Antibody-drug conjugates and their applications

Abstract

The present invention relates to an antibody-drug conjugate and its application, and specifically provides an antibody-drug conjugate, a pharmaceutically acceptable salt, a solvate or a solvate of the salt thereof, wherein the antibody-drug conjugate has a structure shown in Formula I, wherein Ab is an anti-Claudin 18.2 antibody. The antibody-drug conjugate of the present invention has good tumor cell growth inhibition activity both in vivo and in vitro, and has low cytotoxicity, and has good application prospects. Ab‑(L‑D) p Formula Ⅰ.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular, to antibody-drug conjugates and applications thereof. Background Art

[0002] Gastric cancer is one of the most common cancers in the world, with a higher incidence in East Asia, Eastern Europe, and South America, and a lower incidence in North America and Africa. The standard initial treatment for advanced or recurrent gastric cancer is chemotherapy. Although the prognosis of gastric cancer patients has improved significantly due to the development of surgical techniques and perioperative treatment, the 5-year overall survival rate is only 10-15%. Targeted therapy has brought new hope for the treatment of recurrent / advanced gastric cancer. Combining trastuzumab with chemotherapy can bring certain benefits to patients with HER2-positive tumors, but only 15% of patients have HER2-positive expression, and the beneficiary population is limited. In recent years, immunotherapy has brought new hope for the treatment of recurrent / advanced gastric cancer; however, according to the results of the KEYNOTE-12 study, the proportion of PD-L1-positive people suitable for this treatment is only about 40% of patients with recurrent / advanced gastric or gastroesophageal junction adenocarcinoma. Therefore, the development of new gastric cancer treatment drugs is still urgent. Summary of the invention

[0003] The inventors of the present application prepared an anti-Claudin 18.2 antibody-drug conjugate through a large number of experiments and creative work, and confirmed that it has good biological activity, thereby completing the present invention.

[0004] To this end, in the first aspect of the present invention, the present invention provides an antibody drug conjugate, a pharmaceutically acceptable salt, a solvate or a solvate of the salt thereof, wherein the antibody drug conjugate has a structure as shown in Formula I,

[0005] Ab-(LD) p

[0006] Formula I

[0007] in:

[0008] Ab is an anti-Claudin 18.2 antibody, the anti-Claudin 18.2 antibody comprises a heavy chain and a light chain, the heavy chain variable region CDR1 comprises a sequence selected from SEQ ID NO: 2, 10, 18, 26, 34, 42, 68, 76, 84, 92, 100, 108 or 116 or a mutant thereof, the heavy chain variable region CDR2 comprises a sequence selected from SEQ ID NO: 3, 11, 19, 27, 35, 43, 69, 77, 85, 93, 101, 109 or 117 or a mutant thereof, the heavy chain variable region CDR3 comprises a sequence selected from SEQ ID NO: 4, 12, 20, 28, 36, 44, 70, 78, 86, 94, 102, 110 or 118 or a mutant thereof, the light chain variable region CDR1 comprises a sequence selected from SEQ ID NO: NO:50, 58, 124 or 132 or a mutant thereof, the light chain variable region CDR2 comprises a sequence selected from SEQ ID NO:51, 59, 125 or 133 or a mutant thereof, and the light chain variable region CDR3 comprises a sequence selected from SEQ ID NO:52, 60, 126 or 134 or a mutant thereof;

[0009] D is a cytotoxic agent;

[0010] L is a linker, used to connect the anti-Claudin 18.2 antibody and the cytotoxic agent;

[0011] p is 2.0-8.0 (e.g., 2.0-7.0, 2.0-6.0, 2.0-5.0, 2.0-4.0, 3.0-7.0, 3.0-6.0, 3.0-5.0, or 3.0-4.0, or such as 3.0, 4.0, 5.0, 6.0, or 7.0).

[0012] The antibody-drug conjugate of the present invention has good tumor cell growth inhibition activity both in vivo and in vitro, and has good application prospects. The antibody-drug conjugate of the present invention is formed by connecting an anti-Claudin 18.2 antibody and a derivative of Dolastatin toxin, MMAE, through an MC-vc-PAB linker, and its anti-tumor mechanism is as follows: after the antibody-drug conjugate binds to Claudin 18.2 on the surface of tumor cells, it enters the tumor cells through endocytosis and is transported to lysosomes, and then MMAE is released through protease degradation in the lysosomes. After MMAE enters the cytoplasm, it binds to tubulin and inhibits its polymerization, thereby blocking various physiological functions of cells including mitosis in which tubulin participates, thereby inhibiting tumor cell proliferation and causing tumor cell death.

[0013] It should be noted that the "antibody drug conjugate" is a composition containing ADC molecules with the same or different DAR values. Specifically, the present invention provides a composition comprising multiple ADC molecules. In some cases, in the composition, the multiple ADCs respectively contain the same number of drug molecules. In other cases, in the composition, the multiple ADCs respectively contain different numbers of drug molecules.

[0014] The above-mentioned drug-antibody ratio (DAR) refers to the number of drug molecules coupled to the antibody (e.g., p in Formula I). ​​The number of drug molecules contained in the antibody-drug conjugate of the present invention (e.g., p in Formula I) is usually an integer. When the number of drug molecules contained in the antibody-drug conjugate of the present invention (e.g., p in Formula I) is a fraction, the fraction refers to the average number of drug molecules coupled to each antibody in a composition comprising multiple ADC molecules.

[0015] The above-mentioned drug-antibody ratio (DAR) can be verified by conventional means, such as mass spectrometry, ELISA assay, HIC and HPLC. The quantitative distribution of ADC in terms of p can also be determined. In some cases, the separation, purification and verification of homogeneous ADC with a certain p value from ADC with other drug loadings can be achieved by means such as reverse phase HPLC or electrophoresis.

[0016] In some embodiments, the heavy chain variable region CDR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from SEQ ID NO: 2, 10, 18, 26, 34 or 42 or a mutant thereof, the heavy chain variable region CDR2 comprises a sequence selected from SEQ ID NO: 3, 11, 19, 27, 35 or 43 or a mutant thereof, the heavy chain variable region CDR3 comprises a sequence selected from SEQ ID NO: 4, 12, 20, 28, 36 or 44 or a mutant thereof, the light chain variable region CDR1 comprises a sequence selected from SEQ ID NO: 50 or 58 or a mutant thereof, the light chain variable region CDR2 comprises a sequence selected from SEQ ID NO: 51 or 59 or a mutant thereof, and the light chain variable region CDR3 comprises a sequence selected from SEQ ID NO: 52 or 60 or a mutant thereof.

[0017] In some embodiments, the heavy chain variable region CDR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from SEQ ID NO: 68, 76, 84, 92, 100, 108 or 116 or a mutant thereof, the heavy chain variable region CDR2 comprises a sequence selected from SEQ ID NO: 69, 77, 85, 93, 101, 109 or 117 or a mutant thereof, the heavy chain variable region CDR3 comprises a sequence selected from SEQ ID NO: 70, 78, 86, 94, 102, 110 or 118 or a mutant thereof, the light chain variable region CDR1 comprises a sequence selected from SEQ ID NO: 124 or 132 or a mutant thereof, the light chain variable region CDR2 comprises a sequence selected from SEQ ID NO: 125 or 133 or a mutant thereof, and the light chain variable region CDR3 comprises a sequence selected from SEQ ID NO: 126 or 134 or a mutant thereof.

[0018] In some embodiments, the heavy chain variable region CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0019] (1) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,

[0020] (2) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,

[0021] (3) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,

[0022] (4) SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,

[0023] (5) SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,

[0024] (6) SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44,

[0025] (7) SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,

[0026] (8) SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,

[0027] (9) SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86,

[0028] (10) SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,

[0029] (11) SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,

[0030] (12) SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,

[0031] (13) SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118,

[0032] The light chain variable regions CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0033] (1) SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,

[0034] (2) SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,

[0035] (3) SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126,

[0036] (4) SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134.

[0037] In some embodiments, the heavy chain variable region CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0038] (1) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,

[0039] (2) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,

[0040] (3) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,

[0041] (4) SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,

[0042] (5) SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,

[0043] (6) SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44,

[0044] The light chain variable regions CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0045] (1) SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,

[0046] (2) SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60.

[0047] In some embodiments, the heavy chain variable region CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0048] (1) SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,

[0049] (2) SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,

[0050] (9) SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86,

[0051] (4) SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,

[0052] (5) SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,

[0053] (6) SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,

[0054] (7) SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118,

[0055] The light chain variable regions CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0056] (1) SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126,

[0057] (2) SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134.

[0058] In some embodiments, the heavy chain variable region FR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from SEQ ID NO: 5, 13, 21, 29, 37, 45, 71, 79, 87, 95, 103, 111 or 119 or a mutant thereof, the heavy chain variable region FR2 comprises a sequence selected from SEQ ID NO: 6, 14, 22, 30, 38, 46, 72, 80, 88, 96, 104, 112 or 120 or a mutant thereof, and the heavy chain variable region FR3 comprises a sequence selected from SEQ ID NO:

[0059] 7, 15, 23, 31, 39, 47, 73, 81, 89, 97, 105, 113 or 121 or a mutant thereof, the heavy chain variable region FR4 comprises a sequence selected from SEQ ID NO: 8, 16, 24, 32, 40, 48, 74, 82, 90, 98, 106, 114 or 122 or a mutant thereof, the light chain variable region FR1 comprises a sequence selected from SEQ ID NO: 53, 61, 127 or 135 or a mutant thereof, the light chain variable region FR2 comprises a sequence selected from SEQ ID NO: 54, 62, 128 or 136 or a mutant thereof, the light chain variable region FR3 comprises a sequence selected from SEQ ID NO: 55, 63, 129 or 137 or a mutant thereof, and the light chain variable region FR4 comprises a sequence selected from SEQ ID NO: 56, 64, 130 or 138 or a mutant thereof.

[0060] In some embodiments, the heavy chain variable region FR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from SEQ ID NO: 5, 13, 21, 29, 37 or 45 or a mutant thereof, the heavy chain variable region FR2 comprises a sequence selected from SEQ ID NO: 6, 14, 22, 30, 38 or 46 or a mutant thereof, the heavy chain variable region FR3 comprises a sequence selected from SEQ ID NO: 7, 15, 23, 31, 39 or 47 or a mutant thereof, the heavy chain variable region FR4 comprises a sequence selected from SEQ ID NO: 8, 16, 24, 32, 40 or 48 or a mutant thereof, the light chain variable region FR1 comprises a sequence selected from SEQ ID NO: 53 or 61 or a mutant thereof, the light chain variable region FR2 comprises a sequence selected from SEQ ID NO: 54 or 62 or a mutant thereof, the light chain variable region FR3 comprises a sequence selected from SEQ ID NO: 55 or 63 or a mutant thereof, and the light chain variable region FR4 comprises a sequence selected from SEQ ID NO: 56 or 64 or a mutant thereof.

[0061] In some embodiments, the heavy chain variable region FR1 of the anti-Claudin 18.2 antibody comprises a sequence selected from SEQ ID NO: 71, 79, 87, 95, 103, 111 or 119 or a mutant thereof, the heavy chain variable region FR2 comprises a sequence selected from SEQ ID NO: 72, 80, 88, 96, 104, 112 or 120 or a mutant thereof, the heavy chain variable region FR3 comprises a sequence selected from SEQ ID NO: 73, 81, 89, 97, 105, 113 or 121 or a mutant thereof, the heavy chain variable region FR4 comprises a sequence selected from SEQ ID NO: 74, 82, 90, 98, 106, 114 or 122 or a mutant thereof, the light chain variable region FR1 comprises a sequence selected from SEQ ID NO: 127 or 135 or a mutant thereof, the light chain variable region FR2 comprises a sequence selected from SEQ ID NO: 128 or 136 or a mutant thereof, and the light chain variable region FR3 comprises a sequence selected from SEQ ID NO: 1 The light chain variable region FR4 comprises a sequence selected from SEQ ID NO: 130 or 138 or a mutant thereof.

[0062] In some embodiments, the heavy chain variable regions FR1, FR2, FR3, and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0063] (1) SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,

[0064] (2)SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16,

[0065] (3)SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24,

[0066] (4)SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32,

[0067] (5)SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40,

[0068] (6)SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48,

[0069] (7)SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74,

[0070] (8)SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82,

[0071] (9)SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90,

[0072] (10)SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98,

[0073] (11)SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106,

[0074] (12)SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:114,

[0075] (13)SEQ ID NO:119、SEQ ID NO:120、SEQ ID NO:121、SEQ ID NO:122,

[0076] The light chain variable regions FR1, FR2, FR3, and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0077] (1) SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,

[0078] (2) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,

[0079] (3) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130,

[0080] (4) SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138.

[0081] In some embodiments, the heavy chain variable regions FR1, FR2, FR3, and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0082] (1) SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,

[0083] (2) SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,

[0084] (3) SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,

[0085] (4) SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,

[0086] (5) SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40,

[0087] (6) SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, the light chain variable regions FR1, FR2, FR3, and FR4 of the anti-Claudin18.2 antibody are selected from the following sequence combinations:

[0088] (1) SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,

[0089] (2) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64.

[0090] In some embodiments, the heavy chain variable regions FR1, FR2, FR3, and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0091] (1) SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74,

[0092] (2) SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82,

[0093] (3) SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90,

[0094] (4) SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,

[0095] (5) SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,

[0096] (6) SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114,

[0097] (7) SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, the light chain variable regions FR1, FR2, FR3, and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0098] (1) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130,

[0099] (2) SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138. In some embodiments, the heavy chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 1, 9, 17, 25, 33, 41, 67, 75, 83, 91, 99, 107 or 115,

[0100] The light chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 49, 57, 123 or 131.

[0101] In some embodiments, the heavy chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 1, 9, 17, 25, 33 or 41,

[0102] The light chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 49 or 57.

[0103] In some embodiments, the heavy chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 67, 75, 83, 91, 99, 107 or 115,

[0104] The light chain variable region of the anti-Claudin 18.2 antibody is selected from the sequence shown in SEQ ID NO: 123 or 131.

[0105] In some embodiments, the heavy chain variable region and light chain variable region of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0106] (1) SEQ ID NO: 17 and SEQ ID NO: 57;

[0107] (2) SEQ ID NO: 41 and SEQ ID NO: 49;

[0108] (3) SEQ ID NO: 41 and SEQ ID NO: 57;

[0109] (4) SEQ ID NO:115 and SEQ ID NO:131.

[0110] In some embodiments, the sequences of the heavy chain variable region and the light chain variable region of the anti-Claudin 18.2 antibody are SEQ ID NO: 41 and SEQ ID NO: 49, respectively.

[0111] In some embodiments, the heavy chain constant region of the anti-Claudin 18.2 antibody is selected from human IgG (such as IgG1, IgG2, IgG3 or IgG4), IgM, IgA, IgD, IgE constant regions or mutants of the above constant regions, preferably human IgG1;

[0112] The light chain constant region of the anti-Claudin 18.2 antibody is selected from a human lambda constant region, a kappa constant region or a mutant of the above constant region, preferably a human kappa constant region.

[0113] In some embodiments, the amino acid sequence of the heavy chain of the anti-Claudin 18.2 antibody comprises the sequence as shown in SEQ ID NO: 65, or comprises a sequence that is greater than 70%, such as greater than 75%, 80%, 85%, 90%, 95%, 99% identical to the sequence shown in SEQ ID NO: 65;

[0114] The amino acid sequence of the light chain of the anti-Claudin 18.2 antibody comprises the sequence shown in SEQ ID NO: 66, or comprises a sequence with an identity of greater than 70%, for example, greater than 75%, 80%, 85%, 90%, 95%, or 99% to the sequence shown in SEQ ID NO: 66.

[0115] In some embodiments, p is 3.0-4.0.

[0116] In some embodiments, p is 3.0-3.8.

[0117] In some embodiments, p is 3.0, 3.4, 3.5, or 3.8.

[0118] In some embodiments, p is 3.8.

[0119] In some embodiments, the cytotoxic agent is selected from SN-38, gemcitabine, Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), maytansinoids (e.g., Maytansine DM1, Maytansine DM4), calicheamicin, MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin and other toxins, I131, interleukins, tumor necrosis factor, chemokines and nanoparticles.

[0120] In some embodiments, the cytotoxic agent is MMAE.

[0121] The structure of MMAE is:

[0122]

[0123] In some embodiments, the linker is selected from 6-maleimidocaproyl (MC), maleimidopropionyl (MP), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), 4-(N-maleimidomethyl)-cyclohexane-1-carboxyl (MCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), and 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

[0124] In some embodiments, the linker is 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

[0125] In some embodiments, the LD described in Formula I is MC-vc-PAB-MMAE, and its structure is shown below:

[0126]

[0127] In some embodiments,

[0128] Ab includes:

[0129] (a) heavy chain variable region CDR1, CDR2, CDR3 and light chain variable region CDR1, CDR2, CDR3, wherein the sequence of heavy chain variable region CDR1 is shown in SEQ ID NO:42, the sequence of heavy chain variable region CDR2 is shown in SEQ ID NO:43, the sequence of heavy chain variable region CDR3 is shown in SEQ ID NO:44, the sequence of light chain variable region CDR1 is shown in SEQ ID NO:50, the sequence of light chain variable region CDR2 is shown in SEQ ID NO:51, and the sequence of light chain variable region CDR3 is shown in SEQ ID NO:52;

[0130] (b) a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region is as shown in SEQ ID NO: 41, and the sequence of the light chain variable region is as shown in SEQ ID NO: 49; and / or

[0131] (c) a heavy chain and a light chain, wherein the sequence of the heavy chain is shown in SEQ ID NO: 65, and the sequence of the light chain is shown in SEQ ID NO: 66;

[0132] L is MC-vc-PAB; and

[0133] D is MMAE.

[0134] In the second aspect of the present invention, the present invention provides a composition comprising the aforementioned antibody-drug conjugate, a pharmaceutically acceptable salt, solvate or a solvate of the salt thereof.

[0135] In some embodiments, the composition further comprises a known chemotherapeutic drug for treating tumors, such as adriamycin, cyclophosphamide, taxanes [such as paclitaxel (Taxol), docetaxel (Taxotere)], capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Arimidex, Fulon, Aromasin), 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.

[0136] In some embodiments, the composition also contains known immunotherapeutic drugs for treating tumors, such as PD-1 monoclonal antibody (e.g., pembrolizumab, nivolumab), PD-L1 monoclonal antibody (e.g., atezolizumab), TIGIT monoclonal antibody, 4-1BB monoclonal antibody, VEGFR2 monoclonal antibody (e.g., ramucirumab, apatinib), HER2 monoclonal antibody (e.g., trastuzumab, trastuzumab biosimilar, trastuzumab-dkst), etc., or a combination thereof.

[0137] In some embodiments, the composition further comprises an immunosuppressant selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tacrolimus;

[0138] (3) Antimetabolites, such as azathioprine and 6-mercaptopurine, etc.; (4) Polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3, etc.; (5) Alkylating agents, such as cyclophosphamide. Specifically, the immunosuppressants include methylprednisolone, prednisone, azathioprine, prograf, zenapax, sulelac, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.

[0139] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0140] In the third aspect of the present invention, the present invention provides the aforementioned antibody-drug conjugate, its pharmaceutically acceptable salt, solvate or solvate of the salt or the use of the aforementioned composition in the preparation of a medicament for preventing and / or treating a disease associated with Claudin 18.2.

[0141] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, or pancreatic cancer.

[0142] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer.

[0143] In the fourth aspect of the present invention, the present invention provides a method for preventing and / or treating a disease associated with Claudin 18.2, comprising: administering to a subject in need thereof an effective amount of the aforementioned antibody-drug conjugate, a pharmaceutically acceptable salt, solvate or solvate of the salt, or the aforementioned combination.

[0144] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, or pancreatic cancer.

[0145] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer.

[0146] In the fifth aspect of the present invention, the present invention provides the aforementioned antibody-drug conjugate, its pharmaceutically acceptable salt, solvate or solvate of the salt, or the aforementioned composition, which is used for preventing and / or treating diseases associated with Claudin18.2.

[0147] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, or pancreatic cancer.

[0148] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1 The RT-PCR of the embodiment of the present invention shows that the HEK293 stable cell lines express claudin 18.1 and claudin 18.2 respectively; both the HEK293-claudin 18.2 stable cell line and the control KATO III cells can amplify the claudin 18.2-specific characteristic band of 780 bp, while HEK293-claudin 18.1 can only amplify the common fragment of 504 bp;

[0150] Figure 2This is the result of the FACS experiment screening of HEK293-tight junction protein 18.2 high-expressing stable cell lines in the embodiment of the present invention, wherein the black dots are negative controls, and the gray dots are HEK293-tight junction protein 18.2 high-expressing stable cell lines;

[0151] Figure 3 It is the result of the FACS experiment screening NIH3T3-tight junction protein 18.2 high-expression stable cell line in the embodiment of the present invention, wherein the black line is the negative control, the gray shadow is the 3T3-tight junction protein 18.2 stable cell line; NO.32-H is a high-expression 3T3-tight junction protein 18.2 stable cell line, NO.18-M is a medium-expression 3T3-tight junction protein 18.2 stable cell line, and NO.6-L is a low-expression 3T3-tight junction protein 18.2 stable cell line;

[0152] Figure 4 This is a diagram showing the ADCC effect of the Anti-Claudin18.2 antibody of an embodiment of the present invention;

[0153] Figure 5 This is a graph showing the CDC effect of the Anti-Claudin18.2 antibody of an embodiment of the present invention;

[0154] Figure 6 is a hydrophobic interaction chromatogram (HIC) of an antibody drug conjugate of an embodiment of the present invention;

[0155] Figure 7 This is a diagram showing the cell killing effect of different CM311 ADCs in the LT-M11 cell line according to the embodiments of the present invention;

[0156] Figure 8 This is a graph showing the results of the tumor growth inhibition activity of CM311-ADC-1 of the present invention and the control CM311-ADC-2 on the human gastric cancer nude mouse PDX model STO#025;

[0157] Figure 9 This is a result diagram showing the effects of CM311-ADC-1 of the present invention and the control CM311-ADC-2 on the animal body weight of the human gastric cancer nude mouse PDX model STO#025;

[0158] Figure 10 This is a graph showing the results of the tumor growth inhibition activity of CM311-ADC-1 of the present invention and the control CM311-ADC-2 on the human gastric cancer nude mouse PDX model STO#523;

[0159] Figure 11This is a result diagram showing the effects of CM311-ADC-1 of the present invention and the control CM311-ADC-2 on the animal body weight of the human gastric cancer nude mouse PDX model STO#523;

[0160] Figure 12 It is a graph showing the comparison of in vitro cell activities of the antibody-drug conjugate and the antibody of the embodiment of the present invention (QCLog transformation independent fitting graph). DETAILED DESCRIPTION

[0161] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0162] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0163] Claudin protein is a skeleton protein that constitutes the tight junction structure. It is located at the top of the gap between adjacent cells. Its distribution is tissue and organ specific. Its main functions are intercellular adhesion, maintaining cell polarity, regulating paracellular permeability, and participating in the regulation of cell proliferation and differentiation. In tumors, the tight junctions between cells are destroyed and Claudin cannot perform its normal function.

[0164] Claudin 18 is a member of the Claudin family with two different first exons. Therefore, it can produce two isoforms through selective splicing: Claudin 18.1 and Claudin 18.2. These two isoforms are transcribed and amplified in different tissues. Among them, Claudin 18.1 is mainly expressed in lung tissue, while Claudin 18.2 is specifically expressed in gastric tissue. Claudin 18.2 (GenBank accession number: NM_001002026.3) is not expressed in normal tissues other than gastric mucosa, but it is significantly upregulated in a variety of tumors, including 80% of gastrointestinal adenomas, 60% of pancreatic tumors, and some tumors of the bile duct, ovary and lung.

[0165] The term "Claudin 18.2-related disease" refers to a disease in which the expression of Claudin 18.2 in tissue cells is different from (such as exceeding) the normal level. For example, if the expression level of Claudin 18.2 in a tissue cell is higher than the expression level of Claudin 18.2 in a reference or control (i.e., normal tissue cells), it indicates the presence of a Claudin 18.2-related disease in the subject (especially human) from which the tissue cell is derived.

[0166] In the present invention, unless otherwise stated, any numerical range should be understood to include any value or any sub-range within the range.

[0167] In the present invention, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of identical polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. The light chain of an antibody can be divided into two types: κ and λ. The heavy chain can be divided into five types: μ, δ, γ, α or ε. Depending on the heavy chain, antibodies can be divided into five types: IgM, IgD, IgG, IgA and IgE. Within the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the component C1q of the complement system. H and V L The V region can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L) respectively form antibody binding sites. The allocation of amino acids to each region or domain follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917;

[0168] Definition by Chothia et al. (1989) Nature 342:878-883.

[0169] In the present invention, the algorithms used to determine the percentage of sequence identity (homology) and sequence similarity are, for example, BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST 2.0 can be used to determine the percentage of amino acid sequence identity of the present invention, using, for example, the parameters described in the literature or the default parameters. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0170] In the present invention, the amino acid sequence having at least 70% sequence identity with the amino acid sequence includes polypeptide sequences substantially identical to the amino acid sequence, for example, those sequences containing at least 70% sequence identity, preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity when compared with the polypeptide sequence of the present invention using the methods described herein (e.g., BLAST analysis using standard parameters).

[0171] In the present invention, the mutant of the amino acid sequence refers to a sequence having an identity with the amino acid sequence of greater than 70%, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, such as a sequence having 3, 2 or 1 substituted, deleted or added amino acids. Preferably, the substituted, added or deleted amino acids do not exceed 3 amino acids. More preferably, the substituted, added or deleted amino acids do not exceed 2 amino acids. Most preferably, the substituted, added or deleted amino acids do not exceed 1 amino acid.

[0172] A "substitution" variant is a variant in which at least one amino acid residue in the native sequence is removed and inserted into the same position by a different amino acid. The substitution may be single, in which only one amino acid is substituted in the molecule; or may be multiple, in which two or more amino acids are substituted in the same molecule. Multiple substitutions may be located at consecutive sites. Similarly, an amino acid may be substituted by multiple residues, in which such variants include both substitutions and insertions. An "insertion" (or "addition") variant is a variant in which one or more amino acids are inserted into an amino acid at a specific position adjacent to a native sequence. Adjacent amino acids means connected to the α-carboxyl or α-amino functional group of the amino acid. A "deletion" variant is a variant in which one or more amino acids in the native amino acid sequence are removed. Typically, a deletion variant has one or two amino acids deleted in a specific region of its molecule.

[0173] In certain embodiments, less than the theoretical maximum of drug moieties are coupled to the antibody in the coupling reaction. Generally speaking, antibodies do not contain many free and reactive cysteine ​​thiol groups to which drug moieties can be attached; in fact, most cysteine ​​thiol groups in antibodies exist as disulfide bridges. In certain embodiments, antibodies can be reduced under partially or completely reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups.

[0174] In some embodiments, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt, wherein the inorganic acid salt is hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate and carbonate, sulfate or phosphate, and the organic acid salt is formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, α-ketoglutarate, α-glycerophosphate, alkyl sulfonate or aryl sulfonate; preferably, the alkyl sulfonate is methyl sulfonate or ethyl sulfonate; the aryl sulfonate is benzene sulfonate or p-toluene sulfonate.

[0175] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid affording a pharmaceutically acceptable anion.

[0176] As used herein, unless otherwise indicated, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide a compound of the present invention. Prodrugs become active compounds only under biological conditions through this reaction, or they are active in their unreactive form. Prodrugs can generally be prepared using known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, 5th edition).

[0177] In the present invention, solvate refers to these forms of the antibody drug conjugate of the present invention: the antibody drug conjugate forms a complex in a solid or liquid form by coordination with solvent molecules. Hydrate is a specific form of solvate, which has coordinated water molecules. In the present invention, hydrate is a preferred solvate.

[0178] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredients are known or will be apparent to those skilled in the art based on the disclosure of the present invention. As described in REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, EW, ed., Mack Publishing Company, 19th ed. (1995), the method for preparing the pharmaceutical composition includes incorporating appropriate pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed.

[0179] The pharmaceutical composition of the present invention may comprise a pH buffered aqueous solution. Alternatively, a buffer such as phosphate, citrate and other organic acids; an antioxidant including ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide; a protein such as serum albumin, gelatin or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, sucrose, trehalose or dextrin; a chelating agent such as EDTA; a sugar alcohol such as mannitol or sorbitol; a salt-forming counterion such as sodium; and / or a nonionic surfactant such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM .

[0180] The pharmaceutical preparations of the present invention are manufactured by known methods, including conventional mixing, dissolving or lyophilizing methods. The compounds of the present invention can be prepared into pharmaceutical compositions and administered to patients in various ways suitable for the selected mode of administration, for example, orally or parenterally (by intravenous, intramuscular, topical or subcutaneous routes).

[0181] Thus, the compounds of the invention can be systemically administered, for example, orally, in combination with a pharmaceutically acceptable carrier (such as an inert diluent or an assimilable edible carrier). They can be enclosed in hard or soft shell gelatin capsules and can be compressed into tablets. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of swallowable tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions and preparations should contain at least 0.1% of the active compound. The proportions of such compositions and preparations can of course vary and can account for about 1% to about 99% of the weight of a given unit dosage form. In such therapeutically useful compositions, the amount of active compound is such that an effective dosage level can be obtained.

[0182] Tablets, lozenges, pills, capsules, etc. may also contain: binders, such as tragacanth, gum arabic, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, fructose, lactose or aspartame; or flavorings, such as mint, wintergreen oil or cherry flavor. When the unit dosage form is a capsule, in addition to the above type of material, it may also contain a liquid carrier, such as vegetable oil or polyethylene glycol. Various other materials may be present as a coating, or otherwise change the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar, etc. Syrups or elixirs may contain active compounds, sucrose or fructose as sweeteners, methylparaben or propylparaben as preservatives, dyes and flavorings (such as cherry flavor or orange flavor). Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. Additionally, the active compound may be incorporated into sustained-release preparations and sustained-release devices.

[0183] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Aqueous solutions of the active compound or its salts may be prepared, optionally mixed with a nontoxic surfactant. Dispersants in glycerol, liquid polyethylene glycol, triacetin and mixtures thereof, and oils may also be prepared. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0184] The pharmaceutical dosage form suitable for injection or infusion can include a sterile aqueous solution or dispersant or sterile powder containing an active ingredient (optionally encapsulated in liposomes) suitable for the instant preparation of a sterile injectable or infusible solution or dispersant. In all cases, the final dosage form must be sterile, liquid and stable under production and storage conditions. The liquid carrier can be a solvent or liquid dispersion medium, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of a dispersant, or by the use of a surfactant. The effect of preventing microorganisms can be produced by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents, such as sugars, buffers or sodium chloride. The extended absorption of injectable compositions can be produced by using compositions that delay absorption (e.g., aluminum monostearate and gelatin).

[0185] Sterile injectable solutions are prepared by combining the active compound in the required amount in an appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients present in the previously sterile-filtered solution.

[0186] Useful solid carriers include pulverized solids (such as talc, clay, microcrystalline cellulose, silicon dioxide, alumina, etc.). Useful liquid carriers include water, ethanol or ethylene glycol or water-ethanol / ethylene glycol mixtures, in which the compounds of the present invention can be dissolved or dispersed with the help of non-toxic surfactants at effective levels. Adjuvants (such as flavors) and additional antimicrobial agents can be added to optimize the properties for a given use.

[0187] Thickeners (such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified inorganic materials) can also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, etc., for direct application to the user's skin.

[0188] The above-mentioned preparations can be in unit dosage form, which is a physically discrete unit containing a unit dosage, suitable for administration to humans and other mammals. The unit dosage form can be a capsule or tablet, or a plurality of capsules or tablets. Depending on the specific treatment involved, the amount of the unit dosage of the active ingredient can be varied or adjusted between about 0.1 to about 1000 mg or more.

[0189] In addition, it also includes the application of various new drug dosage forms such as emulsion liposomes, microspheres and nanospheres, such as drugs prepared using particulate dispersion systems including polymeric micelles, nanoemulsions, submicroemulsions, microcapsules, microspheres, liposomes and niosomes (also known as non-ionic surfactant vesicles).

[0190] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or side effects resulting from a disease. As used herein, "treat" encompasses any treatment of a patient's disease, including: (a) preventing the disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) inhibiting the symptoms of a disease, i.e., preventing its development; or (c) alleviating the symptoms of a disease, i.e., causing the disease or symptoms to regress.

[0191] In the present invention, "subject" refers to a vertebrate. In certain embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, a mammal refers to a human.

[0192] In the present invention, "effective amount" refers to an amount that is effective in achieving the desired therapeutic or preventive effect at the required dose and time. The "therapeutically effective amount" of the substance / molecule of the present invention may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the substance / molecule to induce the desired response in the individual. The therapeutically effective amount also encompasses the amount in which the therapeutic beneficial effects of the substance / molecule outweigh any toxic or harmful consequences. "Preventively effective amount" refers to an amount that is effective in achieving the desired preventive effect at the required dose and time. Usually, but not necessarily, since the preventive dose is used for the subject before the onset of the disease or in the early stages of the disease, the preventively effective amount will be lower than the therapeutically effective amount. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to a certain extent, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably stop) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with cancer to a certain extent.

[0193] In the present invention, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0194] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from mouse antibodies and the constant region sequences are derived from human antibodies.

[0195] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the complementary determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0196] In addition, in humanization, it is also possible to mutate the amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL, thereby improving one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, the mutations in the CDR are generally no more than one or two. Therefore, the humanized antibodies of the present invention also encompass antibodies comprising 1 or 2 amino acid mutations in the CDR.

[0197] The present invention is further explained below in conjunction with specific embodiments, but these embodiments are not intended to limit the scope of the present invention.

[0198] Example 1 Preparation of humanized antibodies

[0199] 1. Preparation of monoclonal antibody against tight junction protein 18.2

[0200] Multiple strategies were simultaneously applied to immunize Balb / c mice and screen to obtain monoclonal antibodies that specifically bind only to Claudin 18.2 and not to Claudin 18.1, a different splice form of Claudin 18, as follows:

[0201] 1) Construction of a stable cell line expressing tight junction protein 18.2

[0202] The plasmid gene claudin 18.1-puc57-Amp containing the full-length sequence of human claudin 18.1 (UniProtKB-P56856) was synthesized (Suzhou Hongxun Biology, SynbioTech). The plasmid was used as a template, and the upstream primer 5'-ttggcaaagaattgctagatgtccaccaccacatgcc-3' (SEQ ID NO: 171) and the downstream primer 5'-tgttcgggccctcctcgattacacatagtcgtgcttgg-3' (SEQ ID NO: 172) were used to PCR amplify the full-length fragment of human claudin 18.1 (Met1-Val261). The amplified product was enzymatically linked with NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat: M0530L) and cloned into the eukaryotic expression plasmid system. Similarly, a plasmid gene containing the full-length sequence of human tight junction protein 18.2 (UniProtKB-P56856-2) tight junction protein 18.2-puc57-Amp (Suzhou Hongxun Biology, SynbioTech) was synthesized. Using this plasmid as a template, the upstream primer 5'-ttggcaaagaattgctagatggccgtgactgcctgtc-3' (SEQ ID NO: 173) and the downstream primer 5'-tgttcgggccctcctcgattacacatagtcgtgcttgg-3' (SEQ ID NO: 174) were used to PCR amplify the full-length fragment of human tight junction protein 18.2 (Met1-Val261). The amplified product was enzymatically linked with NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat: M0530L) and cloned into the eukaryotic expression plasmid system. NIH3T3 and HEK293 cells were electroporated with this plasmid, and 1-10 μg / mL Puromycin (Gibco, Cat: A1113803) was used to perform step-by-step pressure screening for stable expression cell lines.

[0203] The obtained HEK293-claudin 18.1 and HEK293-claudin 18.2 stable cell lines were first verified by RT-PCR. Total RNA was extracted from positive cloned cells using a Trizol RNA extraction kit, and reverse transcription was performed using a reverse transcription kit (SuperScriptTM First-Strand Synthesis System, Cat: 18080051) and Oligo (dT) primers to obtain a cDNA library. Since the two spliced ​​fragments of tight junction protein, tight junction protein 18.1 and tight junction protein 18.2, differ in the region from the nitrogen terminus to the first extracellular region (Loop 1), primers KNB14 (5'-tgtgcgccaccatggccgtg-3' (SEQ ID NO: 175)) and KNB15 (5'-tggaaggataagattgtacc-3' (SEQ ID NO: 176)) were designed to amplify the region from Loop 1 to the carbon terminus of tight junction protein 18.1 and tight junction protein 18.2 (504 bp); primer KNB16 (5'-tgggtgccattggcctcctg-3' (SEQ ID NO: 177)) was designed to specifically and complementary bind to the nitrogen terminus of tight junction protein 18.2, but not to the nitrogen terminus region of tight junction protein 18.1, and only amplify the full-length fragment from the nitrogen terminus to the carbon terminus of tight junction protein 18.2 (780 bp) to express the KATO of tight junction protein 18.2 III cells (ATCCHTB-103) were used as a positive control. Figure 1 RT-PCR showed that HEK293 stable cell lines expressed claudin 18.1 and claudin 18.2, respectively. Both HEK293-claudin 18.2 stable cell lines and control KATO III cells could amplify a 780 bp characteristic band specific to claudin 18.2, while HEK293-claudin 18.1 could only amplify a 504 bp common fragment.

[0204] The constructed stable cell lines HEK293-claudin 18.2 cells and NIH3T3-claudin 18.2 cells were digested and collected, washed twice with PBS, and 100 μL of 1:200 diluted primary antibody rabbit anti-claudin 18.2 (Abcam, EPR19202, Catalog No.: ab222512) was added to each tube, incubated at 4°C for 60 minutes, and the excess primary antibody solution was washed away with 0.5% BSA / PBS, and then 50 μL of secondary antibody goat anti-rabbit IgGFc-AF647 (Jackson ImmunoResearch, Catalog No.: 111-606-046) was added, incubated at 4°C for 45 minutes, and then the excess secondary antibody was washed away with 0.5% BSA / PBS, and finally the cells were resuspended with 100 μL PBS solution and immediately detected by flow cytometry. The results are shown in Figure 2 , Figure 3 .

[0205] Figure 2 The results of FACS experiments to detect HEK293-Claudin 18.2 stable cell lines transfected with the full-length claudin 18.2 gene are shown. The black dots are untransfected HEK293 cells, and the gray dots are HEK293-Claudin 18.2 stable cell lines. HEK293 cells do not express claudin 18.2, and HEK293-Claudin 18.2 stable lines highly express claudin 18.2 on the cell membrane surface.

[0206] Figure 3 The results of detecting tight junction protein 18.2 in NIH3T3-tight junction protein 18.2 high expression stable cell line by FACS experiment are shown. The black line is the negative control, and the gray shadow is the 3T3-tight junction protein 18.2 stable cell line. NO.32-H is a 3T3-tight junction protein 18.2 stable cell line with high expression of tight junction protein 18.2, NO.18-M is a 3T3-tight junction protein 18.2 stable cell line with medium expression of tight junction protein 18.2, and NO.6-L is a 3T3-tight junction protein 18.2 stable cell line with low expression of tight junction protein 18.2.

[0207] The expanded and grown positive stable cell lines were collected and frozen, among which the NIH3T3-tight junction protein 18.2 stable cell line was used to immunize animals.

[0208] 2) Preparation of anti-claudin 18.2 monoclonal antibodies from hybridomas

[0209] Female Balb / c mice aged 6 to 8 weeks were immunized with 3T3-claudin 18.2 stable cells or plasmid encoding claudin 18.2, or immunized alternately. When immunizing with cells, 1×10 6 3T3-tight junction protein 18.2 stable cells were mixed with Freund's adjuvant or non-Freund's adjuvant and injected into the thigh root and foot pad. Two weeks later, different parts were immunized again. When DNA was used for immunization, 20 μg of plasmid was mixed with 1 μg of CpG and injected directly into the abdomen of mice using a gene gun (Biorad) at 40 psi. Immunization was performed once a week. Three days before the preparation of fusion, 1×10 cells of HEK293-tight junction protein 18.2 high-expressing stable cell line were used. 6 Cells / 50μL / mouse, for tail vein injection shock immunization. Three days later, the mice were killed, and the popliteal lymph nodes, inguinal lymph nodes, and iliac lymph nodes were collected and ground in DMEM to obtain a suspension rich in B cells; the mouse spleen was removed, ground in DMEM and centrifuged to obtain a spleen cell suspension. After taking an appropriate amount of the suspension of lymph nodes and spleen cells and mixing it with SP2 / 0, an electric fusion instrument was used for cell fusion.

[0210] 3) Construction of anti-tight junction protein 18.2 phage antibody library

[0211] The collected mouse spleen and peripheral lymph node cell suspensions were used to extract total RNA using the Trizol RNA extraction kit, and the antibody light and heavy chain cDNA libraries were obtained by reverse transcription using the reverse transcription kit (SuperScript First-Strand Synthesis System, Cat. No. 18080051) using light and heavy chain specific primers. Using the cDNA as a template, the antibody light and heavy chain variable region fragments were PCR amplified using light and heavy chain variable region primers, and then cloned into a phage plasmid vector containing the human antibody light chain constant region Ckappa or the human IgG1 heavy chain constant region CH1 to form a chimeric light chain library and a heavy chain library, respectively. The antibody fragments in the light chain library were double-digested with BspQI and SfiI, and then connected to the heavy chain library to form a mouse chimeric Fab phage display library based on filamentous phage M13, with a library capacity of 1.2×10 10 Take 0.8mL of phage (titer about 1x10 13 / mL) was mixed with 200μL 5% BSA / PBS and 1x10 7 HEK293-tight junction protein 18.1 cells were placed in an ice bath for 1 hour; centrifuged at 1000 rpm for 10 minutes, the supernatant was collected, and the supernatant was mixed with 1x10 6HEK293-Claudin 18.2 cells were mixed, placed in an ice bath for 1 hour, centrifuged at 1000rpm for 3 minutes, and the supernatant was discarded; 1mL 1% BSA / PBS was added, and the cells were washed repeatedly for 5-10 times; 1mL 100mM TEA (triethylamine) was added to lyse the cells, and the cells were incubated at room temperature for 10 minutes, and 0.5mL 1M Tris-HCl, pH7.5 was added to neutralize, and 10mL TG1 E.coli. cells in the logarithmic growth phase were infected, and the cells were allowed to stand at 37°C for 30 minutes. According to the conventional molecular biology process, the phages were recovered, the titer was detected, and the next round of screening was carried out.

[0212] 2. Screening and sequence acquisition of anti-claudin 18.2 specific antibodies

[0213] HEK293-claudin 18.1, HEK293-claudin 18.2 and HEK293 were prestained with 5 μM, 0.5 μM and 0 μM Cell Tracker Green CMFDA Dye (Thermo, Cat. No. C2925) according to the instructions. After washing away the dye, the mixture was mixed at a ratio of 1:1:1 and added to a 96-well plate (2x10 5 Cells / well) and combined with hybridoma supernatant or bacterial induction supernatant. After incubation in ice bath for 1 hour, AlexaFluro647-labeled secondary antibody anti-mouse IgG Fc or anti-human IgG F(ab)'2 (Jackson ImmunoResearch) was added and incubated on ice for 45 minutes. After washing, 100 μL PBS was added to each well to resuspend the cells, and flow cytometry (iQue Screener) was prepared. According to the fluorescence intensity of the FL2 channel, three different cell populations were circled respectively, and the binding of the antibody to be tested to each cell population was detected in the FL4 channel. The screened antibodies bind to HEK293-tight junction protein 18.2 stably transfected cells with high affinity and specificity, while not binding to HEK293-tight junction protein 18.1 stably transfected cells and HEK293 cells. From the hybridoma cells, a total of 320 clones were screened by FACS detection that could bind to tight junction protein 18.2 but not tight junction protein 18.1; after three rounds of panning, 62 clones with high affinity binding to tight junction protein 18.2 but not tight junction protein 18.1 were screened from the phage Fab library.

[0214] The positive clones obtained from the phage library screening were extracted and sequenced, and the variable region sequences were cloned into the heavy chain and light chain constant region vectors for full-length IgG expression. 1 mL of TRNzol was added to the positive cells obtained from the hybridoma for lysis, and the total RNA was extracted by the guanidine isothioate method. Using this as a template, the first-chain cDNA was synthesized, and the variable region DNA sequence corresponding to the hybridoma cell was amplified using the first-chain cDNA as a subsequent template. After sequencing the amplified products, the candidate hybridoma heavy chain and light chain variable region sequences were obtained, as shown below.

[0215] Clone 18D10:

[0216] Heavy chain

[0217] DVQLQESGPGLVKPSQSLSLTCTVT KYSITSNYAVN WIRQFPGNKLEWMG YINYSGNTNYNPSLKS RISITRDTSKNQFFLQLNSVTAEDTATYYCAT SYYGNSFY WGQGTLVTVSA (SEQ ID NO: 139).

[0218] The underlined parts from left to right are CDR1 (SEQ ID NO: 140), CDR2 (SEQ ID NO: 141), and CDR3 (SEQ ID NO: 142);

[0219] The ununderlined parts are FR1 (SEQ ID NO: 143), FR2 (SEQ ID NO: 144), FR3 (SEQ ID NO: 145), and FR4 (SEQ ID NO: 146) from left to right.

[0220] Nucleic acid sequence

[0221] GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTAATTATGCCTGGAACTGGATCCGACAGTTTCCAGGAAACAAACTAGAGTGGATGGGCTACATAAACTACAGTGGGAACACTAACTA TAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTGCTGAGGACACAGCCACATATTATTGTGCAACCTCCTATTATGGTAATTCCTTTATTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO: 178).

[0222] Light chain

[0223] DIVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTE GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNAYSFPWT FGGGTKLEIK (SEQ ID NO: 147).

[0224] The underlined parts from left to right are CDR1 (SEQ ID NO: 148), CDR2 (SEQ ID NO: 149), and CDR3 (SEQ ID NO: 150);

[0225] The ununderlined parts are FR1 (SEQ ID NO: 151), FR2 (SEQ ID NO: 152), FR3 (SEQ ID NO: 153), and FR4 (SEQ ID NO: 154) from left to right.

[0226] Nucleic acid sequence

[0227] GACATTTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCA TCCACTAGGGAGTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGAATGCTTATAGTTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO: 179).

[0228] Clone 18A9:

[0229] Heavy chain

[0230] QVQLQQSGREVVRPGTSVKVSCKPS GYAFTNYLID WVKQRPGQGLEWIG GINPGSGDTVYNEKFK AKATLTADKSSMTANMQLSSLTSDDSAVYFCAR RVRGNSFDS WGQGTLVTVSA (SEQ ID NO: 155).

[0231] The underlined parts from left to right are CDR1 (SEQ ID NO: 156), CDR2 (SEQ ID NO: 157), and CDR3 (SEQ ID NO: 158);

[0232] The ununderlined parts are FR1 (SEQ ID NO: 159), FR2 (SEQ ID NO: 160), FR3 (SEQ ID NO: 161), and FR4 (SEQ ID NO: 162) from left to right.

[0233] Nucleic acid sequence

[0234] CAGGTGCAGCTGCAGCAGTCTGGACGTGAGGTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGCCTTCTGGATACGCCTTCACTAATTACTTGATAGACTGGGTAAAACAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGGGATTAATCCTGGAAGTGGTGACACTGTGTA CAATGAGAAGTTCAAGGCCAAGGCAACACTGACTGCAGACAAATCCTCCATGACTGCCAACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGCGCAAGAAGGGTCCGTGGTAATTCGTTTGATTCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO: 180).

[0235] Light chain

[0236] DIVMSQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTE GVPDRFTGSGSGKDFTLTISSVQAEDLALYYC QNNYFYPLT FGAGTKLELK (SEQ ID NO: 163).

[0237] The underlined parts from left to right are CDR1 (SEQ ID NO: 164), CDR2 (SEQ ID NO: 165), and CDR3 (SEQ ID NO: 166);

[0238] The ununderlined parts are FR1 (SEQ ID NO: 167), FR2 (SEQ ID NO: 168), FR3 (SEQ ID NO: 169), and FR4 (SEQ ID NO: 170) from left to right.

[0239] Nucleic acid sequence

[0240] GACATTTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAATTGTTGATCTACTGGGCA TCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAAAAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCACTTTATTACTGTCAGAATAATTATTTTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO: 181).

[0241] The heavy chain and light chain variable region sequence fragments were PCR amplified, and the heavy chain variable region was cloned into a vector containing a human heavy chain constant region to express the complete IgG1 heavy chain in mammalian cells. Similarly, the light chain variable region was cloned into a vector containing a human light chain constant region to express the complete kappa light chain in mammalian cells. After correct sequencing, it was transfected into HEK293-6E mammalian cells, and IgG1 was secreted into the culture medium after expression. The supernatant was collected and filtered for purification. Protein A chromatography was used to purify IgG, and the culture supernatant was loaded onto a Protein A column of appropriate size, washed with 50mM Tris-HCl pH8.0, 250mM NaCl, and the bound IgG was eluted with 0.1M Glycine-HCl, pH3.0. The protein was ultrafiltered and concentrated using a concentrator (Millipore), OD280 was detected, and the concentration of IgG was determined by spectrophotometry. The purity of IgG was analyzed by SDS-PAGE.

[0242] HEK293-Claudin 18.1 cells, HEK293-Claudin 18.2 cells and HEK293 cells in the logarithmic growth phase were digested and plated at 5 × 10 4Cells / 100 μL were added to a U-shaped 96-well plate, centrifuged at 1100 rpm for 3 minutes, the supernatant was discarded, the cells were gently tapped, 50 μL of gradient diluted antibodies were added to each well (antibody concentration started from 100 nM, 5-fold dilutions in 8 gradients), and incubated at 4°C for 1 hour. After the incubation, 140 μL of 0.5% BSA was added to each well to wash 3 times, and 30 μL / well of secondary antibody AlexaFluro647 anti-human IgG (Jackson ImmunoResearch, Cat. No.: 109-606-170) was added, and incubated at 4°C for 40 minutes. After the incubation, 140 μL of 0.5% BSA was added to each well to wash 3 times, and finally each well was resuspended in 50 μL PBS for flow cytometry (iQue Screener) detection. The results are shown in Table 1, which show that the obtained chimeric Claudin 18.2 IgG1 antibody only recognized HEK293-Claudin 18.2 cells transfected with Claudin 18.2, but had no binding to HEK293 and HEK293-Claudin 18.1.

[0243] Table 1: Binding of tight junction protein 18.2 antibody to stably transfected cell lines (NB means no binding was detected)

[0244]

[0245] 3. Humanization of anti-claudin 18.2 antibody

[0246] Compare the selected monoclonal antibody variable region sequence with the human germline antibody sequence to find the sequence with high homology for CDR transplantation; then use the computer to perform homology modeling, analyze the framework amino acid sequence of the CDR region and its surroundings, and examine its spatial three-dimensional binding mode. By calculating the electrostatic force, van der Waals force, hydrophilicity and entropy value, analyze the key amino acid individuals in each positive monoclonal antibody gene sequence that may interact with the target and maintain the spatial structure, and design the back mutation site on this basis. Analyze and examine the HLA-DR affinity and select the human embryonic framework sequence with low immunogenicity. Analyze the amino acid residues that may be modified during the fermentation process, and design mutations to reduce the possibility of modification.

[0247] A total of different heavy chain derivatives and light chain derivatives were designed. The light and heavy chain derivatives were fully synthesized and cloned into vectors containing the antibody kappa chain constant region Ckappa or the human IgG1 constant region CH1-CH3. The light and heavy chain derivative plasmids of the same parental source were combined and paired, transfected into HEK293.6E cells, expressed for 5-6 days, and the supernatant was purified using a Protein A column.

[0248] The sequence of the humanized antibody is as follows:

[0249] 18D10:

[0250] Heavy chain variable region:

[0251] 18D10VHv1:

[0252] QVQLQESGPGLVKPSETLSLTCTVS KYSITSNYAVN WIRQPPGKGLEWIG YINY SGNTNYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAT SYYGNSFY WGQG TLVTVSS (SEQ ID NO: 1).

[0253] The underlined parts from left to right are CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 3), and CDR3 (SEQ ID NO: 4);

[0254] The ununderlined parts are FR1 (SEQ ID NO: 5), FR2 (SEQ ID NO:

[0255] 6), FR3 (SEQ ID NO: 7), FR4 (SEQ ID NO: 8).

[0256] Nucleic acid sequence

[0257] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCCGGCTACAGCATCACCAGCAACTACGCCTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTCGAGTGGATCGGCTACATCAACTACAGCGGCAACACCAACTA CAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCAGAGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGTGCCACAAGCTACTACGGCAACAGCTTCATCTACTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT(SEQ ID NO: 182).

[0258] 18D10VHv2:

[0259] QVQLQESGPGLVKPSETLSLTCTVS GGSISSNYAWN WIRQPPGKGLEWMG VOICE YSGNTNYNPSLKS RITISRDTSKNQFSLKLSSVTAADTAVYYCAT SYYGNSFY WGQG TLVTVSS (SEQ ID NO: 9).

[0260] The underlined parts from left to right are CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12);

[0261] The ununderlined parts are FR1 (SEQ ID NO: 13), FR2 (SEQ ID NO: 14), FR3 (SEQ ID NO: 15), and FR4 (SEQ ID NO: 16) from left to right.

[0262] Nucleic acid sequence

[0263] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCCGGCGGCAGCATCAGCAGCAACTACGCCTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTCGAGTGGATGGGCTACATCAACTACAGCGGCAACACCAACTA CAACCCCAGCCTGAAGTCCAGAATCACCATCAGCAGAGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGTGCCACAAGCTACTACGGCAACAGCTTCATCTACTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT(SEQ ID NO: 183).

[0264] 18D10VHv3:

[0265] QVQLQESGPGLVKPSETLSLTCTVS GGSISSNYAWN WIRQPPGKGLEWIG YINY SGNTNYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAT SYYGNSFY WGQG TLVTVSS (SEQ ID NO: 17).

[0266] The underlined parts from left to right are CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19), and CDR3 (SEQ ID NO: 20);

[0267] The ununderlined parts are FR1 (SEQ ID NO: 21), FR2 (SEQ ID NO: 22), FR3 (SEQ ID NO: 23), and FR4 (SEQ ID NO: 24) from left to right.

[0268] Nucleic acid sequence

[0269] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCCGGCGGCAGCATCAGCAGCAACTACGCCTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTCGAGTGGATCGGCTACATCAACTACAGCGGCAACACCAACTA CAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCAGAGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGTGCCACAAGCTACTACGGCAACAGCTTCATCTACTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT(SEQ ID NO: 184).

[0270] 18D10VHv4:

[0271] QVQLQESGPGLVKPSETLSLTCTVS GGSISSNYAWN WIRQPPGKGLEWIG YINY SGYTNYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAT SYYGNSFY WGQG TLVTVSS (SEQ ID NO: 25).

[0272] The underlined parts from left to right are CDR1 (SEQ ID NO: 26), CDR2 (SEQ ID NO: 27), and CDR3 (SEQ ID NO: 28);

[0273] The ununderlined parts are FR1 (SEQ ID NO: 29), FR2 (SEQ ID NO: 30), FR3 (SEQ ID NO: 31), and FR4 (SEQ ID NO: 32) from left to right.

[0274] Nucleic acid sequence

[0275] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCCGGCGGCAGCATCAGCAGCAACTACGCCTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTCGAGTGGATCGGCTACATCAACTACAGCGGCTACACCAACTA CAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCAGAGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGTGCCACAAGCTACTACGGCAACAGCTTCATCTACTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT(SEQ ID NO: 185).

[0276] 18D10VHv5:

[0277] QVQLQESGPGLVKPSETLSLTCTVS GGSISSNYAWN WIRQPPGKGLEWIG YINY SGNTAYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAT SYYGNSFY WGQG TLVTVSS (SEQ ID NO: 33).

[0278] The underlined parts from left to right are CDR1 (SEQ ID NO: 34), CDR2 (SEQ ID NO: 35), and CDR3 (SEQ ID NO: 36);

[0279] The ununderlined parts are FR1 (SEQ ID NO: 37), FR2 (SEQ ID NO: 38), FR3 (SEQ ID NO: 39), and FR4 (SEQ ID NO: 40) from left to right.

[0280] Nucleic acid sequence

[0281] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCCGGCGGCAGCATCAGCAGCAACTACGCCTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTCGAGTGGATCGGCTACATCAACTACAGCGGCAACACCGCCTA CAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCAGAGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGTGCCACAAGCTACTACGGCAACAGCTTCATCTACTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT(SEQ ID NO: 186).

[0282] 18D10VHv6:

[0283] QVQLQESGPGLVKPSETLSLTCTVS GGSISSNYAWN WIRQPPGKGLEWIG YYYYY SGNTNYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAT SYYGNSFY WGQG TLVTVSS (SEQ ID NO: 41).

[0284] The underlined parts from left to right are CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 43), and CDR3 (SEQ ID NO: 44);

[0285] The ununderlined parts are FR1 (SEQ ID NO: 45), FR2 (SEQ ID NO: 46), FR3 (SEQ ID NO: 47), and FR4 (SEQ ID NO: 48) from left to right.

[0286] Nucleic acid sequence

[0287] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCCGGCGGCAGCATCAGCAGCAACTACGCCTGGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTCGAGTGGATCGGCTACATCTACTACAGCGGCAACACCAACTA CAACCCCAGCCTGAAGTCCAGAGTGACCATCAGCAGAGACACCAGCAAGAACCAGTTCTCCCTGAAGCTGAGCAGCGTGACAGCCGCCGATACAGCCGTGTACTACTGTGCCACAAGCTACTACGGCAACAGCTTCATCTACTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT(SEQ ID NO: 187).

[0288] Light chain variable region:

[0289] 18D10VLv1:

[0290] DIVMTQSPDSLAVSLGERATINC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLI WASTE GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QNAYSFPWT FGQGTKV EIK (SEQ ID NO: 49).

[0291] The underlined parts from left to right are CDR1 (SEQ ID NO: 50), CDR2 (SEQ ID NO: 51), and CDR3 (SEQ ID NO: 52);

[0292] The ununderlined parts are FR1 (SEQ ID NO: 53), FR2 (SEQ ID NO: 54), FR3 (SEQ ID NO: 55), and FR4 (SEQ ID NO: 56) from left to right.

[0293] Nucleic acid sequence

[0294] GACATCGTGATGACACAGAGCCCTGATAGCCTGGCCGTGTCTCTGGGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACTGGGCC AGCACCAGAGAAAGCGGCGTGCCAGATAGATTCAGCGGCAGCGCCTCTGGAACCGACTTCACCCTGACAATCAGCTCCCTGCAGGCCGAGGATGTGGCCGTGTACTACTGTCAGAACGCCTACAGCTTCCCCTGGACATTCGGCCAGGGCACCAAGGTGGAAATCAAG(SEQ ID NO: 188).

[0295] 18D10VLv2:

[0296] DIVMTQSPDSLAVSLGERATINC KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLI WASTE GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQAYSFPWT FGQGTKV EIK (SEQ ID NO: 57).

[0297] The underlined parts from left to right are CDR1 (SEQ ID NO: 58), CDR2 (SEQ ID NO: 59), and CDR3 (SEQ ID NO: 60);

[0298] The ununderlined parts are FR1 (SEQ ID NO: 61), FR2 (SEQ ID NO: 62), FR3 (SEQ ID NO: 63), and FR4 (SEQ ID NO: 64) from left to right.

[0299] Nucleic acid sequence

[0300] GACATCGTGATGACACAGAGCCCTGATAGCCTGGCCGTGTCTCTGGGAGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCAGATAGATTCAGCGGCAGCGGCTCTGGAACCGACTTCACCCTGACAATCAGCTCCCTGCAGGCCGAGGATGTGGCCGTGTACTACTGTCAGCAGGCCTACAGCTTCCCCTGGACATTCGGCCAGGGCACCAAGGTGGAAATCAAG(SEQ ID NO:189).

[0301] The sequence of the preferred humanized antibody is as follows:

[0302] Heavy chain amino acid sequence:

[0303] QVQLQESGPGLVKPSETLSLTCTVSGGSISSNYAWNWIRQPPGKGLEWIGYIYYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCATSYYGNSFIYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:65).

[0304] Heavy chain nucleic acid sequence

[0305]

[0306] Light chain amino acid sequence:

[0307] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLI YWASTRESGVPDRFSGSGSGTTDFTLTISSSLQAEDVAVYYCQNAYSFPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 66).

[0308] Light chain nucleic acid sequence

[0309] GACATCGTGATGACACAGAGCCCTGATAGCCTGGCCGTGTCTCTGGGAGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCAGATAGATTCAGCGGCAGCGGCTCTGGAACCGACTTCACCCTGACAATCAGCTCCCTGCAGGCCGAGGATGTGGCCGTGTACTACTGTCAGAACGCCTACAGCTTCCCCTGGACATTCGGCCAGGGCACCAAGGTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG(SEQ ID NO:191).

[0310] 18A9:

[0311] Heavy chain variable region:

[0312] 18A9VHv1:

[0313] QVQLVQSGAEVKKPGSSVKVSCKPS GYAFTNYLID WVRQAPGQGLEWMG GIN PGSGDTVYNEKFQ GRVTLTADKSSMTAYMELSSLRSEDTAVYFCAR RVRGNSFDS W GQGTLVTVSS(SEQ ID NO: 67).

[0314] The underlined parts from left to right are CDR1 (SEQ ID NO: 68), CDR2 (SEQ ID NO: 69), and CDR3 (SEQ ID NO: 70);

[0315] The ununderlined parts are FR1 (SEQ ID NO: 71), FR2 (SEQ ID NO: 72), FR3 (SEQ ID NO: 73), and FR4 (SEQ ID NO: 74) from left to right.

[0316] Nucleic acid sequence

[0317] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGTCCTGCAAGCCTTCTGGCTACGCCTTCACCAACTACCTGATCGACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCATCAACCCTGGCAGCGGCGATACAGTGTA CAACGAGAAGTTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTAGCATGACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAAGAGTGCGGGGCAACAGCTTCGATTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT(SEQ ID NO: 192).

[0318] 18A9VHv2:

[0319] QVQLVQSGAEVKKPGSSVKVSCKPS GYFTNYLD WVRQAPGQGLEWMG GIN PGSGDTVYNEKFQ GRVTLTADESTSTAYMELSSLRSEDTAVYFCAR RVRGNSFDS W GQGTLVTVSS (SEQ ID NO: 75).

[0320] The underlined parts from left to right are CDR1 (SEQ ID NO: 76), CDR2 (SEQ ID NO: 77), and CDR3 (SEQ ID NO: 78);

[0321] The ununderlined parts are FR1 (SEQ ID NO: 79), FR2 (SEQ ID NO: 80), FR3 (SEQ ID NO: 81), and FR4 (SEQ ID NO: 82) from left to right.

[0322] Nucleic acid sequence

[0323] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGTCCTGCAAGCCTTCTGGCTACACCTTCACCAACTACCTGATCGACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCATCAACCCTGGCAGCGGCGATACAGTGTACAACGAGAAGTTCCAGGGCAGAGTGACCCTG ACCGCCGACGAGTCTACCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAAGAGTGCGGGGCAACAGCTTCGATTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT (SEQ ID NO: 193).

[0324] 18A9VHv3:

[0325] QVQLVQSGAEVKKPGSSVKVSCKPS GYFTNYLD WVRQAPGQGLEWMG GIN PGSGDTVYNEKFQ GRVTLTADKSSMTAYMELSSLRSEDTAVYYCAR RVRGNSFDS WGQGTLVTVSS (SEQ ID NO: 83).

[0326] The underlined parts from left to right are CDR1 (SEQ ID NO: 84), CDR2 (SEQ ID NO: 85), and CDR3 (SEQ ID NO: 86);

[0327] The ununderlined parts are FR1 (SEQ ID NO: 87), FR2 (SEQ ID NO: 88), FR3 (SEQ ID NO: 89), and FR4 (SEQ ID NO: 90) from left to right.

[0328] Nucleic acid sequence

[0329] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGTCCTGCAAGCCTTCTGGCTACACCTTCACCAACTACCTGATCGACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCATCAACCCTGGCAGCGGCGATACAGTGTA CAACGAGAAGTTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTAGCATGACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTACTGTGCCAGAAGAGTGCGGGGCAACAGCTTCGATTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT(SEQ ID NO: 194).

[0330] 18A9VHv4:

[0331] QVQLVQSGAEVKKPGSSVKVSCKAS GYTFNYLD WVRQAPGQGLEWMG GIN PGSGDTVYNEKFQ GRVTLTADKSSMTAYMELSSLRSEDTAVYFCAR RVRGNSFDS W GQGTLVTVSS (SEQ ID NO: 91).

[0332] The underlined parts from left to right are CDR1 (SEQ ID NO: 92), CDR2 (SEQ ID NO: 93), and CDR3 (SEQ ID NO: 94);

[0333] The ununderlined parts are FR1 (SEQ ID NO: 95), FR2 (SEQ ID NO: 96), FR3 (SEQ ID NO: 97), and FR4 (SEQ ID NO: 98) from left to right.

[0334] Nucleic acid sequence

[0335] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACCTGATCGACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCATCAACCCTGGCAGCGGCGATACAGTGTA CAACGAGAAGTTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTAGCATGACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAAGAGTGCGGGGCAACAGCTTCGATTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT(SEQ ID NO: 195).

[0336] 18A9VHv5:

[0337] QVQLVQSGAEVKKPGSSVKVSCKPS GYFTNYLD WVRQAPGQGLEWMG GIN PGSGDTVYNEKFQ GRVTITADESTSTAYMELSSLRSEDTAVYFCAR RVRGNSFDS W GQGTLVTVSS (SEQ ID NO: 99).

[0338] The underlined parts from left to right are CDR1 (SEQ ID NO: 100), CDR2 (SEQ ID NO: 101), and CDR3 (SEQ ID NO: 102);

[0339] The ununderlined parts are FR1 (SEQ ID NO: 103), FR2 (SEQ ID NO: 104), FR3 (SEQ ID NO: 105), and FR4 (SEQ ID NO: 106) from left to right.

[0340] Nucleic acid sequence

[0341] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGTCCTGCAAGCCTTCTGGCTACACCTTCACCAACTACCTGATCGACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCATCAACCCTGGCAGCGGCGATACAGTGTACAACGAGAAGTTCCAGGGCAGAGTGACCATC ACCGCCGACGAGTCTACCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAAGAGTGCGGGGCAACAGCTTCGATTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT (SEQ ID NO: 196).

[0342] 18A9VHv6:

[0343] QVQLVQSGAEVKKPGASVKVSCKPS GYAFTNYLID WVRQAPGQGLEWMG GIN PGSGDTVYNEKFQ GRVTLTADKSSSTAYMELSSLRSEDTAVYFCAR RVRGNSFDS W GQGTLVTVSS (SEQ ID NO: 107).

[0344] The underlined parts from left to right are CDR1 (SEQ ID NO: 108), CDR2 (SEQ ID NO: 109), and CDR3 (SEQ ID NO: 110);

[0345] The ununderlined parts are FR1 (SEQ ID NO: 111), FR2 (SEQ ID NO: 112), FR3 (SEQ ID NO: 113), and FR4 (SEQ ID NO: 114) from left to right.

[0346] Nucleic acid sequence

[0347] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCAGCGTGAAGGTGTCCTGCAAGCCTTCTGGCTACGCCTTCACCAACTACCTGATCGACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCATCAACCCTGGCAGCGGCGATACAGTGTA CAACGAGAAGTTCCAGGGCAGAGTGACCCTGACCGCCGACAAGTCTAGCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAAGAGTGCGGGGCAACAGCTTCGATTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT(SEQ ID NO: 197).

[0348] 18A9VHv7:

[0349] QVQLVQSGAEVKKPGASVKVSCKPS GYFTNYLD WVRQAPGQGLEWMG GIN PGSGDTVYNEKFQ GRVTLTADTSTSTVYMELSSLRSEDTAVYFCAR RVRGNSFDS W GQGTLVTVSS (SEQ ID NO: 115).

[0350] The underlined parts from left to right are CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), and CDR3 (SEQ ID NO: 118);

[0351] The ununderlined parts are FR1 (SEQ ID NO: 119), FR2 (SEQ ID NO: 120), FR3 (SEQ ID NO: 121), and FR4 (SEQ ID NO: 122) from left to right.

[0352] Nucleic acid sequence

[0353] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCAGCGTGAAGGTGTCCTGCAAGCCTTCTGGCTACACCTTCACCAACTACCTGATCGACTGGGTCCGACAGGCTCCTGGACAGGGACTTGAATGGATGGGCGGCATCAACCCTGGCAGCGGCGATACAGTGTA CAACGAGAAGTTCCAGGGCAGAGTGACCCTGACCGCCGACACCTCTACCAGCACCGTCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTTCTGTGCCAGAAGAGTGCGGGGCAACAGCTTCGATTCTTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT(SEQ ID NO: 198).

[0354] Light chain variable region:

[0355] 18A9VLv1:

[0356] DIVMTQSPDSLAVSLGERATINC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLI WASTE GVPDRFSGSGSGKDFTLTISSLQAEDVAVYYC QNNYFYPLT FGGGTKV EIK (SEQ ID NO: 123).

[0357] The underlined parts from left to right are CDR1 (SEQ ID NO: 124), CDR2 (SEQ ID NO: 125), and CDR3 (SEQ ID NO: 126);

[0358] The ununderlined parts are FR1 (SEQ ID NO: 127), FR2 (SEQ ID NO: 128), FR3 (SEQ ID NO: 129), and FR4 (SEQ ID NO: 130) from left to right.

[0359] Nucleic acid sequence

[0360] GACATCGTGATGACACAGAGCCCTGATAGCCTGGCCGTGTCTCTGGGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTG CTGATCTACTGGGCCAGCACCAGAGAAAGCGGCGTGCCAGATAGATTCAGCGGCAGCGGCTCTGGAAAGGACTTCACCCTGACAATCAGCTCCCTGCAGGCCGAGGATGTGGCCGTGTACTACTGCCAGAACAACTTCTACCCTCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG (SEQ ID NO: 199).

[0361] 18A9VLv2:

[0362] DIVMTQSPDSLAVSLGERATINC KSSQSLLNSGNQKNYLA WYQQKPGQPPKLLI WASTE GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQNYFYPLT FGGGTKV EIK (SEQ ID NO: 131).

[0363] The underlined parts from left to right are CDR1 (SEQ ID NO: 132), CDR2 (SEQ ID NO: 133), and CDR3 (SEQ ID NO: 134);

[0364] The ununderlined parts are FR1 (SEQ ID NO: 135), FR2 (SEQ ID NO: 136), FR3 (SEQ ID NO: 137), and FR4 (SEQ ID NO: 138) from left to right.

[0365] Nucleic acid sequence

[0366] GACATCGTGATGACACAGAGCCCTGATAGCCTGGCCGTGTCTCTGGGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACTGGGCC AGCACCAGAGAAAGCGGCGTGCCAGATAGATTCAGCGGCAGCGGCTCTGGAACCGACTTCACCCTGACAATCAGCTCCCTGCAGGCCGAGGATGTGGCCGTGTACTACTGCCAGCAGAACTACTTCTACCCTCTGACCTTCGGCGGAGGCACCAAGGTGGAAATCAAG(SEQ ID NO: 200).

[0367] Example 2 Pharmacological studies of humanized antibodies

[0368] 1. Affinity determination of humanized antibodies (EC 50 )

[0369] Cells in the logarithmic growth phase were blocked with 3% BSA for 30 minutes and 5 × 10 4 Cells / 100 μL were spread on a U-shaped 96-well plate, centrifuged at 1100 rpm for 3 minutes, the supernatant was discarded, the cells were gently tapped, 50 μL of gradient diluted antibodies were added to each well (antibody concentration started from 100 nM, 5-fold dilutions in 8 gradients), and incubated at 4°C for 1 hour. After the incubation, 140 μL of 0.5% BSA was added to each well to wash 3 times, and 30 μL / well of AF 647APC anti-human secondary antibody was added to incubate at 4°C for 40 minutes. After the incubation, 140 μL of 0.5% BSA was added to each well to wash 3 times, and finally each well was resuspended in 50 μL PBS for iQue (Intellicyt, USA) detection (see Table 2).

[0370] Table 2: Affinity determination results of humanized antibodies

[0371]

[0372]

[0373] 2. ADCC killing activity of CM311 antibody on tumor cells

[0374] Take 30mL of fresh blood into a 50mL centrifuge tube, add 15mL of 1×PBS, mix well, and slowly add to the centrifuge tube that has been added with 20mL of Ficoll.Paque Plu, so that the blood is spread on the surface of Ficoll.Paque Plu. Centrifuge at 20℃, 2000rpm for 30 minutes, discard the top layer of serum, absorb the buffy coat layer (i.e. PBMC), and divide it into 50mL centrifuge tubes, 10mL per tube. Add no less than 30mL of 1×PBS to each tube and mix well. Centrifuge at 4℃, 1300rpm for 10 minutes, discard the supernatant, add 10mL of 1×PBS to wash and count.

[0375] Resuspend the cells in FBS / RPMI 1640 medium and incubate at 37°C, 5% CO2 for 2 hours. Centrifuge at 1300 rpm for 10 minutes, discard the supernatant, resuspend in FBS / RPMI 1640 medium, and inoculate the cells in a U-shaped 96-well plate at 4*10 5 18D10 chimera, 18A9 chimera, anti-tight junction protein 18.2 humanized antibody 18D10, 18A9 (CM311, CM311 is a general term for humanized antibody 18D10 or 18A9) and control antibody anti-KLH dilution (40, 20, 10, 5, 2.5, 1.25 μg / mL), 25 μL / well. Incubate at 37°C, 5% CO2 for 30 minutes. Remove the plate and add Kato III cells, 8×10 3 100 μL / well, 25 μL / well. Incubate at 37℃5% CO2 for 3.5 hours, add 2 μL 10* lysis buffer to the target cell maximum release well, and continue incubation at 37℃5% CO2 for 30 minutes. Take out the well plate, centrifuge at 1000rpm for 3 minutes, take the supernatant to the black ELISA plate, 50 μL / well. Add LDH detection substrate 50 μL / well, incubate at room temperature for 10 minutes, add stop solution 25 μL / well to terminate the reaction. Detect with ELISA reader (Biotek).

[0376] The result is calculated as follows:

[0377]

[0378] The results are as follows Figure 4 As shown, the anti-claudin 18.2 humanized antibodies 18D10 and 18A9 have strong ADCC activity.

[0379] 3. CDC cell killing activity of CM311 antibody on tumor cells

[0380] Take the Kato III cells in the logarithmic growth phase and resuspend them to 1×10 7 / mL. Add CFSE (Sigma, 87444-5MG-F) to a final concentration of 1μM. Incubate at room temperature for 10 minutes and add 3 times the volume of culture medium to terminate the reaction. 4℃

[0381] Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in medium, and inoculate cells into 96-well plates at 1*10 5 / well, 50μL / well. Add the diluted aforementioned 18D10 chimera, 18A9 chimera, anti-tight junction protein 18.2 humanized antibodies 18D10, 18A9 (CM311) and control antibody anti-KLH (concentrations after dilution are 30, 10, 3.33, 1.11, and 0.37μg / mL), 50μL / well. Dilute the complement to 30% with culture medium and add to the well plate, 50μL / well. Incubate at 37°C, 5% CO2 for 2 hours, centrifuge at 1000rpm for 3 minutes, and discard the supernatant. Dilute the PI dye solution 1:200, mix with Sulfate latex (Invitrogen, S37227), and add to a 96-well plate, 100μL / well. Incubate on ice for 10 minutes and detect by FACS. The results are as follows. Figure 5 As shown, the anti-tight junction protein 18.2 humanized antibodies 18D10 and 18A9 have a strong CDC effect.

[0382] Example 3 Preparation and Detection of Antibody Drug Conjugates

[0383] 1. Preparation of Antibody Drug Conjugates

[0384] Take 10 mg of CM311 antibody and replace it in reducing buffer (25mM sodium borate, pH8.0, 25mM NaCl, 5mM EDTA) using a 15mL 30KD ultrafiltration device, and replace it three times; the final volume is about 1mL, transfer it to a new Eppendorf centrifuge tube (weighed), and weigh it; detect the protein concentration and calculate the total protein. Add 2.5 times the molar number of DTT to the antibody, keep it at room temperature for 2 hours, and mix it continuously; use a 15mL 30KD ultrafiltration device to replace it in coupling buffer (50mM Tris, pH7.2, 150mM NaCl, 5mM EDTA), and replace it three times. Take a sample to determine the protein concentration by A280, weigh it, and calculate the total protein; take 10μL of sample and determine the number of free thiol groups by Ellman's method;

[0385] The molar concentration of free thiol groups was calculated using the following formula:

[0386]

[0387] b: Cuvette optical path length (usually 1 cm)

[0388] The number of moles of free thiol groups was calculated based on the molar concentration of free thiol groups and the total protein solution volume.

[0389] Add 1.1 times the molar number of free thiol groups of vc-MMAE, i.e., MC-vc-PAB-MMAE (dissolved in DMSO) to the reduced antibody, mix well, react at room temperature for 2 hours, and mix intermittently. Add 20 times the molar number of vc-MMAE (i.e., MC-vc-PAB-MMAE) of N-acetylcysteine ​​to the reaction system, mix well, and let stand for 5 minutes. Use a 15mL 30KD ultrafiltration device to replace it in the conjugate storage solution (20mM histidine, 3% sucrose, 0.03% Tween-80, pH5.5), replace it three times in total, and obtain the antibody drug conjugate product Anti-Claudin-18.2-ADC (a general term for antibody drug conjugates prepared from humanized antibodies 18D10 or 18A9) and store it at 4°C.

[0390] 2. DAR determination of antibody-drug conjugates

[0391] The drug loading rate (DAR) of the antibody drug conjugate was determined by hydrophobic interaction chromatography (HIC-HPLC).

[0392] The typical spectrum of antibody drug conjugate CM311-18D10-VH6 / VL1-ADC is shown in Figure 6 The average drug loading number DAR was calculated based on the peak area of ​​the spectrum to be 3.8.

[0393] Similarly, the average drug loading number DAR of the antibody drug conjugate CM311-18D10-VH3 / VL2-ADC was 3.5.

[0394] The average drug loading number DAR of the antibody drug conjugate CM311-18D10-VH6 / VL2-ADC is 3.4.

[0395] The average drug loading number DAR of the antibody drug conjugate CM311-18A9-VH7 / VL2-ADC is 3.0.

[0396] It should be noted that the name of the above antibody-drug conjugate is CM311-18D10-VH6 / VL1-ADC, which means that the antibody used in the preparation of the antibody-drug conjugate is CM311-18D10-VH6 / VL1. Further, the antibody name CM311-18D10-VH6 / VL1 indicates that the antibody used in the preparation of the aforementioned antibody-drug conjugate is an antibody of the type 18D10 with a heavy chain variable region of VHv6 and a light chain variable region of VLv1. Corresponding to Table 2 above, it can be seen that the specific antibody used in the preparation of the aforementioned antibody-drug conjugate is h18D10.v16. The names of the remaining three antibody-drug conjugates CM311-18D10-VH3 / VL2-ADC, CM311-18D10-VH6 / VL2-ADC, and CM311-18A9-VH7 / VL2-ADC can be understood with reference to the above. Specifically, the specific antibody used in the preparation of the antibody drug conjugate CM311-18D10-VH3 / VL2-ADC is h18D10.v23 in Table 2; the specific antibody used in the preparation of the antibody drug conjugate CM311-18D10-VH6 / VL2-ADC is h18D10.v26 in Table 2; the specific antibody used in the preparation of the antibody drug conjugate CM311-18A9-VH7 / VL2-ADC is h18A9.v27 in Table 2.

[0397] Example 4 In vitro pharmacodynamic study of antibody-drug conjugates

[0398] After 1-2 generations of cell passage, the revived cell line was first aspirated to a 15 mL centrifuge tube, centrifuged, and the supernatant was discarded; the cell culture bottle was rinsed with 5 mL PBS, and then the cells were digested with 2 mL trypsin, resuspended with culture medium into the previous 15 mL centrifuge tube, centrifuged, and the supernatant was discarded, and then resuspended with culture medium again, and 0.5 mL was taken out for counting with a cell counter. The cells were plated on a 96-well cell culture plate (LT-1C8 cells at 5000 or 10000 cells / well, LT-M11 cells at 5000 cells / well, and BxPC-3 at 3000 cells / well). After culturing for 24 hours, a series of diluted concentrations of antibody-drug conjugate CM311ADC (conjugated with different CM311 monoclonal antibodies) were added and incubated for 96 hours, and then CCK-8 or Presto-Blue colorimetric reagent was added to each well, and the cells were detected with an enzyme marker and four-parameter fitting was performed.

[0399] Experimental reagents and sources:

[0400] Test drug

[0401] Antibodies name concentration storage conditions CM311-18D10-VH3 / VL2-ADC 1.0mg / mL 2-8℃ CM311-18D10-VH6 / VL1-ADC 1.2mg / mL 2-8℃ CM311-18D10-VH6 / VL2-ADC 0.9mg / mL 2-8℃ CM311-18A9-VH7 / VL2-ADC 0.7mg / mL 2-8℃

[0402] Cell lines

[0403]

[0404] Experimental results:

[0405] IC of each CM311ADC 50 The average values ​​are shown in Table 3. Figure 7 Representative graphs showing the killing of LT-M11 cells by different CM311 ADCs.

[0406] Table 3: IC values ​​of different CM311 ADCs in the screened cell lines 50 value

[0407]

[0408] Note: Figure 7 middle:

[0409] Sample 1: CM311-18D10-VH3 / VL2-ADC

[0410] Sample 2: CM311-18D10-VH6 / VL1-ADC

[0411] Sample 3: CM311-18D10-VH6 / VL2-ADC

[0412] Sample 4: CM311-18A9-VH7 / VL2-ADC

[0413] From Table 3 and Figure 7 The results show that different CM311 ADCs showed significant cell killing activity in cell lines with medium and high expression of Claudin18.2, but had no significant cell killing activity in the Claudin18.2-negative BxPC-3 cell line.

[0414] Example 5 In vivo pharmacodynamic study of antibody-drug conjugates

[0415] The anti-tumor activity of CM311 ADC was tested in two gastric cancer PDX models, STO#025 and STO#523, both of which have high Claudin 18 mRNA levels.

[0416] The process of establishing a human gastric cancer nude mouse PDX model is as follows: 3 The tumor tissue was transplanted subcutaneously on the back of BALB / c nude mice. When the tumor volume reached 150-260mm 3The mice were randomly divided into groups, with 5 mice in each group, to ensure uniform tumor volume and body weight among the groups. There were 4 groups in total, including vehicle group, 1mg / kg CM311-ADC-1 group, 3mg / kg CM311-ADC-1 group and 3mg / kg CM311-ADC-2 (non-binding control ADC) group. Among them, CM311-ADC-1 refers to the antibody-drug conjugate CM311-18D10-VH6 / VL1-ADC; compared with CM311-ADC-1, the antibody of CM311-ADC-2 is a human IgG1 isotype control, which has no binding to the target on the surface of tumor cells.

[0417] Data analysis: During the experiment, the tumor volume was measured twice a week. The calculation formula of tumor volume (TV) is: TV = l × w 2 / 2. Where l and w represent the measured length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the measurement results. RTV = V f / V0. V0 is the tumor volume measured at the time of group administration (i.e., Day 0), V f The tumor volume measured on the last day. Relative tumor proliferation rate T / C (%) = (RTV of the drug administration group / RTV of the vehicle group) × 100%. Tumor growth inhibition rate TGI% = (average tumor volume of the vehicle group - average tumor volume of the drug administration group) / average tumor volume of the vehicle group × 100%. When T / C (%) ≤ 40%, and P < 0.05, the test article is considered to have a significant inhibitory effect on tumor growth.

[0418] Experimental results:

[0419] 1) Efficacy study of CM311ADC in human gastric cancer PDX model STO#025 with high expression level of Claudin 18 mRNA

[0420] The experimental results are as follows Figure 8 and Figure 9As shown. After CM311-ADC-1 was administered at a dose of 1 and 3 mg / kg, the relative tumor proliferation rate T / C (%) on Day 28 was 29.86% and 0%, respectively, and the tumor growth inhibition rate TGI% was 70.13% and 100%, respectively. 0 / 5 and 5 / 5 tumors completely regressed, and 2 / 5 and 0 / 5 tumors partially regressed; the T / C (%) of the CM311-ADC-2 (3 mg / kg) group was 67.24%, and the TGI% was 32.76%. The experimental results showed that CM311-ADC-1 (3 mg / kg) and CM311-ADC-1 (1 mg / kg) both had significant activity in inhibiting tumor growth, and CM311-ADC-2 (3 mg / kg) had no significant anti-tumor activity. Tumor-bearing mice had good tolerance to both CM311-ADC-1 and CM311-ADC-2.

[0421] 2) Efficacy study of CM311ADC in human gastric cancer PDX model STO#523 with high expression level of Claudin 18 mRNA

[0422] The experimental results are as follows Figure 10 and Figure 11 As shown. After CM311-ADC-1 was administered at a dose of 1 and 3 mg / kg, the relative tumor proliferation rate T / C (%) on Day 28 was 35.60% and 6.79%, respectively, and the tumor growth inhibition rate TGI% was 64.40% and 93.21%, respectively; the T / C (%) of the CM311-ADC-2 (3 mg / kg) group was 114.81%, and the TGI% was -14.81%. The experimental results showed that both CM311-ADC-1 (3 mg / kg) and CM311-ADC-1 (1 mg / kg) had significant activity in inhibiting tumor growth, and CM311-ADC-2 (3 mg / kg) had no significant anti-tumor activity. Tumor-bearing mice had good tolerance to both CM311-ADC-1 and CM311-ADC-2.

[0423] Example 6 Comparison of in vitro cell activity of antibodies and antibody drug conjugates

[0424] Test method:

[0425] KATO III cell line was plated at 5000 cells / well, and the test samples were added after 24 hours. The test samples were diluted 2.4 times 9 times with a final concentration of 1000 ng / mL, and the reaction time was 96 hours. The fluorescence value was read using a microplate reader after Presto-Blue color development for 60 minutes.

[0426] The samples to be tested are as follows:

[0427] Sample1: CM311, specifically CM311-18D10-VH6 / VL1;

[0428] Sample2: Anti-Claudin-18.2-ADC, specifically CM311-18D10-VH6 / VL1-ADC;

[0429] Sample 3: non-binding control ADC, specifically IgG-L1D1 (IgG-L1D1 is an ADC in which the IgG antibody is coupled to the L1D1 small molecule (i.e. vcMMAE, also known as MC-vc-PAB-MMAE) drug).

[0430] The test results are as follows Figure 12 and as shown in Table 4. Figure 12 As can be seen, Anti-Claudin-18.2-ADC (such as CM311-18D10-VH6 / VL1-ADC) showed strong cell killing effect, EC 50 The value was 16.37 ng / mL; however, neither CM311 (such as CM311-18D10-VH6 / VL1) nor IgG-L1D1 showed obvious cell killing effect.

[0431] Table 4: Comparison of in vitro cell activity of antibodies and antibody drug conjugates (4-parameter fitting results)

[0432] SEQUENCE LISTING <110> Shanghai Meiyake Biotechnology Co., Ltd. Conoya Biopharmaceutical Technology (Chengdu) Co., Ltd. <120> Antibody-drug conjugates and their applications <130> IDC200026 <160> 200 <170> PatentIn version 3.3 <210> 1 <211> 118 <212> PRT <213> Artificial <220> <223> 18D10VHv1 <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asn 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Asn Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 2 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv1 CDR1 <400> 2 Gly Tyr Ser Ile Thr Ser Asn Tyr Ala Trp Asn 1 5 10 <210> 3 <211> 16 <212> PRT <213> Artificial <220> <223> 18D10VHv1 CDR2 <400> 3 Tyr Ile Asn Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 4 <211> 9 <212> PRT <213> Artificial <220> <223> 18D10VHv1 CDR3 <400> 4 Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr 1 5 <210> 5 <211> 25 <212> PRT <213> Artificial <220> <223> 18D10VHv1 FR1 <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 6 <211> 14 <212> PRT <213> Artificial <220> <223> 18D10VHv1 FR2 <400> 6 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> 7 <211> 32 <212> PRT <213> Artificial <220> <223> 18D10VHv1 FR3 <400> 7 Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Thr 20 25 30 <210> 8 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv1 FR4 <400> 8 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 9 <211> 118 <212> PRT <213> Artificial <220> <223> 18D10VHv2 <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Asn Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 10 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv2 CDR1 <400> 10 Gly Gly Ser Ile Ser Ser Asn Tyr Ala Trp Asn 1 5 10 <210> 11 <211> 16 <212> PRT <213> Artificial <220> <223> 18D10VHv2 CDR2 <400> 11 Tyr Ile Asn Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 12 <211> 9 <212> PRT <213> Artificial <220> <223> 18D10VHv2 CDR3 <400> 12 Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr 1 5 <210> 13 <211> 25 <212> PRT <213> Artificial <220> <223> 18D10VHv2 FR1 <400> 13 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 14 <211> 14 <212> PRT <213> Artificial <220> <223> 18D10VHv2 FR2 <400> 14 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met Gly 1 5 10 <210> 15 <211> 32 <212> PRT <213> Artificial <220> <223> 18D10VHv2 FR3 <400> 15 Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Thr 20 25 30 <210> 16 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv2 FR4 <400> 16 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 17 <211> 118 <212> PRT <213> Artificial <220> <223> 18D10VHv3 <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Asn Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 18 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv3 CDR1 <400> 18 Gly Gly Ser Ile Ser Ser Asn Tyr Ala Trp Asn 1 5 10 <210> 19 <211> 16 <212> PRT <213> Artificial <220> <223> 18D10VHv3 CDR2 <400> 19 Tyr Ile Asn Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 20 <211> 9 <212> PRT <213> Artificial <220> <223> 18D10VHv3 CDR3 <400> 20 Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr 1 5 <210> 21 <211> 25 <212> PRT <213> Artificial <220> <223> 18D10VHv3 FR1 <400> 21 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 22 <211> 14 <212> PRT <213> Artificial <220> <223> 18D10VHv3 FR2 <400> 22 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> 23 <211> 32 <212> PRT <213> Artificial <220> <223> 18D10VHv3 FR3 <400> 23 Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Thr 20 25 30 <210> 24 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv3 FR4 <400> 24 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 25 <211> 118 <212> PRT <213> Artificial <220> <223> 18D10VHv4 <400> 25 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Asn Tyr Ser Gly Tyr Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 26 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv4 CDR1 <400> 26 Gly Gly Ser Ile Ser Ser Asn Tyr Ala Trp Asn 1 5 10 <210> 27 <211> 16 <212> PRT <213> Artificial <220> <223> 18D10VHv4 CDR2 <400> 27 Tyr Ile Asn Tyr Ser Gly Tyr Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 28 <211> 9 <212> PRT <213> Artificial <220> <223> 18D10VHv4 CDR3 <400> 28 Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr 1 5 <210> 29 <211> 25 <212> PRT <213> Artificial <220> <223> 18D10VHv4 FR1 <400> 29 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 30 <211> 14 <212> PRT <213> Artificial <220> <223> 18D10VHv4 FR2 <400> 30 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> 31 <211> 32 <212> PRT <213> Artificial <220> <223> 18D10VHv4 FR3 <400> 31 Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Thr 20 25 30 <210> 32 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv4 FR4 <400> 32 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 33 <211> 118 <212> PRT <213> Artificial <220> <223> 18D10VHv5 <400> 33 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Asn Tyr Ser Gly Asn Thr Ala Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 34 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv5 CDR1 <400> 34 Gly Gly Ser Ile Ser Ser Asn Tyr Ala Trp Asn 1 5 10 <210> 35 <211> 16 <212> PRT <213> Artificial <220> <223> 18D10VHv5 CDR2 <400> 35 Tyr Ile Asn Tyr Ser Gly Asn Thr Ala Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 36 <211> 9 <212> PRT <213> Artificial <220> <223> 18D10VHv5 CDR3 <400> 36 Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr 1 5 <210> 37 <211> 25 <212> PRT <213> Artificial <220> <223> 18D10VHv5 FR1 <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 38 <211> 14 <212> PRT <213> Artificial <220> <223> 18D10VHv5 FR2 <400> 38 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> 39 <211> 32 <212> PRT <213> Artificial <220> <223> 18D10VHv5 FR3 <400> 39 Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Thr 20 25 30 <210> 40 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv5 FR4 <400> 40 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 41 <211> 118 <212> PRT <213> Artificial <220> <223> 18D10VHv6 <400> 41 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 42 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv6 CDR1 <400> 42 Gly Gly Ser Ile Ser Ser Asn Tyr Ala Trp Asn 1 5 10 <210> 43 <211> 16 <212> PRT <213> Artificial <220> <223> 18D10VHv6 CDR2 <400> 43 Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 44 <211> 9 <212> PRT <213> Artificial <220> <223> 18D10VHv6 CDR3 <400> 44 Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr 1 5 <210> 45 <211> 25 <212> PRT <213> Artificial <220> <223> 18D10VHv6 FR1 <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser 20 25 <210> 46 <211> 14 <212> PRT <213> Artificial <220> <223> 18D10VHv6 FR2 <400> 46 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> 47 <211> 32 <212> PRT <213> Artificial <220> <223> 18D10VHv6 FR3 <400> 47 Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Thr 20 25 30 <210> 48 <211> 11 <212> PRT <213> Artificial <220> <223> 18D10VHv6 FR4 <400> 48 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 49 <211> 113 <212> PRT <213> Artificial <220> <223> 18D10VLv1 <400> 49 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 50 <211> 17 <212> PRT <213> Artificial <220> <223> 18D10VLv1 CDR1 <400> 50 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 51 <211> 7 <212> PRT <213> Artificial <220> <223> 18D10VLv1 CDR2 <400> 51 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 52 <211> 9 <212> PRT <213> Artificial <220> <223> 18D10VLv1 CDR3 <400> 52 Gln Asn Ala Tyr Ser Phe Pro Trp Thr 1 5 <210> 53 <211> 23 <212> PRT <213> Artificial <220> <223> 18D10VLv1 FR1 <400> 53 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys 20 <210> 54 <211> 15 <212> PRT <213> Artificial <220> <223> 18D10VLv1 FR2 <400> 54 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 55 <211> 32 <212> PRT <213> Artificial <220> <223> 18D10VLv1 FR3 <400> 55 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 20 25 30 <210> 56 <211> 10 <212> PRT <213> Artificial <220> <223> 18D10VLv1 FR4 <400> 56 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 57 <211> 113 <212> PRT <213> Artificial <220> <223> 18D10VLv2 <400> 57 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Ala Tyr Ser Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 58 <211> 17 <212> PRT <213> Artificial <220> <223> 18D10VLv2 CDR1 <400> 58 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 59 <211> 7 <212> PRT <213> Artificial <220> <223> 18D10VLv2 CDR2 <400> 59 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 60 <211> 9 <212> PRT <213> Artificial <220> <223> 18D10VLv2 CDR3 <400> 60 Gln Gln Ala Tyr Ser Phe Pro Trp Thr 1 5 <210> 61 <211> 23 <212> PRT <213> Artificial <220> <223> 18D10VLv2 FR1 <400> 61 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys 20 <210> 62 <211> 15 <212> PRT <213> Artificial <220> <223> 18D10VLv2 FR2 <400> 62 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 63 <211> 32 <212> PRT <213> Artificial <220> <223> 18D10VLv2 FR3 <400> 63 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 20 25 30 <210> 64 <211> 10 <212> PRT <213> Artificial <220> <223> 18D10VLv2 FR4 <400> 64 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 65 <211> 448 <212> PRT <213> Artificial <220> <223> Heavy chain <400> 65 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asn 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 66 <211> 220 <212> PRT <213> Artificial <220> <223> Light chain <400> 66 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Phe Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 220 <210> 67 <211> 118 <212> PRT <213> Artificial <220> <223> 18A9VHv1 <400> 67 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Met Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Val Arg Gly Asn Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 68 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VHv1 CDR1 <400> 68 Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Asp 1 5 10 <210> 69 <211> 16 <212> PRT <213> Artificial <220> <223> 18A9VHv1 CDR2 <400> 69 Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe Gln 1 5 10 15 <210> 70 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VHv1 CDR3 <400> 70 Arg Val Arg Gly Asn Ser Phe Asp Ser 1 5 <210> 71 <211> 25 <212> PRT <213> Artificial <220> <223> 18A9VHv1 FR1 <400> 71 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser 20 25 <210> 72 <211> 14 <212> PRT <213> Artificial <220> <223> 18A9VHv1 FR2 <400> 72 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 73 <211> 33 <212> PRT <213> Artificial <220> <223> 18A9VHv1 FR3 <400> 73 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Met Thr Ala Tyr Met 1 5 10 15 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala 20 25 30 Arg <210> 74 <211> 11 <212> PRT <213> Artificial <220> <223> 18A9VHv1 FR4 <400> 74 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 75 <211> 118 <212> PRT <213> Artificial <220> <223> 18A9VHv2 <400> 75 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Leu Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Val Arg Gly Asn Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 76 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VHv2 CDR1 <400> 76 Gly Tyr Thr Phe Thr Asn Tyr Leu Ile Asp 1 5 10 <210> 77 <211> 16 <212> PRT <213> Artificial <220> <223> 18A9VHv2 CDR2 <400> 77 Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe Gln 1 5 10 15 <210> 78 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VHv2 CDR3 <400> 78 Arg Val Arg Gly Asn Ser Phe Asp Ser 1 5 <210> 79 <211> 25 <212> PRT <213> Artificial <220> <223> 18A9VHv2 FR1 <400> 79 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser 20 25 <210> 80 <211> 14 <212> PRT <213> Artificial <220> <223> 18A9VHv2 FR2 <400> 80 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 81 <211> 33 <212> PRT <213> Artificial <220> <223> 18A9VHv2 FR3 <400> 81 Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met 1 5 10 15 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala 20 25 30 Arg <210> 82 <211> 11 <212> PRT <213> Artificial <220> <223> 18A9VHv2 FR4 <400> 82 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 83 <211> 118 <212> PRT <213> Artificial <220> <223> 18A9VHv3 <400> 83 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Leu Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Met Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Val Arg Gly Asn Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 84 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VHv3 CDR1 <400> 84 Gly Tyr Thr Phe Thr Asn Tyr Leu Ile Asp 1 5 10 <210> 85 <211> 16 <212> PRT <213> Artificial <220> <223> 18A9VHv3 CDR2 <400> 85 Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe Gln 1 5 10 15 <210> 86 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VHv3 CDR3 <400> 86 Arg Val Arg Gly Asn Ser Phe Asp Ser 1 5 <210> 87 <211> 25 <212> PRT <213> Artificial <220> <223> 18A9VHv3 FR1 <400> 87 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser 20 25 <210> 88 <211> 14 <212> PRT <213> Artificial <220> <223> 18A9VHv3 FR2 <400> 88 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 89 <211> 33 <212> PRT <213> Artificial <220> <223> 18A9VHv3 FR3 <400> 89 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Met Thr Ala Tyr Met 1 5 10 15 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 20 25 30 Arg <210> 90 <211> 11 <212> PRT <213> Artificial <220> <223> 18A9VHv3 FR4 <400> 90 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 91 <211> 118 <212> PRT <213> Artificial <220> <223> 18A9VHv4 <400> 91 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Leu Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Met Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Val Arg Gly Asn Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 92 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VHv4 CDR1 <400> 92 Gly Tyr Thr Phe Thr Asn Tyr Leu Ile Asp 1 5 10 <210> 93 <211> 16 <212> PRT <213> Artificial <220> <223> 18A9VHv4 CDR2 <400> 93 Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe Gln 1 5 10 15 <210> 94 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VHv4 CDR3 <400> 94 Arg Val Arg Gly Asn Ser Phe Asp Ser 1 5 <210> 95 <211> 25 <212> PRT <213> Artificial <220> <223> 18A9VHv4 FR1 <400> 95 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 96 <211> 14 <212> PRT <213> Artificial <220> <223> 18A9VHv4 FR2 <400> 96 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 97 <211> 33 <212> PRT <213> Artificial <220> <223> 18A9VHv4 FR3 <400> 97 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Met Thr Ala Tyr Met 1 5 10 15 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala 20 25 30 Arg <210> 98 <211> 11 <212> PRT <213> Artificial <220> <223> 18A9VHv4 FR4 <400> 98 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 99 <211> 118 <212> PRT <213> Artificial <220> <223> 18A9VHv5 <400> 99 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Leu Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Val Arg Gly Asn Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 100 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VHv5 CDR1 <400> 100 Gly Tyr Thr Phe Thr Asn Tyr Leu Ile Asp 1 5 10 <210> 101 <211> 16 <212> PRT <213> Artificial <220> <223> 18A9VHv5 CDR2 <400> 101 Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe Gln 1 5 10 15 <210> 102 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VHv5 CDR3 <400> 102 Arg Val Arg Gly Asn Ser Phe Asp Ser 1 5 <210> 103 <211> 25 <212> PRT <213> Artificial <220> <223> 18A9VHv5 FR1 <400> 103 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser 20 25 <210> 104 <211> 14 <212> PRT <213> Artificial <220> <223> 18A9VHv5 FR2 <400> 104 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 105 <211> 33 <212> PRT <213> Artificial <220> <223> 18A9VHv5 FR3 <400> 105 Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met 1 5 10 15 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala 20 25 30 Arg <210> 106 <211> 11 <212> PRT <213> Artificial <220> <223> 18A9VHv5 FR4 <400> 106 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 107 <211> 118 <212> PRT <213> Artificial <220> <223> 18A9VHv6 <400> 107 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Val Arg Gly Asn Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 108 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VHv6 CDR1 <400> 108 Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Asp 1 5 10 <210> 109 <211> 16 <212> PRT <213> Artificial <220> <223> 18A9VHv6 CDR2 <400> 109 Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe Gln 1 5 10 15 <210> 110 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VHv6 CDR3 <400> 110 Arg Val Arg Gly Asn Ser Phe Asp Ser 1 5 <210> 111 <211> 25 <212> PRT <213> Artificial <220> <223> 18A9VHv6 FR1 <400> 111 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser 20 25 <210> 112 <211> 14 <212> PRT <213> Artificial <220> <223> 18A9VHv6 FR2 <400> 112 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 113 <211> 33 <212> PRT <213> Artificial <220> <223> 18A9VHv6 FR3 <400> 113 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met 1 5 10 15 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala 20 25 30 Arg <210> 114 <211> 11 <212> PRT <213> Artificial <220> <223> 18A9VHv6 FR4 <400> 114 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 115 <211> 118 <212> PRT <213> Artificial <220> <223> 18A9VHv7 <400> 115 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Leu Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Val Arg Gly Asn Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 116 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VHv7 CDR1 <400> 116 Gly Tyr Thr Phe Thr Asn Tyr Leu Ile Asp 1 5 10 <210> 117 <211> 16 <212> PRT <213> Artificial <220> <223> 18A9VHv7 CDR2 <400> 117 Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe Gln 1 5 10 15 <210> 118 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VHv7 CDR3 <400> 118 Arg Val Arg Gly Asn Ser Phe Asp Ser 1 5 <210> 119 <211> 25 <212> PRT <213> Artificial <220> <223> 18A9VHv7 FR1 <400> 119 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser 20 25 <210> 120 <211> 14 <212> PRT <213> Artificial <220> <223> 18A9VHv7 FR2 <400> 120 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 121 <211> 33 <212> PRT <213> Artificial <220> <223> 18A9VHv7 FR3 <400> 121 Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr Met 1 5 10 15 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala 20 25 30 Arg <210> 122 <211> 11 <212> PRT <213> Artificial <220> <223> 18A9VHv7 FR4 <400> 122 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 123 <211> 113 <212> PRT <213> Artificial <220> <223> 18A9VLv1 <400> 123 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Lys Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asn Tyr Phe Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 124 <211> 17 <212> PRT <213> Artificial <220> <223> 18A9VLv1 CDR1 <400> 124 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 125 <211> 7 <212> PRT <213> Artificial <220> <223> 18A9VLv1 CDR2 <400> 125 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 126 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VLv1 CDR3 <400> 126 Gln Asn Asn Tyr Phe Tyr Pro Leu Thr 1 5 <210> 127 <211> 23 <212> PRT <213> Artificial <220> <223> 18A9VLv1 FR1 <400> 127 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys 20 <210> 128 <211> 15 <212> PRT <213> Artificial <220> <223> 18A9VLv1 FR2 <400> 128 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 129 <211> 32 <212> PRT <213> Artificial <220> <223> 18A9VLv1 FR3 <400> 129 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Lys Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 20 25 30 <210> 130 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VLv1 FR4 <400> 130 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 131 <211> 113 <212> PRT <213> Artificial <220> <223> 18A9VLv2 <400> 131 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Asn Tyr Phe Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 132 <211> 17 <212> PRT <213> Artificial <220> <223> 18A9VLv2 CDR1 <400> 132 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Ala <210> 133 <211> 7 <212> PRT <213> Artificial <220> <223> 18A9VLv2 CDR2 <400> 133 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 134 <211> 9 <212> PRT <213> Artificial <220> <223> 18A9VLv2 CDR3 <400> 134 Gln Gln Asn Tyr Phe Tyr Pro Leu Thr 1 5 <210> 135 <211> 23 <212> PRT <213> Artificial <220> <223> 18A9VLv2 FR1 <400> 135 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys 20 <210> 136 <211> 15 <212> PRT <213> Artificial <220> <223> 18A9VLv2 FR2 <400> 136 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 137 <211> 32 <212> PRT <213> Artificial <220> <223> 18A9VLv2 FR3 <400> 137 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 20 25 30 <210> 138 <211> 10 <212> PRT <213> Artificial <220> <223> 18A9VLv2 FR4 <400> 138 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 139 <211> 118 <212> PRT <213> Artificial <220> <223> Clone 18D10 heavy chain <400> 139 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asn 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Asn Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Thr Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 140 <211> 11 <212> PRT <213> Artificial <220> <223> Clone 18D10 Heavy chain CDR1 <400> 140 Gly Tyr Ser Ile Thr Ser Asn Tyr Ala Trp Asn 1 5 10 <210> 141 <211> 16 <212> PRT <213> Artificial <220> <223> Clone 18D10 Heavy chain CDR2 <400> 141 Tyr Ile Asn Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 142 <211> 9 <212> PRT <213> Artificial <220> <223> Clone 18D10 Heavy chain CDR3 <400> 142 Ser Tyr Tyr Gly Asn Ser Phe Ile Tyr 1 5 <210> 143 <211> 25 <212> PRT <213> Artificial <220> <223> Clone 18D10 Heavy chain FR1 <400> 143 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr 20 25 <210> 144 <211> 14 <212> PRT <213> Artificial <220> <223> Clone 18D10 Heavy chain FR2 <400> 144 Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly 1 5 10 <210> 145 <211> 32 <212> PRT <213> Artificial <220> <223> Clone 18D10 Heavy chain FR3 <400> 145 Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln 1 5 10 15 Leu Asn Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Thr 20 25 30 <210> 146 <211> 11 <212> PRT <213> Artificial <220> <223> Cloned 18D10 heavy chain FR4 <400> 146 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10 <210> 147 <211> 113 <212> PRT <213> Artificial <220> <223> Cloned 18D10 light chain <400> 147 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Ala Tyr Ser Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 148 <211> 17 <212> PRT <213> Artificial <220> <223> Clone 18D10 Light chain CDR1 <400> 148 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 149 <211> 7 <212> PRT <213> Artificial <220> <223> Clone 18D10 Light chain CDR2 <400> 149 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 150 <211> 9 <212> PRT <213> Artificial <220> <223> Clone 18D10 Light chain CDR3 <400> 150 Gln Asn Ala Tyr Ser Phe Pro Trp Thr 1 5 <210> 151 <211> twenty three <212> PRT <213> Artificial <220> <223> Clone 18D10 Light chain FR1 <400> 151 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys 20 <210> 152 <211> 15 <212> PRT <213> Artificial <220> <223> Clone 18D10 Light chain FR2 <400> 152 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 153 <211> 32 <212> PRT <213> Artificial <220> <223> Clone 18D10 Light chain FR3 <400> 153 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys 20 25 30 <210> 154 <211> 10 <212> PRT <213> Artificial <220> <223> Clone 18D10 Light chain FR4 <400> 154 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 155 <211> 118 <212> PRT <213> Artificial <220> <223> Clone 18A9 heavy chain <400> 155 Gln Val Gln Leu Gln Gln Ser Gly Arg Glu Val Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Leu Ile Asp Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Met Thr Ala Asn 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Val Arg Gly Asn Ser Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 156 <211> 10 <212> PRT <213> Artificial <220> <223> Clone 18A9 Heavy chain CDR1 <400> 156 Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Asp 1 5 10 <210> 157 <211> 16 <212> PRT <213> Artificial <220> <223> Clone 18A9 Heavy chain CDR2 <400> 157 Gly Ile Asn Pro Gly Ser Gly Asp Thr Val Tyr Asn Glu Lys Phe Lys 1 5 10 15 <210> 158 <211> 9 <212> PRT <213> Artificial <220> <223> Clone 18A9 Heavy chain CDR3 <400> 158 Arg Val Arg Gly Asn Ser Phe Asp Ser 1 5 <210> 159 <211> 25 <212> PRT <213> Artificial <220> <223> Clone 18A9 Heavy chain FR1 <400> 159 Gln Val Gln Leu Gln Gln Ser Gly Arg Glu Val Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Pro Ser 20 25 <210> 160 <211> 14 <212> PRT <213> Artificial <220> <223> Clone 18A9 Heavy chain FR2 <400> 160 Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 <210> 161 <211> 33 <212> PRT <213> Artificial <220> <223> Clone 18A9 Heavy Chain FR3 <400> 161 Ala Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Met Thr Ala Asn Met 1 5 10 15 Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys Ala 20 25 30 Arg <210> 162 <211> 11 <212> PRT <213> Artificial <220> <223> Clone 18A9 Heavy Chain FR4 <400> 162 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10 <210> 163 <211> 113 <212> PRT <213> Artificial <220> <223> Clone 18A9 light chain <400> 163 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Lys Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Asn 85 90 95 Asn Tyr Phe Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 164 <211> 17 <212> PRT <213> Artificial <220> <223> Clone 18A9 light chain CDR1 <400> 164 Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu 1 5 10 15 Thr <210> 165 <211> 7 <212> PRT <213> Artificial <220> <223> Clone 18A9 Light chain CDR2 <400> 165 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 166 <211> 9 <212> PRT <213> Artificial <220> <223> Clone 18A9 Light chain CDR3 <400> 166 Gln Asn Asn Tyr Phe Tyr Pro Leu Thr 1 5 <210> 167 <211> twenty three <212> PRT <213> Artificial <220> <223> Clone 18A9 Light Chain FR1 <400> 167 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys 20 <210> 168 <211> 15 <212> PRT <213> Artificial <220> <223> Clone 18A9 Light Chain FR2 <400> 168 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 169 <211> 32 <212> PRT <213> Artificial <220> <223> Clone 18A9 Light Chain FR3 <400> 169 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Lys Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys 20 25 30 <210> 170 <211> 10 <212> PRT <213> Artificial <220> <223> Clone 18A9 Light Chain FR4 <400> 170 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 1 5 10 <210> 171 <211> 37 <212> DNA <213> Artificial <220> <223> Primers <400> 171 ttggcaaaga attgctagat gtccaccacc acatgcc 37 <210> 172 <211> 38 <212> DNA <213> Artificial <220> <223> Primers <400> 172 tgttcgggcc ctcctcgatt acacatagtc gtgcttgg 38 <210> 173 <211> 37 <212> DNA <213> Artificial <220> <223> Primers <400> 173 ttggcaaaga attgctagat ggccgtgact gcctgtc 37 <210> 174 <211> 38 <212> DNA <213> Artificial <220> <223> Primers <400> 174 tgttcgggcc ctcctcgatt acacatagtc gtgcttgg 38 <210> 175 <211> 20 <212> DNA <213> Artificial <220> <223> Primers <400> 175 tgtgcgccac catggccgtg 20 <210> 176 <211> 20 <212> DNA <213> Artificial <220> <223> Primers <400> 176 tggaaggata agattgtacc 20 <210> 177 <211> 20 <212> DNA <213> Artificial <220> <223> Primers <400> 177 tgggtgccat tggcctcctg 20 <210> 178 <211> 354 <212> DNA <213> Artificial <220> <223> Clone 18D10 heavy chain <400> 178 gatgtgcagc ttcaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ctcaatcacc agtaattatg cctggaactg gatccgacag 120 tttccaggaa acaaactaga gtggatgggc tacataaact acagtgggaa cactaactat 180 aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240 ctgcagttga attctgtgac tgctgaggac acagccacat attattgtgc aacctcctat 300 tatggtaatt cctttattta ctggggccaa gggactctgg tcactgtctc tgca 354 <210> 179 <211> 339 <212> DNA <213> Artificial <220> <223> Clone 18D10 light chain <400> 179 gacattgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120 tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 180 gagtctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatgc ttatagtttt 300 ccgtggacgt tcggtggagg caccaagctg gaaatcaaa 339 <210> 180 <211> 354 <212> DNA <213> Artificial <220> <223> Clone 18A9 heavy chain <400> 180 caggtgcagc tgcagcagtc tggacgtgag gtggtaaggc ctgggacttc agtgaaggtg 60 tcctgcaagc cttctggata cgccttcact aattacttga tagactgggt aaaacagagg 120 cctggacagg gccttgagtg gattggaggg attaatcctg gaagtggtga cactgtgtac 180 aatgagaagt tcaaggccaa ggcaacactg actgcagaca aatcctccat gactgccaac 240 atgcagctca gcagcctgac atctgatgac tctgcggtct atttctgcgc aagaagggtc 300 cgtggtaatt cgtttgattc ctggggccaa gggactctgg tcactgtctc tgca 354 <210> 181 <211> 339 <212> DNA <213> Artificial <220> <223> Clone 18A9 light chain <400> 181 gacattgtga tgtcacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60 atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120 tggtaccagc agaaaccagg gcagcctcct aaattgttga tctactgggc atccactagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg gaaaagattt cactctcacc 240 atcagcagtg tgcaggctga agacctggca ctttattact gtcagaataa ttatttttat 300 ccgctcacgt tcggtgctgg gaccaagctg gagctgaaa 339 <210> 182 <211> 354 <212> DNA <213> Artificial <220> <223> 18D10VHv1 <400> 182 caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60 acctgtaccg tgtccggcta cagcatcacc agcaactacg cctggaactg gatcagacag 120 cctcctggca aaggcctcga gtggatcggc tacatcaact acagcggcaa caccaactac 180 aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240 ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cacaagctac 300 tacggcaaca gcttcatcta ctggggccag ggcacactgg tcaccgtttc ttct 354 <210> 183 <211> 354 <212> DNA <213> Artificial <220> <223> 18D10VHv2 <400> 183 caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60 acctgtaccg tgtccggcgg cagcatcagc agcaactacg cctggaactg gatcagacag 120 cctcctggca aaggcctcga gtggatgggc tacatcaact acagcggcaa caccaactac 180 aaccccagcc tgaagtccag aatcaccatc agcagagaca ccagcaagaa ccagttctcc 240 ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cacaagctac 300 tacggcaaca gcttcatcta ctggggccag ggcacactgg tcaccgtttc ttct 354 <210> 184 <211> 354 <212> DNA <213> Artificial <220> <223> 18D10VHv3 <400> 184 caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60 acctgtaccg tgtccggcgg cagcatcagc agcaactacg cctggaactg gatcagacag 120 cctcctggca aaggcctcga gtggatcggc tacatcaact acagcggcaa caccaactac 180 aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240 ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cacaagctac 300 tacggcaaca gcttcatcta ctggggccag ggcacactgg tcaccgtttc ttct 354 <210> 185 <211> 354 <212> DNA <213> Artificial <220> <223> 18D10VHv4 <400> 185 caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60 acctgtaccg tgtccggcgg cagcatcagc agcaactacg cctggaactg gatcagacag 120 cctcctggca aaggcctcga gtggatcggc tacatcaact acagcggcta caccaactac 180 aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240 ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cacaagctac 300 tacggcaaca gcttcatcta ctggggccag ggcacactgg tcaccgtttc ttct 354 <210> 186 <211> 354 <212> DNA <213> Artificial <220> <223> 18D10VHv5 <400> 186 caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60 acctgtaccg tgtccggcgg cagcatcagc agcaactacg cctggaactg gatcagacag 120 cctcctggca aaggcctcga gtggatcggc tacatcaact acagcggcaa caccgcctac 180 aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240 ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cacaagctac 300 tacggcaaca gcttcatcta ctggggccag ggcacactgg tcaccgtttc ttct 354 <210> 187 <211> 354 <212> DNA <213> Artificial <220> <223> 18D10VHv6 <400> 187 caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60 acctgtaccg tgtccggcgg cagcatcagc agcaactacg cctggaactg gatcagacag 120 cctcctggca aaggcctcga gtggatcggc tacatctact acagcggcaa caccaactac 180 aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240 ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cacaagctac 300 tacggcaaca gcttcatcta ctggggccag ggcacactgg tcaccgtttc ttct 354 <210> 188 <211> 339 <212> DNA <213> Artificial <220> <223> 18D10VLv1 <400> 188 gacatcgtga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60 atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctgacc 120 tggtatcagc agaagcccgg ccagcctcct aagctgctga tctactgggc cagcaccaga 180 gaaagcggcg tgccagatag attcagcggc agcggctctg gaaccgactt caccctgaca 240 atcagctccc tgcaggccga ggatgtggcc gtgtactact gtcagaacgc ctacagcttc 300 ccctggacat tcggccaggg caccaaggtg gaaatcaag 339 <210> 189 <211> 339 <212> DNA <213> Artificial <220> <223> 18D10VLv2 <400> 189 gacatcgtga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60 atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctggcc 120 tggtatcagc agaagcccgg ccagcctcct aagctgctga tctactgggc cagcaccaga 180 gaaagcggcg tgccagatag attcagcggc agcggctctg gaaccgactt caccctgaca 240 atcagctccc tgcaggccga ggatgtggcc gtgtactact gtcagcaggc ctacagcttc 300 ccctggacat tcggccaggg caccaaggtg gaaatcaag 339 <210> 190 <211> 1347 <212> DNA <213> Artificial <220> <223> Heavy chain nucleic acid sequence <400> 190 caggttcagc tgcaagagtc tggacctggc ctggtcaagc ctagcgagac actgagcctg 60 acctgtaccg tgtccggcgg cagcatcagc agcaactacg cctggaactg gatcagacag 120 cctcctggca aaggcctcga gtggatcggc tacatctact acagcggcaa caccaactac 180 aaccccagcc tgaagtccag agtgaccatc agcagagaca ccagcaagaa ccagttctcc 240 ctgaagctga gcagcgtgac agccgccgat acagccgtgt actactgtgc cacaagctac 300 tacggcaaca gcttcatcta ctggggccag ggcacactgg tcaccgtttc ttctgctagc 360 accaagggcc catccgtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga gcccaaatct 660 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 720 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 780 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 900 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 960 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1020 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 1080 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200 1260 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320 tccctctccc tgtctccggg taaatga 1347 <210> 191 <211> 663 <212> DNA <213> Artificial <220> <223> Light chain nucleic acid sequence <400> 191 gacatcgtga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60 atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctgacc 120 tggtatcagc agaagcccgg ccagcctcct aagctgctga tctactgggc cagcaccaga 180 gaaagcggcg tgccagatag attcagcggc agcggctctg gaaccgactt caccctgaca 240 atcagctccc tgcaggccga ggatgtggcc gtgtactact gtcagaacgc ctacagcttc 300 ccctggacat tcggccaggg caccaaggtg gaaatcaagc gaactgtggc tgcaccatct 360 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660 tag 663 <210> 192 <211> 354 <212> DNA <213> Artificial <220> <223> 18A9VHv1 <400> 192 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagc cttctggcta cgccttcacc aactacctga tcgactgggt ccgacaggct 120 cctggacagg gacttgaatg gatgggcggc atcaaccctg gcagcggcga tacagtgtac 180 aacgagaagt tccagggcag agtgaccctg accgccgaca agtctagcat gaccgcctac 240 atggaactga gcagcctgag aagcgaggat accgccgtgt acttctgtgc cagaagagtg 300 cggggcaaca gcttcgattc ttggggccag ggaaccctgg tcaccgtttc ttct 354 <210> 193 <211> 354 <212> DNA <213> Artificial <220> <223> 18A9VHv2 <400> 193 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagc cttctggcta caccttcacc aactacctga tcgactgggt ccgacaggct 120 cctggacagg gacttgaatg gatgggcggc atcaaccctg gcagcggcga tacagtgtac 180 aacgagaagt tccagggcag agtgaccctg accgccgacg agtctaccag caccgcctac 240 atggaactga gcagcctgag aagcgaggat accgccgtgt acttctgtgc cagaagagtg 300 cggggcaaca gcttcgattc ttggggccag ggaaccctgg tcaccgtttc ttct 354 <210> 194 <211> 354 <212> DNA <213> Artificial <220> <223> 18A9VHv3 <400> 194 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagc cttctggcta caccttcacc aactacctga tcgactgggt ccgacaggct 120 cctggacagg gacttgaatg gatgggcggc atcaaccctg gcagcggcga tacagtgtac 180 aacgagaagt tccagggcag agtgaccctg accgccgaca agtctagcat gaccgcctac 240 atggaactga gcagcctgag aagcgaggat accgccgtgt actactgtgc cagaagagtg 300 cggggcaaca gcttcgattc ttggggccag ggaaccctgg tcaccgtttc ttct 354 <210> 195 <211> 354 <212> DNA <213> Artificial <220> <223> 18A9VHv4 <400> 195 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg cttctggcta caccttcacc aactacctga tcgactgggt ccgacaggct 120 cctggacagg gacttgaatg gatgggcggc atcaaccctg gcagcggcga tacagtgtac 180 aacgagaagt tccagggcag agtgaccctg accgccgaca agtctagcat gaccgcctac 240 atggaactga gcagcctgag aagcgaggat accgccgtgt acttctgtgc cagaagagtg 300 cggggcaaca gcttcgattc ttggggccag ggaaccctgg tcaccgtttc ttct 354 <210> 196 <211> 354 <212> DNA <213> Artificial <220> <223> 18A9VHv5 <400> 196 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagc cttctggcta caccttcacc aactacctga tcgactgggt ccgacaggct 120 cctggacagg gacttgaatg gatgggcggc atcaaccctg gcagcggcga tacagtgtac 180 aacgagaagt tccagggcag agtgaccatc accgccgacg agtctaccag caccgcctac 240 atggaactga gcagcctgag aagcgaggat accgccgtgt acttctgtgc cagaagagtg 300 cggggcaaca gcttcgattc ttggggccag ggaaccctgg tcaccgtttc ttct 354 <210> 197 <211> 354 <212> DNA <213> Artificial <220> <223> 18A9VHv6 <400> 197 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgccag cgtgaaggtg 60 tcctgcaagc cttctggcta cgccttcacc aactacctga tcgactgggt ccgacaggct 120 cctggacagg gacttgaatg gatgggcggc atcaaccctg gcagcggcga tacagtgtac 180 aacgagaagt tccagggcag agtgaccctg accgccgaca agtctagcag caccgcctac 240 atggaactga gcagcctgag aagcgaggat accgccgtgt acttctgtgc cagaagagtg 300 cggggcaaca gcttcgattc ttggggccag ggaaccctgg tcaccgtttc ttct 354 <210> 198 <211> 354 <212> DNA <213> Artificial <220> <223> 18A9VHv7 <400> 198 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcgccag cgtgaaggtg 60 tcctgcaagc cttctggcta caccttcacc aactacctga tcgactgggt ccgacaggct 120 cctggacagg gacttgaatg gatgggcggc atcaaccctg gcagcggcga tacagtgtac 180 aacgagaagt tccagggcag agtgaccctg accgccgaca cctctaccag caccgtctac 240 atggaactga gcagcctgag aagcgaggat accgccgtgt acttctgtgc cagaagagtg 300 cggggcaaca gcttcgattc ttggggccag ggaaccctgg tcaccgtttc ttct 354 <210> 199 <211> 339 <212> DNA <213> Artificial <220> <223> 18A9VLv1 <400> 199 gacatcgtga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60 atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctgacc 120 tggtatcagc agaagcccgg ccagcctcct aagctgctga tctactgggc cagcaccaga 180 gaaagcggcg tgccagatag attcagcggc agcggctctg gaaaggactt caccctgaca 240 atcagctccc tgcaggccga ggatgtggcc gtgtactact gccagaacaa ctacttctac 300 cctctgacct tcggcggagg caccaaggtg gaaatcaag 339 <210> 200 <211> 339 <212> DNA <213> Artificial <220> <223> 18A9VLv2 <400> 200 gacatcgtga tgacacagag ccctgatagc ctggccgtgt ctctgggaga gagagccacc 60 atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctggcc 120 tggtatcagc agaagcccgg ccagcctcct aagctgctga tctactgggc cagcaccaga 180 gaaagcggcg tgccagatag attcagcggc agcggctctg gaaccgactt caccctgaca 240 atcagctccc tgcaggccga ggatgtggcc gtgtactact gccagcagaa ctacttctac 300 cctctgacct tcggcggagg caccaaggtg gaaatcaag 339

Claims

1. An antibody drug conjugate, a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: The antibody-drug conjugate has a structure as shown in Formula I, Ab-(LD) p Formula I in: Ab includes: Heavy chain variable region CDR1, CDR2, CDR3 and light chain variable region CDR1, CDR2, CDR3, wherein the sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO:42, the sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO:43, the sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO:44, the sequence of the light chain variable region CDR1 is shown in SEQ ID NO:50, the sequence of the light chain variable region CDR2 is shown in SEQ ID NO:51, and the sequence of the light chain variable region CDR3 is shown in SEQ ID NO:52; L is MC-vc-PAB; D is MMAE; and p is 2.0-8.

0.

2. The antibody drug conjugate of claim 1, a pharmaceutically acceptable salt, a solvate or a solvate of the salt thereof, wherein: Ab comprises: a heavy chain variable region and a light chain variable region, wherein the sequence of the heavy chain variable region is shown in SEQ ID NO: 41, and the sequence of the light chain variable region is shown in SEQ ID NO:

49.

3. The antibody drug conjugate of claim 1, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, wherein: Ab comprises: a heavy chain and a light chain, wherein the sequence of the heavy chain is shown in SEQ ID NO: 65, and the sequence of the light chain is shown in SEQ ID NO:

66.

4. The antibody drug conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: p is 2.0-6.

0.

5. The antibody drug conjugate of claim 4, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: p is 3.0-5.

0.

6. The antibody drug conjugate of claim 5, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: p is 3.0-4.

0.

7. The antibody drug conjugate of claim 6, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, wherein: p is 3.0-3.

8.

8. The antibody drug conjugate of claim 7, or a pharmaceutically acceptable salt, solvate, or solvate of the salt thereof, wherein: p is 3.0, 3.4, 3.5 or 3.

8.

9. The antibody drug conjugate of claim 8, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein: p is 3.

8.

10. A composition comprising the antibody drug conjugate according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, solvate or solvate of the salt thereof.

11. The composition of claim 10, further comprising: At least one of known chemotherapeutic drugs, immunotherapeutic drugs and immunosuppressants for treating tumors; and / or at least one pharmaceutically acceptable carrier, diluent or excipient.

12. Use of the antibody drug conjugate according to any one of claims 1 to 9, its pharmaceutically acceptable salt, solvate or solvate of the salt, or the composition according to claim 10 or 11 in the preparation of a medicament for preventing and / or treating gastric cancer.

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