Binding molecules specific for CD39 and uses thereof
By developing antibodies or antigen-binding moieties that specifically bind CD39, the ATPase activity of CD39 has been significantly reduced, and the technical difficulties of CD39 target inhibition has been solved, and a potential treatment plan for CD39-related diseases has been provided.
Patent Information
- Application Number
- CN202080077131.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Priority Date
- 2019-11-05
- Filing Date
- 2020-11-04
- Publication Date
- 2025-03-14
- Estimated Expiration
- 2040-11-04
AI Technical Summary
The prior art has failed to effectively resolve the inhibition of CD39 targets, resulting in the undeveloped treatment methods for CD39-related diseases.
Provides antibodies or antigen-binding moieties that specifically bind CD39, significantly reducing the ATPase activity of CD39 by including specific HCDR and LCDR sequences.
By inhibiting the activity of CD39, it potentially destroys the immunosuppressive mechanism in the tumor microenvironment, improves anti-tumor effects, and provides new therapeutic approaches for CD39-related diseases.
Smart Images
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Figure GDA0003628573680000251
Abstract
Description
Technical Field
[0001] The present invention relates to antibodies or antigen-binding portions thereof that specifically bind to CD39, as well as uses of the antibodies or antigen-binding portions thereof of the present invention in treating diseases, and therapeutic methods using the antibodies or antigen-binding portions thereof of the present invention. Background of the Invention
[0003] CD39 is a membrane protein that is Ca-dependent 2+ and Mg 2+ It hydrolyzes ATP and ADP to produce AMP. Human CD39 has 510 amino acids, with seven predicted N-linked glycosylation sites, 11 cysteine residues, and two transmembrane regions. CD39 is composed of two transmembrane domains: a small cytoplasmic domain containing NH2- and COOH-terminal segments; and a large extracellular hydrophobic domain composed of five highly conserved domains, termed apyrase conserved regions (ACRs) 1-5, which are key to the enzyme's catabolic activity. The amino acid sequences of ACRs 1 and 5 contain a phosphate-binding motif (DXG), which is crucial for stabilizing the interaction between the enzyme and its nucleotide substrate during phosphate cleavage. In addition, two ACR residues, Glu 174 in ACR 3 and Ser 218 in ACR 4, are essential for the enzyme's function. CD39 has catalytic activity after localization on the cell surface, and its glycosylation is crucial for correct protein folding, membrane targeting and enzyme activity (Antonioli L et al. (2013), Trends Mol Med, 19(6):355–367).
[0004] CD39 is constitutively expressed in the spleen, thymus, lung, and placenta, where it is primarily associated with endothelial cells and immune cell populations such as B cells, natural killer (NK) cells, dendritic cells, Langerhans cells, monocytes, macrophages, mesangial cells, neutrophils, and regulatory T cells (Tregs). CD39 expression is regulated by several proinflammatory cytokines, oxidative stress, and hypoxia, through the transcription factors Sp1, Stat3, and zinc finger protein growth factor-independent 1 transcription factor (Antonioli L et al. (2013), Trends Mol Med, 19(6):355–367).
[0005] Under physiological conditions, the purine mediator ATP is mainly present in the cytoplasm, with a concentration of approximately 1 to 10 mM, while the extracellular ATP concentration is at a lower level, 10 to 100 nM. However, when the body is disturbed, such as inflammation, ischemia, or malignant tumors, a large amount of ATP in the cytoplasm is released outside the cell, acting as a sensory signal and an efferent signal to trigger an immune response. After ATP is released outside the cell, it is hydrolyzed by extracellular CD39 into ADP and AMP. AMP, in conjunction with CD73, generates immunosuppressive adenosine. In this process, CD39 is the rate-limiting enzyme (Faas MM et al. (2017), Mol Aspects Med, 55:9-19). CD39 and CD73 can regulate the functions of immune cells such as lymphocytes, neutrophils, monocytes / macrophages, and dendritic cells (Antonioli L et al. (2013), Trends Mol Med, 19(6):355–367). In the tumor microenvironment, CD39 is highly expressed on the surface of Treg cells, and CD39 is gradually considered to be a specific marker molecule on the surface of Treg cells (Gu J et al. (2017), Cell Mol Immunol, 14(6): 521-528). Adenosine derived from Treg cells acts on the A2A receptor on the surface of lymphocytes (Sundstrom P SH et al. (2016), Cancer Immunol Res, 4(3): 183-193; Ma SR et al. (2017), Mol Cancer, 16(1): 99), inhibiting the proliferation, migration and anti-tumor effect of effector T cells; inhibiting the cytotoxicity of NK cells and the production of cytokines, mediating a series of immunosuppressive effects (Lokshin A et al. (2006), Cancer Res, 66(15): 7758-7765; Hu G et al. (2017), Oncoimmunology, 6(2): e1277305).
[0006] CD39 is highly expressed in many malignant tumors (Allard B et al. (2017), Immunol Rev, 276(1): 121-144; Bastid J et al. (2013), Oncogene, 32(14): 1743-1751). Compared with normal tissues, the expression level of CD39 in tumor tissues such as kidney, lung, ovary, pancreas, and thyroid is significantly increased, suggesting that abnormally high expression of CD39 is associated with the occurrence and development of malignant tumors (Bastid J et al. (2015), Cancer Immunol Res, 3(3): 254-265). In addition, changes in the CD39 / CD73 system may disrupt underlying complex mechanisms, such as immune tolerance to self-antigens driven by Tregs, thereby contributing to the development of certain autoimmune diseases (Karen M. Dwyer et al. (2007), Purinergic Signal, 3(1-2): 171-180).
[0007] Currently, there are no marketed drugs targeting CD39. There is an urgent need to develop CD39 inhibitors and treatments for CD39-related diseases. Summary of the Invention
[0008] The present invention provides antibodies or antigen-binding portions that specifically bind to CD39 and their use in treating diseases.
[0009] In one aspect, the present invention provides an antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 sequences; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences, wherein:
[0010] (a) the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 19, 33 and / or 47 and conservative modifications thereof;
[0011] (b) the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 20, 34 and / or 48 and conservative modifications thereof;
[0012] (c) the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 21, 35 and / or 49 and conservative modifications thereof;
[0013] (d) the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 24, 38 and / or 52 and conservative modifications thereof;
[0014] (e) the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 25, 39, 53 and / or 59 and conservative modifications thereof; and
[0015] (f) The LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 26, 40 and / or 54 and conservative modifications thereof.
[0016] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises:
[0017] 1) (a) comprising HCDR1 of SEQ ID NO: 5, (b) comprising HCDR2 of SEQ ID NO: 6, (c) comprising HCDR3 of SEQ ID NO: 7, (d) comprising LCDR1 of SEQ ID NO: 10, (e) comprising LCDR2 of SEQ ID NO: 11, and (f) comprising LCDR3 of SEQ ID NO: 12;
[0018] 2) (a) HCDR1 comprising SEQ ID NO: 19, (b) HCDR2 comprising SEQ ID NO: 20, (c) HCDR3 comprising SEQ ID NO: 21, (d) LCDR1 comprising SEQ ID NO: 24, (e) LCDR2 comprising SEQ ID NO: 25, and (f) LCDR3 comprising SEQ ID NO: 26;
[0019] 3) (a) HCDR1 comprising SEQ ID NO: 33, (b) HCDR2 comprising SEQ ID NO: 34, (c) HCDR3 comprising SEQ ID NO: 35, (d) LCDR1 comprising SEQ ID NO: 38, (e) LCDR2 comprising SEQ ID NO: 39, and (f) LCDR3 comprising SEQ ID NO: 40;
[0020] 4) (a) HCDR1 comprising SEQ ID NO:47, (b) HCDR2 comprising SEQ ID NO:48, (c) HCDR3 comprising SEQ ID NO:49, (d) LCDR1 comprising SEQ ID NO:52, (e) LCDR2 comprising SEQ ID NO:53, and (f) LCDR3 comprising SEQ ID NO:54;
[0021] 5) (a) comprising HCDR1 of SEQ ID NO: 5, (b) comprising HCDR2 of SEQ ID NO: 6, (c) comprising HCDR3 of SEQ ID NO: 7, (d) comprising LCDR1 of SEQ ID NO: 52, (e) comprising LCDR2 of SEQ ID NO: 59, and (f) comprising LCDR3 of SEQ ID NO: 54; and / or
[0022] 6) (a) HCDR1 comprising SEQ ID NO: 5, (b) HCDR2 comprising SEQ ID NO: 6, (c) HCDR3 comprising SEQ ID NO: 7, (d) LCDR1 comprising SEQ ID NO: 10, (e) LCDR2 comprising SEQ ID NO: 59, and (f) LCDR3 comprising SEQ ID NO: 12.
[0023] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises: (a) HCDR1 comprising SEQ ID NO: 5, (b) HCDR2 comprising SEQ ID NO: 6, (c) HCDR3 comprising SEQ ID NO: 7, (d) LCDR1 comprising SEQ ID NO: 10, (e) LCDR2 comprising SEQ ID NO: 11, and (f) LCDR3 comprising SEQ ID NO: 12.
[0024] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises: (a) HCDR1 comprising SEQ ID NO: 19, (b) HCDR2 comprising SEQ ID NO: 20, (c) HCDR3 comprising SEQ ID NO: 21, (d) LCDR1 comprising SEQ ID NO: 24, (e) LCDR2 comprising SEQ ID NO: 25, and (f) LCDR3 comprising SEQ ID NO: 26.
[0025] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises: (a) HCDR1 comprising SEQ ID NO: 33, (b) HCDR2 comprising SEQ ID NO: 34, (c) HCDR3 comprising SEQ ID NO: 35, (d) LCDR1 comprising SEQ ID NO: 38, (e) LCDR2 comprising SEQ ID NO: 39, and (f) LCDR3 comprising SEQ ID NO: 40.
[0026] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises: (a) HCDR1 comprising SEQ ID NO:47, (b) HCDR2 comprising SEQ ID NO:48, (c) HCDR3 comprising SEQ ID NO:49, (d) LCDR1 comprising SEQ ID NO:52, (e) LCDR2 comprising SEQ ID NO:53, and (f) LCDR3 comprising SEQ ID NO:54.
[0027] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises: (a) HCDR1 comprising SEQ ID NO: 5, (b) HCDR2 comprising SEQ ID NO: 6, (c) HCDR3 comprising SEQ ID NO: 7, (d) LCDR1 comprising SEQ ID NO: 52, (e) LCDR2 comprising SEQ ID NO: 59, and (f) LCDR3 comprising SEQ ID NO: 54.
[0028] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises: (a) HCDR1 comprising SEQ ID NO: 5, (b) HCDR2 comprising SEQ ID NO: 6, (c) HCDR3 comprising SEQ ID NO: 7, (d) LCDR1 comprising SEQ ID NO: 10, (e) LCDR2 comprising SEQ ID NO: 59, and (f) LCDR3 comprising SEQ ID NO: 12.
[0029] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises:
[0030] (i) the heavy chain variable region (VH) comprises an amino acid sequence that is at least 85% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 17, 31, 45, 60 and / or 72 and conservatively modified versions thereof; and
[0031] (ii) the light chain variable region (VL) comprises an amino acid sequence that is at least 85% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 22, 36, 50, 62, 68 and / or 74 and conservatively modified versions thereof.
[0032] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the heavy chain variable region selected from (i); the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the light chain variable region selected from (ii).
[0033] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises:
[0034] 1) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 3, and a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 8;
[0035] 2) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 17, and a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 22;
[0036] 3) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 31, and a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 36;
[0037] 4) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 45, and a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 50;
[0038] 5) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 60, and a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 62;
[0039] 6) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 60, and a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 68; and / or
[0040] 7) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 72, and a light chain variable region (VL) comprising an amino acid sequence at least 85% homologous to the amino acids of SEQ ID NO: 74.
[0041] In certain embodiments, the heavy chain variable region and light chain variable region respectively comprise amino acid sequences that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the heavy chain variable region and light chain variable region selected from 1)-7).
[0042] In certain embodiments, the heavy chain constant region of the antibody is IgG.
[0043] In certain embodiments, the heavy chain constant region of the antibody is selected from IgG1, IgG2 or IgG4.
[0044] In certain embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, Fv, a single chain antibody (scFv), Fab, Fab', Fab'-SH or F(ab')2.
[0045] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain and a light chain, wherein:
[0046] (I) the heavy chain comprises an amino acid sequence that is at least 85% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 27, 41, 55, 64, and / or 76, and conservatively modified versions thereof; and
[0047] (II) The light chain comprises an amino acid sequence having at least 85% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 29, 43, 57, 66, 70 and / or 78 and conservatively modified versions thereof.
[0048] In certain embodiments, the heavy chain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to a heavy chain selected from (I); the light chain comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to a light chain selected from (II).
[0049] In certain embodiments, the antibody, or antigen-binding portion thereof, comprises:
[0050] 1) a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 13, and a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 15;
[0051] 2) a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 27, and a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 29;
[0052] 3) a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 41, and a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 43;
[0053] 4) a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 55, and a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 57;
[0054] 5) a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 64, and a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 66;
[0055] 6) a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 64, and a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 70; and / or
[0056] 7) a heavy chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 76, and a light chain comprising an amino acid sequence at least 85% homologous to the amino acid sequence of SEQ ID NO: 78.
[0057] In certain embodiments, the heavy chain and light chain respectively comprise an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homologous to the heavy chain and light chain selected from 1)-7).
[0058] In another aspect, the present invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable region (VH) consisting of SEQ ID NO: 3 and a light chain variable region (VL) consisting of SEQ ID NO: 8.
[0059] In yet another aspect, the present invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable region (VH) consisting of SEQ ID NO: 17 and a light chain variable region (VL) consisting of SEQ ID NO: 22.
[0060] In yet another aspect, the present invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable region (VH) consisting of SEQ ID NO: 31 and a light chain variable region (VL) consisting of SEQ ID NO: 36.
[0061] In one aspect, the invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable region (VH) consisting of SEQ ID NO: 45 and a light chain variable region (VL) consisting of SEQ ID NO: 50.
[0062] In another aspect, the present invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable region (VH) consisting of SEQ ID NO: 60 and a light chain variable region (VL) consisting of SEQ ID NO: 62.
[0063] In yet another aspect, the present invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable region (VH) consisting of SEQ ID NO: 60 and a light chain variable region (VL) consisting of SEQ ID NO: 68.
[0064] In yet another aspect, the present invention provides an antibody, or antigen-binding portion thereof, comprising a heavy chain variable region (VH) consisting of SEQ ID NO: 72 and a light chain variable region (VL) consisting of SEQ ID NO: 74.
[0065] In certain embodiments, the antibody or antigen-binding portion thereof is a CD39 antagonist.
[0066] In certain embodiments, the CD39 is human CD39 or cynomolgus monkey CD39.
[0067] In certain embodiments, the antibody or antigen-binding portion thereof can reduce the ATPase activity of CD39.
[0068] In one aspect, the present invention provides a nucleic acid composition comprising:
[0069] (I) a first nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 18, 32, 46, 61 and / or 73; and
[0070] (II) A second nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 23, 37, 51, 63, 69 and / or 75.
[0071] In certain embodiments, the nucleic acid composition comprises:
[0072] 1) comprising the first nucleic acid shown in SEQ ID NO: 4, and comprising the second nucleic acid shown in SEQ ID NO: 9;
[0073] 2) comprising the first nucleic acid shown in SEQ ID NO: 18, and comprising the second nucleic acid shown in SEQ ID NO: 23;
[0074] 3) comprising the first nucleic acid shown in SEQ ID NO: 32, and comprising the second nucleic acid shown in SEQ ID NO: 37;
[0075] 4) comprising the first nucleic acid shown in SEQ ID NO: 46, and comprising the second nucleic acid shown in SEQ ID NO: 51;
[0076] 5) comprising the first nucleic acid shown in SEQ ID NO: 61, and comprising the second nucleic acid shown in SEQ ID NO: 63;
[0077] 6) comprising the first nucleic acid shown in SEQ ID NO: 61, and comprising the second nucleic acid shown in SEQ ID NO: 69; and / or
[0078] 7) The first nucleic acid represented by SEQ ID NO: 73 and the second nucleic acid represented by SEQ ID NO: 75.
[0079] In another aspect, the present invention provides an expression vector composition comprising:
[0080] (I) a first expression vector, wherein the first expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 18, 32, 46, 61 and / or 73; and
[0081] (II) A second expression vector, wherein the second expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 23, 37, 51, 63, 69 and / or 75.
[0082] In certain embodiments, the expression vector composition comprises:
[0083] 1) a first expression vector, wherein the first expression vector comprises the nucleotide sequence shown in SEQ ID NO: 4; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence shown in SEQ ID NO: 9;
[0084] 2) a first expression vector, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 18; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 23;
[0085] 3) a first expression vector, wherein the first expression vector comprises the nucleotide sequence shown in SEQ ID NO: 32; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence shown in SEQ ID NO: 37; or
[0086] 4) a first expression vector, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 46; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 51;
[0087] 5) a first expression vector, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 61; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 63;
[0088] 6) a first expression vector, wherein the first expression vector comprises the nucleotide sequence shown in SEQ ID NO: 61; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence shown in SEQ ID NO: 69; and / or
[0089] 7) A first expression vector, wherein the first expression vector comprises the nucleotide sequence shown in SEQ ID NO: 73; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence shown in SEQ ID NO: 75.
[0090] In another aspect, the present invention provides an expression vector comprising:
[0091] (I) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 18, 32, 46, 61 and / or 73; and
[0092] (II) a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 9, 23, 37, 51, 63, 69 and / or 75.
[0093] In certain embodiments, the expression vector comprises:
[0094] 1) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence shown in SEQ ID NO: 4; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence shown in SEQ ID NO: 9;
[0095] 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 18; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 23;
[0096] 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 32; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 37;
[0097] 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 46; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 51;
[0098] 5) comprising the first nucleic acid shown in SEQ ID NO: 61, and comprising the second nucleic acid shown in SEQ ID NO: 63;
[0099] 6) comprising the first nucleic acid shown in SEQ ID NO: 61, and comprising the second nucleic acid shown in SEQ ID NO: 69; and / or
[0100] 7) The first nucleic acid represented by SEQ ID NO: 73 and the second nucleic acid represented by SEQ ID NO: 75.
[0101] In yet another aspect, the present invention provides a nucleic acid composition comprising:
[0102] (I) a first nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, 28, 42, 56, 65 and / or 77; and
[0103] (II) A second nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 16, 30, 44, 58, 67, 71 and / or 79.
[0104] In certain embodiments, the nucleic acid composition comprises:
[0105] 1) comprising the first nucleic acid shown in SEQ ID NO: 14, and comprising the second nucleic acid shown in SEQ ID NO: 16;
[0106] 2) comprising the first nucleic acid shown in SEQ ID NO: 28, and comprising the second nucleic acid shown in SEQ ID NO: 30;
[0107] 3) comprising the first nucleic acid shown in SEQ ID NO: 42, and comprising the second nucleic acid shown in SEQ ID NO: 44;
[0108] 4) comprising the first nucleic acid shown in SEQ ID NO: 56, and comprising the second nucleic acid shown in SEQ ID NO: 58;
[0109] 5) comprising the first nucleic acid shown in SEQ ID NO: 65, and comprising the second nucleic acid shown in SEQ ID NO: 67;
[0110] 6) comprising the first nucleic acid shown in SEQ ID NO: 65, and comprising the second nucleic acid shown in SEQ ID NO: 71; and / or
[0111] 7) The first nucleic acid represented by SEQ ID NO: 77 and the second nucleic acid represented by SEQ ID NO: 79.
[0112] In one aspect, the present invention provides an expression vector composition comprising:
[0113] (I) a first expression vector, wherein the first expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, 28, 42, 56, 65 and / or 77; and
[0114] (II) A second expression vector, wherein the second expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 16, 30, 44, 58, 67, 71 and / or 79.
[0115] In certain embodiments, the expression vector composition comprises:
[0116] 1) a first expression vector, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 14; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 16;
[0117] 2) a first expression vector, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 28; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 30;
[0118] 3) a first expression vector, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 42; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 44;
[0119] 4) a first expression vector, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 56; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 58;
[0120] 5) a first expression vector, wherein the first expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 65; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 67;
[0121] 6) a first expression vector, wherein the first expression vector comprises the nucleotide sequence shown in SEQ ID NO: 65; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence shown in SEQ ID NO: 71; and / or
[0122] 7) A first expression vector, wherein the first expression vector comprises the nucleotide sequence shown in SEQ ID NO: 77; and a second expression vector, wherein the second expression vector comprises the nucleotide sequence shown in SEQ ID NO: 79.
[0123] In another aspect, the present invention provides an expression vector comprising:
[0124] (I) a first nucleic acid sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 14, 28, 42, 56, 65 and / or 77; and
[0125] (II) The second nucleic acid sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 16, 30, 44, 58, 67, 71 and / or 79.
[0126] In certain embodiments, the expression vector comprises:
[0127] 1) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 14; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 16;
[0128] 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 28; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 30;
[0129] 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 42; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 44;
[0130] 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 56; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 58;
[0131] 5) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 65; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence set forth in SEQ ID NO: 67;
[0132] 6) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence shown in SEQ ID NO: 65; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence shown in SEQ ID NO: 71; and / or
[0133] 7) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises the nucleotide sequence shown in SEQ ID NO: 77; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises the nucleotide sequence shown in SEQ ID NO: 79.
[0134] In yet another aspect, the present invention provides a nucleic acid composition comprising:
[0135] (I) a first nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 3, 17, 31, 45, 60 and / or 72; and
[0136] (II) A second nucleic acid comprising a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 8, 22, 36, 50, 62, 68 and / or 74.
[0137] In certain embodiments, the nucleic acid composition comprises:
[0138] 1) the first nucleic acid encoding the heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 3, and the second nucleic acid encoding the light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 8;
[0139] 2) the first nucleic acid encoding the heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 17, and the second nucleic acid encoding the light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 22;
[0140] 3) the first nucleic acid encoding the heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 31, and the second nucleic acid encoding the light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 36;
[0141] 4) the first nucleic acid encoding the heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 45, and the second nucleic acid encoding the light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 50;
[0142] 5) the first nucleic acid encoding the heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 60, and the second nucleic acid encoding the light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 62;
[0143] 6) the first nucleic acid encoding the heavy chain variable region (VH) of the amino acid sequence set forth in SEQ ID NO: 60, and the second nucleic acid encoding the light chain variable region (VL) of the amino acid sequence set forth in SEQ ID NO: 68; and / or
[0144] 7) The first nucleic acid encoding the heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 72, and the second nucleic acid encoding the light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 74.
[0145] In certain embodiments, the nucleic acid composition comprises:
[0146] 1) the first nucleic acid encoding the heavy chain variable region (VH) of the amino acid sequence set forth in SEQ ID NO: 3 as set forth in SEQ ID NO: 4, and the second nucleic acid encoding the light chain variable region (VL) of the amino acid sequence set forth in SEQ ID NO: 8 as set forth in SEQ ID NO: 9;
[0147] 2) the first nucleic acid encoding the heavy chain variable region (VH) of SEQ ID NO: 17 as set forth in SEQ ID NO: 18, and the second nucleic acid encoding the light chain variable region (VL) of SEQ ID NO: 22 as set forth in SEQ ID NO: 23;
[0148] 3) the first nucleic acid encoding the heavy chain variable region (VH) of SEQ ID NO: 31 as set forth in SEQ ID NO: 32, and the second nucleic acid encoding the light chain variable region (VL) of SEQ ID NO: 36 as set forth in SEQ ID NO: 37;
[0149] 4) the first nucleic acid encoding the heavy chain variable region (VH) of SEQ ID NO: 45, as set forth in SEQ ID NO: 46, and the second nucleic acid encoding the light chain variable region (VL) of SEQ ID NO: 50, as set forth in SEQ ID NO: 51;
[0150] 5) the first nucleic acid encoding the heavy chain variable region (VH) of SEQ ID NO: 60 as set forth in SEQ ID NO: 61, and the second nucleic acid encoding the light chain variable region (VL) of SEQ ID NO: 62 as set forth in SEQ ID NO: 63;
[0151] 6) the first nucleic acid encoding the heavy chain variable region (VH) of SEQ ID NO: 60 is as shown in SEQ ID NO: 61, and the second nucleic acid encoding the light chain variable region (VL) of SEQ ID NO: 68 is as shown in SEQ ID NO: 69; and / or
[0152] 7) the first nucleic acid encoding the heavy chain variable region (VH) as shown in SEQ ID NO: 72 is as shown in SEQ ID NO: 73, and the second nucleic acid encoding the light chain variable region (VL) as shown in SEQ ID NO: 74 is as shown in SEQ ID NO: 75.
[0153] In yet another aspect, the present invention provides an expression vector composition comprising:
[0154] (I) a first expression vector, wherein the first expression vector comprises a nucleotide sequence selected from the group consisting of a heavy chain variable region (VH) encoding an amino acid sequence as shown in SEQ ID NO: 3, 17, 31, 45, 60 and / or 72; and
[0155] (II) a second expression vector, wherein the second expression vector contains a nucleotide sequence selected from the light chain variable region (VL) encoding an amino acid sequence as shown in SEQ ID NO: 8, 22, 36, 50, 62, 68 and / or 74.
[0156] In certain embodiments, the expression vector composition comprises:
[0157] 1) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 3; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 8;
[0158] 2) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 17; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 22;
[0159] 3) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 31; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 36;
[0160] 4) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 45; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 50;
[0161] 5) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 60; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 62;
[0162] 6) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 60; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 68; and / or
[0163] 7) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 72; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 74.
[0164] In certain embodiments, the expression vector composition comprises:
[0165] 1) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 4 encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 3; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 9 encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 8;
[0166] 2) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 18 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 17; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 23 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 22;
[0167] 3) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:32 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO:31; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:37 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO:36;
[0168] 4) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:46 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO:45; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:51 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO:50;
[0169] 5) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:61 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO:60; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:63 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO:62;
[0170] 6) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 61 encoding a heavy chain variable region (VH) as shown in SEQ ID NO: 60; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 69 encoding a light chain variable region (VL) as shown in SEQ ID NO: 68; and / or
[0171] 7) a first expression vector, wherein the first expression vector contains a nucleotide sequence as shown in SEQ ID NO: 73 encoding the heavy chain variable region (VH) as shown in SEQ ID NO: 72; and a second expression vector, wherein the second expression vector contains a nucleotide sequence as shown in SEQ ID NO: 75 encoding the light chain variable region (VL) as shown in SEQ ID NO: 74.
[0172] In one aspect, the present invention provides an expression vector comprising:
[0173] (I) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence selected from the group consisting of a heavy chain variable region (VH) encoding an amino acid sequence as set forth in SEQ ID NOs: 3, 17, 31, 45, 60, and / or 72; and
[0174] (II) a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence selected from the light chain variable region (VL) encoding an amino acid sequence as shown in SEQ ID NO: 8, 22, 36, 50, 62, 68 and / or 74.
[0175] In certain embodiments, the expression vector comprises:
[0176] 1) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 3; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 8;
[0177] 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 17; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 22;
[0178] 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 31; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 36;
[0179] 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 45; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 50;
[0180] 5) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 60; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 62;
[0181] 6) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 60; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 68; and / or
[0182] 7) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 72; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 74.
[0183] In certain embodiments, the expression vector comprises:
[0184] 1) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 4 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 3; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 8 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 9;
[0185] 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 18 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 17; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 22 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 23;
[0186] 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:32 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO:31; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:37 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO:36;
[0187] 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:46 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO:45; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:51 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO:50;
[0188] 5) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:61 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO:60; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:63 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO:62;
[0189] 6) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 61 encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 60; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 68 encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 69; and / or
[0190] 7) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) as set forth in SEQ ID NO: 72 and an amino acid sequence as set forth in SEQ ID NO: 73; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) as set forth in SEQ ID NO: 74 and an amino acid sequence as set forth in SEQ ID NO: 75.
[0191] In another aspect, the present invention provides a nucleic acid composition comprising:
[0192] (I) a first nucleic acid comprising a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 13, 27, 41, 55, 64 and / or 76; and
[0193] (II) A second nucleic acid comprising a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 15, 29, 43, 57, 66, 70 and / or 78.
[0194] In certain embodiments, the nucleic acid composition comprises:
[0195] 1) the first nucleic acid encoding the heavy chain of the amino acid sequence set forth in SEQ ID NO: 13, and the second nucleic acid encoding the light chain of the amino acid sequence set forth in SEQ ID NO: 15;
[0196] 2) the first nucleic acid encoding the heavy chain of the amino acid sequence set forth in SEQ ID NO: 27, and the second nucleic acid encoding the light chain of the amino acid sequence set forth in SEQ ID NO: 29;
[0197] 3) the first nucleic acid encoding the heavy chain of the amino acid sequence set forth in SEQ ID NO: 41, and the second nucleic acid encoding the light chain of the amino acid sequence set forth in SEQ ID NO: 43;
[0198] 4) the first nucleic acid encoding the heavy chain of the amino acid sequence set forth in SEQ ID NO: 55, and the second nucleic acid encoding the light chain of the amino acid sequence set forth in SEQ ID NO: 57;
[0199] 5) the first nucleic acid encoding the heavy chain of the amino acid sequence set forth in SEQ ID NO: 64, and the second nucleic acid encoding the light chain of the amino acid sequence set forth in SEQ ID NO: 66;
[0200] 6) the first nucleic acid encoding the heavy chain of the amino acid sequence shown in SEQ ID NO: 64, and the second nucleic acid encoding the light chain of the amino acid sequence shown in SEQ ID NO: 70; and / or
[0201] 7) The first nucleic acid comprises the heavy chain encoding the amino acid sequence shown in SEQ ID NO: 76, and the second nucleic acid comprises the light chain encoding the amino acid sequence shown in SEQ ID NO: 78.
[0202] In certain embodiments, the nucleic acid composition comprises:
[0203] 1) the first nucleic acid encoding the heavy chain of SEQ ID NO: 13, as shown in SEQ ID NO: 14, and the second nucleic acid encoding the light chain of SEQ ID NO: 15, as shown in SEQ ID NO: 16;
[0204] 2) the first nucleic acid encoding the heavy chain of SEQ ID NO: 27 is as shown in SEQ ID NO: 28, and the second nucleic acid encoding the light chain of SEQ ID NO: 29 is as shown in SEQ ID NO: 30;
[0205] 3) the first nucleic acid encoding the heavy chain of SEQ ID NO: 41 as set forth in SEQ ID NO: 42, and the second nucleic acid encoding the light chain of SEQ ID NO: 43 as set forth in SEQ ID NO: 44;
[0206] 4) the first nucleic acid encoding the heavy chain of SEQ ID NO: 55 as set forth in SEQ ID NO: 56, and the second nucleic acid encoding the light chain of SEQ ID NO: 57 as set forth in SEQ ID NO: 58;
[0207] 5) the first nucleic acid encoding the heavy chain of SEQ ID NO: 64 is shown in SEQ ID NO: 65, and the second nucleic acid encoding the light chain of SEQ ID NO: 66 is shown in SEQ ID NO: 67;
[0208] 6) the first nucleic acid encoding the heavy chain of SEQ ID NO: 64 is shown in SEQ ID NO: 65, and the second nucleic acid encoding the light chain of SEQ ID NO: 70 is shown in SEQ ID NO: 71; and / or
[0209] 7) The first nucleic acid encoding the heavy chain of SEQ ID NO: 76 is shown in SEQ ID NO: 77, and the second nucleic acid encoding the light chain of SEQ ID NO: 78 is shown in SEQ ID NO: 79.
[0210] In another aspect, the present invention provides an expression vector composition comprising:
[0211] (I) a first expression vector, wherein the first expression vector comprises a nucleotide sequence selected from the group consisting of a heavy chain encoding an amino acid sequence as set forth in SEQ ID NO: 13, 27, 41, 55, 64, and / or 76; and
[0212] (II) A second expression vector, wherein the second expression vector contains a nucleotide sequence selected from the group encoding the light chain of amino acid sequences shown in SEQ ID NOs: 15, 29, 43, 57, 66, 70 and / or 78.
[0213] In certain embodiments, the expression vector composition comprises:
[0214] 1) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 13; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 15;
[0215] 2) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding the heavy chain of the amino acid sequence set forth in SEQ ID NO: 27; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding the light chain of the amino acid sequence set forth in SEQ ID NO: 29;
[0216] 3) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding the heavy chain of the amino acid sequence set forth in SEQ ID NO: 41; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding the light chain of the amino acid sequence set forth in SEQ ID NO: 43;
[0217] 4) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding the heavy chain having an amino acid sequence as set forth in SEQ ID NO: 55; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding the light chain having an amino acid sequence as set forth in SEQ ID NO: 57;
[0218] 5) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 66;
[0219] 6) a first expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 70; and / or
[0220] 7) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 76; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 78.
[0221] In certain embodiments, the expression vector composition comprises:
[0222] 1) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 14 encoding the heavy chain having an amino acid sequence as set forth in SEQ ID NO: 13; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 16 encoding the light chain having an amino acid sequence as set forth in SEQ ID NO: 15;
[0223] 2) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:28 encoding the heavy chain having an amino acid sequence as set forth in SEQ ID NO:27; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:30 encoding the light chain having an amino acid sequence as set forth in SEQ ID NO:29;
[0224] 3) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:42 encoding the heavy chain having an amino acid sequence as set forth in SEQ ID NO:41; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:44 encoding the light chain having an amino acid sequence as set forth in SEQ ID NO:43;
[0225] 4) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:56 encoding the heavy chain having an amino acid sequence as set forth in SEQ ID NO:55; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:58 encoding the light chain having an amino acid sequence as set forth in SEQ ID NO:57;
[0226] 5) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:65 encoding the heavy chain having an amino acid sequence as set forth in SEQ ID NO:64; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO:67 encoding the light chain having an amino acid sequence as set forth in SEQ ID NO:66;
[0227] 6) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 65 encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 71 encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 70; and / or
[0228] 7) a first expression vector, wherein the first expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 77 encoding the heavy chain of the amino acid sequence as shown in SEQ ID NO: 76; and a second expression vector, wherein the second expression vector comprises a nucleotide sequence as shown in SEQ ID NO: 79 encoding the light chain of the amino acid sequence as shown in SEQ ID NO: 78.
[0229] In yet another aspect, the present invention provides an expression vector comprising:
[0230] (I) a first nucleic acid sequence comprising a nucleotide sequence selected from the group consisting of a heavy chain encoding an amino acid sequence as set forth in SEQ ID NO: 13, 27, 41, 55, 64 and / or 76; and
[0231] (II) A second nucleic acid sequence comprising a nucleotide sequence selected from the group consisting of a light chain encoding an amino acid sequence as shown in SEQ ID NO: 15, 29, 43, 57, 66, 70 and / or 78.
[0232] In certain embodiments, the expression vector comprises:
[0233] 1) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 13; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 15;
[0234] 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 27; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 29;
[0235] 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding the heavy chain of the amino acid sequence set forth in SEQ ID NO: 41; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding the light chain of the amino acid sequence set forth in SEQ ID NO: 43;
[0236] 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 55; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 57;
[0237] 5) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 66;
[0238] 6) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 70; and / or
[0239] 7) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 76; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 78.
[0240] In certain embodiments, the expression vector comprises:
[0241] 1) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 14 encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 13; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 16 encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 15;
[0242] 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:28 encoding the heavy chain of the amino acid sequence as set forth in SEQ ID NO:27; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:30 encoding the light chain of the amino acid sequence as set forth in SEQ ID NO:29;
[0243] 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:42 encoding the heavy chain of the amino acid sequence as set forth in SEQ ID NO:41; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:44 encoding the light chain of the amino acid sequence as set forth in SEQ ID NO:43;
[0244] 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:56 encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO:55; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:58 encoding a light chain having an amino acid sequence as set forth in SEQ ID NO:57;
[0245] 5) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:65 encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO:64; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO:67 encoding a light chain having an amino acid sequence as set forth in SEQ ID NO:66;
[0246] 6) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 65 encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as set forth in SEQ ID NO: 71 encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 70; and / or
[0247] 7) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence as shown in SEQ ID NO: 77 encoding a heavy chain of the amino acid sequence as shown in SEQ ID NO: 76; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence as shown in SEQ ID NO: 79 encoding a light chain of the amino acid sequence as shown in SEQ ID NO: 78.
[0248] In one aspect, the present invention provides a cell comprising the expression vector composition or the expression vector.
[0249] In another aspect, the present invention provides a method for preparing an antibody or an antigen-binding portion thereof, comprising: expressing the antibody or the antigen-binding portion thereof in the cell, and isolating the antibody or the antigen-binding portion thereof from the cell.
[0250] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding portion thereof, and a pharmaceutically acceptable carrier.
[0251] In yet another aspect, the present invention provides a kit comprising the antibody or antigen-binding portion thereof.
[0252] In one aspect, the present invention provides a bispecific antibody or a multispecific antibody comprising the light chain variable region and the heavy chain variable region.
[0253] In another aspect, the present invention provides a single-chain antibody comprising the light chain variable region and the heavy chain variable region.
[0254] In another aspect, the present invention provides an antibody-drug conjugate comprising the light chain variable region and heavy chain variable region.
[0255] In another aspect, the present invention provides a method for treating a disease, comprising administering to a subject in need thereof an effective dose of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate.
[0256] In certain embodiments, the disease is cancer.
[0257] In certain embodiments, the cancer is a solid tumor or a hematological cancer.
[0258] In certain embodiments, the solid tumor is selected from multiple myeloma, melanoma, gastric cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, bladder cancer, kidney cancer, salivary gland cancer, esophageal cancer, glioma, glioblastoma, thyroid cancer, thymic cancer, epithelial cancer, lymphoma, T and / or B cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, testicular cancer, endometrial cancer, prostate cancer and / or brain cancer.
[0259] In certain embodiments, the blood cancer is leukemia.
[0260] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate in preparing a drug.
[0261] In certain embodiments, the medicament is for treating cancer.
[0262] In certain embodiments, the cancer is a solid tumor or a hematological cancer.
[0263] In certain embodiments, the solid tumor is selected from multiple myeloma, melanoma, gastric cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, bladder cancer, kidney cancer, salivary gland cancer, esophageal cancer, glioma, glioblastoma, thyroid cancer, thymic cancer, epithelial cancer, lymphoma, T and / or B cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, testicular cancer, endometrial cancer, prostate cancer and / or brain cancer.
[0264] In certain embodiments, the blood cancer is leukemia.
[0265] In another aspect, the present invention provides the antibody or antigen-binding fragment thereof, and / or the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate for use in treating a disease.
[0266] In certain embodiments, the disease is cancer.
[0267] In certain embodiments, the cancer is a solid tumor or a hematological cancer.
[0268] In certain embodiments, the solid tumor is selected from multiple myeloma, melanoma, gastric cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, bladder cancer, kidney cancer, salivary gland cancer, esophageal cancer, glioma, glioblastoma, thyroid cancer, thymic cancer, epithelial cancer, lymphoma, T and / or B cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, testicular cancer, endometrial cancer, prostate cancer and / or brain cancer.
[0269] In certain embodiments, the blood cancer is leukemia.
[0270] In another aspect, the present invention provides a method for treating a disease, comprising administering to a subject in need thereof an effective dose of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate.
[0271] In certain embodiments, the disease is a CD39-related disease.
[0272] In one aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate in preparing a drug.
[0273] In certain embodiments, the medicament is for treating a CD39-related disease.
[0274] In another aspect, the present invention provides the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate for use in treating a disease.
[0275] In certain embodiments, the disease is a CD39-related disease.
[0276] In another aspect, the present invention provides a method for increasing T cell activity in cancer patients, comprising administering to the cancer patient an effective dose of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate.
[0277] In another aspect, the present invention provides a method for reducing adenosine-mediated inhibition of T cell activity in cancer patients, comprising administering to the cancer patient an effective dose of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate.
[0278] In one aspect, the present invention provides a method for increasing T cell activity in a patient's tumor microenvironment, comprising administering to the cancer patient an effective dose of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate.
[0279] In another aspect, the present invention provides a method for treating or preventing tumors, wherein the method comprises:
[0280] (1) detecting the presence of CD39 polypeptide in cells in the tumor microenvironment, optionally in tumor tissue and / or in adjacent tissue, optionally tumor cells, and
[0281] (2) Based on the expression of CD39 polypeptide in cells in the tumor microenvironment, optionally, when CD39 polypeptide expression is increased compared to a reference level, administering an effective dose of the antibody or antigen-binding fragment thereof, the pharmaceutical composition, the bispecific antibody or multispecific antibody, the single-chain antibody, and / or the antibody-drug conjugate to a subject in need thereof.
[0282] In certain embodiments, the step of detecting the presence of CD39 polypeptide in cells in the tumor microenvironment in (1) comprises obtaining a biological sample from an individual, contacting the cells with an antibody that binds to the CD39 polypeptide, and detecting the expression of CD39 in the cells, wherein the biological sample comprises tumor tissue and / or adjacent tissue.
[0283] Table I Description of the Sequences of the Present Invention
[0284]
[0285]
[0286]
[0287]
[0288]
[0289]
[0290]
[0291]
[0292]
[0293]
[0294]
[0295]
[0296]
[0297]
[0298]
[0299]
[0300]
[0301]
[0302] BRIEF DESCRIPTION OF THE DRAWINGS
[0303] Figure 1 The binding ability of CD39 chimeric antibody to soluble huCD39 protein was determined;
[0304] Figure 2 Demonstrate the ability of CD39 chimeric antibodies to bind to native CD39 protein;
[0305] Figure 3 Demonstrate the ability of CD39 chimeric antibodies to block cell surface ATPase activity;
[0306] Figure 4 The binding ability of humanized CD39 antibody to soluble huCD39 protein was determined;
[0307] Figure 5 Demonstrate the ability of CD39 humanized antibody to bind to cell surface huCD39 protein;
[0308] Figure 6 Demonstrate the ability of CD39 humanized antibody to block cell surface ATPase activity;
[0309] Figure 7 Demonstrated the reversal effect of CD39 humanized antibody on ATP-mediated inhibition of human CD4+ T cell proliferation;
[0310] Figure 8 Demonstrated the reversal effect of CD39 humanized antibody on ATP-mediated inhibition of human CD8+ T cell proliferation;
[0311] Figure 9 showed that humanized CD39 antibodies reversed the ability of CD4+ T cells to release IFN-γ;
[0312] Figure 10 Demonstrate the effect of CD39 humanized antibody-mediated CD39 endocytosis;
[0313] Figure 11 Demonstrate the activation effect of humanized CD39 antibody on DC cells;
[0314] Figure 12 Shows the efficacy evaluation of CD39 humanized antibody in MOLP-8 xenograft model;
[0315] Figure 13 Demonstrated CD39 humanized antibody inhibited MOLP-8 tumor growth in MOLP-8 xenograft model;
[0316] Figure 14 The results showed that humanized CD39 antibody could inhibit the growth of IM-9 tumors in IM-9 xenograft tumor model.
[0317] Detailed description
[0318] definition
[0319] To make this specification easier to understand, some terms are first defined. Other definitions are set out throughout the specification.
[0320] Human CD39, also known as NTPdasel, ENTPD1, ATPDase, and vascular ATP diphosphohydrolase, has an International Commission on Enzyme Designation (ICE) number of EC 3.6.1.5 and exhibits ATPase activity. CD39 hydrolyzes extracellular ATP and ADP into AMP, which is further converted to adenosine by 5' ectonucleotidase. The amino acid sequence of the mature human CD39 polypeptide chain is shown in GenBank under accession number P49961.
[0321] As used herein, the term "antibody" may include complete antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portion") or single chains. In one embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In certain naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, referred to as complementarity determining regions (CDRs), and more conserved regions, referred to as framework regions (FRs), interspersed with each other. Herein, the CDRs in the VH region are abbreviated as HCDRs, i.e., the three CDRs in the VH region can be abbreviated as HCDR1, HCDR2, and HCDR3; the CDRs in the VL region are abbreviated as LCDRs, i.e., the three CDRs in the VL region can be abbreviated as LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
[0322] The heavy chain of the antibody may or may not contain a terminal lysine (K) or a terminal glycine and lysine (GK). Thus, any heavy chain sequence and heavy chain constant region sequence provided herein may end with GK or K, or lack K or GK, regardless of the last amino acid of the sequence. This is because the terminal lysine, and sometimes glycine and lysine, are cleaved off during antibody expression.
[0323] Antibodies typically bind specifically to their cognate antigen with high affinity, ranging from 10 -7 M to 10 -11 The dissociation constant (K D ) is generally considered to be greater than about 10 -6 Any K of M D Indicates nonspecific binding. As used herein, an antibody that "specifically binds" to an antigen refers to an antibody that binds to the antigen with high affinity and is substantially the same antigen, but does not bind to unrelated antigens with high affinity, high affinity meaning having 10 -7 M or less, preferably 10 -8 M or less, even more preferably 5×10 -9M or smaller, and preferably between 10 -8 M and 10 -10 M or smaller K D . An antigen is “substantially identical” to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% or greater sequence identity to the sequence of the given antigen. Immunoglobulins may be from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are divided into multiple subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In certain embodiments, the anti-CD39 antibodies described herein have a human IgG1 or IgG2 subtype. Immunoglobulins (e.g., human IgG1) exist in several isotypes that differ from each other by at most a few amino acids. For example, “antibodies” may include naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies; and single-chain antibodies.
[0324] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, human CD39). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody (e.g., an anti-CD39 antibody described herein) include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments connected at the hinge region by a disulfide bridge; (iii) a Fd fragment, which consists of the VH and CH1 domains; (iv) an Fv fragment, which consists of the VL and VH domains of a single arm of the antibody, (v) a dAb fragment (Ward et al. (1989) Nature 341:544-546), which consists of a VH domain; and (vi) isolated complementarity determining regions (CDRs), or (vii) a combination of two or more isolated CDRs, which may be optionally linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be linked by recombinant methods using a synthetic linker to enable them to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv)); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). These single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These and other potential constructs are described in Chan and Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and screening from these fragments can be used in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.
[0325] The term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region of an antibody of the present invention can be replaced with other amino acid residues from the same side chain family, and the modified antibody can be tested for retained function using the functional assays described herein. Preferably, conservative modifications do not exceed one or two in number.
[0326] "Bispecific" or "bifunctional antibodies" are artificial hybrid antibodies that have two different heavy chain / light chain pairs, thereby generating two antigen-binding sites specific for different antigens. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or linking Fab' fragments. For example, see Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).
[0327] As used herein, the term "monoclonal antibody" refers to an antibody that displays a single binding specificity and affinity for a specific epitope, or an antibody composition in which all antibodies in the antibody composition display a single binding specificity and affinity for a specific epitope. Typically, these monoclonal antibodies will be derived from a single antibody encoding cell or nucleic acid and will proliferate without intentionally introducing any sequence changes. Therefore, the term "human monoclonal antibody" refers to a monoclonal antibody with a variable region and an optional constant region derived from a human germline immunoglobulin sequence. In one embodiment, human monoclonal antibodies are produced by, for example, fusing B cells obtained from transgenic or transchromosomal non-human animals (e.g., transgenic mice with a genome comprising human heavy chain transgenes and light chain transgenes) with hybridomas obtained from immortalized cells.
[0328] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal to human immunoglobulin genes or from hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, such as from transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences and are encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As known in the art (e.g., see Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains antigen binding domains encoded by different genes that are rearranged to form antibodies specific for foreign antigens. In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (called somatic mutations or hypermutations) to increase the antibody's affinity for foreign antigens. The constant region will further change in response to antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid sequences encoding the light and heavy chain immunoglobulin polypeptides in response to an antigen may not be identical to the original germline sequences, but will be substantially identical or similar (i.e., have at least 80% identity).
[0329] "HuMAb" refers to an antibody having a variable region in which the framework and CDR regions in the variable region are all derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The antibodies described herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been transplanted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.
[0330] "Humanized" antibodies refer to antibodies in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids in one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids may be tolerated as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains an antigenic specificity that is similar to that of the original antibody.
[0331] A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.
[0332] A "modified heavy chain constant region" refers to a heavy chain constant region comprising the constant domains CH1, hinge, CH2, and CH3, wherein one or more constant domains are from different isotypes (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the modified constant region comprises a human IgG2 CH1 domain and a human IgG2 hinge and human IgG1 CH3 domain fused to a human IgG1 CH2 domain. In certain embodiments, these modified constant regions also include amino acid modifications within one or more domains relative to the wild-type amino acid sequence.
[0333] As used herein, "isotype" refers to the antibody class encoded by the heavy chain constant region genes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibodies).
[0334] "Isotype" refers to naturally occurring variants within a particular isotype group that differ by a few amino acids (eg, see Jefferis et al. (2009) mAbs 1:1). The antibodies described herein may be of any isotype.
[0335] Unless otherwise indicated elsewhere herein, all amino acid numbering is according to the EU index of the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242).
[0336] "An antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen."
[0337] "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or the biochemical events arising therefrom. Exemplary "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (e.g., ADCC and antibody-dependent cell-mediated phagocytosis (ADCP)), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). These effector functions generally require the combination of an Fc region and a binding domain (e.g., an antibody variable domain).
[0338] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibodies include members of the FcγR family, including allelic variants and alternatively spliced forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA in humans) and one inhibitory receptor (FcγRIIB). The various properties of human FcγRs are summarized in Table A. Most innate effector cell types co-express one or more activating FcγRs and the inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but do not express the inhibitory FcγRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is thought to bind to an activating Fc receptor type equivalent to murine IgG2a.
[0339] Table A. Properties of human FcγRs
[0340]
[0341] "Hinge", "hinge domain" or "hinge region" or "antibody hinge region" refers to the domain of the heavy chain constant region that connects the CH1 domain and the CH2 domain and includes the upper, middle and lower parts of the hinge (Roux et al., J. Immunol. 1998 161: 4083). The hinge provides different flexibility between the binding region and the effector region of the antibody and also provides the site for intermolecular disulfide bonding between the two heavy chain constant regions.
[0342] The term "hinge" includes wild-type hinges as well as variants thereof (e.g., non-naturally occurring hinges or modified hinges). For example, the term "IgG2 hinge" includes wild-type IgG2 hinges and variants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or at most 5, 4, 3, 2, or 1 mutations (e.g., substitutions, deletions, or additions).
[0343] The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge in the heavy chain constant domain. The term "CH1 domain" includes wild-type CH1 domains and variants thereof (e.g., non-naturally occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes wild-type CH1 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, or addition).
[0344] Exemplary CH1 domains include CH1 domains with mutations that alter the biological activity of the antibody (eg, ADCC, CDC, or half-life). Provided herein are CH1 domain modifications that affect the biological activity of an antibody.
[0345] The term "CH2 domain" refers to the heavy chain constant region connecting the hinge and CH3 domain in the heavy chain constant domain. The term "CH2 domain" includes wild-type CH2 domains and variants thereof (e.g., non-naturally occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wild-type CH2 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or up to 5, 4, 3, 2 or 1 mutations (e.g., substitutions, deletions or additions). Exemplary CH2 domains include CH2 domains with mutations that alter the biological activity of the antibody (e.g., ADCC, CDC or half-life).
[0346] The term "CH3 domain" refers to the heavy chain constant region that is the C-terminal end of the CH2 domain in the heavy chain constant domain. The term "CH3 domain" includes wild-type CH3 domains and variants thereof (e.g., non-naturally occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wild-type CH3 domains and variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and / or up to 5, 4, 3, 2 or 1 mutations (e.g., substitutions, deletions or additions). Exemplary CH3 domains include CH3 domains with mutations that alter the biological activity (e.g., ADCC, CDC or half-life) of the antibody. Provided herein are CH3 domain modifications that affect the biological activity of the antibody.
[0347] "CL domain" refers to the constant domain of the light chain. The term "CL domain" includes wild-type CL domain and variants thereof.
[0348] A "native sequence Fc region" or "native sequence Fc" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgG1 Fc region; a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc encompasses multiple isotypes of Fc (e.g., see Jefferis et al. (2009) mAbs 1:1).
[0349] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., CD39) to which an immunoglobulin or antibody specifically binds. Epitopes within protein antigens can be formed from both contiguous amino acids (usually linear epitopes) or non-contiguous amino acids juxtaposed by the tertiary folding of the protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are generally, but not always, retained upon exposure to denaturing solvents, while epitopes formed from tertiary folding are generally lost upon treatment with denaturing solvents. An epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the epitope bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or contiguous peptides (e.g., from CD39) are tested for reactivity with a given antibody (e.g., an anti-CD39 antibody). Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as x-ray crystallography, 2-dimensional nuclear magnetic resonance, and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, ed. (1996)).
[0350] The term "epitope mapping" refers to a process for identifying molecular determinants on an antigen that are involved in antibody-antigen recognition.
[0351] The term "binds to the same epitope" with respect to two or more antibodies means that these antibodies bind to the same stretch of amino acid residues, as determined by a given method. Techniques for determining whether an antibody binds to the "same epitope on CD39" as an antibody described herein include, for example, epitope mapping methods, such as x-ray analysis of crystals of the antigen: antibody complex (which provides atomic resolution of the epitope) and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to antigen fragments (e.g., proteolytic fragments) or mutational variations of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is generally considered to be indicative of epitope composition (e.g., alanine scanning mutagenesis - Cunningham and Wells (1985) Science 244: 1081). In addition, computational combinatorial methods of epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage-displayed peptide libraries.
[0352] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of another antibody to the target. Known competition experiments (such as those described in the Examples) can be used to determine whether two antibodies compete with each other for binding to the target, i.e., whether one antibody inhibits the binding of another antibody to the target and the degree of inhibition. In certain embodiments, an antibody competes with another antibody for binding to the target and inhibits at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the binding. The degree of inhibition or competition may vary depending on which antibody is a "blocking antibody" (i.e., a cold antibody that is first incubated with the target). Competition assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101 / pdb.prot4277 or Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, overlapping epitopes, or adjacent epitopes (e.g., as demonstrated by steric hindrance).
[0353] Other competitive binding assays include: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assays, solid phase direct labeled sandwich analysis (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeled RIA using 1-125 labeling (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).
[0354] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen but not to other antigens. When measured by surface plasmon resonance (SPR) technology using a predetermined antigen (e.g., recombinant human CD39) as an analyte and an antibody as a ligand or by Scatchard analysis of the binding of an antibody to antigen-positive cells in a 2000 surface plasmon resonance instrument, the expression of the antigen is less than 10 -7 M, for example, less than about 10 -8 M, 10 -9 M or 10 -10 1 M or even less equilibrium dissociation constant (KD), and (ii) bind to the predetermined antigen with an affinity at least twice that of its binding to a nonspecific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. Therefore, unless otherwise indicated, an antibody that "specifically binds to human CD39" refers to an antibody that binds to human CD39 with an affinity of at least 10 -7 M or less, for example, less than about 10 -8 M, 10 -9 M or 10 -10An antibody that "cross-reacts with cynomolgus monkey CD39" is one that binds to soluble or cell-bound human CD39 with a KD of 10 -7 M or less, for example, less than 10 -8 M, 10 -9 M or 10 -10 In certain embodiments, antibodies that do not cross-react with CD39 from non-human species exhibit essentially undetectable binding to these proteins in standard binding assays.
[0355] As used herein, the term "Kassoc" or "Ka" is intended to refer to the association rate constant of a particular antibody-antigen interaction, and the term "Kdis" or "Kd" is intended to refer to the dissociation rate constant of a particular antibody-antigen interaction. D " refers to the equilibrium dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed in molar concentration (M). The K of an antibody can be determined using methods well established in the art. D Determine the K value of the antibody D A preferred method is by using surface plasmon resonance, preferably using a biosensor system, such as Surface plasmon resonance system or flow cytometry or Scatchard analysis.
[0356] The term "EC50" refers to the concentration of an antibody or antigen-binding portion thereof that induces 50% of the maximal response, ie, a response halfway between the maximal response and baseline, in the context of an in vitro or in vivo assay using the antibody or antigen-binding fragment thereof.
[0357] As used herein, the term "naturally occurring" as applied to a target refers to the fact that the target can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by man in the laboratory is naturally occurring.
[0358] A "polypeptide" is a chain of at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. A "protein" may comprise one or more polypeptides.
[0359] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA. "Conservative sequence modifications" of the sequences described in the SEQ ID NOs described herein are also provided, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the SEQ ID NOs described herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which amino acid residues are replaced with amino acid residues with similar side chains. Families of amino acid residues with similar side chains are already defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, a predicted nonessential amino acid residue in an anti-CD39 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (e.g., see Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)). Alternatively, in another embodiment, mutations can be randomly introduced along all or part of the anti-CD39 antibody coding sequence by, for example, saturation mutagenesis, and the resulting modified anti-CD39 antibodies can be screened for improved binding activity.
[0360] With respect to nucleic acids, the term "substantial homology" means that two nucleic acids, or designated sequences thereof, are identical in at least about 80% of their nucleotides, typically at least about 90% to 95%, and more preferably at least about 98% to 99.5% of their nucleotides when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). Alternatively, substantial homology exists when segments hybridize under selective hybridization conditions to the complement of a strand.
[0361] With respect to polypeptides, the term "substantial homology" means that two polypeptides or designated sequences thereof, when optimally aligned and compared (with appropriate insertions or deletions of nucleotides), are at least about 80% identical in amino acids, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5%.
[0362] The percent identity between two sequences varies as a function of the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions × 100), where optimal alignment is determined by taking into account the number of gaps, and the length of each gap, that need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting Examples below.
[0363] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) incorporated into the ALIGN program, version 2.0, using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm in the GAP program included in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[0364] The nucleic acid and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. These searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.
[0365] These nucleic acids can be present in intact cells, in cell lysates, or in partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it is purified to remove other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of a chromosome) or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods well known in the art. See F. Ausubel et al., eds. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).
[0366] Nucleic acids (e.g., cDNAs) can be mutated according to standard techniques to provide gene sequences. For coding sequences, these mutations can optionally affect the amino acid sequence. Specifically, DNA sequences that are substantially homologous to or derived from native V sequences, D sequences, J sequences, constant sequences, switch sequences, and other such sequences described herein are contemplated.
[0367] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thereby replicated along with the host genome. In addition, certain vectors are capable of guiding the expression of genes to which they are operably connected. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vectors. However, the present invention also includes expression vectors of other forms providing equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).
[0368] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that these terms are intended to refer not only to a specific individual cell, but also to the progeny of such a cell. Because mutations or environmental influences may cause certain changes in subsequent generations, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.
[0369] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide or hapten. The antigen may be CD39 or a fragment thereof.
[0370] "Immune response" refers to the biological response for exogenous agents in vertebrates, which responds to protect organisms from the effects of these agents and diseases caused therefrom. Immune response is mediated by the cells of the immune system (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells or neutrophils) and by the effect of soluble macromolecules (including antibodies, cytokines and complement) produced by any one of these cells or liver, which results in selective targeting, combination, damage, destruction and / or elimination of invasive pathogens, cells or tissues of infected pathogens, cancerous cells or other abnormal cells or (in the case of autoimmunity or pathological inflammation) normal human cells or tissues from vertebrate bodies. Immune response (response) or reaction (reaction) include, for example, activation or suppression of T cells (such as effector T cells or Th cells, such as CD4+ or CD8+ T cells) or suppression of Treg cells.
[0371] "Immunomodulator" or "immunomodulator" refers to an agent that can participate in regulating, controlling or changing an immune response, such as a component of a signal transduction pathway. "Regulating," "regulating," or "changing" an immune response refers to any change in the activity of an immune system cell or such cell (e.g., an effector T cell). The regulation includes stimulation or suppression of the immune system, which can be manifested as an increase or decrease in the number of different cell types, an increase or decrease in the activity of such cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been discovered, some of which may have enhanced function in the tumor microenvironment. Immunomodulators can be located on the surface of T cells. An "immunomodulatory target" or "immunomodulatory target" is an immunomodulator that is targeted by a substance, agent, moiety, compound, or molecule and whose activity is altered by the binding of the substance, agent, moiety, compound, or molecule. Immunomodulatory targets include, for example, receptors on the cell surface ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").
[0372] The ability of increased stimulation immune response or immune system can be derived from the agonist activity of the enhancement of T cell costimulatory receptors and / or the antagonist activity of the enhancement of inhibitory receptors.The ability of increased stimulation immune response or immune system can be reflected by the increase multiple of EC50 or maximum activity degree in the determination (such as measuring cytokine or chemokine release, cytolytic activity (directly measured on target cells or indirectly measured via detecting CD107a or granzyme) and the change of propagation) of measuring immune response.The ability of stimulation immune response or immune system activity can enhance at least 10%, 30%, 50%, 75%, 2 times, 3 times, 5 times or larger.
[0373] "Immunotherapy" refers to the treatment of a subject having a disease or at risk of contracting a disease or suffering recurrence of a disease by a method involving inducing, enhancing, suppressing or otherwise altering an immune response.
[0374] "Immunostimulatory therapy" or "immunostimulatory therapy" refers to a therapy that increases (induces or enhances) a subject's immune response, eg, to treat cancer.
[0375] "Strengthening an endogenous immune response" means increasing the effectiveness or efficacy of an existing immune response in a subject. This increase in effectiveness and efficacy can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.
[0376] "T effector" ("Teff") cells refer to T cells with cytolytic activity (e.g., CD4+ and CD8+ T cells) and T helper (Th) cells, which secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells).
[0377] As used herein, the term "linked" refers to the association of two or more molecules. The link can be covalent or non-covalent. The link can also be genetically linked (i.e., fused recombinantly). These links can be achieved using a variety of art-recognized techniques, such as chemical coupling and recombinant protein production.
[0378] As used herein, "administering" refers to the physical introduction of a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardial, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein may be administered via non-parenteral routes, such as topical, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.
[0379] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (eg, CD8+ cells) and helper T cells (eg, CD4+ cells). T cell-mediated responses include, for example, T cell toxicity and proliferation.
[0380] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. The CTL response is primarily mediated by CD8+ T cells.
[0381] As used herein, "inhibition" or "neutralization" (e.g., "neutralize CD39," "neutralize the activity of CD39," or "neutralize the enzymatic activity of CD39," etc.) when referring to a CD39 polypeptide refers to a process in which the ATP hydrolysis activity (ATPase) of CD39 is inhibited. This particularly includes inhibiting the CD39-mediated production of AMP and / or ADP, i.e., inhibiting the CD39-mediated breakdown of ATP into AMP and / or ADP. This can be measured, for example, in a cell-based assay that measures the ability of a test compound to directly or indirectly inhibit the conversion of ATP to AMP and / or ADP. For example, as described herein, the disappearance of ATP and / or the production of AMP can be assessed.
[0382] The term "internalization" is used interchangeably with "intracellular internalization" and refers to the molecular, biochemical, and cellular events involved in the transfer of molecules from the extracellular surface of a cell to the intracellular surface of a cell. The processes responsible for the internalization of intracellular molecules are well known and, in particular, involve the internalization of extracellular molecules (such as hormones, antibodies, and small organic molecules); the internalization of membrane-associated molecules (such as cell surface receptors); and complexes of membrane-associated molecules bound to extracellular molecules (e.g., ligands bound to transmembrane receptors or antibodies bound to membrane-associated molecules). Thus, "inducing and / or increasing internalization" includes events in which intracellular internalization is initiated and / or the rate and / or extent of intracellular internalization is increased.
[0383] As used herein, "cancer" refers to a broad class of diseases characterized by uncontrolled abnormal cell growth in the body. Malignant tumors or cells can form due to unregulated cell division, invade neighboring tissues, and metastasize to distant parts of the body via the lymphatic system or bloodstream.
[0384] As used herein, the terms "treat" or "treatment" refer to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, to reverse, alleviate, ameliorate, inhibit, slow, or prevent the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease. Prevention refers to the administration of an agent to a subject not already suffering from the disease to prevent the disease from occurring, or to minimize its effects if the disease occurs.
[0385] "Hematological malignancies" include lymphomas, leukemias, myelomas, or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Exemplary lymphomas include both B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include both Hodgkin's lymphomas and most non-Hodgkin's lymphomas. Non-limiting examples of B-cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlapping with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom macroglobulinemia, nodular marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis. Non-limiting examples of T-cell lymphomas include extranodal T-cell lymphoma, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma. Hematological malignancies also include leukemias, such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and acute lymphoblastic leukemia. Hematological malignancies further include myeloma, such as, but not limited to, multiple myeloma and smoldering multiple myeloma. The term "hematological malignancy" encompasses other blood and / or B-cell or T-cell related cancers.
[0386] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability resulting from the disease. A "prophylactically effective amount" or "prophylactically effective dose" of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering a recurrence of the disease, inhibits the progression or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote regression of a disease or to inhibit the progression or recurrence of a disease can be assessed using a variety of methods known to those skilled in the art, such as assessment in human subjects during clinical trials, assessment in animal model systems that are predictive of efficacy in humans, or assessment by measuring the activity of the agent in in vitro assays.
[0387] For example, an anticancer agent is a drug that slows down the progression of cancer or promotes the regression of cancer in a subject. In a preferred embodiment, a therapeutically effective amount of a drug promotes the regression of cancer to the extent of eliminating the cancer. "Promoting cancer regression" means that the administration of an effective amount of a single drug or a combination of a drug and an anticancer agent in a patient can reduce tumor growth or size, tumor necrosis, reduce the severity of at least one disease symptom, increase the frequency and duration of disease-free periods, prevent damage or disability caused by illness, or otherwise improve disease symptoms. Pharmacological effectiveness refers to the ability of a drug to promote the regression of cancer in a patient. Physiological safety refers to an acceptable low level of toxicity due to drug administration or other adverse physiological effects (adverse effects) at the cell, organ and / or organism level.
[0388] For example, for the treatment of tumors, a therapeutically effective amount or dose of the drug preferably inhibits cell growth or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%, relative to an untreated subject. In the best embodiment, a therapeutically effective amount or dose of the drug completely inhibits cell growth or tumor growth, i.e., preferably inhibits cell growth or tumor growth by 100%. The ability of the compound to inhibit tumor growth can be assessed using the assays described below. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, which inhibition can be measured in vitro by assays known to those skilled in the art. In other preferred embodiments described herein, tumor regression can be observed and can persist for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days.
[0389] The terms "patient" and "subject" refer to any human or non-human animal receiving a prophylactic or therapeutic treatment. For example, the methods and compositions described herein can be used to treat a subject suffering from cancer. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like. Example
[0390] Example 1: Screening and identification of CD39 antibodies
[0391] C57 / BL6 mice were immunized with a recombinant human CD39 extracellular domain protein (huCD39). The first immunization was performed with an emulsion of 50 μg huCD39 protein in complete Freund's adjuvant (intraperitoneal injection), the second immunization was performed with an emulsion of 25 μg huCD39 protein in incomplete Freund's adjuvant (subcutaneous injection), the third immunization was performed with an emulsion of 25 μg huCD39 protein in incomplete Freund's adjuvant (intraperitoneal injection), the fourth immunization was performed with an emulsion of 25 μg huCD39 protein in incomplete Freund's adjuvant (subcutaneous injection), and finally, a final boost immunization was performed with 50 μg huCD39 protein (intraperitoneal injection). Four days after the boost, a portion of the immune splenocytes were fused with SP2 / 0 cells using electrofusion to prepare hybridomas. Primary screening was performed using ELISA and flow cytometry, followed by further enzyme activity blocking activity screening using the huCD39-expressing 293T / 17 cell line (293T / 17-huCD39), and screening using the reversal effect of CD4+ T cell proliferation inhibition. Ultimately, four mouse antibodies with CD39 enzyme activity blocking function were obtained.
[0392] Example 2: Indirect ELISA determination of the binding of chimeric antibodies to huCD39
[0393] The four murine antibody Fv regions screened in Example 1 were fused to the human IgG2 Fc region and constructed into the pcDNA3.1 vector. The antibody proteins were transfected into 293F cells for expression and purified using Protein A affinity chromatography to obtain four chimeric antibodies, 201hIgG2, 216hIgG2, 217hIgG2, and 219hIgG2. The sequences of the four chimeric antibodies are detailed in Table I-1. The affinity of the chimeric antibodies was determined by indirect ELISA: ELISA plates (Corning) were coated with 1 μg / mL huCD39 recombinant protein (Sino Biological) and incubated overnight at 4°C. The next day, the plates were washed five times with PBS buffer and blocked with 200 μL / well of 2% skim milk powder for 1 hour. A range of CD39 chimeric antibodies were incubated at room temperature for 1 hour. The plates were then washed five times with PBST wash buffer (PBS, 0.05% Tween 20), and 100 μL of HRP-labeled secondary antibody was added to each well and incubated at room temperature for 30 minutes. The plates were washed five more times and TMB (Life Technologies) was added for color development for 5 to 10 minutes. 1N HCl was added to terminate the reaction, and the OD value was measured at 450 nm. GraphPad Prism software was used to generate data graphs and calculate affinity data ( Figure 1The results are shown in Table 1. The EC values of the binding activities of the four chimeric antibodies 201hIgG2, 216hIgG2, 217hIgG2, and 219hIgG2 are 50 The values are all 10 -9 M level.
[0394] Table I-1
[0395]
[0396]
[0397] Table 1. Affinity of chimeric antibodies
[0398] Antibody Bottom Top EC50 (nM) 201hIgG2 0.046 1.636 1.395 216hIgG2 0.060 1.427 2.426 217hIgG2 0.070 1.391 4.801 219hIgG2 0.052 1.330 1.690
[0399] Example 3: Flow cytometry determination of the binding of chimeric antibodies to native CD39 on cell surfaces
[0400] Flow cytometry assay: The ability of chimeric antibodies to bind to native CD39 protein on the cell surface was evaluated using the recombinant host cell line 293T / 17-huCD39 cells expressing huCD39 and the recombinant host cell line 293T / 17-cyno CD39 cells expressing cyno CD39. The recombinant cells were resuspended in PBS buffer and 2x10 6 Cells were placed in a 96-well U-shaped plate, and chimeric antibodies with a certain gradient dilution range were incubated in a 4°C refrigerator or on ice for 1 hour, then centrifuged at 4°C 1500rpm for 3 minutes, washed three times with PBS buffer, and then incubated with diluted Alexa Fluor 488-labeled goat anti-human polyclonal antibody (pAb): Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (thermo) in a 4°C refrigerator or on ice for 30 minutes. Finally, the cells were washed three times with PBS as described above and analyzed by MACSQuant flow cytometer. Data graphs were generated and affinity data were statistically analyzed using GraphPad Prism software ( Figure 2 The results are shown in Table 2. The EC values of 293T / 17-huCD39 and 293T / 17-cyno CD39 chimeric antibodies were 50 The values are all 10 -9 M level.
[0401] Table 2. Affinity of chimeric antibodies to cell surface huCD39 antigen
[0402]
[0403] Example 4: Blockade of cell surface ATPase activity by CD39 chimeric antibodies
[0404] This method is based on the 293T / 17-huCD39 and 293T / 17-cyno CD39 cell lines (the pLVX-EF1α-IRES-Puro vector carrying the huCD39 or cynoCD39 gene was transfected into 293T / 17 cells, and cell clones stably expressing huCD39 or cyno CD39 were obtained by puromycin selection). The ability of CD39 antibodies to block cell surface enzyme activity was determined to confirm the biochemical activity of the chimeric antibodies. 293T / 17-huCD39 and 293T / 17-cyno CD39 cells were trypsinized and the cell density was adjusted to 1.6×10 5 / mL, add 50μL / well to a 96-well plate. Add 50μL / well of a certain gradient range of antibodies to the cell wells and incubate at 37°C for 1h. Add 100μL of 50μM ATP to each well and incubate at 37°C for 0.5h. Centrifuge at 1500rpm for 3min, transfer a certain volume of culture supernatant to an opaque 96-well flat-bottom plate (Costar, 3912). Finally, add the corresponding volume of CellTiter Glo reagent in a 1:1 ratio according to the Promega instructions. After equilibration at room temperature for 5 minutes, read the luminescence value on a Perkin-Elmer Envision microplate reader and determine the cell CD39 enzyme activity by measuring the ATP level. Use GraphPadPrism software to generate data graphs and statistically analyze enzyme kinetic data ( Figure 3 The results are shown in Table 3. All antibodies can inhibit the ATPase activity of CD39 on the cell surface. The blocking activity EC of the four chimeric antibodies is 50 Both are at 10 -11 Level (Table 3).
[0405] Table 3. Blocking ability of CD39 chimeric antibodies on cell surface ATPase activity
[0406]
[0407] Example 5: Antibody humanization
[0408] The two murine antibodies obtained in Example 1 were humanized using the CDR transplantation method. Sequence homology and structural similarity between the two murine antibodies and the human antibody were analyzed. Based on this analysis, the CDRs of the murine antibodies were modified and transplanted into a series of human antibody framework regions. Three humanized antibodies were screened and designated h201H3.1+h219L1.1 G2C, h201H3.1+h201L1.1dmut G2C, and M201 HuH1L1(DE)G2C. The sequences of the three humanized antibodies are detailed in Table I-2. The humanized antibodies were constructed into the pcDNA3.1 vector, transfected into 293F cells for expression, and purified using Protein A affinity chromatography.
[0409] Table I-2
[0410]
[0411]
[0412] Example 6: Indirect ELISA determination of binding of humanized antibodies to CD39
[0413] The affinity of humanized antibodies was determined by indirect ELISA: 1 μg / mL huCD39 recombinant protein was coated on an ELISA plate (Corning) and incubated overnight at 4°C. The next day, the plate was washed 5 times with PBS buffer and blocked with 200 μL / well of 2% skim milk powder for 1 hour; a certain dose range of CD39 humanized antibodies was incubated at room temperature for 1 hour; then washed 5 times with PBST wash buffer (PBS, 0.05% Tween 20), 100 μL of HRP-labeled secondary antibody was added to each well, and incubated at room temperature for 30 minutes; the plate was washed 5 times again, TMB (Life Technologies) was added for color development for 5 to 10 minutes; finally, 1N HCl was added to terminate the reaction, and the OD value was measured at 450 nm. Data graphs were generated and affinity data were statistically analyzed using GraphPad Prism software ( Figure 4 The results are shown in Table 4. The EC values of the binding activities of the three humanized antibodies were 50 The values are all 10 -10 M level.
[0414] Table 4. Affinity of humanized antibodies
[0415] Antibody Bottom Top EC50 (nM) h201H3.1+h219L1.1G2C 0.085 0.991 0.709 h201H3.1+h201L1.1dmut G2C 0.109 1.030 0.433 M201HuH1L1(DE)G2C 0.068 1.180 0.514
[0416] Example 7: Flow cytometry determination of the binding of humanized antibodies to native CD39 on cell surface
[0417] Flow cytometry: The ability of humanized antibodies to bind to native CD39 protein on the cell surface was evaluated using 293T / 17-huCD39 cells, a recombinant host cell line expressing huCD39. Resuspend the recombinant cells in PBS buffer and add 2x10 6 Cells were placed in a 96-well U-shaped plate, and humanized antibodies with a certain gradient dilution range were incubated in a 4°C refrigerator or on ice for 1 hour, then centrifuged at 4°C 1500rpm for 3 minutes, washed three times with PBS buffer, and then incubated with diluted Alexa Fluor 488-labeled goat anti-human polyclonal antibody (pAb): Goat anti-Human IgG (H+L) Cross-Adsorbed SecondaryAntibody, Alexa Fluor 488 (thermo) in a 4°C refrigerator or on ice for 30 minutes. Finally, the cells were washed three times with PBS as described above and placed on a MACSQuant flow cytometer for detection and analysis. GraphPad Prism software was used to generate data graphs and statistically analyze affinity data ( Figure 5 The results are shown in Table 5. For the EC of 293T / 17-huCD39 humanized antibody 50 The values are all 10 - 9 M level.
[0418] Table 5. Affinity of humanized antibodies for cell surface huCD39 antigen
[0419]
[0420] Example 8: Blockade of cell surface ATPase activity by humanized CD39 antibodies
[0421] This method is based on the 293T / 17-huCD39 cell line to determine the ability of CD39 antibodies to block cell surface enzyme activity and confirm the biochemical activity of humanized antibodies. 293T / 17-huCD39 cells were trypsinized and the cell density was adjusted to 1.6×10 5 / ml, add 50μL / well to a 96-well plate. Add 50μl of a certain gradient range of antibodies to the cell wells and incubate at 37°C for 1h. Add 100μL of 50μM ATP to each well and incubate at 37°C for 0.5h. Centrifuge at 1500rpm for 3min, transfer a certain volume of culture supernatant to an opaque 96-well flat-bottom plate (Costar, 3912). Finally, add the corresponding volume of CellTiter Glo reagent in a 1:1 ratio according to the Promega instructions. After equilibration at room temperature for 5 minutes, read the luminescence value on a Perkin-Elmer Envision microplate reader and determine the cell CD39 enzyme activity by measuring the ATP level. Use GraphPad Prism software to generate data graphs and statistically analyze enzyme kinetic data ( Figure 6 The results are shown in Table 6. All antibodies can inhibit the ATPase activity of CD39 on the cell surface. The blocking activity of the three humanized antibodies is EC 50 Both are at 10 -11 Level (Table 6).
[0422] Table 6. Blocking ability of CD39 humanized antibodies on cell surface ATPase activity
[0423]
[0424] Example 9: Reversal of ATP-mediated inhibition of human CD4+ T and CD8+ T cell proliferation by humanized CD39 antibody
[0425] This method is based on the in vitro removal of ATP-mediated CD4+ and CD8+ T cell proliferation inhibition by humanized CD39 antibodies and the detection of IFN-γ levels in cell culture supernatants by ELISA. Human peripheral blood mononuclear cells (PBMCs) were revived and labeled with 5 μM CFSE, and the cell concentration was adjusted to 1*10 6 / mL. Anti-CD28 at a final concentration of 0.5μg / mL and human IL-2 at a final concentration of 5ng / mL were added, and 100μL / well was plated in a 96-well plate pre-coated with 2ug / mL anti-CD3. CD39 antibody was added in serial dilutions and incubated at 37°C for 1h; then ATP was added at a final concentration of 20-100μM; after culturing at 37°C for 6-7 days, cells were collected and flow cytometry (Miltenyi) was used to detect the proliferation of CD4+T and CD8+T cells. At the same time, the supernatant was collected and ELISA was used to detect the level of IFN-γ. GraphPad Prism software was used to calculate the proliferation of CD4+T and CD8+T cells ( Figure 7 and Figure 8 ) and IFN-γ level data ( Figure 9The results are shown in Table 7. The activity of CD39 humanized antibodies was 10 -8 ~10 -10 M level (Table 7).
[0426] Table 7. Reversal effect of CD39 antibody on ATP-mediated inhibition of human CD4+ T and CD8+ T cell proliferation
[0427]
[0428] The IFN-γ level in the cell supernatant was detected using an ELISA kit (Dakewe). The results are shown in Table 8. The EC50 values of the three humanized antibodies for stimulating T cells to secrete IFN-γ were all within 10 -8 ~10 -9 M level.
[0429] Table 8. Ability of humanized CD39 antibodies to reverse IFN-γ release by CD4+ T cells
[0430] Antibody Bottom Top EC50 (nM) h201H3.1+h219L1.1G2C -2793 13557 2.455 h201H3.1+h201L1.1d mut G2C 140 7087 6.157 M201HuH1L1(DE)G2C 146 8031 28.420
[0431] Example 10: CD39 humanized antibody-mediated CD39 endocytosis experiment
[0432] Fab-ZAP saporin reagent (Advanced Targeting Systems) was used to detect the endocytosis of humanized CD39 antibodies on IM-9 cells. The antibody was diluted to a certain dose range with 40nM Fab-ZAP human reagent (Advanced Targeting Systems) and incubated at room temperature for 30 minutes to allow Fab-ZAP to bind to the antibody to be tested to form an antibody premix. 50μL of the antibody premix was added to 10,000 cells / well of IM-9 cells and incubated at 37°C, 5% CO2 for 3 days. The cell plate was taken and CTG reagent (Promega) was added to lyse the cells for 2 minutes, and then equilibrated at room temperature for 5 minutes. The luminescence value was measured using an Enspire microplate reader (PerkinElmer), and the cell growth curve was statistically analyzed using GraphPad Prism software. The results are shown in Table 9 and Figure 10 As shown in Figure 2, the three humanized antibodies mediated CD39 endocytosis in a dose-dependent manner, and the IC50 of each antibody was within 10 -11 ~10 -12 M level.
[0433] Table 9. Effects of CD39 humanized antibody-mediated CD39 endocytosis
[0434] Antibody Bottom Top IC50(pM) h201H3.1+h219L1.1G2C -97751 2846167 4.299 h201H3.1+h201L1.1d mut G2C 96175 2439511 8.901 M201HuH1L1(DE)G2C 40891 2289436 10.300
[0435] Example 11: Activation of DC cells by humanized CD39 antibody
[0436] This method mainly uses flow cytometry to detect the expression levels of DCs cell surface molecules CD86 and HLA-DR to determine the activation effect of CD39 humanized antibody on DC cells. Resuscitate and resuspend monocytes and adjust the cell density to 5*10 5 / mL, plated, and stimulated with 1640+10% FBS medium containing M-CSF (50ng / mL) and IL-4 (10ng / mL) at 37°C for 6 days to obtain DC cells; after 6 days, the cell supernatant was discarded, and 1μg / mL of CD39 humanized antibody was added and incubated at 37°C for 1 hour, and then incubated overnight with or without the addition of a certain concentration of ATP. After 24 hours, the cells were collected and used for FACS analysis of CD86 and HLA-DR expression, and statistical data were collected using GraphPad Prism software. The results are shown in Figure 2. Figure 11 As shown, humanized CD39 antibody enhanced the ATP-induced expression of DCs cell surface molecule CD86 alone, as well as the co-expression of CD86 and HLA-DR.
[0437] Example 12: Evaluation of the efficacy of humanized CD39 antibodies in the MOLP-8 model
[0438] MOLP-8 (human multiple myeloma cells) were diluted with PBS: Matrigel 1:1 and cultured at 1×10 7 Cells were subcutaneously inoculated into 6-8 week old female CB-17 SCID mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), with 26 mice in each group. After subcutaneous inoculation, the tumors were grown to a volume of 300 mm. 3 The animals were divided into groups according to the time of birth (the animals with the largest and smallest tumor volumes were excluded from each group) and intraperitoneally (IP) injected with PBS, antibodies h201H3.1+h201L1.1d mut G2C, and M201 HuH1L1(DE)G2C at a dose of 30 mg / kg, once a week (QW), as shown in Table 10.
[0439] Table 10. Administration routes, doses and schedules
[0440]
[0441] On days 1, 3, and 7 after administration, tumor tissues from 6 mice were collected and prepared into single-cell suspensions for enzyme activity detection. The tumors were cut into small pieces and digested with enzymes. After incubation at 37°C for 40 minutes, undigested tissue pieces were removed by filtration using a 70 μm filter. The single-cell suspensions were collected and plated with 5*10 4Cells were plated in a 96-well plate and ATP solution was added at a final concentration of 25 μM. The cells were incubated at 37°C for 30 minutes. A certain volume of culture supernatant was transferred to an opaque 96-well flat-bottom plate (Costar, 3912). Finally, the corresponding volume of CellTiter Glo reagent was added in a 1:1 ratio according to the Promega instructions. After equilibration at room temperature for 5 minutes, the luminescence value was read on a Perkin-Elmer Envision microplate reader. The ATP level was measured to determine the inhibitory effect of the CD39 humanized antibody on the enzyme activity of MOLP-8 tumor cells. The results are shown in Figure 2. Figure 12 As shown, both humanized antibodies had an enzymatic activity blocking effect on MOLP-8 tumor cells at 1, 3, and 7 days after administration.
[0442] Example 13: Growth inhibition of MOLP-8 xenograft tumor model by humanized antibodies
[0443] MOLP-8 (human multiple myeloma cells) were diluted to 1 × 10 8 cells / ml, 0.1 ml / mouse, subcutaneously inoculated into 6-8 week old CB-17SCID mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), half male and half female, randomly divided into groups of 12, with 6 males and 6 females in each group. 7 days after subcutaneous inoculation, PBS, antibodies h201H3.1+h201L1.1d mut G2C and M201 HuH1L1(DE)G2C were injected intraperitoneally (IP) at a dose of 30 mg / kg, twice a week (BIW), as shown in Table 11.
[0444] Table 11. Administration routes, dosages and schedules
[0445]
[0446] The body weight and tumor size of mice were measured twice a week. The formula for calculating tumor size was: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).
[0447] Based on the tumor volume, the tumor growth curve was drawn. Figure 13 It can be seen that antibodies h201H3.1+h201L1.1dmut G2C and M201 HuH1L1(DE)G2C can both inhibit MOLP-8 tumor growth.
[0448] Example 14: Growth inhibition of IM-9 xenograft tumor model by humanized antibodies
[0449] IM-9 (human peripheral blood B lymphocytes) were diluted to 1×108 cells / ml, 0.1ml / mouse, subcutaneously inoculated into 6-8 week old female CB-17SCID mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), 10 mice per group, after subcutaneous inoculation, when the tumor volume reached 50-70mm 3 The patients were divided into groups according to the time of treatment (inclusion criteria: mean tumor volume within ± 3SD, or tumor volume coefficient of variation CV ≤ 30% (CV = standard deviation / mean)) and intraperitoneally (IP) injected with PBS, antibodies h201H3.1+h201L1.1d mut G2C, and M201 HuH1L1(DE)G2C at a dose of 30 mg / kg, twice a week (BIW), as shown in Table 12.
[0450] Table 12. Administration routes, doses, and schedules
[0451]
[0452] The body weight and tumor size of mice were measured twice a week. The formula for calculating tumor size was: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).
[0453] Based on the tumor volume, the tumor growth curve was drawn. Figure 14 It can be seen that antibodies h201H3.1+h201L1.1dmut G2C and M201 HuH1L1(DE)G2C can effectively inhibit IM-9 tumor growth. Sequence Listing <110> Beijing Jacobs Pharmaceuticals Co., Ltd. <120> Binding molecules specific for CD39 and uses thereof <130> BX200APCT <150> PCT / CN2019 / 115505 <151> 2019-11-05 <160> 79 <170> PatentIn version 3.5 <210> 1 <211> 452 <212> PRT <213> Homo sapiens <400> 1 Thr Gln Asn Lys Ala Leu Pro Glu Asn Val Lys Tyr Gly Ile Val Leu 1 5 10 15 Asp Ala Gly Ser Ser His Thr Ser Leu Tyr Ile Tyr Lys Trp Pro Ala 20 25 30 Glu Lys Glu Asn Asp Thr Gly Val Val His Gln Val Glu Glu Cys Arg 35 40 45 Val Lys Gly Pro Gly Ile Ser Lys Phe Val Gln Lys Val Asn Glu Ile 50 55 60 Gly Ile Tyr Leu Thr Asp Cys Met Glu Arg Ala Arg Glu Val Ile Pro 65 70 75 80 Arg Ser Gln His Gln Glu Thr Pro Val Tyr Leu Gly Ala Thr Ala Gly 85 90 95 Met Arg Leu Leu Arg Met Glu Ser Glu Glu Leu Ala Asp Arg Val Leu 100 105 110 Asp Val Val Glu Arg Ser Leu Ser Asn Tyr Pro Phe Asp Phe Gln Gly 115 120 125 Ala Arg Ile Ile Thr Gly Gln Glu Glu Gly Ala Tyr Gly Trp Ile Thr 130 135 140 Ile Asn Tyr Leu Leu Gly Lys Phe Ser Gln Lys Thr Arg Trp Phe Ser 145 150 155 160 Ile Val Pro Tyr Glu Thr Asn Asn Gln Glu Thr Phe Gly Ala Leu Asp 165 170 175 Leu Gly Gly Ala Ser Thr Gln Val Thr Phe Val Pro Gln Asn Gln Thr 180 185 190 Ile Glu Ser Pro Asp Asn Ala Leu Gln Phe Arg Leu Tyr Gly Lys Asp 195 200 205 Tyr Asn Val Tyr Thr His Ser Phe Leu Cys Tyr Gly Lys Asp Gln Ala 210 215 220 Leu Trp Gln Lys Leu Ala Lys Asp Ile Gln Val Ala Ser Asn Glu Ile 225 230 235 240 Leu Arg Asp Pro Cys Phe His Pro Gly Tyr Lys Lys Val Val Asn Val 245 250 255 Ser Asp Leu Tyr Lys Thr Pro Cys Thr Lys Arg Phe Glu Met Thr Leu 260 265 270 Pro Phe Gln Gln Phe Glu Ile Gln Gly Ile Gly Asn Tyr Gln Gln Cys 275 280 285 His Gln Ser Ile Leu Glu Leu Phe Asn Thr Ser Tyr Cys Pro Tyr Ser 290 295 300 Gln Cys Ala Phe Asn Gly Ile Phe Leu Pro Pro Leu Gln Gly Asp Phe 305 310 315 320 Gly Ala Phe Ser Ala Phe Tyr Phe Val Met Lys Phe Leu Asn Leu Thr 325 330 335 Ser Glu Lys Val Ser Gln Glu Lys Val Thr Glu Met Met Lys Lys Phe 340 345 350 Cys Ala Gln Pro Trp Glu Glu Ile Lys Thr Ser Tyr Ala Gly Val Lys 355 360 365 Glu Lys Tyr Leu Ser Glu Tyr Cys Phe Ser Gly Thr Tyr Ile Leu Ser 370 375 380 Leu Leu Leu Gln Gly Tyr His Phe Thr Ala Asp Ser Trp Glu His Ile 385 390 395 400 His Phe Ile Gly Lys Ile Gln Gly Ser Asp Ala Gly Trp Thr Leu Gly 405 410 415 Tyr Met Leu Asn Leu Thr Asn Met Ile Pro Ala Glu Gln Pro Leu Ser 420 425 430 Thr Pro Leu Ser His Ser Thr Tyr Val Ala His His His His His His 435 440 445 His His His His 450 <210> 2 <211> 532 <212> PRT <213> Macaca fascicularis <400> 2 Met Leu Phe Asp Ser Ile Leu Ser Thr Val Gly Leu Ser Lys Leu Val 1 5 10 15 Ser Val Val Ser Ser Pro Ala Ala Ala Leu Ser Lys Ser Asn Val Lys 20 25 30 Thr Phe Cys Ser Lys Asn Ile Leu Ala Ile Leu Gly Phe Ser Ser Ile 35 40 45 Ile Ala Val Ile Ala Leu Leu Ala Val Gly Leu Thr Gln Asn Lys Ala 50 55 60 Leu Pro Glu Asn Ile Lys Tyr Gly Ile Val Leu Asp Ala Gly Ser Ser 65 70 75 80 His Thr Ser Leu Tyr Ile Tyr Lys Trp Pro Ala Glu Lys Glu Asn Asp 85 90 95 Thr Gly Val Val His Gln Val Glu Glu Cys Arg Val Lys Gly Pro Gly 100 105 110 Ile Ser Lys Tyr Val Gln Lys Val Asn Glu Ile Gly Ile Tyr Leu Thr 115 120 125 Asp Cys Met Glu Arg Ala Arg Glu Val Ile Pro Arg Ser Gln His Gln 130 135 140 Glu Thr Pro Val Tyr Leu Gly Ala Thr Ala Gly Met Arg Leu Leu Arg 145 150 155 160 Met Glu Ser Glu Glu Leu Ala Asp Arg Val Leu Asp Val Val Glu Arg 165 170 175 Ser Leu Ser Asn Tyr Pro Phe Asp Phe Gln Gly Ala Arg Ile Ile Thr 180 185 190 Gly Gln Glu Glu Gly Ala Tyr Gly Trp Ile Thr Ile Asn Tyr Leu Leu 195 200 205 Gly Lys Phe Ser Gln Lys Thr Arg Trp Phe Ser Ile Val Pro Tyr Glu 210 215 220 Thr Asn Asn Gln Glu Thr Phe Gly Ala Leu Asp Leu Gly Gly Ala Ser 225 230 235 240 Thr Gln Ile Thr Phe Val Pro Gln Asn Gln Thr Thr Glu Ser Pro Asp 245 250 255 Asn Ala Leu Gln Phe Arg Leu Tyr Gly Lys Asp Tyr Asn Val Tyr Thr 260 265 270 His Ser Phe Leu Cys Tyr Gly Lys Asp Gln Ala Leu Trp Gln Lys Leu 275 280 285 Ala Lys Asp Ile Gln Val Ala Ser Asn Glu Ile Leu Arg Asp Pro Cys 290 295 300 Phe His Pro Gly Tyr Lys Lys Val Val Asn Val Ser Asp Leu Tyr Lys 305 310 315 320 Thr Pro Cys Thr Lys Arg Phe Glu Met Thr Leu Pro Phe Gln Gln Phe 325 330 335 Glu Ile Gln Gly Ile Gly Asn Tyr Gln Gln Cys His Gln Ser Val Leu 340 345 350 Glu Leu Phe Asn Thr Ser Tyr Cys Pro Tyr Ser Gln Cys Ala Phe Asn 355 360 365 Gly Ile Phe Leu Pro Pro Leu Gln Gly Asp Phe Gly Ala Phe Ser Ala 370 375 380 Phe Tyr Phe Val Met Asn Phe Leu Asn Leu Thr Ser Glu Lys Val Ser 385 390 395 400 Gln Glu Lys Val Thr Glu Met Met Lys Lys Phe Cys Ser Gln Pro Trp 405 410 415 Glu Glu Ile Lys Thr Ser Tyr Ala Gly Val Lys Glu Lys Tyr Leu Ser 420 425 430 Glu Tyr Cys Phe Ser Gly Thr Tyr Ile Leu Ser Leu Leu Leu Gln Gly 435 440 445 Tyr His Phe Thr Ala Asp Ser Trp Glu His Ile His Phe Ile Gly Lys 450 455 460 Ile Gln Gly Ser Asp Ala Gly Trp Thr Leu Gly Tyr Met Leu Asn Leu 465 470 475 480 Thr Asn Met Ile Pro Ala Glu Gln Pro Leu Ser Thr Pro Leu Ser His 485 490 495 Ser Thr Tyr Val Phe Leu Met Val Leu Phe Ser Leu Val Leu Val Ile 500 505 510 Val Ala Ile Ile Gly Leu Leu Ile Phe His Lys Pro Ser Tyr Phe Trp 515 520 525 Lys Asp Met Val 530 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Ile Met Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Ala Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 4 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 4 gaggtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactatggaa tgcactgggt tcgtcaggct 120 ccagagaagg ggctggagtg ggttgcatac attagtagtg gcagtagtat catgtactat 180 gcagacacag tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgttc 240 ctgcaaatgg ccagtctgag gtctgaggac acggccatgt attattgtgc aagggacctc 300 tactatgatc acgtccttga ctactggggc caaggcacca ctctcacagt ctcctca 357 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 5 Asp Tyr Gly Met His 1 5 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 6 Tyr Ile Ser Ser Gly Ser Ser Ile Met Tyr Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 7 Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr 1 5 10 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptide <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Arg Gly Tyr 20 25 30 Containing Trp Containing Gln Gln Lys Pro Gly Gly Thr Containing Lys Arg Containing 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Thr Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 9 gataccaga tgacccagtc tccatcctcc tatatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaaattcgt ggttactta ttggcttca gcagaaacca 120 ggtggaacta ttaaacgcct gatctacgcc gcatccactt tagattgg tgtcccaaag 180 aggttcagtg gcagtaggtc tgttcagat tattctca ccatcagcag cttgagtct 240 gaagattttg cagactatta ctgtctacaa tatactagtt atcctcggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 10 Arg Ala Ser Gln Glu Ile Arg Gly Tyr Leu Ile 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 11 Ala Ala Ser Thr Leu Asp Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 12 Leu Gln Tyr Thr Ser Tyr Pro Arg Thr 1 5 <210> 13 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Ile Met Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Ala Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 14 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 14 gaggtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactatggaa tgcactgggt tcgtcaggct 120 ccagagaagg ggctggagtg ggttgcatac attagtagtg gcagtagtat catgtactat 180 gcagacacag tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgttc 240 ctgcaaatgg ccagtctgag gtctgaggac acggccatgt attattgtgc aagggacctc 300 tactatgatc acgtccttga ctactggggc caaggcacca ctctcacagt ctcctcagct 360 agcaccaagg gaccctccgt gtttcctctg gctccttgct ccagatctac ctccgagtct 420 accgccgctc tgggttgtct ggtgaaggac tacttccccg agccagtgac cgtgtcttgg 480 aacagcggag ctctgacatc cggagtgcac acctttccag ccgtgctgca gtcttccggc 540 ctgtattctc tgtcctccgt ggtgaccgtg cttcttcca acttcggcac ccagacctac 600 acttgcaacg tggaccacaa gccctccaac accaaggtgg acaagaccgt ggagcgcaag 660 tgttgcgtcg agtgccctcc ttgcccagct cctccagtgg ccggaccttc tgtgtttctg 720 ttccccccta agcctaagga caccctgatg atctccccgga ccccagaagt gacttgcgtg 780 gtggtggacg tgtctcacga ggaccccgag gtgcagttca attggtacgt ggacggcgtg 840 900. gaggtgcaca acgctaagac caagcccagg gaggagcagt tcaactccac cttccgggtg gtgtcagtgc tgacagtggt gcaccaggat tggctgaacg gcaaggagta caagtgcaag 960 gtgtccaaca agggcctgcc agctcctatc gagagacca tctccaagac caagggccag cccagagagc ctcaggtgta cacactgcct ccttcccggg cccagagac caagaccag 1080 gtgtccctga cttgcctcgt gaagggattc tacccctccg acatcgcagt cgagtggga tccaacggcc agcccgagaa caactacaag accacccctc ctatgctgga ctccgacggc tccttcttcc tgtactccaa gctgaccgtg gacaagtcc gttggcagca gggcaacgtg ttctcttgca gcgtgatgca cgaggccctg cacaaccact acacccaga gagcctgtcc ctgtctcccg gcaag <210> 15 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Arg Gly Tyr 20 25 30 Leu Ile Trp Leu Gln Gln Lys Pro Gly Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Thr Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 16 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 16 gataccaga tgacccagtc tccatcctcc tatatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaaattcgt ggttactta ttggcttca gcagaaacca 120 ggtggaacta ttaaacgcct gatctacgcc gcatccactt tagattgg tgtcccaaag 180 aggttcagtg gcagtaggtc tgttcagat tattctca ccatcagcag cttgagtct 240 gaagattttg cagactatta ctgtctacaa tatactagtt atcctcggac gttcggtgga 300 ggcaccaagc tggaaatcaa aagaaccgtg gccgctcctt ccgtgttcat cttccctccc 360 tccgacgagc agctgaagag cggaacagcc tctgtcgtgt gcctcctgaa caacttctac 420 ccccgggagg ccaaggtcca gtggaaggtg gacaacgctc tgcagagcgg caactctcag 480 gagagcgtga cagagcagga ctccaaggac tccacctact ccctgtcttc caccctgacc 540 ctgtctaagg ccgactacga gaagcacaag gtgtacgctt gcgaggtgac acaccaggga 600 ctgtcctctc cagtgaccaa gtccttcaac cgcggcgagt gt 642 <210> 17 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Pro Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Only Tire Only Only Only Gly Only Only Only Tire Only Only Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Only Arg Asp Leu Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ala Ser 115 <210> 18 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 18 gaggtgcagc tggtggaggtc tgggggaggc ttagtgaagc ctggaggtc cctgaaactc 60 ccctgtgcag cctctggatt cacttcagt gacttgga tgcactggt tcgtcaggct 120 ccagagaagg ggctggagtg ggttgcatac attagtagtg gcagtagtat catctactat 180 gcagacacag tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgttc 240 ctgcaaatga ccagtctgag gtctgaggac acggccatgt attactgtgc aagggacctc 300 tactatgatc acgtccttga ctattggggc caaggcacca ctctcacagt cgcctca 357 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 19 Asp Tyr Gly Met His 1 5 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 20 Tyr Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 21 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 21 Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr 1 5 10 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30 Leu Ile Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Asn Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 23 gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaaattagt ggttacttaa tctggcttca gcagaaacca 120 gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180 aggttcagtg gcaataggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240 gaagattttg cagactatta ctgtctacaa tatgctagtt atcctcggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 24 Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ile 1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 25 Ala Ala Dear Thr Leu Asp Dear 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 26 Leu Gln Tyr Ala Ser Tyr Pro Arg Thr 1 5 <210> 27 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Pro Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Ile Ile Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ala Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 28 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 28 gaggtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 ccctgtgcag cctctggatt cactttcagt gactatggaa tgcactgggt tcgtcaggct 120 ccagagaagg ggctggagtg ggttgcatac attagtagtg gcagtagtat catctactat 180 gcagacacag tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgttc 240 ctgcaaatga ccagtctgag gtctgaggac acggccatgt attactgtgc aagggacctc 300 tactatgatc acgtccttga ctattggggc caaggcacca ctctcacagt cgcctcagct 360 agcaccaagg gaccctccgt gtttcctctg gctccttgct ccagatctac ctccgagtct 420 accgccgctc tgggttgtct ggtgaaggac tacttccccg agccagtgac cgtgtcttgg 480 aacagcggag ctctgacatc cggagtgcac acctttccag ccgtgctgca gtcttccggc 540 ctgtattctc tgtcctccgt ggtgaccgtg ccttcttcca acttcggcac ccagacctac 600 acttgcaacg tggaccacaa gccctccaac accaaggtgg acaagaccgt ggagcgcaag tgttgcgtcg agtgccctcc ttgcccagct cctccagtgg ccggaccttc tgtgtttctg 720 ttccccccta agcctaagga caccctgatg atctccccgga ccccagaagt gacttgcgtg 780 gtggtggacg tgtctcacga ggaccccgag gtgcagttca attggtacgt ggacggcgtg 840 900. gaggtgcaca acgctaagac caagcccagg gaggagcagt tcaactccac cttccgggtg gtgtcagtgc tgacagtggt gcaccaggat tggctgaacg gcaaggagta caagtgcaag 960 gtgtccaaca agggcctgcc agctcctatc gagagacca tctccaagac caagggccag cccagagagc ctcaggtgta cacactgcct ccttcccggg cccagagac caagaccag 1080 gtgtccctga cttgcctcgt gaagggattc tacccctccg acatcgcagt cgagtggga tccaacggcc agcccgagaa caactacaag accaccccctc ctagctgga ctccgacggc 1200 tccttcttcc tgtactccaa gctgaccgtgzgaagtccc gttggcagca gggcaacgtg 1260 ttctcttgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gagcctgtcc 1320 ctgtctcccg gcaag 1335 <210> 29 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 29 Asp Ile Gln Met Thr Gln Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Will Be Left Thr Cys Arg Only Gln Glu And Gly Tyr 20 25 30 Containing Trp Containing Gln Gln Lys Pro Asp Gly Thr Containing Lys Arg 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Asn Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 30 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 30 gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaaattagt ggttacttaa tctggcttca gcagaaacca 120 gatggacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180 aggttcagtg gcaataggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240 gaagattttg cagactatta ctgtctacaa tatgctagtt atcctcggac gttcggtgga 300 ggcaccaagc tggaaatcaa aagaaccgtg gccgctcctt ccgtgttcat cttccctccc 360 tccgacgagc agctgaagag cggaacagcc tctgtcgtgt gcctcctgaa caacttctac 420 ccccgggagg ccaaggtcca gtggaaggtg gacaacgctc tgcagagcgg caactctcag 480 gagagcgtga cagagcagga ctccaaggac tccacctact ccctgtcttc caccctgacc 540 ctgtctaagg ccgactacga gaagcacaag gtgtacgctt gcgaggtgac acaccaggga 600 ctgtcctctc cagtgaccaa gtccttcaac cgcggcgagt gt 642 <210> 31 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Val Ile Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Ser Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 32 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 32 gaggtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactatggaa tgcactgggt tcgtcaggct 120 ccagagaagg ggctggagtg ggttgcatac attagtagcg gcagtagtgt catctactat 180 gtagacacag tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgttc 240 ctgcaaatga ccagtctgag gtctgaggac acggccatgt attactgtgc aagggacctc 300 tactatgatc acgtccttga ctcctggggc caaggcacca ctctcacagt ctcctca 357 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 33 Asp Tyr Gly Met His 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 34 Tyr Ile Ser Ser Gly Ser Ser Val Ile Tyr Tyr Val Asp Thr Val Lys 1 5 10 15 Gly <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 35 Asp Leu Tyr Tyr Asp His Val Leu Asp Ser 1 5 10 <210> 36 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 36 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Gly Gly Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 37 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 37 gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaaattggt ggttacttaa gctggcttca gcagaaacca 120 gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180 aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240 gaagattttg cagactatta ctgtctacaa tatgctagtt atcctcggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 38 Arg Ala Ser Gln Glu Ile Gly Gly Tyr Leu Ser 1 5 10 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 39 Ala Ala Ser Thr Leu Asp Ser 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 40 Leu Gln Tyr Ala Ser Tyr Pro Arg Thr 1 5 <210> 41 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Val Ile Tyr Tyr Val Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Ser Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 42 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 42 gaggtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactatggaa tgcactgggt tcgtcaggct 120 ccagagaagg ggctggagtg ggttgcatac attagtagcg gcagtagtgt catctactat 180 gtagacacag tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgttc 240 ctgcaaatga ccagtctgag gtctgaggac acggccatgt attactgtgc aagggacctc 300 tactatgatc acgtccttga ctcctggggc caaggcacca ctctcacagt ctcctcagct 360 agcaccaagg gaccctccgt gtttcctctg gctccttgct ccagatctac ctccgagtct 420 accgccgctc tgggttgtct ggtgaaggac tacttccccg agccagtgac cgtgtcttgg 480 aacagcggag ctctgacatc cggagtgcac acctttccag ccgtgctgca gtcttccggc 540 ctgtattctc tgtcctccgt ggtgaccgtg ccttcttcca acttcggcac ccagacctac 600 acttgcaacg tggaccacaa gccctccaac accaaggtgg acaagaccgt ggagcgcaag 660 tgttgcgtcg agtgccctcc ttgcccagct cctccagtgg ccggaccttc tgtgtttctg 720 ttccccccta agcctaagga caccctgatg atctcccgga ccccagaagt gacttgcgtg 780 gtggtggacg tgtctcacga ggaccccgag gtgcagttca attggtacgt ggacggcgtg 840 gaggtgcaca acgctaagac caagcccagg gaggagcagt tcaactccac cttccgggtg 900 gtgtcagtgc tgacagtggt gcaccaggat tggctgaacg gcaaggagta caagtgcaag 960 gtgtccaaca agggcctgcc agctcctatc gagaagacca tctccaagac caagggccag 1020 cccagagagc ctcaggtgta cacactgcct cttcccggg aggagatgac caagaaccag 1080 gtgtccctga cttgcctcgt gaagggattc tacccctccg acatcgcagt cgagtggggaa 1140 tccaacggcc agcccgagaa caactacaag accacccctc ctatgctgga ctccgacggc 1200 tccttcttcc tgtactccaa gctgaccgtg gacaagtccc gttggcagca gggcaacgtg 1260 ttctcttgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gagcctgtcc 1320 ctgtctcccg gcaag 1335 <210> 43 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 43 Asp Ile Gln Put Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Gly Gly Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 44 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 44 gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaaattggt ggttacttaa gctggcttca gcagaaacca 120 gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180 aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240 gaagattttg cagactatta ctgtctacaa tatgctagtt atcctcggac gttcggtgga 300 ggcaccaagc tggaaatcaa aagaaccgtg gccgctcctt ccgtgttcat cttccctccc 360 tccgacgagc agctgaagag cggaacagcc tctgtcgtgt gcctcctgaa caacttctac 420 ccccgggagg ccaaggtcca gtggaaggtg gandacgctc tgcagagcgg caacctcag 480 gagagcgtga cagagcagga ctccaaggac tccacctact ccctgtctc caccctgacc 540 ctgtctaagg ccgactacga gagcacaag gtgtacgctt gcgaggtgac acaccaggga 600 ctgtccctc cagtgaccaa gtccttcaac cgcggcgagt gt 642 <210> 45 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Only Tyr Ile Serve Gly Serve Only Arg Tyr Tyr Only Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Create Create 115 <210> 46 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 46 gaggtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactatggaa tgcattgggt tcgtcaggct 120 ccagagaagg ggctggagtg ggttgcatac attagtagtg gcagtagtat ccgctactat 180 gcagacacag tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgttc 240 ctgcaaatga ccagtctgcg gtctgaggac acggccatat attactgtgc aagggacctc 300 tactatgatc acgtccttga ctactggggc caaggcacca ctctcacagt ctcctca 357 <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 47 Asp Tyr Gly Met His 1 5 <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 48 Tyr Ile Ser Ser Gly Ser Ser Ile Arg Tyr Tyr Ala Asp Thr Val Lys 1 5 10 15 Gly <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 49 Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr 1 5 10 <210> 50 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 50 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Will Be Leu Thr Cys Arg Will Be Gln Glu Will Be Gly Tyr 20 25 30 Asn Trp and Gln Gln Lys Pro Asp Gly Thr with Lys Arg 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 51 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 51 gataccaga tgacccagtc tccatcctcc tatatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaagttagt ggttacttaa actggcttca gcagaagcca 120 gatgaacta ttaaacgcct gatctacgcc gcatccact tagattctgg tgtcccaag 180 aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240 gaagattttg cagactatta ctgtctacaa tatgctagtt atcctcggac gttcggtgga 300 ggtaccaagg tggaaatcaa g 321 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 52 Arg Ala Ser Gln Glu Val Ser Gly Tyr Leu Asn 1 5 10 <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 53 Ala Ala Dear Thr Leu Asp Dear 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 54 Leu Gln Tyr Ala Ser Tyr Pro Arg Thr 1 5 <210> 55 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Ile Arg Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 56 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 56 gaggtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt gactatggaa tgcattgggt tcgtcaggct 120 ccagagaagg ggctggagtg ggttgcatac attagtagtg gcagtagtat ccgctactat 180 gcagacacag tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgttc 240 ctgcaaatga ccagtctgcg gtctgaggac acggccatat attactgtgc aagggacctc 300 tactatgatc acgtccttga ctactggggc caaggcacca ctctcacagt ctcctcagct 360 agcaccaagg gaccctccgt gtttcctctg gctccttgct ccagatctac ctccgagtct 420 accgccgctc tgggttgtct ggtgaaggac tacttccccg agccagtgac cgtgtcttgg 480 aacagcggag ctctgacatc cggagtgcac acctttccag ccgtgctgca gtcttccggc 540 ctgtattctc tgtcctccgt ggtgaccgtg ccttcttcca acttcggcac ccagacctac 600 acttgcaacg tggaccacaa gccctccaac accaaggtgg acaagaccgt ggagcgcaag tgttgcgtcg agtgccctcc ttgcccagct cctccagtgg ccggaccttc tgtgtttctg 720 ttccccccta agcctaagga caccctgatg atctccccgga ccccagaagt gacttgcgtg 780 gtggtggacg tgtctcacga ggaccccgag gtgcagttca attggtacgt ggacggcgtg 840 900. gaggtgcaca acgctaagac caagcccagg gaggagcagt tcaactccac cttccgggtg gtgtcagtgc tgacagtggt gcaccaggat tggctgaacg gcaaggagta caagtgcaag 960 gtgtccaaca agggcctgcc agctcctatc gagagacca tctccaagac caagggccag cccagagagc ctcaggtgta cacactgcct ccttcccggg cccagagac caagaccag 1080 gtgtccctga cttgcctcgt gaagggattc tacccctccg acatcgcagt cgagtggga tccaacggcc agcccgagaa caactacaag accacccctc ctatgctgga ctccgacggc tccttcttcc tgtactccaa gctgaccgtg gacaagtcc gttggcagca gggcaacgtg ttctcttgca gcgtgatgca cgaggccctg cacaaccact acacccaga gagcctgtcc ctgtctcccg gcaag 1335 <210> 57 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 57 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Val Ser Gly Tyr 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 58 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 58 gataccaga tgacccagtc tccatcctcc tatatctgcct ctctgggaga aagagtcagt 60 ctcacttgtc gggcaagtca ggaagttagt ggttacttaa actggcttca gcagaagcca 120 gatgaacta ttaaacgcct gatctacgcc gcatccact tagattctgg tgtcccaag 180 aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240 gaagattttg cagactatta ctgtctacaa tatgctagtt atcctcggac gttcggtgga 300 ggtaccaagg tggaaatcaa gagaaccgtg gccgctcctt ccgtgttcat cttccctccc 360 tccgacgagc agctgaagag cggaacagcc tctgtcgtgt gcctcctgaa caacttctac 420 ccccgggagg ccaaggtcca gtggaaggtg gacaacgctc tgcagagcgg caactctcag 480 gagagcgtga cagagcagga ctccaaggac tccacctact ccctgtcttc caccctgacc 540 ctgtctaagg ccgactacga gaagcacaag gtgtacgctt gcgaggtgac acaccaggga 600 ctgtcctctc cagtgaccaa gtccttcaac cgcggcgagt gt 642 <210> 59 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 59 Ala Ala Ser Thr Leu Glu Ser 1 5 <210> 60 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 60 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Ile Met Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 61 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 61 caagtgcagc tcgtcgaaag cggaggaggc gtggtgcagc ccggaaggtc tctgagactg 60 agctgtgctg ccagcggctt cactttcagc gactacggca tgcactgggt cagacaagcc 120 cccggcaagg gactggaatg ggtcgcttac atcagctccg gcagcagcat catgtactac 180 gccgacacag tgaagggaag gttcacaatc tctagggaca acagcaagaa cacactctat 240 ctgcagatga actccctcag agccgaggat acagctgtgt actactgcgc tagggatctg 300 tactacgacc acgtgctcga ttactggggc caaggcacaa cagtgacagt gagcagc 357 <210> 62 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 62 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Val Ser Gly Tyr 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 63 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 63 gataccaga tgactcagag cccaagctct ctgagcgcca gcgtgggaga taggtcaca 60 atcacttgta gggccagcca agaggtgagc ggctatctga attggctcca gcagaaaccc 120 ggcaagcca tcaagact gatctatgcc gccagcactc tggagtccgg agtgccatct 180 aggttcagcg gcagcagaag cggcagcgac tacactca caatcagctc cctccagcca 240 gaagacttcg ccacttacta ctgtctgcag tatgccagct acccaggac ttcggacag 300 ggtaccagg tggagatcaa a 321 <210> 64 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 64 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Gly Ser Ser Ile Met Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 65 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 65 60. caagtgcagc tcgtcgaaag cggaggaggc gtggtgcagc ccggaaggtc tctgagactg agctgtgctg ccagcggctt cactttcagc gactacggca tgcactgggt cagacaagcc cccggcaagg gactggaatg ggtcgcttac atcagctccg gcagcagcat catgtactac gccgacacag tgaagggaag gttcacaatc tctagggaca acagcaagaa cacactctat ctgcagatga actccctcag agccgaggat acagctgtgt actactgcgc tagggatctg 360. tactacgacc acgtgctcga ttactggggc caaggcacaa cagtgacagt gagcagcgct agcaccaagg gaccctccgt gtttcctctg gctccttgct ccagatctac ctccgagtct accgccgctc tgggttgtct ggtgaaggac tacttccccg agccagtgac cgtgtcttgg 480 aacagcggag ctctgacatc cggagtgcac acctttccag ccgtgctgca gtcttccggc 540 ctgtattctc tgtcctccgt ggtgaccgtg ccttcttcca acttcggcac ccagacctac 600 acttgcaacg tggaccacaa gccctccaac accaaggtgg acaagaccgt ggagcgcaag tgttgcgtcg agtgccctcc ttgcccagct cctccagtgg ccggaccttc tgtgtttctg 720 ttccccccta agcctaagga caccctgatg atctccccgga ccccagaagt gacttgcgtg 780 gtggtggacg tgtctcacga ggaccccgag gtgcagttca attggtacgt ggacggcgtg 840 900. gaggtgcaca acgctaagac caagcccagg gaggagcagt tcaactccac cttccgggtg gtgtcagtgc tgacagtggt gcaccaggat tggctgaacg gcaaggagta caagtgcaag 960 gtgtccaaca agggcctgcc agctcctatc gagagacca tctccaagac caagggccag cccagagagc ctcaggtgta cacactgcct ccttcccggg cccagagac caagaccag 1080 gtgtccctga cttgcctcgt gaagggattc tacccctccg acatcgcagt cgagtggga tccaacggcc agcccgagaa caactacaag accacccctc ctatgctgga ctccgacggc tccttcttcc tgtactccaa gctgaccgtg gacaagtcc gttggcagca gggcaacgtg ttctcttgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gagcctgtcc 1320 ctgtctcccg gcaag 1335 <210> 66 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 66 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Val Ser Gly Tyr 20 25 30 Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 67 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 67 gataccaga tgactcagag cccaagctct ctgagcgcca gcgtgggaga taggtcaca 60 atcacttgta gggccagcca agaggtgagc ggctatctga attggctcca gcagaaaccc 120 ggcaaggcca tcaagagact gatctatgcc gccagcactc tggagtccgg agtgccatct 180 aggttcagcg gcagcagaag cggcagcgac tacactctca caatcagctc cctccagcca 240 gaagacttcg ccacttacta ctgtctgcag tatgccagct acccaaggac ttcggacag 300 ggtaccaagg tggagatcaa aagaaccgtg gccgctcctt ccgtgttcat cttccctccc 360 tccgacgagc agctgaagag cggaacagcc tctgtcgtgt gcctcctgaa caacttctac 420 ccccgggagg ccaaggtcca gtggaaggtg gacaacgctc tgcagagcgg caactctcag 480 gagagcgtga cagagcagga ctccaaggac tccacctact ccctgtcttc caccctgacc 540 ctgtctaagg ccgactacga gaagcacaag gtgtacgctt gcgaggtgac acaccaggga 600 ctgtcctctc cagtgaccaa gtccttcaac cgcggcgagt gt 642 <210> 68 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 68 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr and Thr Cys Arg Only Serve as Gln Glu and Arg Gly Tyr 20 25 30 Lion With Trp Lion Gln Gln Lys Pro Gly Lys Ala And Lys Arg Lys 35 40 45 Tyr Ala Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Thr Thr Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 69 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 69 gataccaga tgacacagtc ccctagctct ctgtccgcca gcgtgggaga taggtgaca 60 atcacttgta gggccagcca agagattagg ggctatctga tctggctgca gcagaaaccc 120 ggcaaggcca tcaagaggct gatctacgcc gccagcactc tggagagcgg agtcccaagc 180 agattttccg gcagccgctc cggcagcgat tacactca caatcagctc tctgcagcca 240 gaggactcg ccacttacta ctgtctgcag tacacaagct acccaggac attcggccaa 300 ggcactaagg tggagatcaa a 321 <210> 70 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 70 Asp With Gln Met Thr Gln Serving Pro Serving Leu Serving Ala Serving Val Gly 1 5 10 15 Asp Arg Val Thr and Thr Cys Arg Only Serve as Gln Glu and Arg Gly Tyr 20 25 30 Lion With Trp Lion Gln Gln Lys Pro Gly Lys Ala And Lys Arg Lys 35 40 45 Tyr Ala Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Thr Thr Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 71 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 71 gacatccaga tgacacagtc ccctagctct ctgtccgcca gcgtgggaga tagggtgaca 60 atcacttgta gggccagcca agagattagg ggctatctga tctggctgca gcagaaaccc 120 ggcaaggcca tcaagaggct gatctacgcc gccagcactc tggagagcgg agtcccaagc 180 agattttccg gcagccgctc cggcagcgat tacactctca caatcagctc tctgcagcca 240 gaggacttcg ccacttacta ctgtctgcag tacacaagct acccaaggac attcggccaa 300 ggcactaagg tggagatcaa aagaaccgtg gccgctcctt ccgtgttcat cttccctccc 360 tccgacgagc agctgaagag cggaacagcc tctgtcgtgt gcctcctgaa caacttctac 420 ccccgggagg ccaaggtcca gtggaaggtg gacaacgctc tgcagagcgg caactctcag 480 gagagcgtga cagagcagga ctccaaggac tccacctact ccctgtcttc caccctgacc 540 ctgtctaagg ccgactacga gaagcacaag gtgtacgctt gcgaggtgac acaccaggga 600 ctgtcctctc cagtgaccaa gtccttcaac cgcggcgagt gt 642 <210> 72 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 72 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Gly Ser Ser Ile Met Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Leu Thr Val Ser Ser 115 <210> 73 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 73 gaggtgcagc tggtggagag cggcggcggc ctggtgcagc cggcggcag cctgagactg 60 agctgcgccg ccagcggctt caccttcagc gactacggca tgcactgggt gagacaggcc 120 cccggcaagg gcctggagtg ggtgagctac atcagcagcg gcagcagcat catgtactac 180 gccgacaccg tgaagggcag attcaccatc agcagagaca acgccaagaa cagcctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagagacctg 300 tactacgacc acgtgctgga ctactggggc cagggcaccc tgctgaccgt gagcagc 357 <210> 74 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 74 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Arg Gly Tyr 20 25 30 Leu Ile Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Thr Thr Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 75 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 75 gataccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60 atcacctgca gagccagcca ggagatcaga ggtacctga tctggctgca gcagaagccc 120 ggcggcgcca tcaagact gattacgcc gccagcaccc tggagagcgg cgtgcccagc 180 agattcagcg gcagcagaag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgcctgcag tacaccacct accxagac ctcggcggc 300 g321 g 321 <210> 76 <211> 445 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 76 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Gly Ser Ser Ile Met Tyr Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Leu Tyr Tyr Asp His Val Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 210 215 220 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 290 295 300 Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 77 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 77 60. gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgagactg agctgcgccg ccagcggctt caccttcagc gactacggca tgcactgggt gagacaggcc cccggcaagg gcctggagtg ggtgagctac atcagcagcg gcagcagcat catgtactac gccgacaccg tgaagggcag attcaccatc agcagagaca acgccaagaa cagcctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc cagagacctg 360. tactacgacc acgtgctgga ctactggggc cagggcaccc tgctgaccgt gagcagcgct agcaccaagg gaccctccgt gtttcctctg gctccttgct ccagatctac ctccgagtct accgccgctc tgggttgtct ggtgaaggac tacttccccg agccagtgac cgtgtcttgg 480 aacagcggag ctctgacatc cggagtgcac acctttccag ccgtgctgca gtcttccggc 540 ctgtattctc tgtcctccgt ggtgaccgtg ccttcttcca acttcggcac ccagacctac 600 acttgcaacg tggaccacaa gccctccaac accaaggtgg acaagaccgt ggagcgcaag tgttgcgtcg agtgccctcc ttgcccagct cctccagtgg ccggaccttc tgtgtttctg 720 ttccccccta agcctaagga caccctgatg atctccccgga ccccagaagt gacttgcgtg 780 gtggtggacg tgtctcacga ggaccccgag gtgcagttca attggtacgt ggacggcgtg 840 900. gaggtgcaca acgctaagac caagcccagg gaggagcagt tcaactccac cttccgggtg gtgtcagtgc tgacagtggt gcaccaggat tggctgaacg gcaaggagta caagtgcaag 960 gtgtccaaca agggcctgcc agctcctatc gagagacca tctccaagac caagggccag cccagagagc ctcaggtgta cacactgcct ccttcccggg cccagagac caagaccag 1080 gtgtccctga cttgcctcgt gaagggattc tacccctccg acatcgcagt cgagtggga tccaacggcc agcccgagaa caactacaag accacccctc ctatgctgga ctccgacggc tccttcttcc tgtactccaa gctgaccgtg gacaagtcc gttggcagca gggcaacgtg ttctcttgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gagcctgtcc 1320 ctgtctcccg gcaag 1335 <210> 78 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 78 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Arg Gly Tyr 20 25 30 Leu Ile Trp Leu Gln Gln Lys Pro Gly Gly Ala Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Thr Ser Tyr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 79 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 79 gataccaga tgacccagag ccccagcagc ctgagcgcca gcgtgggcga cagagtgacc 60 atcacctgca gagccagcca ggagatcaga ggtacctga tctggctgca gcagaagccc 120 ggcggcgcca tcaagagact gatctacgcc gccagcaccc tggagagcgg cgtgcccagc 180 agattcagcg gcagcagaag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240 gaggacttcg ccacctacta ctgcctgcag tacaccagct accccagaac cttcggcggc 300 ggtaccaagg tggagatcaa gagaaccgtg gccgctcctt ccgtgttcat cttccctccc 360 tccgacgagc agctgaagag cggaacagcc tctgtcgtgt gcctcctgaa caacttctac 420 ccccgggagg ccaaggtcca gtggaaggtg gacaacgctc tgcagagcgg caactctcag 480 gagagcgtga cagagcagga ctccaaggac tccacctact ccctgtcttc caccctgacc 540 ctgtctaagg ccgactacga gaagcacaag gtgtacgctt gcgaggtgac acaccaggga 600 ctgtcctctc cagtgaccaa gtccttcaac cgcggcgagt gt 642
Claims
1. An antibody or antigen-binding portion thereof that binds to CD39, wherein: The antibody or its antigen-binding portion comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 sequences; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the antibody or its antigen-binding portion comprises: 1) (a) HCDR1 of SEQ ID NO:5, (b) HCDR2 of SEQ ID NO:6, (c) HCDR3 of SEQ ID NO:7, (d) LCDR1 of SEQ ID NO:10, (e) LCDR2 of SEQ ID NO:11, and (f) LCDR3 of SEQ ID NO:12; or 2) (a) HCDR1 of SEQ ID NO:5, (b) HCDR2 of SEQ ID NO:6, (c) HCDR3 of SEQ ID NO:7, (d) LCDR1 of SEQ ID NO:10, (e) LCDR2 of SEQ ID NO:59, and (f) LCDR3 of SEQ ID NO:
12.
2. The antibody or antigen-binding portion thereof that binds to CD39 according to claim 1, wherein The antibody or antigen-binding portion thereof comprises: 1) a heavy chain variable region (VH), whose amino acid sequence is shown in SEQ ID NO: 3, and a light chain variable region (VL), whose amino acid sequence is shown in SEQ ID NO: 8; 2) a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 60, and a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 62; 3) a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 60, and a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 68; or 4) a heavy chain variable region (VH), whose amino acid sequence is shown in SEQ ID NO: 72, and a light chain variable region (VL), whose amino acid sequence is shown in SEQ ID NO:
74.
3. The antibody or antigen-binding portion thereof that binds to CD39 according to claim 1 or 2, wherein The heavy chain constant region of the antibody is IgG.
4. The antibody or antigen-binding portion thereof that binds to CD39 according to claim 1 or 2, wherein The heavy chain constant region of the antibody is selected from IgG1, IgG2 or IgG4.
5. The antibody or antigen-binding portion thereof that binds to CD39 according to claim 1 or 2, wherein The antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, Fab, Fab', Fab'-SH or F(ab')2.
6. The antibody or antigen-binding portion thereof that binds to CD39 according to claim 1 or 2, wherein The antibody is a human modified antibody.
7. The antibody or antigen-binding portion thereof that binds to CD39 according to claim 1 or 2, wherein The antibody is a Fv or a single chain antibody (scFv).
8. The antibody or antigen-binding portion thereof that binds to CD39 according to claim 1, wherein The antibody or antigen-binding portion thereof comprises: 1) a heavy chain, the amino acid sequence of which is shown in SEQ ID NO: 13, and a light chain, the amino acid sequence of which is shown in SEQ ID NO: 15; 2) a heavy chain having an amino acid sequence as shown in SEQ ID NO:64, and a light chain having an amino acid sequence as shown in SEQ ID NO:66; 3) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 64, and a light chain having an amino acid sequence as shown in SEQ ID NO: 70; or 4) a heavy chain, the amino acid sequence of which is shown in SEQ ID NO: 76, and a light chain, the amino acid sequence of which is shown in SEQ ID NO:
78.
9. The antibody or antigen-binding portion thereof that binds to CD39 according to any one of claims 1, 2 or 8, wherein The antibody or antigen-binding portion thereof is a CD39 antagonist.
10. The antibody or antigen-binding portion thereof that binds to CD39 according to claim 9, wherein The CD39 is human CD39 or cynomolgus monkey CD39.
11. The antibody or antigen-binding portion thereof that binds to CD39 according to any one of claims 1, 2 or 8, wherein The antibody or the antigen-binding portion thereof can reduce the activity of ATPase of CD39.
12. A nucleic acid composition comprising: 1) a first nucleic acid, wherein The first nucleic acid comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 3, and a second nucleic acid, wherein the second nucleic acid comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 8; 2) a first nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 60, and a second nucleic acid, wherein the second nucleic acid comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 62; 3) a first nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 60, and a second nucleic acid, wherein the second nucleic acid comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 68; or 4) a first nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 72, and a second nucleic acid, wherein the second nucleic acid comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:
74.
13. The nucleic acid composition according to claim 12, comprising: 1) the first nucleic acid contains the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 4, and the second nucleic acid contains the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 9; 2) the first nucleic acid contains the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 61, and the second nucleic acid contains the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 63; 3) the first nucleic acid contains the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 61, and the second nucleic acid contains the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 69; or 4) The first nucleic acid contains the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 73, and the second nucleic acid contains the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:
75.
14. An expression vector composition comprising: 1) A first expression vector, wherein: The first expression vector contains a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 3; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain variable region (VL) as shown in SEQ ID NO: 8; 2) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 60; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 62; 3) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 60; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 68; or 4) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain variable region (VH) as shown in SEQ ID NO: 72; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain variable region (VL) as shown in SEQ ID NO:
74.
15. The expression vector composition according to claim 14, comprising: 1) A first expression vector, wherein: The first expression vector contains a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 4; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 9; 2) a first expression vector, wherein the first expression vector contains the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 61; and a second expression vector, wherein the second expression vector contains the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 63; 3) a first expression vector, wherein the first expression vector contains the nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 61; and a second expression vector, wherein the second expression vector contains the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 69; and / or 4) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 73; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO:
75.
16. An expression vector comprising: 1) A first nucleic acid sequence, wherein: The first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 3; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 8; 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as set forth in SEQ ID NO: 60; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as set forth in SEQ ID NO: 62; 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 60; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO: 68; or 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region (VH) having an amino acid sequence as shown in SEQ ID NO: 72; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region (VL) having an amino acid sequence as shown in SEQ ID NO:
74.
17. The expression vector according to claim 16, comprising: 1) A first nucleic acid sequence, wherein: The first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 4; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 9; 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 61; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 63; 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 61; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO: 69; or 4) a first nucleic acid sequence, wherein the first nucleic acid sequence contains a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 73; and a second nucleic acid sequence, wherein the second nucleic acid sequence contains a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO:
75.
18. A nucleic acid composition comprising: 1) a first nucleic acid, wherein The first nucleic acid comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 13, and a second nucleic acid, wherein the second nucleic acid comprises a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 15; 2) a first nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64, and a second nucleic acid, wherein the second nucleic acid comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 66; 3) a first nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64, and a second nucleic acid, wherein the second nucleic acid comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 70; or 4) a first nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a heavy chain of the amino acid sequence shown in SEQ ID NO: 76, and a second nucleic acid, wherein the second nucleic acid comprises a nucleotide sequence encoding a light chain of the amino acid sequence shown in SEQ ID NO:
78.
19. The nucleic acid composition according to claim 18, comprising: 1) The first nucleic acid contains a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 14, and the second nucleic acid contains a nucleotide sequence encoding a light chain as shown in SEQ ID NO: 16; 2) the first nucleic acid contains a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 65, and the second nucleic acid contains a nucleotide sequence encoding a light chain as shown in SEQ ID NO: 67; 3) the first nucleic acid contains the nucleotide sequence encoding the heavy chain as shown in SEQ ID NO: 65, and the second nucleic acid contains the nucleotide sequence encoding the light chain as shown in SEQ ID NO: 71; or 4) The first nucleic acid contains the nucleotide sequence encoding the heavy chain as shown in SEQ ID NO: 77, and the second nucleic acid contains the nucleotide sequence encoding the light chain as shown in SEQ ID NO:
79.
20. An expression vector composition comprising: 1) A first expression vector, wherein: The first expression vector contains a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 13; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 15; 2) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 64; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 66; 3) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 64; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 70; or 4) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 76; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO:
78.
21. The expression vector composition according to claim 20, comprising: 1) A first expression vector, wherein: The first expression vector contains a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 14; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain as shown in SEQ ID NO: 16; 2) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 65; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain as shown in SEQ ID NO: 67; 3) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 65; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain as shown in SEQ ID NO: 71; or 4) a first expression vector, wherein the first expression vector contains a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 77; and a second expression vector, wherein the second expression vector contains a nucleotide sequence encoding a light chain as shown in SEQ ID NO:
79.
22. An expression vector comprising: 1) A first nucleic acid sequence, wherein: The first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 13; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 15; 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 64; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as set forth in SEQ ID NO: 66; 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain having an amino acid sequence as shown in SEQ ID NO: 64; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain having an amino acid sequence as shown in SEQ ID NO: 70; or 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain of an amino acid sequence as shown in SEQ ID NO: 76; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain of an amino acid sequence as shown in SEQ ID NO:
78.
23. The expression vector according to claim 22, comprising: 1) A first nucleic acid sequence, wherein: The first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 14; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain as shown in SEQ ID NO: 16; 2) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 65; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain as shown in SEQ ID NO: 67; 3) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 65; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain as shown in SEQ ID NO: 71; or 4) a first nucleic acid sequence, wherein the first nucleic acid sequence comprises a nucleotide sequence encoding a heavy chain as shown in SEQ ID NO: 77; and a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a nucleotide sequence encoding a light chain as shown in SEQ ID NO:
79.
24. A cell comprising the expression vector composition of any one of claims 14-15 and 20-21 or the expression vector of any one of claims 16-17 and 22-23.
25. A method for preparing an antibody or antigen-binding portion thereof that binds to CD39, comprising: The antibody or antigen-binding portion thereof is expressed in the cell of claim 24, and the antibody or antigen-binding portion thereof is isolated from the cell.
26. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof that binds to CD39 according to any one of claims 1 to 11, and a pharmaceutically acceptable carrier.
27. A kit comprising the antibody or antigen-binding portion thereof that binds to CD39 according to any one of claims 1 to 11.
28. A bispecific antibody or multispecific antibody comprising the light chain variable region and the heavy chain variable region according to claim 1 or 2.
29. A single-chain antibody comprising the light chain variable region and heavy chain variable region according to claim 1 or 2.
30. An antibody-drug conjugate comprising the light chain variable region and the heavy chain variable region of claim 1 or 2.
31. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the pharmaceutical composition according to claim 26, the bispecific antibody or multispecific antibody according to claim 28, the single-chain antibody according to claim 29, and / or the antibody-drug conjugate according to claim 30 in the preparation of a medicament for treating CD39-related cancer, wherein the cancer is a solid tumor selected from human multiple myeloma and / or B-cell lymphoma.
32. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the pharmaceutical composition according to claim 26, the bispecific antibody or multispecific antibody according to claim 28, the single-chain antibody according to claim 29, and / or the antibody-drug conjugate according to claim 30 in the preparation of a medicament for increasing T cell activity in cancer patients, reducing adenosine-mediated inhibition of T cell activity in cancer patients, or increasing T cell activity in the patient's tumor microenvironment, wherein the cancer is a solid tumor selected from human multiple myeloma and / or B-cell lymphoma.
Citation Information
Patent Citations
Compositions and methods for treating cancer
CN110382544A
Anti-CD39 antibodies
WO2017157948A1