPV, which can be used in the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0434] However, of greatest interest are spinal cells, and their cultivation (tissue culture) has become routine. Examples of mammalian host cells that can be used include SV40-transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, 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 testicular supporting 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 cancer cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In certain embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293 and derivatives thereof.

[0435] Host cells are transformed with the above-mentioned expression or cloning vectors that can produce anti-CD39 antibodies and cultured in a conventional nutrient medium that is modified to be suitable for inducing promoters, selecting transformed cells or amplifying genes encoding target sequences. In another embodiment, the antibodies can be prepared by homologous recombination methods known in the art. In certain embodiments, the host cells are capable of producing the antibodies or antigen-binding fragments thereof of the present invention.

[0436] The present invention also provides a method for expressing the antibody or antigen-binding fragment thereof of the present invention, comprising culturing the host cell provided by the present invention under the condition of expressing the vector of the present invention. The host cell used to produce the antibody or antigen-binding fragment thereof in the present invention can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM, (Sigma)), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) can be used to culture the host cell. In addition, any medium described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4767704; 4657866; 4927762; 4560655; or 5122469; WO 90 / 03430; WO87 / 00195; or U.S. Pat. No. Re. 30985 can be used as a culture medium for the host cells. These culture media can be supplemented with necessary hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride and phosphate), buffers (such as HEPES), nucleotides (such as adenylate and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds with final concentrations usually in the micromolar range), and glucose or an energy source equivalent thereto. The culture medium may also contain any other necessary additives known in the art at appropriate concentrations. The conditions of the culture medium, such as temperature, pH value and the like, are those previously used for the host cell selected for expression and are well known to ordinary technicians.

[0437] When using recombinant technology, antibodies can be produced intracellularly in the periplasmic space, or directly secreted into the matrix. If the antibody is produced intracellularly, the first step is to remove the particle debris (host cell or cracked fragments), for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for separating antibodies secreted into the periplasmic space of Escherichia coli. Briefly, the cell paste is thawed for more than 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. When the antibody is secreted into the matrix, a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device, is usually first used to concentrate the supernatant from the expression system. Any of the aforementioned steps may include a protease inhibitor, such as PMSF, to inhibit proteolysis, and may include antibiotics to prevent the growth of accidental contaminants.

[0438] The anti-CD39 antibody or antigen-binding fragment thereof prepared from the cells can be purified by purification methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography column, ammonium sulfate precipitation, salting out and affinity chromatography, wherein affinity chromatography is the preferred purification technique.

[0439] In certain embodiments, protein A fixed to a solid phase is used for immunoaffinity purification of the antibody and its antigen-binding fragment. The type of the antibody and the presence of any immunoglobulin Fc domain in the antibody determine whether protein A is suitable as an affinity ligand. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is suitable for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 1567 1575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than agarose. If the antibody contains a CH3 domain, Bakerbond ABX can be used. TM The antibody was purified by HPLC-MS / MS (JT Baker, Phillipsburg, NJ). Other protein purification techniques may also be determined based on the antibody to be obtained, such as fractionation in an ion exchange column, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose chromatography based on anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0440] Following any preliminary purification steps, the mixture comprising the antibody of interest and impurities can be subjected to low pH hydrophobic interaction chromatography using a wash buffer having a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., from about 0 to 0.25 M salt concentration).

[0441] Pharmaceutical composition

[0442] The present invention further provides a pharmaceutical composition comprising the anti-CD39 antibody or antigen-binding fragment thereof of the present invention, and one or more pharmaceutically acceptable carriers.

[0443] The pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed in the present invention may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous solvents, non-aqueous solvents, antimicrobial substances, isotonic substances, buffers, antioxidants, anesthetics, suspending agents / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components well known in the art or various combinations thereof.

[0444] Applicable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Applicable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl gallate. As disclosed in the present invention, one or more antioxidants such as methionine are included in the composition comprising the antibody or its antigen-binding fragment disclosed in the present invention, which can reduce the oxidation of the antibody or its antigen-binding fragment. The reduction of oxidation can prevent or reduce the reduction of binding affinity, thereby improving antibody stability and extending shelf life. Therefore, in certain embodiments, the pharmaceutical composition provided by the present invention comprises one or more antibodies or their antigen-binding fragments described in the present invention and one or more antioxidants such as methionine. The present invention further provides various methods for preventing oxidation of the antibodies or antigen-binding fragments thereof, extending their shelf life and / or improving their activity, for example, by mixing the antibodies or antigen-binding fragments thereof provided in the present invention with one or more antioxidants (e.g., methionine).

[0445] Further, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous media such as fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinyl pyrrolidone, emulsifiers such as polysorbate 80 (Tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents as carriers may be added to the pharmaceutical compositions in multiple dose containers and include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, glucose, glycerol or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting agents, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

[0446] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, saccharin sodium, cellulose, magnesium carbonate, etc.

[0447] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, for example, a liquid solvent, a suspending agent, an emulsifier, or a solid form suitable for producing a liquid solvent, a suspending agent, or an emulsifier. Injectable formulations can include sterile and / or pyrogen-free solutions, sterile dried solubles that are combined with solvents before use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions for injection, sterile dried insoluble products that are combined with media before use, and sterile and / or pyrogen-free emulsions. The solvent can be an aqueous phase or a non-aqueous phase.

[0448] In certain embodiments, the unit dose of the injection preparation is packaged in an ampoule, a tube or a syringe with a needle. As is known in the art, all preparations for injection should be sterile and pyrogen-free.

[0449] In certain embodiments, a sterile lyophilized powder can be prepared by dissolving the antibody or antigen-binding fragment thereof disclosed in the present invention in a suitable solvent. The solvent may contain a component that can improve the stability of the powder or a reconstituted solution obtained from the powder, or improve other pharmacological components of the powder or the reconstituted solution. Applicable excipients include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other applicable substances. The solvent may contain a buffer, such as a citrate buffer, a sodium phosphate or potassium phosphate buffer or other buffer known to those skilled in the art, and in one embodiment, the pH of the buffer is neutral. The dissolution is subsequently sterilized by filtration under standard conditions known in the art, and then lyophilized to obtain an ideal formulation. In one embodiment, the resulting solvent is dispensed into small tubes and lyophilized. Each small tube can accommodate a single dose or multiple doses of the anti-CD39 antibody or antigen-binding fragment thereof or a composition thereof. The amount loaded in each small tube can be slightly higher than that required for each dose or multiple doses (e.g., 10% excess) to ensure accurate sampling and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at a temperature ranging from about 4°C to room temperature.

[0450] The lyophilized powder is reconstituted with water for injection to obtain a preparation for injection. In one embodiment, the lyophilized powder can be added to sterile pyrogen-free water or other applicable liquid carriers for reconstitution. The exact amount is determined by the selected therapy and can be determined based on empirical values.

[0451] Reagent test kit

[0452] In certain embodiments, the present invention provides a kit comprising an antibody or antigen binding fragment thereof provided by the present invention and / or a pharmaceutical composition provided by the present invention. In certain embodiments, the present invention provides a kit comprising an antibody or antigen binding fragment thereof provided by the present invention and a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from chemotherapeutics, anticancer drugs, radiotherapeutics, immunotherapeutics, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators and cytokines.

[0453] If desired, such a kit may further include one or more of the various conventional pharmaceutical kit components, such as a container with one or more pharmaceutically acceptable carriers, additional containers, etc., which will be apparent to those skilled in the art. Instructions (as inserts or labels) indicating the amounts of the components to be administered, instructions for administration, and / or instructions for mixing the components may also be included in the kit.

[0454] How to use

[0455] The present invention also provides a method for treating a disease, disorder or condition associated with CD39 in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the present invention and / or a pharmaceutical composition according to the present invention. In certain embodiments, the subject is a human.

[0456] In some embodiments, the disease, disorder, or condition associated with CD39 is characterized by expression or overexpression of CD39.

[0457] In some embodiments, the disease, disorder or condition associated with CD39 is cancer. In some embodiments, the cancer is a cancer expressing CD39. The "CD39-expressing" cancer used in the present invention refers to a cancer characterized by expressing CD39 in cancer cells or immune cells or immunosuppressive cells infiltrating tumors, and the level of CD39 expressed in cancer cells or immune cells or immunosuppressive cells infiltrating tumors is significantly higher than the expected level of normal cells. A variety of methods can be used to determine the presence or content 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 an antigen-binding fragment thereof, which binds to and detects the expressed CD39 protein. Alternatively, CD39 can also be detected at the level of nucleic acid expression using methods such as qPCR, reverse transcriptase PCR, microarrays, SAGE, FISH, etc. In some embodiments, the test sample is from a cancer cell or tissue or an immune cell infiltrating a tumor. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same person from whom the test sample was obtained. For example, a reference sample can be a non-diseased sample adjacent or nearby to a test sample (eg, a tumor).

[0458] 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, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small intestine cancer, urethra 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 cancer, skin cancer In some embodiments, the cancer is leukemia, lymphoma, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, or breast cancer.

[0459] 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 levels significantly higher than those normally found on non-cancerous or non-immunosuppressive cells.

[0460] In some embodiments, the disease, disorder or condition is an autoimmune disease or infection. In some embodiments, the autoimmune disease is immune thrombocytopenia, systemic scleroderma, 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.

[0461] In another aspect, a method of treating a disease, disorder or condition in an individual who would benefit from modulation of CD39 activity is provided, the method comprising administering to an individual in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof and / or a pharmaceutical composition as described herein. In certain embodiments, the disease, disorder or condition is a disease, disorder or condition associated with CD39 as defined above.

[0462] The therapeutically effective dose of the antibody or antigen-binding fragment thereof of the present invention depends on various factors known in the art, such as body weight, age, past medical history, current treatment, health status of the subject and potential for cross infection, allergy, hypersensitivity and side effects, as well as route of administration and degree of tumor development. A person skilled in the art (e.g., a doctor or veterinarian) may proportionally reduce or increase the dose according to these or other conditions or requirements.

[0463] In certain embodiments, antibodies or antigen binding fragments as described herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the dosage can vary with the course of treatment. For example, in certain embodiments, the initial dosage can be higher than the subsequent dosage. In certain embodiments, the dosage is adjusted during the course of treatment based on the response of the subject being administered.

[0464] Dosage regimens may be adjusted to achieve the optimum response (eg, therapeutic response). For example, a single dose may be administered or multiple divided doses may be administered over a period of time.

[0465] The antibodies or antigen-binding fragments thereof disclosed in the present invention can be administered by administration methods known in the art, such as parenteral administration (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection, including intravenous infusion, intramuscular injection or intradermal injection) or non-parenteral administration routes (e.g., oral administration, nasal administration, sublingual administration, rectal administration or topical administration).

[0466] In some embodiments, the antibody or antigen-binding fragment thereof disclosed in the present invention can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibody or antigen-binding fragment thereof disclosed in the present invention can be administered in combination with a second therapeutic agent (e.g., a chemotherapeutic agent, an anticancer agent, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic 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, or a cytokine).

[0467] The term "immunotherapy" as used herein refers to a therapy that stimulates the immune system to fight a disease (e.g., cancer) or generally enhances the immune system. Examples of immunotherapy include, but are not limited to, checkpoint regulators, adoptive cell transfer, cytokines, oncolytic viruses, and therapeutic vaccines.

[0468] "Targeted therapy" is a therapy that acts on specific molecules involved in cancer, such as specific proteins that are present in cancer cells but not in normal cells or are more abundant in cancer cells, or target molecules in the cancer microenvironment that help cancer grow and survive. Targeted therapy targets the therapeutic agent to the tumor, sparing normal tissue from the effects of the therapeutic agent.

[0469] In some such embodiments, when the antibodies or antigen-binding fragments thereof disclosed in the present invention are used in combination with one or more additional therapeutic agents, they can be administered simultaneously with the one or more additional therapeutic agents. In some such embodiments, the antibodies or antigen-binding fragments thereof and the additional therapeutic agents can be administered simultaneously as part of the same pharmaceutical composition. However, the antibodies or antigen-binding fragments thereof "used in combination" with other therapeutic agents do not need to be administered simultaneously or in the same composition as the therapeutic agent. The meaning of "used in combination" in the present invention also includes that the antibodies or antigen-binding fragments thereof administered before or after another therapeutic agent are also considered to be "used in combination" with the therapeutic agent, even if the antibodies or antigen-binding fragments thereof and the second substance are administered by different administration methods. Where possible, the other therapeutic agents used in combination with the antibodies or antigen-binding fragments thereof disclosed in the present invention can be administered according to the methods of the product instructions of the other therapeutic agents, or according to the Surgeon's Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)), or according to other methods known in the art.

[0470] In another aspect, the present invention further provides a method for regulating CD39 activity in CD39-positive cells, comprising exposing CD39-positive cells to an antibody or antigen-binding fragment thereof provided by the present invention. In some embodiments, the CD39-positive cells are immune cells.

[0471] In another aspect, the present invention provides a method for detecting the presence or content of CD39 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof and / or the pharmaceutical composition provided by the present invention, and determining the presence or content of CD39 in the sample.

[0472] In another aspect, the present invention provides a method for diagnosing a disease, disorder or condition associated with CD39 in an individual, comprising: a) contacting a sample obtained from the individual with the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition provided by the present invention; b) determining the presence or amount of CD39 in the sample; and c) correlating the presence or amount of CD39 with the presence or condition of the disease, disorder or condition associated with CD39 in the individual.

[0473] In another aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention and / or the pharmaceutical composition provided by the present invention, which is optionally conjugated with a detectable moiety, and can be used to detect a disease, disorder or condition associated with CD39. The kit may further include instructions for use.

[0474] In another aspect, the present invention also provides the use of the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition provided by the present invention in the preparation of a medicament for treating, preventing or alleviating a disease, disorder or condition associated with CD39 in an individual, or in the preparation of a diagnostic reagent for diagnosing a disease, disorder or condition associated with CD39.

[0475] On the other hand, the present invention provides a method for treating, preventing or alleviating a disease that can be treated by reducing the ATPase activity of CD39 in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or an antigen-binding fragment thereof provided by the present invention and / or a pharmaceutical composition provided by the present invention. For example, the antibody or antigen-binding fragment thereof provided by the present invention can be administered to reduce the ATPase activity of cancer cells expressing CD39 or immune cells or immunosuppressive cells that infiltrate tumors. In some embodiments, the subject is human. In some embodiments, the subject has a disease, disorder or condition selected from the group consisting of cancer, autoimmune disease and infection.

[0476] In another aspect, the present invention provides a method for 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 the antibody or antigen-binding fragment thereof described in the present invention and / or the pharmaceutical composition described in the present invention.

[0477] The following examples are intended to better illustrate the present invention and should not be construed as limiting the scope of the present invention. All of the following specific compositions, materials and methods, in whole or in part, are within the scope of the present invention. These specific compositions, materials and methods are not intended to limit the present invention, but are only intended to illustrate that specific embodiments are within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials and methods without creative work and without departing from the scope of the present invention. It should be understood that the various modifications made to the method of the present invention may still be included within the scope of the present invention. The inventor intends to include such changes within the scope of the present invention.

[0478] Example

[0479] Example 1. Preparation of materials

[0480] 1.1 Generation of reference antibodies

[0481] Anti-CD39 reference antibodies were generated based on the disclosed sequences. Antibody 9-8B is disclosed in patent application WO2016 / 073845A1, and its heavy chain and light chain variable region sequences are SEQ ID NOs: 46 and 48, respectively, in this application. Antibody T895 is disclosed as antibody 31895 in patent application WO 2019 / 027935A1, and its heavy chain and light chain variable region sequences are SEQ ID NOs: 55 and 57, respectively, in this application. Antibody I394 is disclosed in patent application WO 2018 / 167267A1, and its heavy chain and light chain variable region sequences are SEQ ID NOs: 113 and 114, respectively, in this application. The heavy chain variable region and light chain variable region of antibodies 9-8B, T895 and I394 are shown in Table 10 below. DNA sequences encoding reference antibodies were cloned and expressed in Expi293 cells (Invitrogen). Cell culture medium was collected and centrifuged to remove cell pellets. The harvested supernatant was purified using a protein A affinity chromatography column (Mabselect Sure, GE Healthcare) to obtain a reference antibody preparation.

[0482] Table 10. Variable region amino acid sequences of three reference antibodies

[0483]

[0484]

[0485] 1.2 Establishment of CD39 stable expression cell lines in humans, cynomolgus monkeys and mice

[0486] The DNA sequences encoding full-length human CD39 (NP_001767.3), cynomolgus CD39 (XP_015311944.1) and mouse CD39 (NP_033978.1) were cloned into expression vectors and then transfected and expressed in HEK293 cells. Transfected cells expressing human CD39, cynomolgus CD39 and mouse CD39 were cultured in selective medium. Single cell clones stably expressing human CD39, cynomolgus CD39 or mouse CD39 were isolated by limiting dilution. Cells were then screened by FACS using anti-human CD39 antibody (BD, Cat#555464), anti-cynomolgus CD39 antibody (9-8B), and anti-mouse CD39 antibody (Biolegend, Cat#143810).

[0487] In a similar manner, CHOK1 cells transfected with human CD39, cynomolgus CD39 or mouse CD39 expression plasmids (Invitrogen) were cultured in selection medium. Single cell clones stably expressing human CD39, cynomolgus CD39 or mouse CD39 were isolated by limiting dilution, and then cells were screened by FACS using the anti-human CD39 antibody, the anti-cynomolgus CD39 antibody or the anti-mouse CD39 antibody.

[0488] The stable cell lines were named HEK293-hCD39, HEK293-cynoCD39, HEK293-mCD39, CHOK1-hCD39, CHEK1-cynoCD39, and CHOK1-mCD39, and all of them showed high expression and ATPase activity.

[0489] 1.3 Production of recombinant proteins

[0490] The DNA sequence encoding the extracellular region (ECD) of human CD39 was cloned into an expression vector and transfected into HEK293 cells to express the ECD recombinant protein.

[0491] Example 2. Production of antibodies

[0492] 2.1 Immunization and hybridoma generation and screening

[0493] To generate antibodies against CD39, Balb / c and SJL / J mice (SLAC) were immunized with recombinantly expressed human CD39 antigen or fragments thereof or DNA encoding full-length human CD39 and / or cells expressing human CD39. During the immunization procedure, plasma and serum samples were obtained by bleeding from the tail vein or retroorbital to monitor the immune response. Mice with sufficient titers of anti-CD39 antibodies were used for fusion experiments. Splenocytes and / or lymph node cells from immunized mice were isolated and fused with mouse myeloma cell lines (SP2 / 0). Hybridomas that can produce CD39-specific antibodies were screened by ELISA analysis with human CD39 ECD recombinant protein or by Acumen analysis (TTP Labtech) with CHOK1-hCD39 cells that stably express human CD39. The specificity of the hybridoma clones to hCD39 was confirmed by FACS and enzyme activity blocking experiments, and they were subcloned to obtain stable hybridoma clones. After 1-2 rounds of subcloning, monoclonal hybridomas were expanded to produce antibodies and stored frozen.

[0494] The antibody-secreting hybridomas are subcloned by limiting dilution. Stable subclones are cultured in vitro to produce antibodies for characterization in tissue culture medium. After 1-2 rounds of subcloning, monoclonal hybridomas are expanded to produce antibodies.

[0495] After culturing for about 14 days, the hybridoma cell culture medium was collected and purified by protein A affinity chromatography column (GE). The hybridoma clone antibodies were named mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 and mAb35, respectively.

[0496] Example 3. Antibody Characterization

[0497] 3.1 Antibodies

[0498] Hybridoma clones mAb13, mAb14, mAb19, mAb21, mAb23, mAb34 and mAb35 were characterized in a series of binding and functional assays described in detail below.

[0499] 3.2 Binding affinity to human CD39, cynomolgus monkey CD39 and mouse CD39

[0500] 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 cells lacking CD39 expression (CHOK1-blank) as a negative control.

[0501] CHOK1-hCD39, CHOK1-cCD39, CHOK1-mCD39, and CHOK1-blank cells were cultured in culture medium according to ATCC procedures. The cells were collected and plated at 3 × 10 6 Resuspend the cells in blocking buffer at a density of 10 cells / ml. 5 The cells were transferred to a 96-well FACS plate at a density of 10 cells / well. The plate was centrifuged and washed twice with FACS buffer (PBS, 1% FBS, 0.05% Tween-20). Starting at 30 μg / ml, 4-fold serial dilutions of anti-CD39 antibody were prepared in FACS buffer. Reference antibody 9-8B and mouse / human IgG control were used as positive and negative controls, respectively. The cells were resuspended in 100 μL / well of the diluted antibody and the plate was incubated at 4°C for 60 minutes. The plate was washed with FACS buffer and Alexa Fluor 500 was added. Labeled secondary antibody (1:1000 in FACS buffer) was added to each well and incubated at 4°C for 30 min. The plate was washed with FACS buffer and the cells were resuspended in 100 μL / well of PBS. TMCells were analyzed and mean fluorescence intensity was determined. A complete binding curve for CD39 expressing cells was generated by testing a range of antibody concentrations. The apparent affinity of each antibody was determined using Prism software.

[0502] Similarly, cells SK-MEL-5, SK-MEL-28 or MOLP-8 expressing human CD39 were incubated with gradient concentrations of anti-CD39 antibodies at 4°C for 30 minutes. The cells were washed 3 times with FACS buffer and then incubated with fluorescently labeled secondary antibodies (goat anti-mouse IgG or goat anti-human IgG) at 4°C for 30 minutes. The cells were washed 3 times, then resuspended in FACS buffer and analyzed by flow cytometry on BD Celesta. GraphPad Prism 8.02 was used to plot and analyze the data.

[0503] The binding affinities of the seven purified hybridoma antibodies compared to the known anti-CD39 antibody 9-8B are shown in Table 11. All hybridoma antibodies bound to human and cynomolgus CD39 in a dose-dependent manner, but no hybridoma antibody recognized mouse CD39 in FACS analysis.

[0504]

[0505] 3.3 Detection of ATPase inhibition

[0506] SK-MEL-5 and MOLP-8 cells expressing CD39 were washed with PBS buffer and incubated with gradient antibodies 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 the orthophosphate products from ATP degradation were detected using a Malachite Green Phosphate Detection Kit (R&D system, Catalog # DY996) according to the manufacturer's manual. Isotypes 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% signal in this assay.

[0507] As summarized in Table 11, all seven purified hybridoma antibodies had good ATPase inhibitory activity compared to reference antibody 9-8B.

[0508] 3.4 Analysis of ATP-mediated T cell proliferation inhibition

[0509] Human T cells labeled with CSFE and stimulated with anti-CD3 and anti-CD28 were incubated with anti-CD39 antibody or isotype control in the presence of ATP. The proliferation of T cells was analyzed in FACS by CSFE dilution. mIgG2a was used as isotype control.

[0510] The T cell proliferation activity of the selected anti-CD39 antibodies mAb21 and mAb23 was as follows Figure 1 and are summarized in Table 11. 50 is the concentration of the indicated antibody that achieves 50% signal in this assay. Both antibodies enhanced T cell proliferation in a dose-dependent manner, ie, both antibodies blocked ATP-mediated inhibition of T cell proliferation.

[0511] 3.5 Epitope Binning

[0512] Anti-CD39 antibody was labeled with Alex488 labeling kit and diluted in a series of concentrations before mixing with CHOK1-hCD39 cells to detect EC binding using FACS. 80 The blocking efficacy of unlabeled antibodies on labeled antibodies was tested. Briefly, CHOK1-hCD39 monocytes were prepared to a density of 2 x 10 6 / ml, and inoculated into 96-well plates at 50 μl / well, then mixed with gradient antibodies to a final volume of 100 μl, and then diluted to twice the EC 80 The concentration of Alex488 labeled antibody was added in equal volumes. The 96-well plate was incubated at 4 ° C for 1 hour, centrifuged and washed 3 times with 200 μl FACS buffer. FACS analysis was performed on a FACScelesta machine, and the data were analyzed by Flowjo software. Compared with the non-competitive wells (only Alex488 labeled antibody), the blocking percentage was calculated, and the competition rate was higher than 80% and was classified into the same epitope group.

[0513] The competition results are shown in Table 12. Based on the competition results, the four anti-CD39 hybridoma antibodies (mAb14, mAb19, mAb21, mAb23) can be divided into four different epitope groups, as shown in Table 11. Specifically, anti-CD39 antibodies mAb19 and mAb21 compete for highly similar epitopes and are classified as epitope group I, as shown in Table 11. mAb14 does not compete with any other antibodies tested and is classified as epitope group IV, as shown in Table 11. mAb23 shows cross-competition with mAb19 and mAb21 and is classified as epitope group II in Table 11.

[0514] Table 12. Summary of epitope groups of anti-CD39 hybridoma antibodies

[0515]

[0516] 3.6 Hybridoma Sequencing

[0517] RNA was isolated from monoclonal hybridoma cells and reverse transcribed into cDNA using a commercial kit. The heavy and light chain variable regions were then amplified using the cDNA as a template using primers from the Mouse Ig-Primer Set (Novagen). The PCR products of the correct size were collected and purified and then ligated with a suitable plasmid vector. The ligated products were transformed into DH5α competent cells. Clones were screened and the inserts were analyzed by DNA sequencing.

[0518] The variable region sequences of the hybridoma antibodies are shown in Table 2 below.

[0519] Example 4. Production and characterization of chimeric antibodies

[0520] 4.1. Generation and production of chimeric antibodies

[0521] DNA encoding the variable regions of 4 selected hybridoma antibodies (mAb14, mAb19, mAb21 and mAb23) was synthesized and subcloned into an expression vector containing human IgG constant genes in advance. The vector was transfected into mammalian cells for recombinant protein expression, and the expressed antibodies were purified using a protein A affinity chromatography column. The resulting chimeric antibodies are referred to as c14, c19, c21 and c23 in this application, wherein the prefix "c" means "chimeric", and the numbers represent hybridoma antibody clones, for example, the number "14" means that it comes from the hybridoma antibody mAb14.

[0522] 4.2. Characterization of chimeric antibodies

[0523] The activity of the four purified chimeric antibodies in blocking ATP-mediated T cell proliferation inhibition was tested (similar to the method described in Example 3.4). Figure 2 As shown, anti-CD39 chimeric antibodies c14, c19, c21 and c23 blocked CD4 in a dose-dependent manner (at a concentration range of 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM and 0.001 nM). + Inhibition of T cell proliferation. CFSE-CD4 + T and hIgG4 were used as positive and negative controls, respectively, for ATP-mediated T cell proliferation.

[0524] The purified four chimeric antibodies were further tested for their ability to enhance ATP-induced dendritic cell (DC) activation and maturation in the presence of ATP. ATP induces DC maturation by stimulating the P2Y11 receptor of monocyte-derived DCs.

[0525] 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 4 different doses of anti-CD39 chimeric antibodies in the presence of ATP for another 24 hours. DC maturation was then evaluated by FACS analysis of the expression of CD86, CD83, and HLA-DR.

[0526] Figure 3 Shown are the levels of CD39 on the surface of DCs detected by FACS. Figure 4 A to 4C show the CD86 ( Figure 4 A) CD83 Figure 4 B) and HLA-DR ( Figure 4 C) Expression. ATP-induced DC maturation showed increased expression of CD86, CD83, and HLA-DR compared with vehicle treatment. The four anti-CD39 antibodies c14, c19, c21, and c23 all significantly promoted ATP-induced DC maturation.

[0527] The antitumor activity of the chimeric antibody was also tested in vivo. NOD-SCID mice were subcutaneously inoculated with tumor cells (10 x 10 6 ), for tumor development. When the average tumor size reaches about 80mm 3 Mice were randomized at 4 pm. Treatment was started on the same day of randomization at a dose of 30 mg / kg twice a week. After randomization, tumor volume was measured twice a week with a caliper in two dimensions and expressed in mm using the following formula: 3 Volume is represented as: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the width of the tumor (the longest tumor dimension perpendicular to L). Dosing was performed in a laminar flow cabinet, and tumor and body weight were measured. Two-way ANOVA was performed using Graphpad Prism.

[0528] Tumor growth results of chimeric anti-CD39 antibody c23 Figure 5 As shown. Human IgG1 and IgG4 types of c23 were obtained and detected. Compared with the vehicle group, both c23-hIgG4 and c23-hIgG1 chimeric antibodies showed anti-tumor effects, and there was no significant difference between c23-hIgG4 and c23-hIgG1.

[0529] Example 5. Humanization and affinity maturation of antibodies

[0530] 5.1 Humanization

[0531] Chimeric antibodies c23 and c14 were selected as humanized clones. The antibody sequences were aligned with human germline sequences to determine the best fitting model. Based on the homology with the original mouse antibody sequence, the best matching human germline sequence was selected as the template for humanization. The CDRs from the mouse antibody sequence were then grafted onto the template together with residues that maintained the upper core and central core structures of the antibody. Optimized mutations were introduced into the framework region to produce variants of the humanized heavy chain variable region and variants of the humanized light chain variable region, which were mixed and matched to provide multiple humanized antibody clones. After grafting and mutation, the humanized antibodies maintained similar binding affinity to cells expressing human CD39. The humanized antibodies were further evaluated by CD39 ATPase inhibition assays and in vitro immune cell activation assays. Some humanized antibodies were also studied in vivo.

[0532] A total of 31 humanized antibody clones of c23 were obtained, mixing and matching 7 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 7 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). The 31 humanized antibody clones were named hu23.H1L1, hu23.H1L2, etc., as shown in Table 9 above and Tables 13, 14 and 15 below, where the prefix "hu" means "humanized" and the suffix such as "H1L1" indicates a humanized antibody clone numbered c23 having a hu23.VH_1 variable region variant and a hu23.VL_1 variable region variant.

[0533] Table 13. Heavy chain variable region and light chain variable region of humanized antibody of c23

[0534]

[0535] Table 14. Heavy chain variable region and light chain variable region of humanized antibody of c23

[0536]

[0537] Table 15. Heavy chain variable region and light chain variable region of humanized antibody of c23

[0538]

[0539] Similarly, a total of 16 humanized antibodies of c14 were obtained, mixing and matching 4 variants of humanized c14 heavy chain variable regions (i.e., hu14.VH_1, hu14.VH_2, hu14.VH_3, and hu14.VH_4) and 4 variants of humanized c14 light chain variable regions (i.e., hu14.VL_1, hu14.VL_2, hu14.VL_3, and hu14.VL_4). The 16 humanized antibody clones were named hu14.H1L1, hu14.H1L2, and so on, as shown in Table 16 below.

[0540] Table 16. Heavy chain variable regions and light chain variable regions of 16 humanized antibodies of c14

[0541]

[0542] Several humanized antibody clones of c23 were also obtained by yeast display. In brief, the mouse heavy and light chain sequences were aligned with an internal database of human antibody sequences. The templates IGHV1-3*01 and IGKV3-11*01 with the highest homology were selected for heavy and light chain CDR grafting, respectively. Reverse mutations were identified by a high-throughput method using yeast display. Specifically, positions that contribute to CDR conformation (vernier region residues) were identified, and a reverse mutation library was created by incorporating templates and mouse residues at each position during DNA synthesis. The final candidates were determined by sequencing the primary binders of the human CD39 protein. The humanized antibodies of c23 obtained by yeast display were named hu23.201 (VH / VL with SEQ ID NO: 146 / 111), hu23.203 (VH / VL with SEQ ID NO: 146 / 112), hu23.207 (VH / VL with SEQ ID NO: 147 / 111) and hu23.211 (VH / VL with SEQ ID NO: 39 / 63).

[0543] The humanized antibodies in Tables 13, 14, 15 and 16 were recombinantly produced and subsequently subjected to binding affinity testing, showing that they were able to retain the specificity of binding to human CD39. Those antibodies with relatively high affinity were further evaluated in functional experiments including CD39 blocking assays and in vitro immune cell activation assays.

[0544] Specifically, Biacore (GE) was used to characterize the binding affinity of humanized antibodies hu23.H5L5, hu23.201, hu14.H1L1, and reference antibodies I394 and T895 to human CD39. In brief, the antibodies to be tested were captured into a CM5 chip (GE) using a Human Antibody Capture Kit (GE). The 6xHis-labeled human CD39 antigen was diluted several times and injected at 30 μl / min for 180 seconds. The buffer flow was maintained for 400 seconds. The chip was regenerated using 3M MgCl2. The association and dissociation curves were fitted to a 1:1 binding model, and the Ka / Kd / K of each antibody was calculated. D Table 17 below summarizes the data for the affinity of the antibodies tested.

[0545] Table 17. Binding affinity of antibodies to human CD39 as tested by Biacore assay.

[0546] Antibody ka(1 / Ms) kd(1 / s) <![CDATA[K D (M)]]> hu23.H5L5 8.22E+04 1.60E-03 1.95E-08 hu23.201 6.75E+04 1.62E-03 2.40E-08 hu14.H1L1 9.03E+05 4.55E-03 5.03E-09 I394 2.03E+05 1.26E-03 6.21E-09 T895 1.33E+05 1.39E-01 1.04E-06

[0547] In addition, according to the manufacturer's instructions, the anti-human CD39 binding affinity of humanized antibodies hu23.H5L5 and hu14.H1L2 and reference antibodies I394, T895 and 9-8B was characterized using the Octet assay (Creative Biolabs). In short, the antibody was coupled to the sensor, and then the sensor was immersed in a CD39 gradient solution (starting concentration of 200nM, 2-fold dilution, a total of 8 doses). Their binding reactions were measured in real time, and the results were globally fitted. The affinity data of the antibodies detected are summarized in Table 18 below.

[0548] Table 18. Binding affinity of antibodies to human CD39 as tested by Octet assay

[0549]

[0550]

[0551] In addition, an NG motif (N55G56) that is easy to deamidate was identified in the HCDR2 of the humanized antibody clone of the c23 antibody (e.g., hu23.H5L5). In order to remove the deamidation site, different mutations were introduced into N55 or G56, and it was found that N55 and G56 could each mutate into a variety of residues, but still retain the specificity of binding to human CD39. For example, it was found that when N55 was replaced by a single point of G, S or Q, the antibody binding affinity was retained and had no negative impact on its binding to human CD39. Similarly, when G56 was replaced by A or D, the mutant antibody also retained its specific binding and binding affinity to human CD39. It is expected that other mutations will also play the same role.

[0552] 5.2 Binding specificity detection

[0553] The binding specificity of the purified humanized antibody hu23.H5L5 to ENTPDase family members was detected by ELISA assay. In brief, ENTPD1 (i.e., CD39) and ENTPD 2 / 3 / 5 / 6 proteins were coated on a 96-well ELISA plate and placed at 4°C overnight. The next day, the ELISA plate was washed and blocked with 200 μl / well of blocking buffer (a mixture of PBS containing 1% BSA and 0.05% Tween20) for 2 hours. The hu23.H5L5 gradient solution was then transferred to the wells and stained with anti-hIgG-HRP. After the plate was washed, the plate was developed with TMB substrate and the reaction was terminated with 2N HCl. OD450 was recorded using a plate reader and graphed using GraphpadPrism. The binding specificity of hu23.H5L5 is shown in Figure 2. Figure 6 As shown. Figure 6 A: It can be seen that the humanized antibody hu23.H5L5 specifically binds to human CD39, but does not bind to any of the ENTPD 2 / 3 / 5 / 6 proteins. Figure 6 B shows that the negative control hIgG4 did not bind to any of the ENTPD 1 / 2 / 3 / 5 / 6 proteins.

[0554] 5.3. Characterization of humanized antibodies

[0555] The binding affinity of the c23 humanized antibodies was tested 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 Figure 7 A, 7B and Figure 8 In. EC 50 The indicated antibody concentration is that at which the signal in this assay reaches 50%.

[0556] Table 19. Binding activity of c23 humanized antibody to MOLP8 cells

[0557]

[0558]

[0559] ND: Not detected under the experimental conditions.

[0560] Table 20. Binding activity of humanized antibody c23 to MOLP8

[0561] Antibody hu23.201 hu23.203 hu23.207 hu23.211 c23 hIgG4 <![CDATA[EC 50 (nM)]]> 1.289 0.429 2.246 1.557 1.279 ND

[0562] ND: Not detected under the experimental conditions.

[0563] Selected c23 humanized antibodies were tested on SK-MEL-28 cells for ATPase inhibition assay (as described in Example 3.3). Fig. 9 Inhibition plots for the indicated antibodies are shown in A and 9B and are summarized in Table 21. hu23.H5L5 and hu23.201 were selected for further validation.

[0564] Table 21. ATPase inhibitory activity of humanized antibody of c23 on SK-MEL-28 cells

[0565] Antibody hu23.H7L1 hu23.H7L5 hu23.H7L6 hu23.H1L2 hu23.H1L4 hu23.H2L2 c23 <![CDATA[IC 50 (nM)]]> 0.147 0.144 0.121 0.964 0.59 0.767 0.158 Antibody hu23.H4L4 hu23.H5L5 hu23.H5L6 hu23.201 hu23.203 hu23.211 c23 <![CDATA[IC 50 (nM)]]> 0.487 0.122 0.13 0.264 0.32 0.386 0.2092

[0566] Using a similar method as described in Example 3.2, the binding affinity of the humanized antibody of c14 was tested by FACS using MOLP-8 cells expressing human CD39.

[0567] The humanized antibody clones of c14 that showed good binding affinity were Fig.10 Table 22 summarizes the EC 50 value.

[0568] Table 22. Binding activity of humanized antibody of c14 to MOLP8 cells

[0569] Antibody hu14.H1L1 hu14.H2L2 hu14.H3L1 hu14.H3L3 hu14.H3L4 hu14.H4L4 c14 <![CDATA[EC 50 (nM)]]> 7.212 6.908 5.952 6.088 6.046 5.459 17.52

[0570] 5.4. Epitope Binning

[0571] The competitive binding of the selected humanized antibodies was tested (the method is described in Example 3.5). The epitope grouping results of humanized antibodies hu23.H5L5 and hu14.H1L1 and the reference antibody are shown in Fig.19 As shown in A.

[0572] According to the competition results (such as Fig.19 A), the two humanized anti-CD39 antibodies hu23.H5L5 and hu14.H1L1 can be divided into two different epitope groups (see Fig.19 B). Specifically, anti-CD39 antibody hu23.H5L5 competed with reference antibodies I394, T895, and 9-8B for highly similar epitopes and was classified as epitope group I. hu14.H1L1, c34, and c35 did not compete with any of the other antibodies tested except for partial competition with T895 and were therefore classified as epitope group II.

[0573] 5.5 Characterization of optimized humanized antibodies

[0574] 5.5.1 Blockade of CD39 by hu23.H5L5 enhances human T cell proliferation in the presence of extracellular ATP (eATP) Cell proliferation.

[0575] Human PBMCs stimulated with anti-CD3 and anti-CD28 antibodies were incubated with 25 nM humanized anti-CD39 antibody hu23.H5L5 and vehicle, respectively, in the presence of ATP. Cell culture supernatants were collected and used to detect IL-2 and IFN-γ secretion, respectively. On day 5, CD4 + T and CD8 + Proliferation of T cells.

[0576] like Fig.11 As shown in A to 11D, hu23.H5L5 significantly enhanced CD4 + and CD8 + T cells proliferate and activate their production of IL-2 and IFN-γ. Fig.11 As shown in A, 11B and 11D, hu23.H5L5 showed significantly higher activity than I394 in enhancing T cell activation in PBMCs.

[0577] Isolation of human CD8 from healthy donor PBMC + T cells were then labeled with cell proliferation dye, activated with anti-CD3 stands and anti-CD28 antibodies, and then treated with different doses of humanized anti-CD39 antibody hu23.H5L5 or reference antibody I394 for 5 days. On day 3, after starting CD39 blocking treatment, 200 μM ATP was added to the cells. On day 5, CD8 + T cell proliferation percentage (%), CD25 + Cell percentage (%) and viable cell percentage (%).

[0578] like Fig.23 As shown in A to 23C, hu23.H5L5 significantly reversed the inhibition of human CD8 + T cell proliferation.

[0579] Following a similar method as described in Example 3.2, the binding affinity of humanized antibodies hu23.H5L5 and hu14.H1L1 was tested on different cells by FACS.

[0580] Fig.12 A to 12E show the effects of antibodies hu23.H5L5 and hu14.H1L1 on SK-MEL-5 ( Fig.12 A) SK-MEL-28( Fig.12 B) MOLP-8( Fig.12 C) CHOK1-cynoCD39 ( Fig.12 D) and CHOK1-mCD39( Fig.12 E) binding affinity. Reference antibodies T895 and I394 were tested in parallel as control antibodies. Fig.12 As shown in Table 23, antibodies hu23.H5L5 and hu14.H1L1 both bound to cells expressing human CD39 and cynomolgus CD39 in a dose-dependent manner and had similar EC at subnanomolar or nanomolar levels. 50 Affinity. None of them recognized mouse CD39 in FACS studies. The difference in maximum signal (mean fluorescence intensity, MFI) between cells for each antibody may be due to their different expression levels.

[0581] Table 23. Antibody affinity EC detected by FACS 50 (nM)

[0582] cell hu23.H5L5 hu14.H1L1 T895 I394 SK-MEL-5 0.69 7.88 0.21 0.49 SK-MEL-28 0.99 29.14 0.36 0.94 MOLP-8 0.14 0.35 0.11 0.11 CHO-K1 / cynoCD39 3.375 ND 4.192 2.708 CHO-K1 / mCD39 ND ND ND ND

[0583] ND: Not detected under the experimental conditions.

[0584] Fig.13 It showed that hu23.H5L5 blocked the proliferation of SK-MEL-5 cells ( Fig.13 A) or MOLP-8 cells ( Fig.13 B) CD39 ATPase activity on the ATPase activity of the reference antibodies T895 and I394, similar to the results (method as described in Example 3.3). The results are summarized in Table 24.

[0585] hu23.H5L5 shows enzymatic blocking IC in SK-MEL-5 cells 50 The value is 70 pM, showing enzymatic blocking IC in MOLP-8 cells 50 The value was 330 pM, which is similar to or slightly better than reference antibodies T895 and I394. However, hu14.H1L1 did not reach saturation blocking on either cell line, identifying 9-8B as a non-blocker in this assay.

[0586] Table 24. ATPase activity inhibition (IC 50 )(nM)

[0587] <![CDATA[IC 50 (nM)]]> hu23.H5L5 hu14.H1L1 T895 I394 9-8B SK-MEL-5 0.07 NA 0.09 0.10 ND MOLP-8 0.33 26.51 0.51 0.21 ND

[0588] NA: Not available

[0589] ND: Not detected under the experimental conditions.

[0590] 5.5.2 Blockade of CD39 by hu23.H5L5 enhances ATP-mediated monocyte activation.

[0591] The humanized antibody hu23.H5L5 was also tested in an ATP-mediated monocyte activation assay. ATP-mediated proinflammatory activity plays an important role in regulating the function of multiple immune cell types, including monocytes. To evaluate 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 different concentrations ranging from 0.2 nM to 100 nM in the presence of ATP. It was shown that hu23.H5L5 can effectively induce monocyte activation at a concentration of 0.2 nM (i.e., the lowest concentration detected). CD80 ( Fig.14 A) CD86( Fig.14 B) and CD40 ( Fig.14 C) to evaluate the activation of monocytes (hu23.H5L5 at a concentration of 50 nM). Reference anti-CD39 antibodies I394 and T895 were used as controls, and hIgG4 was used as an isotype control.

[0592] The results are shown in Fig.14 ATP stimulation alone demonstrated upregulation of CD80 and CD86, indicating monocyte activation. Humanized anti-CD39 antibody hu23.H5L5 further enhanced ATP-mediated monocyte activation, as demonstrated by upregulation of CD80, CD86, and CD40, at levels comparable to reference antibody I394. Reference antibody T895 had no significant effect on ATP-induced activated monocytes.

[0593] 5.5.3 Blockade of CD39 by hu23.H5L5 enhances ATP-mediated DC activation.

[0594] Selected humanized antibodies hu23.H5L5 were also tested in an ATP-mediated DC activation assay (following a similar method as described in Example 4.2). Briefly, DC maturation was assessed by analyzing CD83 expression by FACS assay. ATP induced DC maturation by showing increased expression of CD83 ( Fig.15A). Starting from levels as low as 0.2 nM, hu23.H5L5 increased CD83 expression in a dose-dependent manner, and at an antibody level of 0.6 nM, hu23.H5L5 significantly increased CD83 expression. This was more potent than either of the reference antibodies T895 and I394.

[0595] To further evaluate the effect of ATP-mediated DC activation on T cell activation, ATP-activated DCs were washed and then incubated with allogeneic T cells for mixed lymphocyte reaction (MLR). T cell proliferation ( Fig.15 B) and IFN-γ produced by activated T cells ( Fig.15 C).

[0596] Compared with reference antibodies I394 and T895, the anti-CD39 antibody hu23.H5L5 had a dose-dependent and significant effect on promoting ATP-induced DC maturation, with reference I394 showing similar but slightly weaker activity and T895 having a very mild effect. Fig.15 As shown in B and 15C, in the MLR assay, the anti-CD39 blocking antibody hu23.H5L5 enhanced ATP-mediated MoDC maturation, resulting in higher T cell proliferation and IFN-γ production.

[0597] 5.5.4 Blockade of CD39 by hu23.H5L5 promotes IL1β release from human macrophages induced by LPS stimulation.

[0598] Isolation of human CD14 from healthy human PBMC + T cells were then added at 2x10 6 The density of each well will enrich for CD14 + Monocytes were seeded into 6-well plates and incubated with 100 ng / mL human GM-CSF for 6 days to generate M1-like macrophages. In vitro differentiated macrophages were treated with increasing doses of hu23.H5L5 or reference antibody I394 for 1 hour, then stimulated with 10 ng / mL LPS for 3 hours, and then incubated with 800 μM ATP for 2 hours. IL-1β in cell culture supernatants was quantified by ELISA.

[0599] The results are as follows Fig. 20 As shown. Asterisks indicate significant differences between conditions. Fig. 20 As shown, hu23.H5L5 significantly promoted the release of IL1β from human macrophages induced by LPS stimulation, and in promoting the release of IL1β from human macrophages induced by LPS stimulation, hu23.H5L5 showed significantly higher activity than the reference antibody I394.

[0600] 5.6 In vivo studies

[0601] The effects of humanized antibodies hu23.H5L5 and hu14.H1L1 were determined on MOLP-8 xenograft mice according to the method described in Example 4.2.

[0602] The results are shown in Fig.16 All anti-CD39 antibodies inhibited tumor growth compared with the vehicle group. The efficacy observed for I394 was slightly weaker than that of the other antibodies, including hu23.H5L5 and hu14.H1L1.

[0603] According to the method described in Example 4.2, the anti-tumor effect of humanized antibody hu23.H5L5 at different doses (0.03 mg / kg, 0.3 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg, intraperitoneal administration, twice a week, 6 doses) was also investigated in a PBMC transplant animal model (NCG mice, inoculated with MOLP-8 cells, 5M / mouse).

[0604] The results are shown in Fig.21 In. Fig.21 As shown, the humanized antibody hu23.H5L5 effectively inhibited tumor growth at all doses tested.

[0605] The inventors also determined whether the anti-tumor effect of anti-CD39 antibodies depends on NK cells or macrophages. Anti-asialo-GM1 NK depletion treatment was started on day 7, 20 μl / mouse intraperitoneally, once every 5 days. Macrophage depletion treatment with clodronate liposomes was also started on days 7 and 9, 200 μl / mouse intravenously, once a week. Blood sample analysis data showed that the agent can significantly remove mononuclear phagocytes or NK.

[0606] In NK cells ( Fig.17 ) or macrophages ( Fig.18 ) were depleted, the tumor growth inhibitory effect of hu23.H5L5 was abolished, indicating that the anti-tumor effect of anti-CD39 antibodies depends on NK cells and macrophages.

[0607] Specifically, Fig.17 As shown in Figure 2, anti-asialo-GM1 slightly enhanced tumor growth at the late stage compared with vehicle. Moreover, the combination of hu23.H5L5 and anti-asialo-GM1 completely abolished the tumor growth inhibitory effect of hu23.H5L5 compared with the hu23.H5L5 treated group. Fig.18 As shown, 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.

[0608] Example 6. Epitope Mapping

[0609] To determine the epitope of the anti-CD39 antibody, the inventors designed CD39 mutants and defined them by substitutions of amino acids exposed on the surface of the human CD39 molecule. The mutants were cloned into an expression vector fused with a C-terminal EGFP sequence and transfected into HEK-293F cells, as shown in Table 25 below. The target amino acid mutations are indicated using the numbering of UniProtKB-P49961 (ENTP1_HUMAN), which is the wild-type amino acid sequence of human CD39, and its sequence is shown in SEQ ID NO: 162. For example, V77G indicates that the valine at position 77 of SEQ ID NO: 162 is replaced by glycine.

[0610] Table 25. Human CD39 mutants

[0611]

[0612]

[0613] Briefly, human CD39 mutants were generated by gene synthesis and cloned into the expression vector pCMV3-GFPSpark. Vectors containing confirmed mutant sequences were prepared and transfected into HEK293F cells. Three days after transfection, cells were collected and EGFP was detected to confirm transgene expression. A series of doses (starting from 100nM, 3-fold dilution, 11 points) of antibodies were tested by FACS in the 20 mutants generated and stained with anti-hIgG labeled with AlexFluor647. Antibody binding is described as relative binding of AlexFluor647 intensity divided by GFP intensity. Results are shown in Fig. 22 middle.

[0614] like Fig. 22 As shown, the humanized antibody hu23.H5L5 lost binding to mutants KW27-6 and KW27-20, but did not lose binding to other mutants. Mutant KW27-6 has amino acid substitutions at residues Q96, N99, E143 and R147, indicating that one or more or all of the above residues of the mutant are critical for the core epitope of hu23.H5L5; mutant KW27-20 has amino acid substitutions at residues R138, M139 and E142, indicating that one or more or all of the above residues of the mutant are also critical for the core epitope of hu23.H5L5.

[0615] like Fig. 22As shown, the chimeric antibody c34 lost binding to mutant KW27-16, but not to other mutants. Mutant KW27-16 has amino acid substitutions at residues K5, E100 and D107, indicating that one or more or all of the above residues of the mutant are critical for the core epitope of c34.

[0616] like Fig. 22 As shown, the chimeric antibody c35 lost binding to mutant KW27-2, but not to other mutants. Mutant KW27-2 had amino acid substitutions at residues V81, E82, R111, and V115, indicating that one or more or all of the above residues of the mutant are critical for the core epitope of c35.

[0617] like Fig. 22 As shown, reference antibody T895 lost binding to mutant KW27-20, but did not lose binding to other mutants. Mutant KW27-20 has amino acid substitutions at residues R138, M139 and E142, indicating that one or more or all of the above residues of the mutant are critical for the core epitope of T895.

[0618] like Fig. 22 As shown, the reference antibody I394 lost binding to mutants KW27-6 and KW27-20, but did not lose binding to other mutants. Mutant KW27-6 has amino acid substitutions at residues Q96, N99, E143 and R147, indicating that one or more or all of the above residues of the mutant are critical for the core epitope of I394; mutant KW27-20 has amino acid substitutions at residues R138, M139 and E142, indicating that one or more or all of the above residues of the mutant are also critical for the core epitope of I394.

[0619] like Fig. 22 As shown, reference antibody 9-8B lost binding to mutant KW27-6, but did not lose binding to other mutants. Mutant KW27-6 has amino acid substitutions at residues Q96, N99, E143 and R147, indicating that one or more or all of the above residues of the mutant are critical for the core epitope of 9-8B.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to human CD39, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein: (a) the sequence of HCDR1 is shown in SEQ ID NO: 4, the sequence of HCDR2 is shown in SEQ ID NO: 10, and the sequence of HCDR3 is shown in SEQ ID NO: 16; the sequence of LCDR1 is shown in SEQ ID NO: 22, the sequence of LCDR2 is shown in SEQ ID NO: 28, and the sequence of LCDR3 is shown in SEQ ID NO: 34; or (b) the sequence of HCDR1 is shown in SEQ ID NO: 4, the sequence of HCDR2 is shown in SEQ ID NO: 134, and the sequence of HCDR3 is shown in SEQ ID NO: 16; the sequence of LCDR1 is shown in SEQ ID NO: 22, the sequence of LCDR2 is shown in SEQ ID NO: 28, and the sequence of LCDR3 is shown in SEQ ID NO: 34; or (c) the sequence of HCDR1 is shown in SEQ ID NO: 4, the sequence of HCDR2 is shown in SEQ ID NO: 135, and the sequence of HCDR3 is shown in SEQ ID NO: 16; the sequence of LCDR1 is shown in SEQ ID NO: 22, the sequence of LCDR2 is shown in SEQ ID NO: 28, and the sequence of LCDR3 is shown in SEQ ID NO: 34; or (d) the sequence of the HCDR1 is shown in SEQ ID NO: 4, the sequence of the HCDR2 is shown in SEQ ID NO: 137, and the sequence of the HCDR3 is shown in SEQ ID NO: 16; the sequence of the LCDR1 is shown in SEQ ID NO: 22, the sequence of the LCDR2 is shown in SEQ ID NO: 28, and the sequence of the LCDR3 is shown in SEQ ID NO: 34; or (e) the sequence of the HCDR1 is shown in SEQ ID NO: 4, the sequence of the HCDR2 is shown in SEQ ID NO: 138, and the sequence of the HCDR3 is shown in SEQ ID NO: 16; the sequence of the LCDR1 is shown in SEQ ID NO: 22, the sequence of the LCDR2 is shown in SEQ ID NO: 28, and the sequence of the LCDR3 is shown in SEQ ID NO: 34; or (f) the sequence of HCDR1 is shown in SEQ ID NO: 4, the sequence of HCDR2 is shown in SEQ ID NO: 139, and the sequence of HCDR3 is shown in SEQ ID NO: 16; the sequence of LCDR1 is shown in SEQ ID NO: 22, the sequence of LCDR2 is shown in SEQ ID NO: 28, and the sequence of LCDR3 is shown in SEQ ID NO: 34; or (g) the sequence of the HCDR1 is shown as SEQ ID NO: 4, the sequence of the HCDR2 is shown as SEQ ID NO: 136, and the sequence of the HCDR3 is shown as SEQ ID NO: 16; the sequence of the LCDR1 is shown as SEQ ID NO: 22, the sequence of the LCDR2 is shown as SEQ ID NO: 28, and the sequence of the LCDR3 is shown as SEQ ID NO:

34.

2. The antibody or antigen-binding fragment thereof according to claim 1, further comprising heavy chains HFR1, HFR2, HFR3 and HFR4, and light chains LFR1, LFR2, LFR3 and LFR4, wherein: (a) The HFR1 is composed of X 19 VxDV 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 The sequence composition shown is (b) The HFR2 is composed of WVX 34 QX 35 PGX 36 X 37 LEWX 38 X 39 The sequence composition shown is (c) The HFR3 is composed of 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 The sequence composition shown is (d) The HFR4 is made of WGQGTX 57 The sequence composition shown in VTVSS is (e) the LFR1 is composed of the sequence shown as X3IVX4TQSPATLX5X6SPGERX7TX8X9C, (f) The LFR2 is composed of WYQQKPGQX 10 PX 11 The sequence composition shown in LLIY, (g) The LFR3 is composed of GX 12 PX 13 RFSGSGSGTX 14 X 15 TLTISSX 16 EPEDFAVYX 17 The sequence shown in C, and (h) The LFR4 is made of FGX 18 The sequence shown in GTKLEIK is composed of Where 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; X 10 A or S; X 11 is R or K; X 12 is I or V; X 13 A or T; X 14 Is D or S; X 15 is F or Y; X 16 L, M or V; X 17 is Y or F; X 18 is G or Q; X 19 It is Q or E; X 20 is E or Q; X 21 is G or A; X 22 is G or E; X 23 L or V; X 24 is V or K; X 25 is G or A; X 26 L, M or V; X 27 is R or K; X 28 is V or L; X 29 A or K; X 30 is F or Y; X 31 is N or T; X 32 Is F or L; X 33 is S or K; X 34 is R or K; X 35 A or S; X 36 It is K or Q; X 37 is R or G; X 38 is M, I or V; X 39 is G or A; X 40 is R or K; X 41 is V, A or F; X 42 Is I or L; X 43 is S or T; X 44 is R or A; X 45 is D or T; X 46 is K, A or S; X 47 is S or N; X 48 is L, V or A; X 49 M or L; X 50 It is Q or E; X 51 M or L; X 52 is S, I or N; X 53 is R or K; X 54 is S or T; X 55 A or T; X 56 is R, N or T; and X 57 It is T or L.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein: (a) The HFR1 is represented by EVQLVESGGGLVKPGGSX 61 The sequence composition shown in RLSCAASGFTFS, (b) The HFR2 is composed of WVRQX 62 PGKGLEWVX 63 The sequence composition shown is (c) The HFR3 is composed of RFTISRDDSKNTX 64 The sequence shown in YLQMNSLKTEDTAVYYCTT is composed of (d) the HFR4 consists of the sequence shown as WGQGTTVTVSS, (e) The LFR1 is composed of EIVX 65 TQSPATLSX 66 SPGERX 67 The sequence composition shown in TLSC, (f) The LFR2 is composed of WYQQKPGQX 68 The sequence composition shown in PRLLIY, (g) The LFR3 is composed of GIPARFSGSGSGTDFTLTISSX 69 EPEDFAVYX 70 The sequence shown in C, and (h) the LFR4 consists of the sequence shown as FGGGTKLEIK, Where X 61 L or M; X 62 A or S; X 63 is G or A; X 64 L or V; X 65 It is L or M; X 66 L or V; X 67 A or V; X 68 A or S; X 69 L or V; X 70 It is Y or F.

4. The antibody or antigen-binding fragment thereof according to claim 2, wherein: (a) The HFR1 is composed of 71 VQLVQSGAEVKKPGASVKX 72 SCKASGYX 73 The sequence composition shown in LK, (b) The HFR2 is composed of WVX 74 QX 75 LEWX 76 The sequence shown in G is composed of (c) The HFR3 is composed of X 77 X 78 TX 79 TX 80 DTSX 81 X 82 TAYX 83 ELX 84 SLRSEDTAVYYCAX 85 The sequence composition shown is (d) The HFR4 is made of WGQGTX 57 The sequence composition shown in VTVSS is (e) The LFR1 is composed of 86 IVLTQSPATLX 87 X 88 SPGERX 89 TX 90 X 91 The sequence composition shown in C, (f) The LFR2 is composed of WYQQKPGQX 10 PX 11 The sequence composition shown in LLIY, (g) The LFR3 is composed of GX 92 PX 93 RFSGSGSGTX 94 X 95 TLTISSX 96 The sequence composition is shown in EPEDFAVYYC, and (h) the LFR4 consists of the sequence shown as FGQGTKLEIK, Where X 10 , X 11 and X 57 As defined in claim 2, X 71 It is Q or E; X 72 is V or L; X 73 N or T; X 74 is R or K; X 75 is R or G; X 76 is M or I; X 77 is R or K; X 78 is V or A; X 79 Is I or L; X 80 is R or A; X 81 A or S; X 82 is S or N; X 83 M or L; X 84 is S or I; X 85 is R or N; X 86 is E or Q; X 87 is S or T; X 88 L or A; X 89 A or V; X 90 is L or I; X 91 is S or T; X 92 is I or V; X 93 A or T; X 94 Is D or S; X 95 is F or Y; X 96 It is L or M.

5. The antibody or antigen-binding fragment thereof according to claim 2, wherein: The HFR1 consists of a sequence selected from the group consisting of SEQ ID NOs: 84-86, 115, 119-120 and 131; The HFR2 is selected from the group consisting of Composition: SEQ ID NO: 87-90 and 121-123; The HFR3 is selected from the following sequence Composition: SEQ ID NO: 91-97, 116-117 and 124-125; The HFR4 is composed of a sequence selected from the group consisting of Composition: SEQ ID NO: 79 and 118; The LFR1 is composed of a sequence selected from the group consisting of Composition: SEQ ID NO: 98-103 and 127-129; The LFR2 is composed of a sequence selected from the group consisting of Composition: SEQ ID NO: 104, 105 and 130; The LFR3 consists of a sequence selected from the group consisting of SEQ ID NOs: 106-110 and 132-133, and The LFR4 consists of a sequence selected from the group consisting of SEQ ID NOs: 83 and 153.

6. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of a sequence selected from the group consisting of SEQ ID NO: 47, 60, 62, 64, 66, 140, 141, 142, 146, 147, 39, and homologous sequences having at least 80% sequence identity thereto but still retaining specific binding affinity to human CD39; The light chain variable region is composed of a sequence selected from the group consisting of SEQ ID NO: 56, 61, 63, 65, 67, 143, 144, 145, 111, 112, 63, and homologous sequences having at least 80% sequence identity thereto but still maintaining specific binding affinity to human CD39, wherein: (a) the amino acid sequence of HCDR1 is as shown in DTFLH; and (b) the amino acid sequence of the HCDR2 is selected from the group consisting of SEQ ID NOs: 10, 134, 135, 136, 137, 138 and 139; and (c) the amino acid sequence of HCDR3 is shown as SPYYYGSGYRIFDV; and (d) the amino acid sequence of LCDR1 is shown as SAFSSVNYMH; and (e) the amino acid sequence of LCDR2 is as shown in TTSNLAS; and (f) The amino acid sequence of LCDR3 is shown as QQRSTYPFT.

7. The antibody or antigen-binding fragment thereof according to claim 1, comprising: (a) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 47, and the light chain variable region consists of the sequence shown in SEQ ID NO: 56, or (b) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 60, and the light chain variable region consists of the sequence shown in SEQ ID NO: 63, or (c) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 62, and the light chain variable region consists of the sequence shown in SEQ ID NO: 63, or (d) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 64, and the light chain variable region consists of the sequence shown in SEQ ID NO: 63, or (e) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 66, and the light chain variable region consists of the sequence shown in SEQ ID NO: 63, or (f) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 60, and the light chain variable region consists of the sequence shown in SEQ ID NO: 65, or (g) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 62, and the light chain variable region consists of the sequence shown in SEQ ID NO: 65, or (h) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 64, and the light chain variable region consists of the sequence shown in SEQ ID NO: 65, or (i) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 66, and the light chain variable region consists of the sequence shown in SEQ ID NO: 65, or (j) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 60, and the light chain variable region consists of the sequence shown in SEQ ID NO: 67, or (k) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 62, and the light chain variable region consists of the sequence shown in SEQ ID NO: 67, or (l) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 64, and the light chain variable region consists of the sequence shown in SEQ ID NO: 67, or (m) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 66, and the light chain variable region consists of the sequence shown in SEQ ID NO: 67, or (n) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 140, and the light chain variable region consists of the sequence shown in SEQ ID NO: 61, or (o) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 141, and the light chain variable region consists of the sequence shown in SEQ ID NO: 61, or (p) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 142, and the light chain variable region consists of the sequence shown in SEQ ID NO: 61, or (q) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 140, and the light chain variable region consists of the sequence shown in SEQ ID NO: 143, or (r) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 141, and the light chain variable region consists of the sequence shown in SEQ ID NO: 143, or (s) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 142, and the light chain variable region consists of the sequence shown in SEQ ID NO: 143, or (t) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 140, and the light chain variable region consists of the sequence shown in SEQ ID NO: 144, or (u) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 141, and the light chain variable region consists of the sequence shown in SEQ ID NO: 144, or (v) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 142, and the light chain variable region consists of the sequence shown in SEQ ID NO: 144, or (w) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 140, and the light chain variable region consists of the sequence shown in SEQ ID NO: 145, or (x) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 141, and the light chain variable region consists of the sequence shown in SEQ ID NO: 145, or (y) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 142, and the light chain variable region consists of the sequence shown in SEQ ID NO: 145, or (z) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 146, and the light chain variable region consists of the sequence shown in SEQ ID NO: 111, or (aa) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 146, and the light chain variable region consists of the sequence shown in SEQ ID NO: 112, or (bb) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 147, and the light chain variable region consists of the sequence shown in SEQ ID NO: 111, or (cc) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the sequence shown in SEQ ID NO: 39, and the light chain variable region consists of the sequence shown in SEQ ID NO:

63.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, further comprising one or more amino acid residue substitutions or modifications, but still maintaining specific binding affinity to human CD39, wherein the one or more amino acid residue substitutions or modifications occur in one or more non-CDR sequences in the heavy chain variable region or the light chain variable region.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, further comprising an Fc region.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the Fc region comprises the Fc region of human immunoglobulin (Ig). 11 . The antibody or antigen-binding fragment thereof according to claim 10 , wherein the Fc region of human immunoglobulin (Ig) comprises the Fc region of human IgG. 12 . 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.

13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the Fc region derived from human IgG1 comprises L234A and L235A mutations.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is humanized.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is a monoclonal antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody or a fusion protein.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is a diabody, Fab, Fab', F(ab')2, 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 or multispecific antibody.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which can be used at a rate of no more than 10 -8 EC of M 50 Specifically binds to human CD39, the EC 50 Determined by FACS detection.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which has one or more properties selected from the group consisting of: (a) Binds specifically to human CD39 but not mouse CD39 as determined by FACS assay; (b) not more than 10 -8 EC of M 50 Specifically binds to cynomolgus monkey CD39, the EC 50 Determined by FACS assay; (c) not more than 10 -7 M of K D The K D Values ​​were determined by Biacore assay; (d) not more than 10 -8 M of K D The K D Values ​​were determined by Octet analysis; (e) With an IC value not exceeding 50 nM 50 Inhibiting ATPase activity in cells expressing CD39, the IC 50 Determined by ATPase activity assay; (f) capable of enhancing ATP-mediated monocyte activation at a concentration of no more than 10 nM as determined by FACS analysis of the expression of CD80, CD86 and / or CD40; (g) is capable of enhancing ATP-mediated T cell activation in PBMCs at a concentration not exceeding 25 nM, as measured by IL-2 secretion, IFN-γ secretion, CD4 + or CD8 + T cell proliferation assay; (h) capable of enhancing ATP-mediated dendritic cell (DC) activation at a concentration not exceeding 25 nM as measured by CD83 expression as assayed by FACS, or by the ability of activated DC to promote T cell proliferation, or by the ability of activated DC to promote IFN-γ production in a mixed lymphocyte reaction (MLR) assay; (i) Ability to block adenosine-induced CD4 + Inhibition of T cell proliferation, the concentrations being measured by FACS assay; (j) capable of inhibiting tumor growth in a mammal in a NK cell- or macrophage-dependent manner; (k) Able to reverse the inhibition of human CD8 by eATP + T cell proliferation, through T cell proliferation, CD25 + Cell and live cell population assays; and (l) capable of enhancing the release of IL1β from human macrophages induced by LPS stimulation at a concentration not exceeding 50 nM, as measured by ELISA assay.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is bispecific. 20 . The antibody or antigen-binding fragment thereof according to claim 19 , which is capable of specifically binding to a second antigen other than CD39 or a second epitope on CD39.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is linked to one or more conjugate moieties.

22. The antibody or antigen-binding fragment thereof according to claim 21, wherein The conjugate moieties include clearance modifiers, chemotherapeutic agents, toxins, radioisotopes, luminescent labels, enzyme substrate labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents, purification moieties or other anti-cancer drugs.

23. The antibody or antigen-binding fragment thereof according to claim 21, wherein The conjugate moiety includes a lanthanide or a fluorescent label.

24. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 and one or more pharmaceutically acceptable carriers.

25. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-20.

26. A vector comprising the isolated polynucleotide according to claim 25.

27. A host cell comprising the vector according to claim 26.

28. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20 and / or the pharmaceutical composition according to claim 24, and a second therapeutic agent.

29. A method for expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, comprising culturing the host cell according to claim 27 under conditions for expressing the vector according to claim 26.

30. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 and / or a pharmaceutical composition according to claim 24 in the preparation of a medicament for treating, preventing or alleviating a disease, disorder or condition associated with CD39 in a subject, wherein the disease, disorder or condition is selected from the group consisting of gastric cancer, lung cancer, pancreatic cancer, colon cancer, prostate cancer, melanoma, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, T or B cell lymphoma.

31. The use according to claim 30, wherein the disease is melanoma or multiple myeloma.

32. The use according to claim 30, wherein the subject has been identified as having cancer cells expressing CD39 or tumor-infiltrating immune cells or immunosuppressive cells.

33. The use according to claim 32, wherein the level of CD39 is significantly higher than the level normally found on non-cancerous cells or non-immunosuppressive cells.

34. The use according to claim 30, wherein the subject is a human.

35. The use according to claim 30, wherein the drug is administered orally, intranasally, intravenously, subcutaneously, sublingually or intramuscularly.

36. The use of claim 30, further comprising administering a therapeutically effective amount of a second therapeutic agent.

37. The use of claim 36, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, anticancer agents, radiotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, targeted therapeutic agents, cell therapeutic agents, gene therapeutic agents, hormone therapeutic agents, antiviral agents, antibiotics, analgesics, antioxidants, metal chelators, and cytokines.

38. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 and / or the pharmaceutical composition according to claim 24 in the preparation of a diagnostic reagent for detecting the presence or content of CD39 in a sample.

39. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 and / or a pharmaceutical composition according to claim 24 in the preparation of a diagnostic agent for diagnosing a disease, disorder or condition associated with CD39 in a subject, wherein the diagnosis comprises: a) contacting a sample obtained from the subject with the antibody or antigen-binding fragment thereof and / or the pharmaceutical composition; b) determining the presence or amount of CD39 in the sample; c) correlating the presence or amount of CD39 with the presence or status of a disease, disorder or condition associated with CD39 in the subject.

40. A kit for detecting CD39, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 and / or the pharmaceutical composition according to claim 24.

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