Antibodies for treating tumors and use thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to antibodies for treating tumors and the use thereof. Provided are a ROR2-binding humanized antibody or an antigen-binding fragment thereof, and an ROR2-binding antibody or an antigen-binding fragment thereof. The antibodies can be used for treating ROR2-positive tumors, such as breast cancer, colon cancer, pancreatic cancer and blood cancer, have good anti-cancer activity, and have good clinical application and drug development values.

Description

An antibody for treating tumors and its application

[0001] Priority information

[0002] This application claims priority to Chinese Patent Application No. 202410825134.0, filed on June 21, 2024, entitled "An antibody for treating tumors and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedicine, and more specifically, to an antibody for treating tumors and its application. Background Technology

[0004] Cancer is a major disease affecting human survival and development. Immunotherapy, including tumor-targeting antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a new hope in the fight against cancer. Immunotherapy, represented by PD-1 / L1, has shown great potential, but even with PD-1 / L1 therapy, which currently has the broadest approved indications, many more patients still cannot benefit from it.

[0005] Therefore, the market for anti-tumor drugs urgently needs further development, especially the development of drugs for treating ROR2-positive tumors, as its abnormally high expression is common in cancers such as breast cancer, colon cancer, pancreatic cancer, and hematological malignancies, and it is very easy for these tumors to worsen. Summary of the Invention

[0006] The present invention aims to at least partially solve one of the technical problems in the related art.

[0007] Therefore, a first aspect of the present invention provides a humanized antibody or antigen-binding fragment thereof that binds to ROR2. According to an embodiment of the present invention, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the CDR sequence of the heavy chain variable region and the CDR sequence of the light chain variable region are selected from any one of the following or amino acid sequences having at least 95% identity with them:

[0008] Heavy chain variable region CDR sequences: SEQ ID NO: 1~18;

[0009] Light chain variable region CDR sequence: SEQ ID NO: 19~36.

[0010] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment comprises:

[0011] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:1, 2, and 3 or amino acid sequences having at least 95% identity with SEQ ID NO:1, 2, and 3; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:19, 20, and 21 or amino acid sequences having at least 95% identity with SEQ ID NO:19, 20, and 21; or

[0012] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:4, 5, and 6 or amino acid sequences having at least 95% identity with SEQ ID NO:4, 5, and 6; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:22, 23, and 24 or amino acid sequences having at least 95% identity with SEQ ID NO:22, 23, and 24; or

[0013] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:7, 8, and 9 or amino acid sequences having at least 95% identity with SEQ ID NO:7, 8, and 9; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:25, 26, and 27 or amino acid sequences having at least 95% identity with SEQ ID NO:25, 26, and 27; or

[0014] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:10, 11, and 12 or amino acid sequences having at least 95% identity with SEQ ID NO:10, 11, and 12; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:28, 29, and 30 or amino acid sequences having at least 95% identity with SEQ ID NO:28, 29, and 30; or

[0015] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:13, 14, and 15 or amino acid sequences having at least 95% identity with SEQ ID NO:13, 14, and 15; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:31, 32, and 33 or amino acid sequences having at least 95% identity with SEQ ID NO:31, 32, and 33; or

[0016] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences, respectively, as shown in SEQ ID NO:16, 17, and 18 or amino acid sequences having at least 95% identity with SEQ ID NO:16, 17, and 18; and the light chain variable regions CDR1, CDR2, and CDR3 sequences, respectively, as shown in SEQ ID NO:34, 35, and 36 or amino acid sequences having at least 95% identity with SEQ ID NO:34, 35, and 36.

[0017] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment specifically recognizes ROR2.

[0018] In other embodiments of the present invention, the humanized antibody or its antigen-binding fragment contains a heavy chain framework region sequence, the heavy chain framework region sequence including FR1, FR2, FR3 and FR4;

[0019] Optionally, the FR1 has the amino acid sequence shown in SEQ ID NO:37 or has an L2V mutation amino acid sequence compared to the amino acid sequence shown in SEQ ID NO:37; and / or

[0020] The FR2 has the amino acid sequence shown in SEQ ID NO:39; and / or

[0021] The FR3 has the amino acid sequence shown in any one of SEQ ID NO:40-42; and / or

[0022] The FR4 has the amino acid sequence shown in SEQ ID NO:43.

[0023] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment has a heavy chain variable region as shown in any one of SEQ ID NO: 44-46.

[0024] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment contains a light chain framework region sequence, the light chain framework region sequence including FR1, FR2, FR3 and FR4;

[0025] Optionally, the FR1 has the amino acid sequence shown in any one of SEQ ID NO:47-49; and / or

[0026] The FR2 has the amino acid sequence shown in SEQ ID NO:50; and / or

[0027] The FR3 has the amino acid sequence shown in SEQ ID NO:51 or, compared to the amino acid sequence shown in SEQ ID NO:51, has an L2R mutated amino acid sequence; and / or

[0028] The FR4 has the amino acid sequence shown in SEQ ID NO:53.

[0029] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment has a light chain variable region as shown in any one of SEQ ID NO: 54-56.

[0030] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment contains a heavy chain framework region sequence, the heavy chain framework region sequence including FR1, FR2, FR3 and FR4;

[0031] Optionally, the FR1 has the amino acid sequence shown in SEQ ID NO:57 or has an A25S mutation amino acid sequence compared to the amino acid sequence shown in SEQ ID NO:57; and / or

[0032] The FR2 has the amino acid sequence shown in SEQ ID NO:59; and / or

[0033] The FR3 has the amino acid sequence shown in SEQ ID NO:60 or, compared to the amino acid sequence shown in SEQ ID NO:60, has a K16T mutated amino acid sequence; and / or

[0034] The FR4 has the amino acid sequence shown in SEQ ID NO:62.

[0035] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment has a heavy chain variable region as shown in any one of SEQ ID NO:63-65.

[0036] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment contains a light chain framework region sequence, the light chain framework region sequence including FR1, FR2, FR3 and FR4;

[0037] Optionally, the FR1 has the amino acid sequence shown in any one of SEQ ID NO:66-68; and / or

[0038] The FR2 has the amino acid sequence shown in SEQ ID NO:69 or, compared to the amino acid sequence shown in SEQ ID NO:69, has a W15L mutated amino acid sequence; and / or

[0039] The FR3 has the amino acid sequence shown in any one of SEQ ID NO:71-73; and / or

[0040] The FR3 has the amino acid sequence shown in SEQ ID NO:74.

[0041] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment has a light chain variable region as shown in any one of SEQ ID NO:75-77.

[0042] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment has a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NO: 44-46 and a light chain variable region having an amino acid sequence shown in any one of SEQ ID NO: 54-56; or

[0043] The humanized antibody or its antigen-binding fragment has a heavy chain variable region having an amino acid sequence shown in any one of SEQ ID NO: 63-65 and a light chain variable region having an amino acid sequence shown in any one of SEQ ID NO: 75-77.

[0044] In some embodiments of the present invention, the humanized antibody or its antigen-binding fragment further comprises a heavy chain constant region, at least a portion of which is derived from a human IgG antibody or a mutant thereof.

[0045] A second aspect of the present invention provides an antibody or antigen-binding fragment thereof that binds to ROR2. In some embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the CDR sequence of the heavy chain variable region and the CDR sequence of the light chain variable region are selected from any one of the following or amino acid sequences having at least 95% identity with them:

[0046] Heavy chain variable region CDR sequence:

[0047] (1) X1YTFT SYX2MHWV, where X1 is G or empty, X2 is L or Y, and X1 and X2 do not satisfy the following: X1 is empty, X2 is L;

[0048] (2)X3X4X5X6X7X8INPYNX9X 10 TX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 Where X3 is L or empty, X4 is E or empty, X5 is W or empty, X6 is I or empty, X7 is G or empty, X8 is Y or empty, X9 is D or G, X 10 For G or S, X 11 X is K, N, or empty.12 Y or empty, X 13 X is A, N, or empty. 14 E, Q, or empty, X 15 For K or empty, X 16 F or empty, X 17 For K, Q, or empty, X 18 For D, G, or empty, X 19 For K, R, or empty, X 20 A or empty, X 21 For T or empty,

[0049] And X3-X 21 The following conditions must not be met simultaneously: X3 is L, X4 is E, X5 is W, X6 is I, X7 is G, X8 is Y, X9 is D or G, X 10 For G or S, X 11 For K or N, X 12 Let Y and X be the numbers. 13 If N or empty, X 14 E or empty, X 15 Let K, X 16 Let F, X 17 For K or empty, X 18 For D or empty, X 19 For K or empty, X 20 Let A and X be the two numbers. 21 Let T be the value of T.

[0050] (3)ARSD VYYGVRFAYX 22 X 23 X 24 X 25 , where X 22 For W or empty, X 23 G or empty, X 24 Q or empty, X 25 G or empty,

[0051] And X 22 -X 25 The following conditions must not be met simultaneously: X 22 For W, X 23 For G, X 24 For Q, X 25 G;

[0052] (4) The sequences shown in SEQ ID NO: 78-140;

[0053] Light chain variable region CDR sequence:

[0054] (5)X 26 X 27 X 28 X 29 X 30SVTYX 31 X 32 X 33 X 34 X 35 , where X 26 C or empty, X 27 S or R or empty, X 28 A or empty, X 29 S or empty, X 30 S or Q or empty, X 31 For T or empty, X 32 Y or empty, X 33 For W or empty, X 34 Y or empty, X 35 Q or empty,

[0055] And the condition is X 26 -X 35 The following conditions must not be met simultaneously: X 26 Let C, X 27 For S or R, X 28 Let A and X be the two numbers. 29 Let S and X be the values ​​of S and X. 30 For S or Q, X 31 Let T, X 32 Let Y and X be the numbers. 33 For W, X 34 Let Y and X be the numbers. 35 For Q;

[0056] (6)X 36 X 37 DTSX 38 X 39 X 40 X 41 , where X 36 For I or empty, X 37 Y or empty, X 38 If N or empty, X 39 For L, R, or empty, X 40 A or empty, X 41 For T, S, or empty.

[0057] And the condition is X 36 -X 41 The following conditions are not met: X 36 For I, X 37 Let Y and X be the numbers. 38 Let N, X 39 For L or empty, X 40 Let A and X be the two numbers. 41 S or empty;

[0058] (7)X 42 QQWSSYPFTX 43 X 44X 45 X 46 , where X 42 C or empty, X 43 F or empty, X 44 G or empty, X 45 S or Q or empty, X 46 G or empty,

[0059] And the condition is X 42 -X 46 The following conditions are not met: X 42 Let C, X 43 Let F, X 44 For G, X 45 S or empty, X 46 G;

[0060] (8) The sequences shown in SEQ ID NO: 141-203.

[0061] In some embodiments of the present invention, the CDR sequence of the heavy chain variable region and the CDR sequence of the light chain variable region are selected from any one of the following or amino acid sequences having at least 95% identity with them:

[0062] Heavy chain variable region CDR sequence: SEQ ID NO: 204-215;

[0063] Light chain variable region CDR sequence: SEQ ID NO: 216-227.

[0064] In some embodiments of the present invention, the antibody or its antigen-binding fragment comprises:

[0065] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:78, 79, and 80 or amino acid sequences having at least 95% identity with SEQ ID NO:78, 79, and 80; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:141, 142, and 143 or amino acid sequences having at least 95% identity with SEQ ID NO:141, 142, and 143; or

[0066] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:81, 82, and 83 or amino acid sequences having at least 95% identity with SEQ ID NO:81, 82, and 83; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:144, 145, and 146 or amino acid sequences having at least 95% identity with SEQ ID NO:144, 145, and 146; or

[0067] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:84, 85, and 86 or amino acid sequences having at least 95% identity with SEQ ID NO:84, 85, and 86; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:147, 148, and 149 or amino acid sequences having at least 95% identity with SEQ ID NO:147, 148, and 149; or

[0068] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:87, 88, and 89 or amino acid sequences having at least 95% identity with SEQ ID NO:87, 88, and 89; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:150, 151, and 152 or amino acid sequences having at least 95% identity with SEQ ID NO:150, 151, and 152; or

[0069] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:90, 91, and 92 or amino acid sequences having at least 95% identity with SEQ ID NO:90, 91, and 92; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:153, 154, and 155 or amino acid sequences having at least 95% identity with SEQ ID NO:153, 154, and 155; or

[0070] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:93, 94, and 95 or amino acid sequences having at least 95% identity with SEQ ID NO:93, 94, and 95; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:156, 157, and 158 or amino acid sequences having at least 95% identity with SEQ ID NO:156, 157, and 158; or

[0071] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:96, 97, and 98 or amino acid sequences having at least 95% identity with SEQ ID NO:96, 97, and 98; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:159, 160, and 161 or amino acid sequences having at least 95% identity with SEQ ID NO:159, 160, and 161; or

[0072] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:99, 100, and 101 or amino acid sequences having at least 95% identity with SEQ ID NO:99, 100, and 101; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:162, 163, and 164 or amino acid sequences having at least 95% identity with SEQ ID NO:162, 163, and 164; or

[0073] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:102, 103, and 104 or amino acid sequences having at least 95% identity with SEQ ID NO:102, 103, and 104; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:165, 166, and 167 or amino acid sequences having at least 95% identity with SEQ ID NO:165, 166, and 167; or

[0074] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:105, 106, and 107 or amino acid sequences having at least 95% identity with SEQ ID NO:105, 106, and 107; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:168, 169, and 170 or amino acid sequences having at least 95% identity with SEQ ID NO:168, 169, and 170; or

[0075] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:108, 109, and 110 or amino acid sequences having at least 95% identity with SEQ ID NO:108, 109, and 110; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:171, 172, and 173 or amino acid sequences having at least 95% identity with SEQ ID NO:171, 172, and 173; or

[0076] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:111, 112, and 113 or amino acid sequences having at least 95% identity with SEQ ID NO:111, 112, and 113; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:174, 175, and 176 or amino acid sequences having at least 95% identity with SEQ ID NO:174, 175, and 176; or

[0077] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:114, 115, and 116 or amino acid sequences having at least 95% identity with SEQ ID NO:114, 115, and 116; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:177, 178, and 179 or amino acid sequences having at least 95% identity with SEQ ID NO:177, 178, and 179; or

[0078] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:117, 118, and 119 or amino acid sequences having at least 95% identity with SEQ ID NO:117, 118, and 119; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:180, 181, and 182 or amino acid sequences having at least 95% identity with SEQ ID NO:180, 181, and 182; or

[0079] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:120, 121, and 122 or amino acid sequences having at least 95% identity with SEQ ID NO:120, 121, and 122; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:183, 184, and 185 or amino acid sequences having at least 95% identity with SEQ ID NO:183, 184, and 185; or

[0080] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:123, 124, and 125 or amino acid sequences having at least 95% identity with SEQ ID NO:123, 124, and 125; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:186, 187, and 188 or amino acid sequences having at least 95% identity with SEQ ID NO:186, 187, and 188; or

[0081] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:126, 127, and 128 or amino acid sequences having at least 95% identity with SEQ ID NO:126, 127, and 128; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:189, 190, and 191 or amino acid sequences having at least 95% identity with SEQ ID NO:189, 190, and 191; or

[0082] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:129, 130, and 131 or amino acid sequences having at least 95% identity with SEQ ID NO:129, 130, and 131; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:192, 193, and 194 or amino acid sequences having at least 95% identity with SEQ ID NO:192, 193, and 194; or

[0083] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:132, 133, and 134 or amino acid sequences having at least 95% identity with SEQ ID NO:132, 133, and 134; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:195, 196, and 197 or amino acid sequences having at least 95% identity with SEQ ID NO:195, 196, and 197; or

[0084] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:135, 136, and 137 or amino acid sequences having at least 95% identity with SEQ ID NO:135, 136, and 137; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:198, 199, and 200 or amino acid sequences having at least 95% identity with SEQ ID NO:198, 199, and 200; or

[0085] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:138, 139, and 140 or amino acid sequences having at least 95% identity with SEQ ID NO:138, 139, and 140; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:201, 202, and 203 or amino acid sequences having at least 95% identity with SEQ ID NO:201, 202, and 203; or

[0086] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:204, 205, and 206 or amino acid sequences having at least 95% identity with SEQ ID NO:204, 205, and 206; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:216, 217, and 218 or amino acid sequences having at least 95% identity with SEQ ID NO:216, 217, and 218; or

[0087] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:207, 208, and 209 or amino acid sequences having at least 95% identity with SEQ ID NO:207, 208, and 209; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:219, 220, and 221 or amino acid sequences having at least 95% identity with SEQ ID NO:219, 220, and 221; or

[0088] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:210, 211, and 212 or amino acid sequences having at least 95% identity with SEQ ID NO:210, 211, and 212; the light chain variable regions CDR1, CDR2, and CDR3 sequences respectively, as shown in SEQ ID NO:222, 223, and 224 or amino acid sequences having at least 95% identity with SEQ ID NO:222, 223, and 224; or

[0089] The heavy chain variable regions CDR1, CDR2, and CDR3 sequences, respectively, are shown in SEQ ID NO:213, 214, and 215 or amino acid sequences having at least 95% identity with SEQ ID NO:213, 214, and 215; and the light chain variable regions CDR1, CDR2, and CDR3 sequences, respectively, are shown in SEQ ID NO:225, 226, and 227 or amino acid sequences having at least 95% identity with SEQ ID NO:225, 226, and 227.

[0090] In some embodiments of the present invention, the antibody or its antigen-binding fragment has a heavy chain variable region of any of the amino acid sequences shown in SEQ ID NO:228-252.

[0091] In some embodiments of the present invention, the antibody or its antigen-binding fragment has a light chain variable region of any of the amino acid sequences shown in SEQ ID NO:253-277.

[0092] In some embodiments of the present invention, the antibody or its antigen-binding fragment has a heavy chain variable region of the amino acid sequence shown in any one of SEQ ID NO:228-252 and a light chain variable region of the amino acid sequence shown in any one of SEQ ID NO:253-277.

[0093] A third aspect of the present invention provides a nucleic acid molecule. In some embodiments of the present invention, the nucleic acid molecule encodes the humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect, or the antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect.

[0094] A fourth aspect of the present invention provides an expression vector. In some embodiments of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect.

[0095] In some embodiments of the present invention, the expression vector is a eukaryotic expression vector.

[0096] A fifth aspect of the present invention provides a recombinant cell. In some embodiments of the present invention, the recombinant cell carries the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect, or expresses the humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect or the antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect.

[0097] In some embodiments of the present invention, the recombinant cells are obtained by introducing the expression vector described in the fourth aspect into a host cell.

[0098] It should be noted that the recombinant cells described in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this invention are preferably prokaryotic or eukaryotic cells, and the recombinant cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0099] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the humanized antibody or its antigen-binding fragment that binds ROR2 as described in the first aspect of this application, or the antibody or its antigen-binding fragment that binds ROR2 as described in the second aspect. Those skilled in the art will readily understand that the suitable expression conditions include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal expression conditions for the humanized antibody or its antigen-binding fragment that binds ROR2 as described in the first aspect, or the antibody or its antigen-binding fragment that binds ROR2 as described in the second aspect, based on the specific environment of their laboratory.

[0100] In some embodiments of the present invention, the recombinant cells are eukaryotic cells;

[0101] Optionally, the recombinant cells are mammalian cells.

[0102] A sixth aspect of the present invention provides a pharmaceutical composition. In some embodiments of the present invention, the pharmaceutical composition comprises a humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect, an antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect, a nucleic acid molecule as described in the third aspect, an expression vector as described in the fourth aspect, or a recombinant cell as described in the fifth aspect.

[0103] According to embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc. Specific examples may be water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and one or more combinations thereof. In many cases, the pharmaceutical composition includes isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Of course, a pharmaceutically acceptable carrier may also include trace amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, to prolong the shelf life or potency of the antibody or its antigen-binding fragment.

[0104] For example, the humanized antibody or antigen-binding fragment thereof that binds to ROR2, or the antibody or antigen-binding fragment thereof that binds to ROR2, described in this invention, can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms, such as liquids, semi-solids, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions. The humanized antibody or antigen-binding fragment thereof that binds to ROR2, or the antibody or antigen-binding fragment thereof that binds to ROR2, can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0105] In some embodiments, the pharmaceutical composition comprises combinations that are separate in time and / or space, provided that they can work together to achieve the objectives of the invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0106] The seventh aspect of this invention provides the use of the humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect, the antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, the recombinant cell as described in the fifth aspect, and the pharmaceutical composition as described in the sixth aspect in the preparation of a medicament. In some embodiments of this invention, the medicament is used to treat tumors.

[0107] In some embodiments of the present invention, the tumor is a ROR2-positive tumor.

[0108] In some embodiments of the present invention, the tumor includes breast cancer, colon cancer, pancreatic cancer, and blood cancer.

[0109] An eighth aspect of the present invention provides a kit for detecting ROR2. In some embodiments of the present invention, the kit comprises a humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect, or an antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect.

[0110] The kits provided by this invention can be used, for example, in immunoblotting, immunoprecipitation, and other kits involving the detection of ROR2 antigens and antibodies using their specific binding properties. These kits may contain any one or more of the following: an antagonist, a ROR2 antibody, or a drug reference material; a protein purification column; an immunoglobulin affinity purification buffer; a cell assay diluent; instructions or literature, etc. ROR2 antibodies or their antigen-binding fragments can be used in various types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for related diseases by detecting the serum or blood of a subject. Such related diseases may include tumors, such as one or more of breast cancer, colon cancer, pancreatic cancer, and hematologic malignancies.

[0111] The ninth aspect of the present invention provides the use of the humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect, the antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, or the recombinant cells as described in the fifth aspect in the preparation of a kit for detecting ROR2.

[0112] A tenth aspect of the present invention provides a chimeric antigen receptor against ROR2. In some embodiments of the present invention, the chimeric antigen receptor against ROR2 includes an extracellular region, a transmembrane region, and an intracellular region.

[0113] The extracellular region includes an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment is the humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect, or the antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect.

[0114] The eleventh aspect of this invention provides a method for treating tumors. In some embodiments of this invention, the method comprises administering to a subject a pharmaceutically effective amount of at least one of the following: the humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect, the antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cells described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect.

[0115] In some embodiments of the present invention, the tumor is a ROR2-positive tumor.

[0116] In some embodiments of the present invention, the tumor includes breast cancer, colon cancer, pancreatic cancer, and blood cancer.

[0117] The twelfth aspect of this invention provides a method for diagnosing whether a subject has a tumor. In some embodiments of this invention, the method includes detecting a test sample from the subject using at least one of the following: a humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect, an antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect, a nucleic acid molecule as described in the third aspect, an expression vector as described in the fourth aspect, recombinant cells as described in the fifth aspect, or a kit as described in the eighth aspect.

[0118] In some embodiments of the present invention, the tumor is a ROR2-positive tumor.

[0119] In some embodiments of the present invention, the tumor includes breast cancer, colon cancer, pancreatic cancer, and blood cancer.

[0120] In some embodiments of the present invention, the sample to be tested includes at least one of the following: tissue, cells, blood, serum, plasma, saliva, sweat, feces, or urine.

[0121] The thirteenth aspect of the present invention provides the use of at least one of the following in treating tumors: the humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect; the antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect; the nucleic acid molecule as described in the third aspect; the expression vector as described in the fourth aspect; the recombinant cell as described in the fifth aspect; or the pharmaceutical composition as described in the sixth aspect.

[0122] In some embodiments of the present invention, the tumor is a ROR2-positive tumor.

[0123] In some embodiments of the present invention, the tumor includes breast cancer, colon cancer, pancreatic cancer, and blood cancer.

[0124] The fourteenth aspect of the present invention provides the use of at least one of the following in diagnosing whether a subject has a tumor: the humanized antibody or antigen-binding fragment thereof that binds ROR2 as described in the first aspect; the antibody or antigen-binding fragment thereof that binds ROR2 as described in the second aspect; the nucleic acid molecule as described in the third aspect; the expression vector as described in the fourth aspect; the recombinant cells as described in the fifth aspect; or the kit as described in the eighth aspect.

[0125] In some embodiments of the present invention, the tumor is a ROR2-positive tumor.

[0126] In some embodiments of the present invention, the tumor includes breast cancer, colon cancer, pancreatic cancer, and blood cancer.

[0127] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0128] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0129] Figure 1 shows the ELISA results of different murine antibodies binding to ROR2 antigen according to embodiments of the present invention;

[0130] Figure 2 shows the SDS-PAGE identification results of the purified humanized antibodies B16 and A12 provided according to an embodiment of the present invention;

[0131] Figure 3 shows the OD450 values ​​of the ELISA method provided according to an embodiment of the present invention for detecting the binding of humanized antibodies B16 and A12 to the extracellular domain antigen of ROR2;

[0132] Figure 4 shows the affinity kinetics determination results of the B16 and A12 humanized antibodies provided according to embodiments of the present invention;

[0133] Figure 5 shows the results of flow cytometry detection of ROR2 protein expression levels on the surface of T-47D (T47D) and MDA-MB-231 (231) cells using different concentrations of humanized antibodies B16 and A12 provided in the embodiments of the present invention;

[0134] Figure 6 shows the results of ROR2 antigen endocytosis mediated by B16 and A12 humanized antibodies provided according to an embodiment of the present invention;

[0135] Figure 7 shows the effect of the B16 humanized antibody provided according to an embodiment of the present invention on the primary cell invasion ability of breast cancer T47D cells and pancreatic cancer patient-derived tumor transplant tissue (PDX);

[0136] Figure 8 shows the effect of the B16 humanized antibody provided according to an embodiment of the present invention on PDX colorectal cancer tumor cells.

[0137] Figure 9 shows the effect of the B16 humanized antibody provided according to an embodiment of the present invention on PDX colorectal cancer tumor cells;

[0138] Figure 10 shows the effect of the humanized B16 antibody (e.g., B16-H1L1) provided according to embodiments of the present invention on the colony formation of acute myeloid leukemia (AML) cells. The left image is a representative diagram of AML cell lines forming colonies on a methylcellulose semi-solid matrix containing either the control antibody (Control) or B16-H1L1 (50 μg / mL), with a scale bar of 100 μm. The right bar chart represents the average number of colonies formed for each sample (n=3), statistically analyzed using a t-test. A represents the OCI-AML5 (ROR2-positive) cell line, and B represents the THP-1 stable cell line overexpressing ROR2.

[0139] Figure 11 shows that treatment with the anti-ROR2 humanized antibody B16-H1L1 provided according to an embodiment of the present invention significantly inhibited tumor growth and metastasis, where A is a representative photograph obtained in the invasion assay after treatment with the anti-ROR2 humanized antibody B16-H1L1 or the control antibody overexpressing ROR2 pancreatic cancer PANC-1 cells; the histogram represents the mean ± SD of the mean number of invasive cells per field of view; B is the tumor burden determined by the IVIS imaging system; 5 x 10 4 Luciferase-transfected ROR2-overexpressing PANC-1 cells were injected into the pancreas of immunodeficient NCG mice; B16-H1L1 was administered at a dose of 10 mg / kg body weight twice weekly, with imaging performed twice weekly; the line plot on the right shows the results of luciferase signal quantification to represent tumor burden size; C represents the tumor burden of the pancreas, liver, and spleen in mice with pancreatic cancer cells treated with B16-H1L1 or a control antibody, assessed by in vitro bioluminescence imaging; the dot plot represents the mean luciferase signal ± SD in mice. Detailed Implementation

[0140] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0141] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0142] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0143] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0144] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0145] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0146] Terms and Definitions

[0147] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0148] In this article, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases.

[0149] In this paper, certain regions within the variable region exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the location where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0150] In this document, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment," and can include a portion of a complete antibody, generally an antigen-binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated," "PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol).

[0151] In this article, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (such as a mouse) with the constant region of an antibody from another species (such as a human).

[0152] In this article, the term "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv backbone (FR)) amino acid sequences in the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with the non-CDR amino acid sequences in the constant and variable regions of an antibody from another species (e.g., human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when all non-CDR amino acid sequences in both the constant and variable regions are humanized, it is called a humanized antibody. The humanization method can be performed using conventional antibody engineering techniques, and will not be elaborated upon here.

[0153] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0967284, EP 1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0154] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0155] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0156] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0157] In this document, the term "treatment" means used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0158] In this document, the term "diagnosis" refers to the identification, revelation, ascertainment, and / or definition of the localization of a pathological state, disease, or condition. In some specific embodiments, the pharmaceutical compositions of the present invention, when administered to a subject or exposed to samples from a subject, aid in the diagnosis of cancer, tumor formation, or condition.

[0159] In many embodiments, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has received or is currently receiving any form of treatment. As used herein, the terms "subject" or "patient" refer to a mammalian subject or patient. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, poultry, goats, and sheep. In some embodiments, the subject is a human. In some embodiments, the subject is a person suspected of having a tumor.

[0160] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0161] The sequence information involved in this invention is shown in Table 1 below:

[0162] Table 1 Note: *CDR stands for complementarity determining region; **FR stands for framework region.

[0163] Table 2 summarizes the proportion of humanized sequences in each region of the humanized antibodies B16-H1L1, B16-H2L2, B16-H3L3, and A12-H1L1, A12-H2L2, and A12-H3L3, as well as the proportion of humanized sequences in the complete antibodies. For example, the 82 / 89 and 92.1% proportions of the backbone region in the heavy chain variable region of B16-H1L1 indicate that the backbone region of the heavy chain variable region of B16-H1L1 antibody contains 89 amino acids, of which 82 are humanized sequences, and the proportion of humanized sequences in the backbone region of the heavy chain variable region of B16-H1L1 is 92.1%. The percentages of complementarity-determining regions (CDRs) in the B16-H1L1 heavy chain variable region are 24 / 31 and 77.4%, respectively. In the B16-H1L1 antibody, the CDRs consist of 31 amino acids, of which 24 are human sequences, representing 77.4% of the total CDRs. Similarly, the percentages of the entire B16-H1L1 heavy chain are 436 / 450 and 96.9%, respectively. In the B16-H1L1 antibody, the heavy chain consists of 450 amino acids, of which 436 are human sequences, representing 96.9% of the total heavy chain. This indicates a high level of antibody humanization. The remaining antibody data in the table are annotated as above.

[0164] Table 2

[0165] Example 1: Expression and purification of chimeric antibodies against ROR2

[0166] pCDNA3.1 plasmid (purchased from Guangzhou Aiji Biotechnology Co., Ltd.) expressing the human ROR2 protein extracellular fragment and a 6x his tag was transfected into HEK293F cells. Cells were cultured in FreeStyle medium, and the culture supernatant was collected after 4 days. The supernatant was purified using a nickel column and concentrated by ultrafiltration to obtain the human ROR2 extracellular fragment. Five BALB / c mice were immunized with the human ROR2 extracellular fragment and adjuvant. Mice that produced high-titer anti-ROR2 serum were selected, and spleen cells were obtained and RNA was extracted. The RNA was reverse transcribed into cDNA and amplified by PCR using specific primers to obtain a phage Fab antibody library. Candidate clones that could bind to the immobilized human ROR2 protein extracellular domain were screened using ELISA, sequenced, and expressed as full-length antibodies in HEK293F cells. Antibodies were purified by Protein A affinity chromatography: B1, B3, B5, B6, B8, B9, B10, B17, B18, B21, B23, B26, B28, B31, and A7. The amino acid sequences of each antibody are shown in Table 1.

[0167] The binding ability of candidate clonal antibodies to the extracellular domain of human ROR2 protein was detected by ELISA. ELISA plates were coated with 2 μg / mL human ROR2 protein extracellular domain, blocked with 5% milk, and incubated with different concentrations of candidate antibodies at room temperature for 1 hour. The plates were then detected with HRP-labeled goat anti-mouse secondary antibody and TMB colorimetric assay. The absorbance at 450 nm was read by an ELISA reader. The results are shown in Figure 1. All the screened candidate clonal antibodies could bind to the extracellular domain of human ROR2.

[0168] Example 2: Expression and purification of humanized anti-ROR2 monoclonal antibody

[0169] Based on the amino acid sequences of the previously obtained B16 chimeric antibody (B16) and A12 chimeric antibody (A12), the modified antibodies were used. The amino acid sequences of the B16 and A12 chimeric antibodies are shown in Table 1. The expression sequences of the six modified humanized antibodies, B16-H1L1, B16-H2L2, B16-H3L3 and A12-H1L1, A12-H2L2, A12-H3L3 (see Table 1), were cloned into PSV0 vectors (purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd.) and transfected into CHO cells for antibody expression. After 6 days of culture, the cell supernatant was collected and purified and concentrated using a Protein A column. The purified protein was identified by SDS-PAGE gel chromatography, and the results are shown in Figure 2. The results show that the purified antibodies have high purity.

[0170] In Figure 2A, from left to right, the first, second, third, and fourth columns represent the non-reducing SDS-PAGE gel results of humanized antibodies B16-H1L1, B16-H2L2, B16-H3L3, and A12 chimeric antibodies with a loading amount of 1 μg, respectively; the fifth and sixth columns represent the standards with loading amounts of 1 μg and 0.5 μg, respectively; the seventh column represents the protein marker; and the eighth, ninth, tenth, and eleventh columns represent the reducing SDS-PAGE gel results of humanized antibodies A12-H1L1, A12-H2L2, A12-H3L3, and A12 chimeric antibodies with a loading amount of 2 μg, respectively. In Figure 2B, from left to right, columns 1 through 5 represent the non-reducing SDS-PAGE gel results for humanized antibodies B16-H1L1, B16-H2L2, B16-H3L3, and A12 chimeric antibodies and standards, respectively, with a loading amount of 1 μg. Column 6 represents the protein markers. Columns 7 through 10 represent the reducing SDS-PAGE gel results for humanized antibodies A12-H1L1, A12-H2L2, A12-H3L3, and A12 chimeric antibodies, respectively, with a loading amount of 2 μg. As can be seen from Figure 2, the reducing and non-reducing humanized antibody bands in the SDS-PAGE gel are consistent in size with those of normal antibodies, and the band gray values ​​are close to those of the standards, indicating that the purified three B16 humanized antibodies and three A12 humanized antibodies all have high purity.

[0171] Example 3: ELISA verification of the affinity of humanized antibodies B16 and A12 for the extracellular domain of ROR2 protein.

[0172] The extracellular fragments of ROR2 and ROR1 were coated onto 96-well plates overnight and blocked with 5% milk. Purified antibodies (10 μg / mL) were then added to different wells for ELISA detection. The experimental procedures were standard in the field. The OD450 values ​​of the humanized antibodies binding to the ROR2 extracellular fragment are shown in Figure 3A. It can be seen that the humanized antibodies B16-H1L1, B16-H2L2, B16-H1L2, B16-H1L3, and A12-H1L1, A12-H2L2, A12-H3L3 showed good affinity even at low concentrations, indicating that humanized antibodies can be used in ELISA. As a negative control, the OD450 values ​​of the humanized antibodies binding to the ROR1 extracellular fragment are shown in Figure 3B. UC961 is the antibody that recognizes the ROR1 extracellular fragment, indicating that the above-mentioned humanized antibodies have good specificity.

[0173] It should be noted that the heavy chain of antibody B16-H1L2 has the same sequence as the heavy chain of antibody B16-H1L1, and the light chain of antibody B16-H1L2 has the same sequence as the light chain of antibody B16-H2L2; the heavy chain of antibody B16-H1L3 has the same sequence as the heavy chain of antibody B16-H1L1, and the light chain of antibody B16-H1L3 has the same sequence as the light chain of antibody B16-H3L3.

[0174] Example 4: Protein-protein interaction assay to determine affinity kinetics of humanized antibodies B16 and A12

[0175] B16 chimeric antibody (B16), A12 chimeric antibody (A12), B16 humanized antibodies (B16-H1L1, B16-H2L2, B16-H3L3), and A12 humanized antibodies (A12-H1L1, A12-H2L2, A12-H3L3) were diluted to a concentration of 10 μg / mL and loaded onto the protein interaction analyzer (Fortebio Octet RED96) sensor, following standard procedures in the field. The ROR2 extracellular antigen was then prepared into three concentration gradients: 20 nM, 5 nM, and 1.25 nM. Affinity with different antibodies was measured, and the dissociation constant (K0) was calculated. D (Value). As shown in Figure 4, the K values ​​of six humanized antibodies. D The values ​​all reached the nM level, indicating that they have a high affinity for the ROR2 antigen.

[0176] Example 5: Flow cytometry detection of antibody binding ability

[0177] The three purified B16 humanized antibodies and three A12 humanized antibodies obtained above were subjected to flow cytometry staining, following standard procedures in the field, to detect T47D cells (antibody concentrations of 0.1, 1, and 10 μg / mL, respectively, see Figure 5). When T47D cells (purchased from ATCC, American Center for Type Culture Collection) were detected with different concentrations of humanized antibodies B16-H1L1, B16-H1L2, B16-H1L3 and A12-H1L1, A12-H2L2 and A12-H3L3 antibodies, the fluorescence intensity of the flow cytometry showed a gradient change, and high sensitivity was achieved even at a low antibody concentration of 1 μg / mL.

[0178] Example 6: Flow cytometry comparison of binding ability between humanized antibodies and commercial antibodies

[0179] Humanized antibodies B16-H1L1 and A12-H1L1 were conjugated with Alexa Flour-488 dye, and their interaction with the commercial antibody hROR2 Alexa was detected by flow cytometry. The binding ability of 488-conjugated antibody (catalog number: FAB20641G, R&R) to the cell surface ROR2 protein was measured. The results are shown in Figure 6. At a concentration of 10 μg / mL, the fluorescence signal intensity of the B16-H1L1 and A12-H1L1 antibodies was significantly higher than that of the commercial antibody, indicating that they have a stronger binding ability to the ROR2 protein.

[0180] Example 7: The endocytosis effect of humanized antibodies

[0181] T47D cells were co-incubated with different humanized antibodies (3 μg / mL) on ice for 30 minutes, then divided into two groups and incubated at 4℃ or 37℃ for 0.5 or 3 hours respectively. After incubation with secondary antibody (Alexa647 Anti-human IgG, 1:500 dilution) on ice for 30 minutes, the expression level of ROR2 protein was determined by flow cytometry. The endocytosis rate of the humanized antibodies was calculated using the following formula: 100 - (fluorescence intensity after incubation at 37℃ - fluorescence intensity after incubation at 4℃) * 100 (both subtracting the fluorescence intensity of the control). As shown in Figure 7, more than 50% of the antibodies from B16-H1L1, B16-H1L2, and B16-H1L3 were endocytosed into the cells after 3 hours, while A12-H1L1 showed better endocytosis, with more than 70% of the antibodies endocytosed within 0.5 hours.

[0182] Example 8: Effect of B16 humanized antibody on the invasive ability of breast cancer cell line T47D

[0183] Pancreatic cancer PDX tumor tissue grown from T47D cells or isolated single cells from immunodeficient mice was pre-incubated with B16 chimeric and humanized antibody or control IgG for half an hour (50 μg / mL). The cells were then added to Transwell chambers lined with matrix gel (10:1). The cell suspension was diluted with growth factor-free and serum-free MBGM, and NIH3T3 cell-specific culture medium (collected from NIH3T3 cells cultured for 24 hours without serum) was added to the lower chamber. After 24 hours, the number of cells that had penetrated the matrix gel and adhered to the bottom of the wells was observed and counted. The results are shown in Figure 8. The results show that after treatment with the B16 humanized antibody, the number of breast cancer cell lines that penetrated the matrix gel was significantly reduced, indicating that the humanized antibody can significantly reduce their invasive ability.

[0184] Example 9: Effects of B16 humanized antibody on PDX colorectal cancer tumor cells

[0185] The spheroidization assay involved culturing tumor cells into single cells in MEGM medium rich in growth factors in a low-adhesion culture plate and observing the ability of each single cell to form spheroids. Stronger spheroidization ability indicated stronger stemness of the tumor cells. When 50 μg / mL of humanized B16 antibody was added to MEGM medium containing colon cancer PDX tumor cells (initial cell count of 5000 or 20000), after two weeks of culture, the number of spheroids formed in the antibody-treated group was significantly less than that in the untreated group and the control IgG group. This demonstrates that the humanized B16 antibody of this invention can effectively inhibit the spheroidization ability of T47D colon cancer PDX tumor cells. The results are shown in Figures 9A and 9B. To verify whether the B16 chimeric antibody has an inhibitory effect on tumor growth in vivo, a colon cancer PDX mouse model was first established. Mice were then divided into antibody-treated and control groups. Mice in the antibody-treated group were injected intravenously with B16-H1L1 antibody (10 mg / kg), and the size of the tumor tissue was measured regularly. The growth curve is shown in Figure 9C. The results showed that B16-H1L1 antibody could significantly inhibit the growth of PDX colorectal cancer tumors in immunodeficient mice. The tumor weight of the experimental group after B16-H1L1 treatment was also significantly smaller than that of the control group (Figure 9D and E).

[0186] Example 10: Effect of B16 humanized antibody on clonogenicity of acute myeloid leukemia cells

[0187] Human acute myeloid leukemia cells OCI-AML5 (purchased from Shanghai BLUEFBIO Life Science) were counted on a hemocytometer, with 50 cells per well seeded into 96-well plates. 90 μL of MethoCult was added to each well. TM H4434 Classic, 7 μL IMDM medium, 1 μL penicillin / streptomycin, 2 μL fetal bovine serum; three replicate wells for each experiment.

[0188] During the culturing period from day 3 to day 14, daily observations were performed using an inverted fluorescence microscope for photographing and counting. The results, shown in Figure 10, indicate that cells treated with the humanized B16 antibody B16-H1L1 formed fewer colonies, suggesting that B16-H1L1 can inhibit the colony formation of acute myeloid leukemia cells.

[0189] Example 11: Treatment with the anti-ROR2 humanized antibody B16-H1L1 significantly inhibited tumor growth and metastasis.

[0190] 1X10 5ROR2-overexpressing PNAC1 pancreatic cancer cells (purchased from Wuhan Pronosei Biotechnology Co., Ltd.) were incubated on ice for 1 hour with 50 μg / mL of anti-ROR2 antibody or control antibody. Then, the cells were added to the upper chamber of a Transwell containing matrix gel. 600 μL of DMEM containing 10% fetal bovine serum (FBS) was added to the lower chamber. The cells were incubated at 37°C for 6–18 hours in a 5% CO2 incubator. The culture medium in the chamber was aspirated, and the cells were washed once with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes, then stained with 0.1% crystal violet for 20–40 minutes. After staining, the cells were washed twice with PBS to remove residual crystal violet. The cells were gently wiped away from the upper layer of the chamber filter membrane with a cotton swab, allowed to air dry, and then randomly selected fields of view under a microscope to count the cells and calculate the average value. Each experimental group was set up in triplicate.

[0191] 5X10 4 PNAC1 cells overexpressing ROR2 and labeled with luciferase were injected into the pancreas of 4-6 week old NCG mice. The following day, each mouse was intraperitoneally injected with Luciferase luminescent substrate, and the cellular luminescence signal was detected using an IVIS200 imaging system. Based on the bioluminescence signal, the mice were randomly divided into two groups: a treatment group (n=5) and a control group (n=5). Antibody (10 mg / kg) was injected via tail vein twice a week, with bioluminescence signals monitored weekly. The survival of mice in both groups was observed. Around week four, the mice were sacrificed, and the pancreas, spleen, and liver were harvested. Luciferase activity was detected in real-time using the IVIS200 imaging system, and the signal was quantified.

[0192] The results, as shown in Figure 11, indicate that the humanized B16 antibody B16-H1L1 can inhibit the growth and metastasis of pancreatic cancer in mice.

[0193] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

[0194] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0195] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A humanized antibody or antigen binding fragment thereof that binds ROR2, characterized in that, The humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, CDR sequences of the heavy chain variable region and CDR sequences of the light chain variable region are selected from any one of the following or an amino acid sequence having at least 95% identity thereto: Heavy chain variable region CDR sequences: SEQ ID NOs: 1-18; Light chain variable region CDR sequences: SEQ ID NOs: 19-36.

2. The humanized antibody or antigen-binding fragment thereof of claim 1, wherein, The humanized antibody or antigen-binding fragment thereof comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 1, 2 and 3, respectively; light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 19, 20 and 21, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 19, 20 and 21, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 4, 5 and 6, respectively; light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 22, 23 and 24, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 22, 23 and 24, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 7, 8 and 9, respectively; light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 25, 26 and 27, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 25, 26 and 27, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 10, 11 and 12, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 10, 11 and 12, respectively; light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 28, 29 and 30, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 28, 29 and 30, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 13, 14 and 15, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 13, 14 and 15, respectively; light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 31, 32 and 33, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 31, 32 and 33, respectively; or heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 16, 17 and 18, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 16, 17 and 18, respectively; light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 34, 35 and 36, respectively, or amino acid sequences having at least 95% identity to SEQ ID NOs: 34, 35 and 36, respectively. heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 16, 17, and 18; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 34, 35, and 36.

3. The humanized antibody or antigen-binding fragment thereof of claim 1, wherein, The humanized antibody or antigen-binding fragment thereof specifically recognizes ROR2.

4. The humanized antibody or antigen-binding fragment thereof of claim 1, wherein, The humanized antibody or antigen-binding fragment thereof comprises heavy chain framework region sequences comprising FR1, FR2, FR3, and FR4; Optionally, the FR1 has the amino acid sequence set forth in SEQ ID NO: 37 or an amino acid sequence having the L2V mutation as compared to the amino acid sequence set forth in SEQ ID NO: 37; and / or the FR2 has the amino acid sequence set forth in SEQ ID NO: 39; and / or the FR3 has the amino acid sequence set forth in any one of SEQ ID NOs: 40-42; and / or the FR4 has the amino acid sequence set forth in SEQ ID NO:

43.

5. The humanized antibody or antigen-binding fragment thereof of claim 4, wherein, The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as set forth in any one of SEQ ID NOs: 44-46.

6. The humanized antibody or antigen-binding fragment thereof of claim 1, wherein, The humanized antibody or antigen-binding fragment thereof comprises light chain framework region sequences comprising FR1, FR2, FR3, and FR4; Optionally, the FR1 has the amino acid sequence set forth in any one of SEQ ID NOs: 47-49; and / or the FR2 has the amino acid sequence set forth in SEQ ID NO: 50; and / or the FR3 has the amino acid sequence set forth in SEQ ID NO: 51 or an amino acid sequence having the L2R mutation as compared to the amino acid sequence set forth in SEQ ID NO: 51; and / or the FR4 has the amino acid sequence set forth in SEQ ID NO:

53.

7. The humanized antibody or antigen-binding fragment thereof of claim 6, wherein, The humanized antibody or antigen-binding fragment thereof has a light chain variable region with an amino acid sequence as set forth in any one of SEQ ID NOs: 54-56.

8. The humanized antibody or antigen-binding fragment thereof of claim 1, wherein, The humanized antibody or antigen-binding fragment thereof comprises heavy chain framework region sequences comprising FR1, FR2, FR3, and FR4; Optionally, the FR1 has the amino acid sequence set forth in SEQ ID NO: 57 or an amino acid sequence having the A25S mutation as compared to the amino acid sequence set forth in SEQ ID NO: 57; and / or the FR2 has the amino acid sequence set forth in SEQ ID NO: 59; and / or the FR3 has the amino acid sequence set forth in SEQ ID NO: 60 or an amino acid sequence having the K16T mutation as compared to the amino acid sequence set forth in SEQ ID NO: 60; and / or the FR4 has the amino acid sequence set forth in SEQ ID NO:

62.

9. The humanized antibody or antigen-binding fragment thereof of claim 8, wherein, The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as set forth in any one of SEQ ID NOs: 63-65.

10. The humanized antibody or antigen-binding fragment thereof of claim 1, wherein, The humanized antibody or antigen-binding fragment thereof contains a light chain framework region sequence comprising FR1, FR2, FR3, and FR4; Optionally, the FR1 has an amino acid sequence as set forth in any one of SEQ ID NOs: 66-68; and / or the FR2 has an amino acid sequence as set forth in SEQ ID NO: 69 or an amino acid sequence with a W15L mutation compared to the amino acid sequence as set forth in SEQ ID NO: 69; and / or the FR3 has an amino acid sequence as set forth in any one of SEQ ID NOs: 71-73; and / or the FR3 has an amino acid sequence as set forth in SEQ ID NO:

74.

11. The humanized antibody or antigen-binding fragment thereof of claim 10, wherein, The humanized antibody or antigen-binding fragment thereof has a light chain variable region with an amino acid sequence as set forth in any one of SEQ ID NOs: 75-77.

12. The humanized antibody or antigen-binding fragment thereof of claim 1, wherein, The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as set forth in any one of SEQ ID NOs: 44-46 and a light chain variable region with an amino acid sequence as set forth in any one of SEQ ID NOs: 54-56; or The humanized antibody or antigen-binding fragment thereof has a heavy chain variable region with an amino acid sequence as set forth in any one of SEQ ID NOs: 63-65 and a light chain variable region with an amino acid sequence as set forth in any one of SEQ ID NOs: 75-77.

13. The humanized antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein, The humanized antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, at least a portion of which is from a human IgG antibody or a mutant thereof.

14. An antibody or antigen-binding fragment thereof that binds ROR2, characterized in that, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the CDR sequences of the heavy chain variable region and the CDR sequences of the light chain variable region being selected from any one of the following or an amino acid sequence with at least 95% identity thereto: Heavy chain variable region CDR sequences: (1) X1YTFTSYX2MHWV, wherein X1 is G or null, X2 is L or Y, and X1, X2 do not satisfy the following: X1 is null, X2 is L; (2) X3X4X5X6X7X8INPYNX9X 10 T X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 wherein X3 is L or null, X4 is E or null, X5 is W or null, X6 is I or null, X7 is G or null, X8 is Y or null, X9 is D or G, X 10 is G or S, X 11 is K or N or null, X 12 is Y or null, X 13 is A or N or null, X 14 is E or Q or null, X 15 is K or null, X 16 is F or null, X 17 is K or Q or null, X 18 is D or G or null, X 19 is K or R or null, X 20 is A or null, X 21 is T or null, And X3-X 21 The following conditions must not be met simultaneously: X3 is L, X4 is E, X5 is W, X6 is I, X7 is G, X8 is Y, X9 is D or G, X 10 For G or S, X 11 For K or N, X 12 Let Y and X be the numbers. 13 If N or empty, X 14 E or empty, X 15 Let K, X 16 Let F, X 17 For K or empty, X 18 For D or empty, X 19 For K or empty, X 20 Let A and X be the two numbers. 21 Let T be the value of T. (3) ARSD VYYGVRFAY X 22 X 23 X 24 X 25 wherein X 22 is W or null, X 23 is G or null, X 24 is Q or null, X 25 is G or null, and X 22 - X 25 Not simultaneously satisfy the following: X 22 is W, X 23 is G, X 24 is Q, X 25 is G; (4) sequences as set forth in SEQ ID NOs: 78-140; Light chain variable region CDR sequences: (5) X 26 X 27 X 28 X 29 X 30 SVTYX 31 X 32 X 33 X 34 X 35 wherein X 26 is C or null, X 27 is S or R or null, X 28 is A or null, X 29 is S or null, X 30 is S or Q or null, X 31 is T or null, X 32 is Y or null, X 33 is W or null, X 34 is Y or null, X 35 is Q or null, and provided that X 26 - X 35 Not simultaneously satisfied: X 26 is C, X 27 is S or R, X 28 is A, X 29 is S, X 30 is S or Q, X 31 is T, X 32 is Y, X 33 is W, X 34 is Y, X 35 is Q; (6) X 36 X 37 DTSX 38 X 39 X 40 X 41 wherein X 36 is I or null, X 37 is Y or null, X 38 is N or null, X 39 is L or R or null, X 40 is A or null, X 41 is T or S or null, and provided that X 36 - X 41 does not satisfy the following: X 36 is I, X 37 is Y, X 38 is N, X 39 is L or null, X 40 is A, X 41 is S or null; (7) X 42 QQWSSYPFTX 43 X 44 X 45 X 46 wherein X 42 is C or null, X 43 is F or null, X 44 is G or null, X 45 is S or Q or null, X 46 is G or null, and provided that X 42 - X 46 does not satisfy the following: X 42 is C, X 43 is F, X 44 is G, X 45 is S or null, X 46 is G; (8) sequences as set forth in SEQ ID NOs: 141-203.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein, The CDR sequences of the heavy chain variable region and the CDR sequences of the light chain variable region are selected from any one of the following or an amino acid sequence with at least 95% identity thereto: Heavy chain variable region CDR sequences: SEQ ID NOs: 204-215; Light chain variable region CDR sequences: SEQ ID NOs: 216-227.

16. The antibody or antigen-binding fragment thereof of claim 14, wherein, The antibody or antigen-binding fragment thereof comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 78, 79, and 80, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 78, 79, and 80; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 141, 142, and 143, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 141, 142, and 143; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 81, 82, and 83, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 81, 82, and 83; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 144, 145, and 146, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 144, 145, and 146; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 84, 85, and 86, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 84, 85, and 86; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 147, 148, and 149, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 147, 148, and 149; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 87, 88, and 89, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 87, 88, and 89; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 150, 151, and 152, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 150, 151, and 152; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 90, 91, and 92, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 90, 91, and 92; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 153, 154, and 155, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 153, 154, and 155; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 90, 91, and 92, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 90, 91, and 92; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 153, 154, and 155, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 153, 154, and 155; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 96, 97, and 98; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 159, 160, and 161, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 159, 160, and 161; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 96, 97, and 98; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 159, 160, and 161, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 159, 160, and 161; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 96, 97, and 98; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 159, 160, and 161, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 159, 160, and 161; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 96, 97, and 98; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 159, 160, and 161, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 159, 160, and 161; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 96, 97, and 98; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 159, 160, and 161, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 159, 160, and 161; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 96, 97, and 98; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 159, 160, and 161, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 159, 160, and 161; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 108, 109, and 110, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 108, 109, and 110; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 171, 172, and 173, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 171, 172, and 173; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 111, 112, and 113, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 111, 112, and 113; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 174, 175, and 176, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 174, 175, and 176; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 114, 115, and 116, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 114, 115, and 116; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 177, 178, and 179, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 177, 178, and 179; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 117, 118, and 119, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 117, 118, and 119; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 180, 181, and 182, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 180, 181, and 182; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 120, 121, and 122, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 120, 121, and 122; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 183, 184, and 185, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 183, 184, and 185; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 123, 124, and 125, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 123, 124, and 125; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 186, 187, and 188, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 186, 187, and 188; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 126, 127, and 128, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 126, 127, and 128; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 189, 190, and 191, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 189, 190, and 191; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 129, 130, and 131, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 129, 130, and 131; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 192, 193, and 194, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 192, 193, and 194; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 132, 133, and 134; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 195, 196, and 197, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 195, 196, and 197; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 135, 136, and 137, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 135, 136, and 137; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 198, 199, and 200, respectively, or as an amino acid sequence at least 95% identical to SEQ ID NOs: 198, 199, and 200; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 138, 139, and 140, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 138, 139, and 140; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 201, 202, and 203, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 201, 202, and 203; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 204, 205, and 206, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 204, 205, and 206; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 216, 217, and 218, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 216, 217, and 218; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 207, 208, and 209, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 207, 208, and 209; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 219, 220, and 221, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 219, 220, and 221; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 210, 211, and 212, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 210, 211, and 212; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 222, 223, and 224, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 222, 223, and 224; or heavy chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 213, 214, and 215, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 213, 214, and 215; light chain variable region CDR1, CDR2, CDR3 sequences set forth in SEQ ID NOs: 225, 226, and 227, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs: 225, 226, and 227.

17. The antibody or antigen-binding fragment thereof of claim 14, wherein, the antibody or antigen-binding fragment thereof has a heavy chain variable region of an amino acid sequence set forth in any one of SEQ ID NOs: 228-252.

18. The antibody or antigen-binding fragment thereof of claim 14, wherein, the antibody or antigen-binding fragment thereof has a light chain variable region of an amino acid sequence set forth in any one of SEQ ID NOs: 253-277.

19. The antibody or antigen-binding fragment thereof of claim 14, wherein, said antibody or antigen-binding fragment thereof has a heavy chain variable region of the amino acid sequence of any one of SEQ ID NOs: 228-252 and a light chain variable region of the amino acid sequence of any one of SEQ ID NOs: 253-277.

20. A nucleic acid molecule, characterized in that, said nucleic acid molecule encodes the humanized antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13 or the antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19.

21. An expression vector comprising the nucleic acid of claim 20. carrying the nucleic acid molecule according to claim 20.

22. The expression vector of claim 21, wherein, said expression vector is a eukaryotic expression vector.

23. A recombinant cell, wherein, said recombinant cell carries the nucleic acid molecule according to claim 19 or the expression vector according to claim 20 or 21 or expresses the humanized antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13 or the antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19.

24. The recombinant cell of claim 23, wherein, said recombinant cell is obtained by introducing the expression vector according to claim 21 or 22 into a host cell.

25. The recombinant cell of claim 24, wherein, said recombinant cell is a eukaryotic cell; optionally, said recombinant cell is a mammalian cell.

26. A pharmaceutical composition comprising, a humanized antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13 or an antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19 or a nucleic acid molecule according to claim 20 or an expression vector according to claim 21 or 22 or a recombinant cell according to any one of claims 23 to 25.

27. Use of a humanized antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13 or an antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19 or a nucleic acid molecule according to claim 20 or an expression vector according to claim 21 or 22 or a recombinant cell according to any one of claims 23 to 25 or a pharmaceutical composition according to claim 26 for the manufacture of a medicament for the treatment of a tumor.

28. Use according to claim 27, characterized in that, said tumor is a ROR2-positive tumor; optionally, said tumor is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, blood cancer.

29. A kit for detecting ROR2, characterized by, said kit comprises a humanized antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13 or an antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19.

30. Use of a humanized antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13 or an antibody or antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19 or a nucleic acid molecule according to claim 20 or an expression vector according to claim 21 or 22 or a recombinant cell according to any one of claims 23 to 25 for the manufacture of a kit for the detection of ROR2.

31. A chimeric antigen receptor against ROR2, characterized in that, comprises an extracellular region, a transmembrane region and an intracellular region, The extracellular region comprises an antibody or an antigen-binding fragment which is a humanized antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13 or an antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19.

32. A method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-31. comprising administering to the subject a pharmaceutically effective amount of at least one of: the humanized antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13, the antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19, the nucleic acid molecule according to claim 20, the expression vector according to claim 21 or 22, the recombinant cell according to any one of claims 23 to 25 or the pharmaceutical composition according to claim 26.

33. The method of claim 32, wherein, The tumor is a ROR2-positive tumor; Optionally, the tumor is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, blood cancer.

34. A method of diagnosing whether a subject has a tumor, comprising, The method comprises detecting a test sample from the subject using at least one of: the humanized antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13, the antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19, the nucleic acid molecule according to claim 20, the expression vector according to claim 21 or 22, the recombinant cell according to any one of claims 23 to 25 or the kit according to claim 29.

35. The method of claim 34, wherein, The tumor is a ROR2-positive tumor; Optionally, the tumor is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, blood cancer.

36. Use of at least one of the humanized antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13, the antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19, the nucleic acid molecule according to claim 20, the expression vector according to claim 21 or 22, the recombinant cell according to any one of claims 23 to 25 or the pharmaceutical composition according to claim 26 for the treatment of a tumor.

37. The use according to claim 36, characterized in that The tumor is a ROR2-positive tumor; Optionally, the tumor is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, blood cancer.

38. Use of at least one of the humanized antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 1 to 13, the antibody or an antigen-binding fragment thereof binding to ROR2 according to any one of claims 14 to 19, the nucleic acid molecule according to claim 20, the expression vector according to claim 21 or 22, the recombinant cell according to any one of claims 23 to 25 or the kit according to claim 29 for diagnosing whether a subject has a tumor.

39. The use according to claim 38, characterized in that The tumor is a ROR2-positive tumor; Optionally, the tumor is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, blood cancer.

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