Conjugated antibody-drugs and their uses.

Abstract

DEPCT6627 / 06 / 2566 This invention relates to antibody-drug conjugates and their uses, and specifically. Provide the conjugated antibody-drug, its pharmaceutically acceptable salt and solvate, or the solvate of... The conjugated antibody-drug salt has a structure represented by the formula I, where Ab is the anti-clodine. 18.2. The conjugated antibody-drug of this invention has an inhibitory activity on cell growth. Beneficial tumor control (both internal and external), low toxicity, and good potential for use. Ab-(LD)p Formula I;

Description

Antibody-drug conjugates and their applications 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 worldwide, with higher incidence in East Asia, Eastern Europe, and South America, and lower incidence in North America and Africa. Chemotherapy is the standard initial treatment for advanced or recurrent gastric cancer. Although prognosis for gastric cancer patients has improved significantly due to advances in surgical techniques and perioperative care, the 5-year overall survival rate is only 10-15%. Targeted therapies have offered new hope for the treatment of recurrent / advanced gastric cancer. Combining trastuzumab with chemotherapy has shown some benefit in patients with HER2-positive tumors, but only 15% of patients have HER2 expression, limiting the potential for benefit. In recent years, immunotherapy has offered new hope for the treatment of recurrent / advanced gastric cancer; however, according to the results of the KEYNOTE-12 study, only approximately 40% of patients with PD-L1-positive gastric or gastroesophageal junction adenocarcinoma are eligible for this treatment. Therefore, the development of new therapeutic agents for gastric cancer remains urgent.

[0003] Summary of the Invention

[0004] The inventors of the present application prepared an anti-Claudin 18.2 antibody-drug conjugate through extensive experiments and creative work, and confirmed that the conjugate has good biological activity, thereby completing the present invention.

[0005] To this end, in the first aspect of the present invention, the present invention provides an antibody drug conjugate, a pharmaceutically acceptable salt, solvate or solvate of the salt thereof, wherein the antibody drug conjugate has the structure shown in Formula I,

[0006] Ab-(LD) p

[0007] Formula I

[0008] in:

[0009] 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, and 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;

[0010] D is a cytotoxic agent;

[0011] L is a linker used to connect the anti-Claudin 18.2 antibody and the cytotoxic agent;

[0012] 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).

[0013] The antibody-drug conjugate of the present invention exhibits excellent tumor cell growth inhibition activity both in vitro and in vivo, and has promising application prospects. The antibody-drug conjugate of the present invention is composed of an anti-Claudin 18.2 antibody and MMAE, a derivative of Dolastatin, linked via an MC-vc-PAB linker. Its anti-tumor mechanism of action is as follows: After binding to Claudin 18.2 on the surface of tumor cells, the antibody-drug conjugate is internalized and transported to lysosomes. MMAE is then degraded by proteases within the lysosomes, releasing MMAE. After entering the cytoplasm, MMAE binds to tubulin and inhibits its polymerization, thereby blocking various cellular physiological functions involved in tubulin, including mitosis, thereby inhibiting tumor cell proliferation and leading to tumor cell death.

[0014] 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 compositions comprising multiple ADC molecules. In some cases, the multiple ADCs in the composition each contain the same number of drug molecules. In other cases, the multiple ADCs in the composition each contain a different number of drug molecules.

[0015] The drug-antibody ratio (DAR) refers to the number of drug molecules conjugated to the antibody (e.g., p in Formula I). ​​The number of drug molecules contained in the antibody-drug conjugates of the present invention (e.g., p in Formula I) is generally an integer. When the number of drug molecules contained in the antibody-drug conjugates of the present invention (e.g., p in Formula I) is a fraction, the fraction refers to the average number of drug molecules conjugated to each antibody in a composition comprising multiple ADC molecules.

[0016] The drug-antibody ratio (DAR) can be verified by conventional means, such as mass spectrometry, ELISA assays, HIC, and HPLC. The quantitative distribution of ADC in terms of p can also be determined. In some cases, separation, purification, and verification of homogeneous ADCs with a certain p value from ADCs with other drug loads can be achieved by means such as reverse phase HPLC or electrophoresis.

[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: 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.

[0018] 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.

[0019] 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:

[0020] (1) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,

[0021] (2) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,

[0022] (3) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,

[0023] (4) SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,

[0024] (5) SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,

[0025] (6) SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44,

[0026] (7) SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,

[0027] (8) SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,

[0028] (9) SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86,

[0029] (10) SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,

[0030] (11) SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,

[0031] (12) SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,

[0032] (13) SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118,

[0033] The light chain variable regions CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0034] (1) SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,

[0035] (2) SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,

[0036] (3) SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126,

[0037] (4) SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134.

[0038] 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:

[0039] (1) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,

[0040] (2) SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,

[0041] (3) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,

[0042] (4) SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,

[0043] (5) SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,

[0044] (6) SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44,

[0045] The light chain variable regions CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0046] (1) SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,

[0047] (2) SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60.

[0048] 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:

[0049] (1) SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,

[0050] (2) SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78,

[0051] (9) SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86,

[0052] (4) SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,

[0053] (5) SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102,

[0054] (6) SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,

[0055] (7) SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118,

[0056] The light chain variable regions CDR1, CDR2, and CDR3 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0057] (1) SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126,

[0058] (2) SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134.

[0059] 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, the heavy chain variable region FR3 comprises a sequence selected from SEQ ID NO: 7, 15, 23, 31, 39, 47, 73, 81, 89, 97, 105, 113 or 121 or a mutant thereof, and the heavy chain variable region FR4 comprises a sequence selected from SEQ ID NO: 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: 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,

[0088] The light chain variable regions FR1, FR2, FR3, and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0089] (1) SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,

[0090] (2) SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64.

[0091] 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:

[0092] (1) SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74,

[0093] (2) SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82,

[0094] (3) SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90,

[0095] (4) SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,

[0096] (5) SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,

[0097] (6) SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114,

[0098] (7) SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122,

[0099] The light chain variable regions FR1, FR2, FR3, and FR4 of the anti-Claudin 18.2 antibody are selected from the following sequence combinations:

[0100] (1) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130,

[0101] (2) SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138.

[0102] 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,

[0103] 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.

[0104] 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,

[0105] 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.

[0106] 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,

[0107] 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.

[0108] 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:

[0109] (1) SEQ ID NO: 17 and SEQ ID NO: 57;

[0110] (2) SEQ ID NO: 41 and SEQ ID NO: 49;

[0111] (3) SEQ ID NO: 41 and SEQ ID NO: 57;

[0112] (4) SEQ ID NO: 115 and SEQ ID NO: 131.

[0113] 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.

[0114] 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, IgA constant region or mutants of the above constant regions, preferably human IgG1;

[0115] 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 mutants of the above constant regions, preferably a human kappa constant region.

[0116] In some embodiments, the amino acid sequence of the heavy chain of the anti-Claudin 18.2 antibody comprises the sequence shown in SEQ ID NO: 65, or comprises a sequence that is greater than 70%, e.g., greater than 75%, 80%, 85%, 90%, 95%, or 99% identical to the sequence shown in SEQ ID NO: 65;

[0117] 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 that is greater than 70% identical to the sequence shown in SEQ ID NO: 66, for example, greater than 75%, 80%, 85%, 90%, 95%, or 99%.

[0118] In some embodiments, p is 3.0-4.0.

[0119] In some embodiments, p is 3.0-3.8.

[0120] In some embodiments, p is 3.0, 3.4, 3.5, or 3.8.

[0121] In some embodiments, p is 3.8.

[0122] 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.

[0123] In some embodiments, the cytotoxic agent is MMAE.

[0124] The structure of MMAE is:

[0125]

[0126] 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).

[0127] In some embodiments, the linker is 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

[0128] In some embodiments, the LD described in Formula I is MC-vc-PAB-MMAE, which has the following structure:

[0129]

[0130] In some embodiments,

[0131] Ab includes:

[0132] (a) 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 set forth in SEQ ID NO:42, the sequence of the heavy chain variable region CDR2 is set forth in SEQ ID NO:43, the sequence of the heavy chain variable region CDR3 is set forth in SEQ ID NO:44, the sequence of the light chain variable region CDR1 is set forth in SEQ ID NO:50, the sequence of the light chain variable region CDR2 is set forth in SEQ ID NO:51, and the sequence of the light chain variable region CDR3 is set forth in SEQ ID NO:52;

[0133] (b) 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; and / or

[0134] (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;

[0135] L is MC-vc-PAB; and

[0136] D is MMAE.

[0137] 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 solvate of the salt thereof.

[0138] In some embodiments, the composition further comprises a known chemotherapy 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.

[0139] In some embodiments, the composition further comprises 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.

[0140] In some embodiments, the composition further comprises an immunosuppressant selected from the group consisting of: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporine and tacrolimus; (3) antimetabolites, such as azathioprine and 6-mercaptopurine; (4) polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3; and (5) alkylating agents, such as cyclophosphamide. Specifically, the immunosuppressant is, for example, methylprednisolone, prednisone, azathioprine, Prograf, Zenapax, Sule, cyclosporine, tacrolimus, rapamycin, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, and the like.

[0141] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0142] In a third aspect, the present invention provides the aforementioned antibody-drug conjugate, its pharmaceutically acceptable salt, solvate or solvate of the salt, or use of the aforementioned composition in the preparation of a medicament for preventing and / or treating diseases associated with Claudin 18.2.

[0143] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, or pancreatic cancer.

[0144] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer.

[0145] In the fourth aspect of the present invention, the present invention provides a method for preventing and / or treating diseases associated with Claudin 18.2, comprising: administering to a subject in need thereof a preventive and / or therapeutically effective amount of the aforementioned antibody-drug conjugate, a pharmaceutically acceptable salt, solvate or solvate of the salt, or the aforementioned composition.

[0146] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, or pancreatic cancer.

[0147] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer.

[0148] 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, for use in preventing and / or treating diseases associated with Claudin 18.2.

[0149] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer, gastroesophageal junction adenocarcinoma, or pancreatic cancer.

[0150] In some embodiments, the disease associated with Claudin 18.2 is gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0151] FIG1 is an RT-PCR result of an embodiment of the present invention showing that HEK293 stably transfected cell lines express claudin 18.1 and claudin 18.2, respectively; both the HEK293-claudin 18.2 stably transfected cell line and the control KATO III cells can amplify a 780 bp characteristic band specific to claudin 18.2, while HEK293-claudin 18.1 can only amplify a common fragment of 504 bp;

[0152] FIG2 is the result of FACS experiment screening HEK293-claudin 18.2 high-expressing stable cell lines according to an embodiment of the present invention, wherein black dots are negative controls and gray dots are HEK293-claudin 18.2 high-expressing stable cell lines;

[0153] FIG3 shows the results of a FACS experiment screening NIH3T3-claudin 18.2 high-expressing stably transfected cell line in an embodiment of the present invention, wherein the black line is a negative control, the gray shadow is a 3T3-claudin 18.2 stably transfected cell line; NO.32-H is a 3T3-claudin 18.2 high-expressing stably transfected cell line, NO.18-M is a 3T3-claudin 18.2 medium-expressing stably transfected cell line, and NO.6-L is a 3T3-claudin 18.2 low-expressing stably transfected cell line;

[0154] FIG4 is a graph showing the ADCC effect of the Anti-Claudin18.2 antibody according to an embodiment of the present invention;

[0155] FIG5 is a graph showing the CDC effect of the Anti-Claudin18.2 antibody according to an embodiment of the present invention;

[0156] FIG6 is a hydrophobic interaction chromatogram (HIC) of an antibody-drug conjugate according to an embodiment of the present invention;

[0157] FIG7 is a graph showing the cell killing effects of different CM311 ADCs in LT-M11 cell lines according to an embodiment of the present invention;

[0158] FIG8 is a graph showing the results of the tumor growth inhibition activity of CM311-ADC-1 of an embodiment of the present invention and the control CM311-ADC-2 on the human gastric cancer nude mouse PDX model STO#025;

[0159] FIG9 is a graph showing the effects of CM311-ADC-1 and CM311-ADC-2 on the body weight of the human gastric cancer nude mouse PDX model STO#025;

[0160] FIG10 is a graph showing the results of the tumor growth inhibition activity of CM311-ADC-1 of an embodiment of the present invention and the control CM311-ADC-2 on the human gastric cancer nude mouse PDX model STO#523;

[0161] FIG11 is a graph showing the effects of CM311-ADC-1 and CM311-ADC-2 on the body weight of STO#523, a human gastric cancer nude mouse PDX model;

[0162] FIG12 is a graph showing the comparison of in vitro cell activity between the antibody-drug conjugate and the antibody according to an embodiment of the present invention (QC Log transformation independent fitting graph). DETAILED DESCRIPTION

[0163] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0164] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0165] Claudin is a skeletal protein that forms tight junctions. It is located at the apical side of the intercellular space between adjacent cells. Its distribution is tissue-organ specific. Its main functions include intercellular adhesion, maintaining cell polarity, regulating paracellular permeability, and participating in the regulation of cell proliferation and differentiation. In tumors, tight junctions between cells are disrupted, and Claudin cannot perform its normal functions.

[0166] Claudin 18, a member of the claudin family, has two distinct first exons. This allows for alternative splicing to produce two isoforms: claudin 18.1 and claudin 18.2. These two isoforms are transcribed and amplified in different tissues. Claudin 18.1 is primarily expressed in the lung, while claudin 18.2 is specifically expressed in the stomach. Claudin 18.2 (GenBank accession number: NM_001002026.3) is not expressed in normal tissues other than the gastric mucosa. However, it is significantly upregulated in various tumors, including 80% of gastrointestinal adenomas, 60% of pancreatic tumors, and some bile duct, ovarian, and lung tumors.

[0167] The term "Claudin 18.2-associated disease" refers to a disease in which the expression of Claudin 18.2 in tissue cells differs from (e.g., exceeds) normal levels. 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-associated disease in the subject (particularly a human) from which the tissue cell is derived.

[0168] In the present invention, unless otherwise stated, any numerical range should be understood to include any value or any sub-range within the range.

[0169] 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 chains of antibodies can be divided into two types: κ and λ. Heavy chains 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 consists of a heavy chain variable region (V H ) and 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 LThe 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 C1q component of the complement system. H and V L The V domain can be further subdivided into regions of hypervariability, 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 amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) respectively form the antibody binding site. The assignment of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0170] In the present invention, algorithms used to determine percent 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 percent identity of the amino acid sequences of the present invention, using, for example, the parameters described in the literature or the default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0171] In the present invention, the amino acid sequence having at least 70% sequence identity with an amino acid sequence includes polypeptide sequences that are substantially identical to the amino acid sequence, for example, those sequences that contain 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 to the polypeptide sequence of the present invention using the methods described herein (e.g., BLAST analysis using standard parameters).

[0172] In the present invention, a variant of an amino acid sequence refers to a sequence having greater than 70% identity to the amino acid sequence, such as greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, for example, a sequence having 3, 2, or 1 amino acid substitutions, deletions, or additions. Preferably, the number of amino acids substituted, added, or deleted does not exceed 3. More preferably, the number of amino acids substituted, added, or deleted does not exceed 2. Most preferably, the number of amino acids substituted, added, or deleted does not exceed 1.

[0173] "Substitution" variants are variants in which at least one amino acid residue in the native sequence has been removed and a different amino acid has been inserted in the same position. The substitutions can be single, in which only one amino acid is substituted in the molecule, or multiple, in which two or more amino acids are substituted in the same molecule. Multiple substitutions can be located at consecutive sites. Likewise, one amino acid can be substituted by multiple residues, in which case such variants include both substitutions and insertions. "Insertion" (or "addition") variants are variants in which one or more amino acids are inserted adjacent to an amino acid at a specific position in a native sequence. Adjacent to an amino acid means linked to the α-carboxyl or α-amino functional group of the amino acid. "Deletion" variants are variants in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of the molecule.

[0174] In certain embodiments, less than the theoretical maximum number 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 partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups.

[0175] 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; and the aryl sulfonate is benzene sulfonate or p-toluene sulfonate.

[0176] 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.

[0177] 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 after this reaction under biological conditions, 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).

[0178] In the present invention, solvates refer to forms of the antibody-drug conjugates of the present invention that form complexes in solid or liquid form by coordination with solvent molecules. Hydrates are a specific form of solvates that have coordinated water molecules. In the present invention, hydrates are preferred solvates.

[0179] Methods for preparing various pharmaceutical compositions containing a certain amount of active ingredient are known or will be apparent to those skilled in the art based on the present disclosure. 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 suitable pharmaceutical excipients, carriers, diluents, etc., which are non-toxic to the cells or mammals exposed thereto at the dosages and concentrations employed.

[0180] The pharmaceutical compositions 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 .

[0181] 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 the patient in various ways suitable for the selected mode of administration, for example, orally or parenterally (by intravenous, intramuscular, topical or subcutaneous routes).

[0182] Therefore, the compounds of the present 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% 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 achieved.

[0183] Tablets, lozenges, pills, capsules, etc. may also contain: binders such as gum 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, it may contain, in addition to the above types of materials, a liquid carrier such as a vegetable oil or polyethylene glycol. Various other materials may be present as a coating or otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar. Syrups or elixirs may contain the active compound, sucrose or fructose as a sweetener, methylparaben or propylparaben as a preservative, a dye, and a flavoring (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 can be incorporated into sustained-release preparations and devices.

[0184] The active compound can also be administered intravenously or intraperitoneally by infusion or injection. Aqueous solutions of the active compound or its salts can be prepared, optionally mixed with a nontoxic surfactant. Dispersions in glycerol, liquid polyethylene glycol, triacetin, and mixtures thereof, and oils can also be prepared. Under ordinary storage and use conditions, these formulations contain a preservative to prevent microbial growth.

[0185] Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersants or sterile powders containing active ingredients (optionally encapsulated in liposomes) suitable for instant preparations of sterile injectable or infusible solutions or dispersants. 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 dispersants, or by the use of surfactants. 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. Prolonged absorption of injectable compositions can be produced by using compositions that delay absorption (e.g., aluminum monostearate and gelatin).

[0186] Sterile injectable solutions are prepared by combining the active compound in the required amount in an appropriate solvent with various of the 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 ingredient present in a previously sterile-filtered solution.

[0187] Useful solid carriers include pulverized solids (such as talc, clay, microcrystalline cellulose, silicon dioxide, aluminum oxide, 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 in an effective amount, optionally with the aid of non-toxic surfactants. Adjuvants (such as flavors) and additional antimicrobial agents can be added to optimize the properties for a given use.

[0188] 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 application directly to the user's skin.

[0189] The above-mentioned formulations can be presented in unit dosage form, which is a physically discrete unit containing a unit dose 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 active ingredient in a unit dose can be varied or adjusted from about 0.1 to about 1000 mg or more.

[0190] 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 lipid vesicles (niosomes) (also known as non-ionic surfactant vesicles).

[0191] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, 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 causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the onset of a disease or symptom in a patient who is susceptible to the disease or symptom but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., arresting its development; or (c) relieving the symptoms of a disease, i.e., causing regression of the disease or symptom.

[0192] In the present invention, "subject" refers to a vertebrate. In certain embodiments, the 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, the mammal refers to a human.

[0193] In the present invention, an "effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired therapeutic or preventive effect. 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 elicit the desired response in the individual. A therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the substance / molecule outweigh any toxic or deleterious consequences. A "prophylactically effective amount" refers to an amount that is effective at the necessary dosage and time to achieve the desired preventive effect. Typically, but not necessarily, a prophylactic dose is used in subjects before the onset of disease or in the early stages of the disease, so the prophylactic effective amount will be lower than the therapeutically effective amount. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to a certain extent, preferably stop) cancer cell infiltration 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.

[0194] 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.

[0195] 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 a mouse antibody and the constant region sequences are derived from a human antibody.

[0196] "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) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0197] 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 to thereby improve one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their impact on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests as described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. In addition, the mutations in the CDRs are typically no more than one or two. Therefore, the humanized antibodies of the present invention also encompass antibodies comprising one or two amino acid mutations in the CDRs.

[0198] The present invention will be further explained below with reference to specific examples, but these examples are not intended to limit the scope of the present invention.

[0199] Example 1 Preparation of humanized antibodies

[0200] 1. Preparation of anti-claudin 18.2 monoclonal antibody

[0201] Multiple strategies were used simultaneously to immunize Balb / c mice and screen for monoclonal antibodies that specifically bind to Claudin 18.2 and not to Claudin 18.1, a different splice form of Claudin 18. The specific strategies are as follows:

[0202] 1) Construction of a stable cell line expressing tight junction protein 18.2

[0203] A plasmid containing the full-length sequence of human claudin 18.1 (UniProtKB-P56856) (Claudin 18.1-puc57-Amp) was synthesized (SynbioTech, Suzhou). Using this plasmid as a template, the full-length human claudin 18.1 fragment (Met1-Val261) was amplified by PCR using the upstream primer 5'-ttggcaaagaattgctagatgtccaccaccacatgcc-3' (SEQ ID NO: 171) and the downstream primer 5'-tgttcgggccctcctcgattacacatagtcgtgcttgg-3' (SEQ ID NO: 172). The amplified product was ligated using NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat: M0530L) and cloned into a eukaryotic expression plasmid system. Similarly, a plasmid containing the full-length sequence of human claudin 18.2 (UniProtKB-P56856-2) (Claudin 18.2-puc57-Amp) was synthesized (SynbioTech, Suzhou). Using this plasmid as a template, the full-length human claudin 18.2 fragment (Met1-Val261) was amplified by PCR using the upstream primer 5'-ttggcaaagaattgctagatggccgtgactgcctgtc-3' (SEQ ID NO: 173) and the downstream primer 5'-tgttcgggccctcctcgattacacatagtcgtgcttgg-3' (SEQ ID NO: 174). The amplified product was ligated using NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat: M0530L) and cloned into a eukaryotic expression plasmid system. NIH3T3 and HEK293 cells were electroporated with this plasmid, and 1-10 μg / mL Puromycin (Gibco, Cat: A1113803) was used for stepwise pressure screening of stable expression cell lines.

[0204] The resulting HEK293-Claudin 18.1 and HEK293-Claudin 18.2 stably transfected cell lines were first verified using RT-PCR. Total RNA was extracted from positive clones using a Trizol RNA extraction kit, and cDNA libraries were generated by reverse transcription using an oligo(dT) primer using a reverse transcription kit (SuperScript™ First-Strand Synthesis System, Cat: 18080051). Since the two spliced ​​fragments of claudin, claudin 18.1 and claudin 18.2, differ in the region from the N-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 (504 bp) between Loop 1 and the C-terminus of claudin 18.1 and claudin 18.2; primer KNB16 (5'-tgggtgccattggcctcctg-3' (SEQ ID NO: 177)) was designed to specifically and complementary bind to the N-terminus of claudin 18.2 and not bind to the N-terminal region of claudin 18.1, thereby amplifying only the full-length fragment (780 bp) from the N-terminus to the C-terminus of claudin 18.2 to express the KATO of claudin 18.2. III cells (ATCC HTB-103) served as a positive control. The results are shown in Figure 1. RT-PCR showed that the HEK293 stably transfected cell lines expressed both Claudin 18.1 and Claudin 18.2. Both the HEK293-Claudin 18.2 stably transfected cell line and the control KATO III cells amplified a 780 bp characteristic band specific for Claudin 18.2, while only a 504 bp common fragment was amplified in the HEK293-Claudin 18.1 cell line.

[0205] The stably transfected HEK293 and NIH3T3 cells were digested and harvested, washed twice with PBS, and 100 μL of a 1:200 diluted primary antibody, rabbit anti-Claudin 18.2 (Abcam, EPR19202, Catalog No. ab222512), was added to each tube. The cells were incubated at 4°C for 60 minutes. Excess primary antibody was removed with 0.5% BSA / PBS, and 50 μL of secondary antibody, goat anti-rabbit IgG Fc-AF647 (Jackson ImmunoResearch, Catalog No. 111-606-046), was added. The cells were incubated at 4°C for 45 minutes. Excess secondary antibody was then removed with 0.5% BSA / PBS, and the cells were resuspended in 100 μL of PBS and immediately analyzed by flow cytometry. The results are shown in Figures 2 and 3.

[0206] Figure 2 shows the results of a FACS assay for HEK293-Claudin 18.2 stably transfected cells transfected with the full-length Claudin 18.2 gene. Black dots represent untransfected HEK293 cells, while gray dots represent HEK293-Claudin 18.2 stably transfected cells. HEK293 cells do not express Claudin 18.2, while HEK293-Claudin 18.2 stably transfected cells highly express Claudin 18.2 on their cell membranes.

[0207] Figure 3 shows the results of a FACS assay for detecting claudin 18.2 in NIH3T3-Claudin 18.2-overexpressing stably transfected cell lines. The black line represents the negative control, and the gray shadow represents the 3T3-Claudin 18.2-overexpressing stably transfected cell lines. No. 32-H represents a 3T3-Claudin 18.2-overexpressing stably transfected cell line, No. 18-M represents a 3T3-Claudin 18.2-overexpressing stably transfected cell line, and No. 6-L represents a 3T3-Claudin 18.2-overexpressing stably transfected cell line.

[0208] The expanded and grown positive stably transfected cell lines were collected and frozen, among which the NIH3T3-cleavage protein 18.2 stably transfected cell line was used to immunize animals.

[0209] 2) Preparation of anti-claudin 18.2 monoclonal antibodies from hybridomas

[0210] Female Balb / c mice aged 6 to 8 weeks were immunized with 3T3-claudin 18.2 stably transfected cells or plasmid encoding claudin 18.2, or immunized alternately. When cells were used for immunization, 1×10 63T3-Claudin 18.2 stably transfected cells were mixed with Freund's adjuvant or non-Freund's adjuvant and injected into the thigh root and footpad. Two weeks later, immunization was repeated at different sites. When DNA was used for immunization, 20 μg of plasmid was mixed with 1 μg of CpG and injected directly into the abdomen of the mouse using a gene gun (Biorad) at 40 psi. Immunization was performed once a week. Three days before fusion, 1×10 cells of HEK293-Claudin 18.2 stably transfected cells were used. 6 Immunization was performed by tail vein injection with 50 μL of cells per mouse. Three days later, mice were sacrificed, and popliteal, inguinal, and iliac lymph nodes were harvested and ground in DMEM to obtain a B cell-rich suspension. The spleens were removed, ground in DMEM, and centrifuged to obtain a splenocyte suspension. An appropriate amount of the lymph node and splenocyte suspension was mixed with SP2 / 0 and fused using an electroporation instrument.

[0211] 3) Construction of anti-tight junction protein 18.2 phage antibody library

[0212] The total RNA was extracted from the collected mouse spleen and peripheral lymph node cell suspension using a Trizol RNA extraction kit, and reverse transcription was performed using a reverse transcription kit (SuperScript First-Strand Synthesis System, Cat. No. 18080051) using light and heavy chain specific primers to obtain antibody light and heavy chain cDNA libraries, respectively. 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 enzyme-digested and 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 heavy chain library, respectively. The antibody fragments in the light chain library were double-digested with BspQI and SfiI and then ligated into 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 200 μL 5% BSA / PBS and 1x10 7 HEK293-claudin 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 and incubated on ice for 1 hour. The mixture was centrifuged at 1000 rpm for 3 minutes, and the supernatant was discarded. 1 mL of 1% BSA / PBS was added and the cells were washed repeatedly 5-10 times. The cells were lysed by adding 1 mL of 100 mM TEA (triethylamine) at room temperature for 10 minutes. After neutralization with 0.5 mL of 1M Tris-HCl, pH 7.5, the cells were infected with 10 mL of TG1 E. coli cells in the logarithmic growth phase and incubated at 37°C for 30 minutes. Phages were recovered, titered, and the next round of screening was performed according to standard molecular biology protocols.

[0213] 2. Screening and sequence acquisition of anti-claudin 18.2 specific antibodies

[0214] HEK293-claudin 18.1, HEK293-claudin 18.2, and HEK293 were pre-stained 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 cells were mixed at a ratio of 1:1:1 and added to a 96-well plate (2 × 10 5 Cells were plated at 100 μL per well (100 μL / well) and bound to hybridoma supernatants or bacterial induction supernatants. After incubation on ice for 1 hour, AlexaFluro647-labeled secondary antibodies, anti-mouse IgG Fc or anti-human IgG F(ab)'2 (Jackson ImmunoResearch), were added and incubated on ice for 45 minutes. After washing, cells were resuspended in 100 μL of PBS per well and prepared for flow cytometry (iQue Screener). Three different cell populations were circled based on fluorescence intensity in the FL2 channel, and the binding of the test antibodies to each cell population was measured in the FL4 channel. The screened antibodies bound to HEK293 cells stably transfected with Claudin 18.2 with high affinity and specificity, while not binding to HEK293 cells stably transfected with Claudin 18.1 or HEK293 cells. From hybridoma cells, a total of 320 clones were screened by FACS detection that could bind to Claudin 18.2 but not Claudin 18.1; after three rounds of panning, 62 clones with high affinity binding to Claudin 18.2 but not Claudin 18.1 were screened from the phage Fab library.

[0215] Positive clones screened from the phage library were extracted and sequenced, and the variable region sequences were cloned into heavy and light chain constant region vectors for full-length IgG expression. Positive cells from hybridomas were lysed with 1 mL of TRNzol, and total RNA was extracted using the guanidine isothiocyanate method. This RNA was used as a template for synthesizing first-strand cDNA, which was then used as a template for amplifying the variable region DNA sequences corresponding to the hybridoma cells. Sequencing of the amplified products yielded the candidate hybridoma heavy and light chain variable region sequences, as shown below.

[0216] Clone 18D10:

[0217] heavy chain

[0218]

[0219] The underlined parts from left to right are CDR1 (SEQ ID NO: 140), CDR2 (SEQ ID NO: 141), and CDR3 (SEQ ID NO: 142);

[0220] 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.

[0221] Nucleic acid sequence

[0222]

[0223] light chain

[0224]

[0225] The underlined parts from left to right are CDR1 (SEQ ID NO: 148), CDR2 (SEQ ID NO: 149), and CDR3 (SEQ ID NO: 150);

[0226] 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.

[0227] Nucleic acid sequence

[0228]

[0229] Clone 18A9:

[0230] heavy chain

[0231]

[0232] The underlined parts from left to right are CDR1 (SEQ ID NO: 156), CDR2 (SEQ ID NO: 157), and CDR3 (SEQ ID NO: 158);

[0233] 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.

[0234] Nucleic acid sequence

[0235]

[0236] light chain

[0237]

[0238] The underlined parts from left to right are CDR1 (SEQ ID NO: 164), CDR2 (SEQ ID NO: 165), and CDR3 (SEQ ID NO: 166);

[0239] 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.

[0240] Nucleic acid sequence

[0241]

[0242]

[0243] The heavy and light chain variable region sequence fragments were PCR amplified, and the heavy chain variable region was cloned into a vector containing the 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 the human light chain constant region to express the complete kappa light chain in mammalian cells. After correct sequencing, the fragments were transfected into HEK293-6E mammalian cells. The expressed IgG1 was secreted into the culture medium, and the supernatants were collected, filtered, and purified. IgG was purified using Protein A chromatography. The culture supernatant was loaded onto an appropriately sized Protein A column, washed with 50 mM Tris-HCl, pH 8.0, 250 mM NaCl, and bound IgG was eluted with 0.1 M Glycine-HCl, pH 3.0. The protein was concentrated by ultrafiltration using a Millipore concentrator, and the OD280 was measured. The IgG concentration was determined spectrophotometrically. The purity of the IgG was analyzed by SDS-PAGE.

[0244] 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 4 100 μL of cells were added to a U-shaped 96-well plate and centrifuged at 1100 rpm for 3 minutes. The supernatant was discarded and the cells were gently flicked to disperse. 50 μL of serially diluted antibodies (8 5-fold dilutions starting at 100 nM) were added to each well and incubated at 4°C for 1 hour. After incubation, the cells were washed three times with 140 μL of 0.5% BSA per well. 30 μL / well of secondary antibody Alexa Fluor 647 anti-human IgG (Jackson ImmunoResearch, Cat. No. 109-606-170) was added and incubated at 4°C for 40 minutes. After incubation, the cells were washed three times with 140 μL of 0.5% BSA per well. Finally, each well was resuspended in 50 μL of PBS for flow cytometry analysis (iQue Screener). The results are shown in Table 1, which showed that the obtained chimeric Claudin 18.2 IgG1 antibody only recognized HEK293-Claudin 18.2 cells transfected with Claudin 18.2, but did not bind to HEK293 and HEK293-Claudin 18.1.

[0245] Table 1: Binding of Claudin 18.2 Antibodies to Stably Transfected Cell Lines (NB indicates no binding detected)

[0246]

[0247] 3. Humanization of anti-claudin 18.2 antibody

[0248] The variable region sequences of the selected monoclonal antibodies were aligned with human germline antibody sequences to identify sequences with high homology for CDR transplantation. Computer-assisted homology modeling was then performed to analyze the CDR regions and surrounding framework amino acid sequences to examine their spatial binding patterns. By calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy, key amino acids within each positive monoclonal antibody gene sequence that may interact with the target and maintain the spatial structure were identified. Back-mutation sites were then designed based on this information. HLA-DR affinity was analyzed to select human germline framework sequences with low immunogenicity. Amino acid residues that may be modified during fermentation were identified, and mutations were designed to reduce the likelihood of such modifications.

[0249] A total of different heavy chain derivatives and light chain derivatives were designed. After the full sequences of the light and heavy chain derivatives were synthesized, they were cloned into vectors containing the antibody kappa chain constant region Ckappa or the human IgG1 constant region CH1-CH3. After the light and heavy chain derivative plasmids of the same parental source were combined and paired, they were transfected into HEK293.6E cells and expressed for 5-6 days. The supernatant was collected and purified on a Protein A column.

[0250] The sequence of the humanized antibody is as follows:

[0251] 18D10:

[0252] Heavy chain variable region:

[0253] 18D10VHv1:

[0254]

[0255] The underlined parts from left to right are CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 3), and CDR3 (SEQ ID NO: 4);

[0256] The ununderlined parts are FR1 (SEQ ID NO: 5), FR2 (SEQ ID NO: 6), FR3 (SEQ ID NO: 7), and FR4 (SEQ ID NO: 8) from left to right.

[0257] Nucleic acid sequence

[0258]

[0259] 18D10VHv2:

[0260]

[0261] The underlined parts from left to right are CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12);

[0262] 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.

[0263] Nucleic acid sequence

[0264]

[0265] 18D10VHv3:

[0266]

[0267] The underlined parts from left to right are CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19), and CDR3 (SEQ ID NO: 20);

[0268] 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.

[0269] Nucleic acid sequence

[0270]

[0271] 18D10VHv4:

[0272]

[0273] The underlined parts from left to right are CDR1 (SEQ ID NO: 26), CDR2 (SEQ ID NO: 27), and CDR3 (SEQ ID NO: 28);

[0274] 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.

[0275] Nucleic acid sequence

[0276]

[0277]

[0278] 18D10VHv5:

[0279]

[0280] The underlined parts from left to right are CDR1 (SEQ ID NO: 34), CDR2 (SEQ ID NO: 35), and CDR3 (SEQ ID NO: 36);

[0281] 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.

[0282] Nucleic acid sequence

[0283]

[0284] 18D10VHv6:

[0285]

[0286] The underlined parts from left to right are CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 43), and CDR3 (SEQ ID NO: 44);

[0287] 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.

[0288] Nucleic acid sequence

[0289]

[0290] Light chain variable region:

[0291] 18D10VLv1:

[0292]

[0293] The underlined parts from left to right are CDR1 (SEQ ID NO: 50), CDR2 (SEQ ID NO: 51), and CDR3 (SEQ ID NO: 52);

[0294] 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.

[0295] Nucleic acid sequence

[0296]

[0297] 18D10VLv2:

[0298]

[0299] The underlined parts from left to right are CDR1 (SEQ ID NO: 58), CDR2 (SEQ ID NO: 59), and CDR3 (SEQ ID NO: 60);

[0300] 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.

[0301] Nucleic acid sequence

[0302]

[0303] The preferred humanized antibody sequence is as follows:

[0304] Heavy chain amino acid sequence:

[0305]

[0306] Heavy chain nucleic acid sequence

[0307]

[0308] Light chain amino acid sequence:

[0309]

[0310] Light chain nucleic acid sequence

[0311]

[0312]

[0313] 18A9:

[0314] Heavy chain variable region:

[0315] 18A9VHv1:

[0316]

[0317] The underlined parts from left to right are CDR1 (SEQ ID NO: 68), CDR2 (SEQ ID NO: 69), and CDR3 (SEQ ID NO: 70);

[0318] 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.

[0319] Nucleic acid sequence

[0320]

[0321] 18A9VHv2:

[0322]

[0323] The underlined parts from left to right are CDR1 (SEQ ID NO: 76), CDR2 (SEQ ID NO: 77), and CDR3 (SEQ ID NO: 78);

[0324] 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.

[0325] Nucleic acid sequence

[0326]

[0327] 18A9VHv3:

[0328]

[0329] The underlined parts from left to right are CDR1 (SEQ ID NO: 84), CDR2 (SEQ ID NO: 85), and CDR3 (SEQ ID NO: 86);

[0330] 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.

[0331] Nucleic acid sequence

[0332]

[0333] 18A9VHv4:

[0334]

[0335] The underlined parts from left to right are CDR1 (SEQ ID NO: 92), CDR2 (SEQ ID NO: 93), and CDR3 (SEQ ID NO: 94);

[0336] 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.

[0337] Nucleic acid sequence

[0338]

[0339] 18A9VHv5:

[0340]

[0341] The underlined parts from left to right are CDR1 (SEQ ID NO: 100), CDR2 (SEQ ID NO: 101), and CDR3 (SEQ ID NO: 102);

[0342] 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.

[0343] Nucleic acid sequence

[0344]

[0345] 18A9VHv6:

[0346]

[0347] The underlined parts from left to right are CDR1 (SEQ ID NO: 108), CDR2 (SEQ ID NO: 109), and CDR3 (SEQ ID NO: 110);

[0348] 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.

[0349] Nucleic acid sequence

[0350]

[0351] 18A9VHv7:

[0352]

[0353] The underlined parts from left to right are CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), and CDR3 (SEQ ID NO: 118);

[0354] 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.

[0355] Nucleic acid sequence

[0356]

[0357] Light chain variable region:

[0358] 18A9VLv1:

[0359]

[0360] The underlined parts from left to right are CDR1 (SEQ ID NO: 124), CDR2 (SEQ ID NO: 125), and CDR3 (SEQ ID NO: 126);

[0361] 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.

[0362] Nucleic acid sequence

[0363]

[0364] 18A9VLv2:

[0365]

[0366] The underlined parts from left to right are CDR1 (SEQ ID NO: 132), CDR2 (SEQ ID NO: 133), and CDR3 (SEQ ID NO: 134);

[0367] 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.

[0368] Nucleic acid sequence

[0369]

[0370] Example 2 Pharmacological Studies of Humanized Antibodies

[0371] 1. Affinity determination of humanized antibodies (EC 50 )

[0372] Cells in the logarithmic growth phase were blocked with 3% BSA for 30 minutes and 5×10 4 100 μL of cells were plated in a U-shaped 96-well plate and centrifuged at 1100 rpm for 3 minutes. The supernatant was discarded and the cells were gently flicked to disperse. 50 μL of serially diluted antibodies (8 5-fold dilutions starting at 100 nM) were added to each well and incubated at 4°C for 1 hour. After incubation, the cells were washed three times with 140 μL of 0.5% BSA per well. AF 647APC anti-human secondary antibody was added at 30 μL / well and incubated at 4°C for 40 minutes. After incubation, the cells were washed three times with 140 μL of 0.5% BSA per well. Finally, each well was resuspended in 50 μL of PBS for iQue (Intellicyt, USA) detection (see Table 2).

[0373] Table 2: Affinity determination results of humanized antibodies

[0374]

[0375]

[0376] 2. ADCC cytotoxicity of CM311 antibody against tumor cells

[0377] Transfer 30 mL of fresh blood to a 50 mL centrifuge tube, add 15 mL of 1× PBS, mix thoroughly, and slowly add to a centrifuge tube containing 20 mL of Ficoll-Paque Plus, allowing the blood to spread over the surface of the Ficoll-Paque Plus. Centrifuge at 20°C, 2000 rpm for 30 minutes. Discard the top layer of serum, aspirate the buffy coat (i.e., PBMCs), and aliquot into 50 mL centrifuge tubes, 10 mL per tube. Add at least 30 mL of 1× PBS to each tube and mix thoroughly. Centrifuge at 4°C, 1300 rpm for 10 minutes, discard the supernatant, add 10 mL of 1× PBS, and count the cells.

[0378] 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 seed the cells in a U-shaped 96-well plate at 4 x 10 5 Add the aforementioned 18D10 chimera, 18A9 chimera, anti-claudin 18.2 humanized antibodies 18D10 and 18A9 (CM311, CM311 is a collective term for humanized antibodies 18D10 or 18A9), and control antibody anti-KLH dilutions (40, 20, 10, 5, 2.5, and 1.25 μg / mL) at 25 μL / well. Incubate at 37°C, 5% CO2 for 30 minutes. Remove the plate and add Kato III cells (8×10 3 Incubate at 37°C, 5% CO2 for 3.5 hours. Add 2 μL of 10x lysis buffer to the wells showing maximum target cell release and continue incubation at 37°C, 5% CO2 for 30 minutes. Remove the plate and centrifuge at 1000 rpm for 3 minutes. Transfer the supernatant to a black microplate and add 50 μL / well. Add 50 μL / well of LDH detection substrate and incubate at room temperature for 10 minutes. Terminate the reaction by adding 25 μL / well of stop solution. Detect using a microplate reader (Biotek).

[0379] The result is calculated as follows:

[0380]

[0381] The results are shown in FIG4 , showing that the anti-claudin 18.2 humanized antibodies 18D10 and 18A9 have strong ADCC activity.

[0382] 3. CDC cell killing activity of CM311 antibody against tumor cells

[0383] Kato III cells in the logarithmic growth phase were resuspended to 1×10 7Cells / mL. Add CFSE (Sigma, 87444-5MG-F) to a final concentration of 1 μM. Incubate at room temperature for 10 minutes and terminate the reaction by adding 3 volumes of culture medium. Centrifuge at 1000 rpm at 4°C for 5 minutes, discard the supernatant, resuspend in culture medium, and seed the cells into a 96-well plate at 1 x 10 5 50 μL / well. Add the diluted 18D10 chimera, 18A9 chimera, anti-claudin 18.2 humanized antibodies 18D10 and 18A9 (CM311), and control anti-KLH (concentrations after dilution: 30, 10, 3.33, 1.11, and 0.37 μg / mL) at 50 μL / well. Dilute complement to 30% in culture medium and add 50 μL / well to the plate. Incubate at 37°C, 5% CO2 for 2 hours. Centrifuge at 1000 rpm for 3 minutes and discard the supernatant. Dilute the PI staining solution 1:200, mix with Sulfate latex (Invitrogen, S37227), and add 100 μL / well to a 96-well plate. Incubate on ice for 10 minutes and analyze by FACS. The results are shown in FIG5 , showing that the anti-claudin 18.2 humanized antibodies 18D10 and 18A9 have a strong CDC effect.

[0384] Example 3 Preparation and Detection of Antibody Drug Conjugates

[0385] 1. Preparation of Antibody Drug Conjugates

[0386] Take 10 mg of CM311 antibody and replace it with reducing buffer (25mM sodium borate, pH 8.0, 25mM NaCl, 5mM EDTA) using a 15mL 30KD ultrafiltration device for three replacements; the final volume is about 1mL, transfer to a new Eppendorf centrifuge tube (weighed), and weigh it; test the protein concentration and calculate the total protein amount. Add 2.5 times the molar number of DTT to the antibody, incubate at room temperature for 2 hours, and mix continuously; use a 15mL 30KD ultrafiltration device to replace it with coupling buffer (50mM Tris, pH 7.2, 150mM NaCl, 5mM EDTA) for three replacements. Take a sample to determine the protein concentration using A280, weigh it, and calculate the total protein amount; take 10μL of sample and determine the number of free thiol groups using Ellman's method;

[0387] The molar concentration of free thiol groups was calculated using the following formula:

[0388]

[0389] b: Cuvette optical path length (usually 1 cm)

[0390] The number of moles of free thiol groups was calculated based on the molar concentration of free thiol groups and the total volume of protein solution.

[0391] To the reduced antibody, add 1.1 times the molar number of free sulfhydryl groups of vc-MMAE (i.e., MC-vc-PAB-MMAE) (dissolved in DMSO). Mix thoroughly and react at room temperature for 2 hours with intermittent mixing. Add 20 times the molar number of vc-MMAE (i.e., MC-vc-PAB-MMAE) of N-acetylcysteine ​​to the reaction mixture, mix thoroughly, and let it stand for 5 minutes. Use a 15 mL 30KD ultrafiltration device to replace the solution in conjugate storage solution (20 mM histidine, 3% sucrose, 0.03% Tween-80, pH 5.5). Repeat three replacements to 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), which should be stored at 4°C.

[0392] 2. DAR determination of antibody-drug conjugates

[0393] The drug loading rate (DAR) of the antibody drug conjugate was determined by hydrophobic interaction chromatography (HIC-HPLC).

[0394] The spectrum of a typical antibody drug conjugate CM311-18D10-VH6 / VL1-ADC is shown in Figure 6. Based on the peak area of ​​the spectrum, the average drug loading number DAR was calculated to be 3.8.

[0395] Similarly, the average drug loading number DAR of the antibody drug conjugate CM311-18D10-VH3 / VL2-ADC was 3.5.

[0396] The average drug loading rate (DAR) of the antibody-drug conjugate CM311-18D10-VH6 / VL2-ADC is 3.4.

[0397] The average drug loading rate (DAR) of the antibody-drug conjugate CM311-18A9-VH7 / VL2-ADC was 3.0.

[0398] It should be noted that the name of the above antibody-drug conjugate, such as CM311-18D10-VH6 / VL1-ADC, indicates that the antibody used to prepare the antibody-drug conjugate is CM311-18D10-VH6 / VL1. Furthermore, the antibody name CM311-18D10-VH6 / VL1 indicates that the antibody used to prepare the aforementioned antibody-drug conjugate is an antibody of the 18D10 class having 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 to prepare the aforementioned antibody-drug conjugate is h18D10.v16. The names of the other 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; and the specific antibody used in the preparation of the antibody drug conjugate CM311-18A9-VH7 / VL2-ADC is h18A9.v27 in Table 2.

[0399] Example 4 In vitro pharmacodynamic study of antibody drug conjugates

[0400] After 1-2 passages of cell culture, the revived cell lines were passaged. The supernatant was aspirated into a 15 mL centrifuge tube, centrifuged, and the supernatant discarded. The cell culture flask was rinsed with 5 mL of PBS, and the cells were trypsinized with 2 mL of trypsin and resuspended in culture medium into the same 15 mL centrifuge tube. The cells were centrifuged, the supernatant discarded, and resuspended again in culture medium. 0.5 mL of the cell suspension was counted using a cell counter. The cells were plated onto 96-well cell culture plates (5,000 or 10,000 cells / well for LT-1C8 cells, 5,000 cells / well for LT-M11 cells, and 3,000 cells / well for BxPC-3 cells). After 24 hours of culture, serially diluted concentrations of the antibody-drug conjugate CM311 ADC (conjugated to different CM311 monoclonal antibodies) were added and incubated for 96 hours. CCK-8 or Presto-Blue colorimetric reagent was then added to each well. The cells were detected using a microplate reader and four-parameter fitting was performed.

[0401] Experimental reagents and sources:

[0402] Test drug

[0403]

[0404] cell lines

[0405]

[0406] Experimental results:

[0407] Each CM311ADC IC 50 The average values ​​are shown in Table 3. Figure 7 is a representative graph showing the killing of LT-M11 cells by different CM311 ADCs.

[0408] Table 3: IC values ​​of different CM311 ADCs in the screened cell lines 50 value

[0409]

[0410]

[0411] Note: In Figure 7:

[0412] Sample 1: CM311-18D10-VH3 / VL2-ADC

[0413] Sample 2: CM311-18D10-VH6 / VL1-ADC

[0414] Sample 3: CM311-18D10-VH6 / VL2-ADC

[0415] Sample 4: CM311-18A9-VH7 / VL2-ADC

[0416] From the results in Table 3 and Figure 7, it can be seen that different CM311 ADCs exhibited significant cell killing activity in cell lines with moderate and high expression of Claudin18.2, but had no significant cell killing activity in the Claudin18.2-negative BxPC-3 cell line.

[0417] Example 5 In vivo pharmacodynamic study of antibody drug conjugates

[0418] The anti-tumor activity of CM311 ADC was tested in two gastric cancer PDX models, STO#025 and STO#523, both of which have elevated Claudin 18 mRNA levels.

[0419] 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. 3At the time of the study, mice were randomly divided into four groups, each containing five mice, to ensure uniform tumor volume and body weight among the groups. These groups included vehicle, 1 mg / kg CM311-ADC-1, 3 mg / kg CM311-ADC-1, and 3 mg / kg CM311-ADC-2 (non-binding control ADC). CM311-ADC-1 refers to the antibody-drug conjugate CM311-18D10-VH6 / VL1-ADC; CM311-ADC-2, compared to CM311-ADC-1, uses a human IgG1 isotype control that does not bind to targets on the tumor cell surface.

[0420] Data analysis: During the experiment, 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 length and width of the tumor, respectively. The relative tumor volume (RTV) is calculated based on the measurement results. RTV = V f / V0. Where V0 is the tumor volume measured at the time of group administration (i.e. Day 0), V f = tumor volume measured on the last day. Relative tumor growth rate (T / C) (%) = (RTV of the drug group / RTV of the vehicle group) × 100%. Tumor growth inhibition rate (TGI) % = (mean tumor volume of the vehicle group - mean tumor volume of the drug group) / mean tumor volume of the vehicle group × 100%. A test article was considered to have a significant inhibitory effect on tumor growth when T / C (%) ≤ 40% and P < 0.05.

[0421] Experimental results:

[0422] 1) Efficacy study of CM311ADC in the human gastric cancer PDX model STO#025 with high Claudin 18 mRNA expression

[0423] The experimental results are shown in Figures 8 and 9. Following administration of CM311-ADC-1 at doses of 1 and 3 mg / kg, the relative tumor growth 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, respectively, experienced complete regression, and 2 / 5 and 0 / 5 tumors experienced partial regression. In the CM311-ADC-2 (3 mg / kg) group, the T / C was 67.24% and the TGI was 32.76%. These results demonstrate that both CM311-ADC-1 (3 mg / kg) and CM311-ADC-1 (1 mg / kg) exhibited significant tumor growth inhibition activity, while CM311-ADC-2 (3 mg / kg) showed no significant antitumor activity. Both CM311-ADC-1 and CM311-ADC-2 were well tolerated by tumor-bearing mice.

[0424] 2) Efficacy study of CM311ADC in the human gastric cancer PDX model STO#523 with high Claudin 18 mRNA expression

[0425] The experimental results are shown in Figures 10 and 11. Following administration of CM311-ADC-1 at doses of 1 and 3 mg / kg, the relative tumor growth rates (T / C%) on Day 28 were 35.60% and 6.79%, respectively, and the tumor growth inhibition rates (TGI%) were 64.40% and 93.21%, respectively. In the CM311-ADC-2 (3 mg / kg) group, the T / C% was 114.81% and the TGI% was -14.81%. These results demonstrate that both CM311-ADC-1 (3 mg / kg) and CM311-ADC-1 (1 mg / kg) exhibited significant tumor growth inhibition activity, while CM311-ADC-2 (3 mg / kg) showed no significant antitumor activity. Both CM311-ADC-1 and CM311-ADC-2 were well tolerated by tumor-bearing mice.

[0426] Example 6 Comparison of in vitro cellular activity of antibodies and antibody-drug conjugates

[0427] Test method:

[0428] KATO III cells were plated at 5,000 cells / well and the test samples were added 24 hours later. The test samples were diluted 2.4-fold nine times to a final concentration of 1,000 ng / mL and incubated for 96 hours. Fluorescence was read using a microplate reader after 60 minutes of Presto-Blue color development.

[0429] The samples to be tested are as follows:

[0430] Sample 1: CM311, specifically CM311-18D10-VH6 / VL1;

[0431] Sample 2: Anti-Claudin-18.2-ADC, specifically CM311-18D10-VH6 / VL1-ADC;

[0432] 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).

[0433] The test results are shown in Figure 12 and Table 4. As can be seen from Figure 12, Anti-Claudin-18.2-ADC (such as CM311-18D10-VH6 / VL1-ADC) showed a 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.

[0434] Table 4: Comparison of in vitro cell activity of antibodies and antibody drug conjugates (4-parameter fitting results)

[0435] Curve Fit: 4-Parameter

[0436]

Claims

DEPCT6627 / 06 / 25661. Conjugated antibody against a drug, its pharmaceutically acceptable salt or its solvate, or the solvate of such salt, the conjugated antibody against a drug has the structure shown in formula IAb-(LD)pFormulaI, where Ab is the anti-clodine18.2 antibody, the anti-clodine18.2 antibody consists of heavy and light chains and the CDR1 heavy chain variant region consists of sequences selected from the sequences shown in SEQIDNO:2,10,18,26,34,42,68,76,84,92,100,108 or 116 or its mutagens, the CDR2 ... The heavy variant region of CDR3 consists of sequences selected from the sequences shown in SEQID NO: 3, 11, 19, 27, 35, 43, 69, 77, 85, 93, 101, 109, or 117, or their mutations. The light variant region of CDR1 consists of sequences selected from the sequences shown in SEQID NO: 50, 58, 124, or 132, or their mutations. The light variant region of CDR2 consists of sequences selected from the sequences shown in SEQID NO: 51, 59, or their mutations.125 or 133 or its mutants and the CDR3 light variant region comprised of sequences selected from the sequences as shown in SEQIDNO:52,60,126 or 134 or its mutants; D is the cytotoxic agent; L is the linker for the conjugation of the anti-clodine 18.2 antibody and the cytotoxic agent; p is 2.0-8.

02. Conjugated antibody with drug of claim 1, its pharmaceutically acceptable salt, or the solvate of such salt in which the CDR1 heavy variant region of the anti-clodine 18.2 antibody is comprised. The CDR2 heavy variant region consists of sequences selected from the sequences shown in SEQID NO: 2, 10, 18, 26, 34, or 42, or their mutations. The CDR3 heavy variant region consists of sequences selected from the sequences shown in SEQID NO: 4, 12, 20, 28, 36, or 44, or their mutations. The CDR1 light variant region consists of sequences selected from the sequences shown in SEQID NO: 50 or 58, or their mutations.The CDR2 light-chain variant region consists of sequences selected from the sequences shown in SEQID NO: 51 or 59 or their mutants; and the CDR3 light-chain variant region consists of sequences selected from the sequences shown in SEQID NO: 52 or 60 or their mutants; or the CDR1 heavy-chain variant region of the anti-clodine18.2 antibody consists of sequences selected from the sequences shown in SEQID NO: 68, 76, 84, 92, 100, 108 or 116 or their mutants. Its CDR2 heavy variant region consists of sequences selected from the sequences shown in SEQID NO: 69, 77, 85, 93, 101, 109, or 117, or its mutants; its CDR3 heavy variant region consists of sequences selected from the sequences shown in SEQID NO: 70, 78, 86, 94, 102, 110, or 118, or its mutants; its CDR1 light variant region consists of sequences selected from the sequences shown in SEQID NO: 124 or 132, or its mutants.The CDR2 light variant region consists of sequences selected from the sequences shown in SEQIDNO:125 or 133 or their mutants, and the CDR3 light variant region consists of sequences selected from the sequences shown in SEQIDNO:126 or 134 or their mutants; or the CDR1, CDR2 and CDR3 heavy variant regions of the anti-clodine18.2 antibody were selected from combinations of the following sequences:(1)SEQIDNO:2,SEQIDNO:3,SEQIDNO:4(2)SEQIDNO:10,SEQIDNO:11,SEQIDNO:12(3)SEQIDNO:18,SEQIDNO:19,SEQIDNO:20(4)SEQIDNO:26,SEQIDNO:27,SEQIDNO:28(5)SE QIDNO:34,SEQIDNO:35,SEQIDNO:36(6)SEQIDNO:42,SEQIDNO:43,SEQIDNO:44(7)SEQIDNO:68,SEQIDNO:69,SEQIDNO:70(8)SEQIDNO:76,SEQIDNO: 77,SEQIDNO:78(9)SEQIDNO:84,SEQIDNO:85,SEQIDNO:86(10)SEQIDNO:92,SEQIDNO:93,SEQIDNO:94(11)SEQIDNO:100,SEQIDNO:101,SEQIDNO:102 (12)SEQIDNO:108,SEQIDNO:109,SEQIDNO:110(13)SEQIDNO:116,SEQIDNO:117,SEQIDNO:118CDR1,CDR2 and CDR3 light variant regions of anti-clodine18.2 antibodies were selected from the combination of the following sequences:(1)SEQIDNO:50,SEQIDNO:51,SEQIDNO:52(2)SEQIDNO:58,SEQIDNO:59,SEQIDNO:60(3)SEQIDNO:124,SEQIDNO:125,SEQIDNO:126(4)SEQIDNO:132,SEQIDNO:133,SEQIDNO:134 The popular CDR1,CDR2 and CDR3 heavy chain variation regions of the anti-clodine18.2 antibody were selected from the combination of the following sequences:(1)SEQIDNO:2,SEQIDNO:3,SEQIDNO:4(2)SEQID NO:10,SEQIDNO:11,SEQIDNO:12(3)SEQIDNO:18,SEQIDNO:19,SEQIDNO:20(4)SEQIDNO:26,SEQIDNO:27,SEQIDNO:28(5)SEQIDNO:34,SEQIDNO:35,SEQIDNO:36(6)SEQIDNO:42,SEQIDNO:43,SEQIDNO:44CDR1,CDR2 and CDR3 light chain variation regions of anti-clodine18.2 antibodies were selected. In addition to the combination of the following sequences (1)SEQIDNO:50,SEQIDNO:51,SEQIDNO:52(2)SEQIDNO:58,SEQIDNO:59,SEQIDNO:60; or the popular CDR1,CDR2 and CDR3 heavy variable regions of the anti-clodine18.2 antibody was selected from the combination of the following sequences (1)SEQIDNO:68,SEQIDNO:59,SEQIDNO:60; DNO:69,SEQIDNO:70(2)SEQIDNO:76,SEQIDNO:77,SEQIDNO:78(3)SEQIDNO:84,SEQIDNO:85,SEQIDNO:86(4)SEQIDNO:92,SEQIDNO:93,SEQIDNO: 94(5)SEQIDNO:100,SEQIDNO:101,SEQIDNO:102(6)SEQIDNO:108,SEQIDNO:109,SEQIDNO:110(7)SEQIDNO:116,SEQIDNO:117,SEQIDNO:118CDR1,The CDR2 and CDR3 light variant regions of the anti-clodine18.2 antibody were selected from the combination of the following sequences: (1) SEQIDNO:124, SEQIDNO:125, SEQIDNO:126; (2) SEQIDNO:132, SEQIDNO:133, SEQIDNO:134; 3. The drug conjugate of claim 1, its pharmaceutically acceptable salt, or the solvate of such salt in which the FR1 heavy variant region of the anti-clodine18.2 antibody is composed of a sequence selected from the sequences shown in SEQIDNO:5,13,21,29,37,45,71,79,87,95,103,111 or 119 or variants. Its variants, FR2, the heavy variant region consists of sequences selected from the sequences shown in SEQIDNO:6,14,22,30,38,46,72,80,88,96,104,112 or 120 or its variants; FR3, the heavy variant region consists of sequences selected from the sequences shown in SEQIDNO:7,15,23,31,39,47,73,81,89,97,105,113 or 121 or its variants; FR4, the heavy variant region consists of sequences selected from the sequences shown in SEQIDNO:8,16,24,32,40,48,74,82,90,98,106,114 or 122 or its variants.FR1 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 53, 61, 127, or 135, or their variants; FR2 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 54, 62, 128, or 136, or their variants; FR3 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 55, 63, 129, or 137, or their variants; and FR4 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 56, 64, 130, or 138, or their variants; the preferred variable is F. R1 Heavy Chain Variant Region of the anti-clodine18.2 antibody consists of sequences selected from the sequences shown in SEQIDNO:5,13,21,29,37 or 45 or its mutants; FR2 Heavy Chain Variant Region consists of sequences selected from the sequences shown in SEQIDNO:6,14,22,30,38 or 46 or its mutants; FR3 Heavy Chain Variant Region consists of sequences selected from the sequences shown in SEQIDNO:7,15,23,31,39 or 47 or its mutants; FR4 Heavy Chain Variant Region consists of sequences selected from the sequences shown in SEQIDNO:8,16,24,32,FR1 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 53 or 61 or their mutations; FR2 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 54 or 62 or their mutations; FR3 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 55 or 63 or their mutations; and FR4 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 56 or their mutations. 64 or its mutants; or the popular FR1 heavy chain variant region of the anti-clodine18.2 antibody composed of sequences selected from the sequences shown in SEQIDNO: 71, 79, 87, 95, 103, 111 or 119 or its mutants; FR2 heavy chain variant region composed of sequences selected from the sequences shown in SEQIDNO: 72, 80, 88, 96, 104, 112 or 120 or its mutants; FR3 heavy chain variant region composed of sequences selected from the sequences shown in SEQIDNO: 73, 81, 89, 97, 105, 113 or 121 or its mutants.FR4 is a heavy variant region consisting of sequences selected from the sequences shown in SEQID NO: 74, 82, 90, 98, 106, 114, or 122, or their variants; FR1 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 127 or 135, or their variants; FR2 is a light variant region consisting of sequences selected from SEQID NO: 128 or 136, or their variants; FR3 is a light variant region consisting of sequences selected from the sequences shown in SEQID NO: 129 or 137, or their variants; and FR4 is a light variant region consisting of... The sequences selected were from the sequences shown in SEQIDNO:130 or 138 or its mutants; or the preferred FR1, FR2, FR3, FR4 heavy variable regions of the anti-clodine18.2 antibody was selected from the combination of the following sequences: (1)SEQIDNO:5,SEQIDNO:6,SEQIDNO:7,SEQIDNO:8(2)SEQ IDNO:13,SEQIDNO:14,SEQIDNO:15,SEQIDNO:16(3)SEQIDNO:21,SEQIDNO:22,SEQIDNO:23,SEQIDNO:24(4)SEQIDNO:29,SEQIDNO:30,SEQIDNO:31,SE QIDNO:32(5)SEQIDNO:37,SEQIDNO:38,SEQIDNO:39,SEQIDNO:40(6)SEQIDNO:45,SEQIDNO:46,SEQIDNO:47,SEQIDNO:48(7)SEQIDNO:71,SEQIDNO:72,SEQIDNO:73,SEQIDNO:74(8)SEQIDNO:79,SEQIDNO:80,SEQIDNO:81,SEQIDNO:82(9)SEQIDNO:87,SEQIDNO:88,SEQIDNO:89,SEQIDNO:90(10)SEQIDNO :95,SEQIDNO:96,SEQIDNO:97,SEQIDNO:98(11)SEQIDNO:103,SEQIDNO:104,SEQIDNO:105,SEQIDNO:106(12)SEQIDNO:111,SEQIDNO:112,SEQIDNO:11 3,SEQIDNO:114(13)SEQIDNO:119,SEQIDNO:120,SEQIDNO:121,SEQIDNO:122FR1,FR2,FR3 and FR4 light variant regions of anti-clodine18.2 antibodies were selected from the combination of the following sequences:(1)SEQIDNO:53,SEQIDNO:54,SEQIDNO:55,SEQIDNO:56(2)SEQIDNO:61,SEQIDNO:62,SEQIDNO :63,SEQIDNO:64(3)SEQIDNO:127,SEQIDNO:128,SEQIDNO:129,SEQIDNO:130(4)SEQIDNO:135,SEQIDNO:136,SEQIDNO:137,SEQIDNO:138 The more popular FR1,FR2,FR3 and FR4 heavy chain variation regions of anti-clodine18.2 antibodies were selected from the combination of the following sequences:(1)SEQIDNO:5 ,SEQIDNO:6,SEQIDNO:7,SEQIDNO:8(2)SEQIDNO:13,SEQIDNO:14,SEQIDNO:15,SEQIDNO:16(3)SEQIDNO:21,SEQIDNO:22,SEQIDNO:23,SEQIDNO:24(4) SEQIDNO:29,SEQIDNO:30,SEQIDNO:31,SEQIDNO:32(5)SEQIDNO:37,SEQIDNO:38,SEQIDNO:39,SEQIDNO:40(6)SEQIDNO:45,SEQIDNO:46,SEQIDNO:47,The light variant regions of anti-clodine18.2 antibodies, SEQIDNO:48FR1,FR2,FR3 and FR4, were selected from the following combinations of sequences:(1)SEQIDNO:53,SEQIDNO:54,SEQIDNO:55,SEQIDNO:56(2)SEQIDNO:61,SEQIDNO:62,SEQIDNO:63,SEQIDNO:64; or more commonly, FR1,FR2,FR3 and FR4, the heavy variant regions of anti-clodine18.2 antibodies, were selected from the following combinations of sequences:(1)SEQIDNO:71,SEQIDNO:72,SEQIDNO:73,SEQIDNO:74(2)SEQIDNO:79,SEQIDNO:80,SEQIDNO:81,SEQIDNO:82(3)SEQIDNO:87,SEQIDNO:88,SEQIDNO:8 9,SEQIDNO:90(4)SEQIDNO:95,SEQIDNO:96,SEQIDNO:97,SEQIDNO:98(5)SEQIDNO:103,SEQIDNO:104,SEQIDNO:105,SEQIDNO:106(6)SEQID NO:111,SEQIDNO:112,SEQIDNO:113,SEQIDNO:114(7)SEQIDNO:119,SEQIDNO:120,SEQIDNO:121,SEQIDNO:122FR1,FR2,FR3 and FR4 area The light chain conjugate of anti-clodine18.2 antibody was selected from the combination of the following sequences:(1)SEQIDNO:127,SEQIDNO:128,SEQIDNO:129,SEQIDNO:130(2)SEQIDNO:135,SEQIDNO:136,SEQIDNO:137,SEQIDNO:1384.The drug conjugate of claim1, salt,Its pharmaceutically acceptable solvate or the solvate of such salt in which the heavy chain variant regions of the anti-clodine 18.2 antibody were selected from the sequences shown in SEQID NO: 1, 9, 17, 25, 33, 41, 67, 75, 83, 91, 99, 107 or 115; the light chain variant regions of the anti-clodine 18.2 antibody were selected from the sequences shown in SEQID NO: 49, 57, 123 or 131; the preferred heavy chain variant regions of the anti-clodine 18.2 antibody were selected from the sequences shown in SEQID NO: QIDNO:1,9,17,25,33 or 41. The light chain variant region of anti-clodine18.2 antibody was selected from the sequences shown in SEQIDNO:49 or 57; or, more commonly, the heavy chain variant region of anti-clodine18.2 antibody was selected from the sequences shown in SEQIDNO:67,75,83,91,99,107 or 115. The light chain variant region of anti-clodine18.2 antibody was selected from the sequences shown in SEQIDNO:123 or 1315. Conjugated antibody against the drug of claim 1, salt,Its pharmaceutically acceptable solvate or the solvate of such salt in which the heavy chain variable region and light chain variable region of the anti-clodine18.2 antibody were selected from the combination of the following sequences: (1) SEQIDNO:17 and SEQIDNO:57, (2) SEQIDNO:41 and SEQIDNO:49, (3) SEQIDNO:41 and SEQIDNO:57, (4) SEQIDNO:115 and SEQIDNO:

131. The preferred sequences of the heavy chain variable region and light chain variable region of the anti-clodine18.2 antibody are SEQIDNO:41 and SEQIDNO:49, respectively.

6. Conjugated antibody against the drug of claim 1, salt, solvate. Its pharmacodynamically acceptable or solvate of such salt in which the heavy chain fixed region of anti-clodine 18.2 antibody has been selected from the fixed region of human IgG (i.e., IgG1, IgG2, IgG3, or IgG4), IgM, IgA, IgD, IgA, or mutagenesis of the above fixed regions, preferably human IgG1; the light chain fixed region of anti-clodine 18.2 antibody has been selected from the fixed region of human lambda, the fixed region of kappa, or mutagenesis of the above fixed regions, preferably human kappa; 7. Conjugated antibody against the drug of claim 1, salt,Its pharmaceutically acceptable solvate or the solvate of such salt in which the amino acid sequence of the heavy chain of the anti-clodine18.2 antibody is composed of the sequence as shown in SEQIDNO:65 or a sequence which has greater than 70% similarity, i.e., greater than 75%, 80%, 85%, 90%, 95%, 99% with SEQIDNO:65; the amino acid sequence of the light chain of the anti-clodine18.2 antibody is composed of the sequence as shown in SEQIDNO:66 or a sequence which has greater than 70% similarity, i.e., greater than 75%, 80%, 85%, 90%, 95%, 99% with SEQIDNO:

66.

8. Conjugated antibody to the drug of claim 1, salt, pharmaceutically acceptable solvate. The pharmaceutically acceptable conjugate antibody of the drug of claim 1, its pharmaceutically acceptable solvate or the solvate of such salt in which the cytotoxic substance has been selected is SN-38, gemcytabine, monomethyl aristatin E (MMAE), monomethyl aristatin F (MMAF), methansinoid (e.g. methansin DM1, methansin DM4), kaliciamycin,MGBA (e.g., duocarmycin), doxorubicin, ricin, diphtheria toxin and other toxins, I131, interleukin, tumor necrosis factor, chemokines and nanoparticles, and the popular cytotoxic agent is MMAE10. Conjugated antibody against the drug of claim 1, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate or solvate of such salt in which linkers have been selected from 6-maleimidocaproil (MC), maleimidopropionil (MP), N-succinimidyl 4-(2-pyridylthio)valerate (SPP), 4-(N-maleimidomethyl)-cyclohexan-1-formyl (MCC), N-succinimidyl (4-iodo-acetyl)aminobento The popular linker is 6-maleimidocappoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB).

11. Conjugated antibody against the drug of claim 1, its pharmaceutically acceptable salt, solvate or solvate of such salt in which the Ab includes (a) CDR1, CDR2, CDR3 heavy chain variant regions and CDR1, CDR2, CDR3 light chain variant regions in which the sequence of the CDR1 heavy chain variant region is shown in SEQID NO:42, the sequence of the CDR2 heavy chain variant region is shown in SEQID NO:43,(b) Heavy wire variation area and light wire variation area where the sequence of the heavy wire variation area is shown in SEQID NO:44, the sequence of the light wire variation area is shown in SEQID NO:50, the sequence of the light wire variation area is shown in SEQID NO:51 and the sequence of the light wire variation area is shown in SEQID NO:52; and / or (c) Heavy wire and light wire variation area where the sequence of the heavy wire variation area is shown in SEQID NO:41 and the sequence of the light wire variation area is shown in SEQID NO:49; and / or (c) Heavy wire and light wire where the sequence of the heavy wire is shown in SEQID NO:65 and the sequence of the light wire is shown in SEQID NO:

52. SEQIDNO:66L is MC-vc-PAB; and D is MMAE12. The composition shall include a conjugated antibody to a drug of any of the claims 1-11, its pharmaceutically acceptable salt, its solvate, or its solvate of such salt; by alternative, it shall be further composed of at least one chemotherapy, immunotherapy, or immunosuppressant drug known for the treatment of tumors; or by alternative, at least one pharmaceutically acceptable carrier, diluent, or drug filler.

13. The use of a conjugated antibody to a drug of any of the claims 1-11, its salt,The pharmaceutically acceptable solvate of it or the solvate of such salt or the components of claim 12 in the preparation of a drug for the prevention and / or treatment of diseases associated with claudin 18.

2. The most popular diseases associated with claudin 18.2 are gastric cancer, adenocarcinoma of the esophageal junction and pancreatic cancer; the more popular disease associated with claudin 18.2 is gastric cancer.

14. Method for the prevention and / or treatment of diseases associated with claudin 18.2 which includes: administration to the patient as needed of an effective preventive and / or therapeutic dose of the conjugated antibody against any of claims 1-11, salt, its pharmaceutically acceptable solvate or the solvate of such salt or the components of claim 12. The most popular diseases associated with claudin 18.2 are gastric cancer, adenocarcinoma of the esophageal junction and pancreatic cancer; the more popular disease associated with claudin 18.2 is gastric cancer.

15. Conjugated antibody against any of claims 1-11, salt,Its pharmaceutically acceptable solvate or its solvate of such salt or components of claim 12 for use in the prevention and / or treatment of diseases associated with claudin 18.

2. The most common diseases associated with claudin 18.2 are gastric cancer, adenocarcinoma of the esophageal junction, and pancreatic cancer.

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