TW202330620A - An antibody or antigen-binding fragment thereof targeting ror1 and use thereof - Google Patents

Abstract

The present invention relates to antibodies that specifically recognize ROR1, methods of preparation thereof, and uses thereof. The present invention also relates to antibody-coupled drugs (ADCs) targeting ROR1 and compositions containing the molecule. In addition, the present invention relates to the therapeutic and diagnostic uses of these antibodies, antibody fragments, and antibody-coupled drugs.

Description

An antibody targeting ROR1 or an antigen-binding fragment thereof and its application

The present invention relates to antibodies that specifically recognize ROR1, their preparation methods and uses. The invention also relates to antibody drug conjugates (ADCs) targeting ROR1 and compositions containing the molecules. Furthermore, the present invention relates to the therapeutic and diagnostic uses of these antibodies, antibody fragments and antibody drug conjugates.

Antibody-Drug Conjugates (ADC) are derivatives of traditional antibody drugs. They are composed of monoclonal antibody drugs targeting specific antigens and small molecule cytotoxic drugs coupled by linkers. They are both antibody drugs. With its specific targeting and killing effect of traditional small molecule drugs, its main indication is malignant tumors. ADCs have quickly become a hot topic in the research and development of anti-tumor drugs due to their excellent tumor-killing effects. However, the toxicity of ADC is usually high, so the specificity of the target antigen is required. Targets suitable for the development of ADC are generally highly expressed in tumors and low or barely expressed in healthy tissues; the antigen is up-regulated in tumor cells. The surface receptor can promote tumor growth or survival, and the target antigen should have internalization properties.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1, also known as receptor-associated neurotrophic tyrosine kinase 1, NTRKR1) and receptor tyrosine kinase-like orphan receptor 2 (ROR2) are single-pass transmembrane proteins belonging to the receptor tyrosine kinase (RTK) family. Their extracellular components It consists of an immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD domain and KRD domain). The intracellular structure consists of a tyrosine kinase domain and two serine-rich domains. It consists of a threonine- or threonine-rich domain and a proline-rich domain. ROR1 and ROR2 participate in the non-canonical Wnt signaling pathway through the FZD domain binding ligand Wnt5a (Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, Koshida I, Suzuki K, Yamada G, Schwabe GC et al (2003). Genes Cells 8: 645-654; Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al (2008). Proc Natl Acad Sci USA 105: 3047-3052; Paganoni S, Bernstein J, Ferreira A (2010). Neuroscience 165: 1261-1274). ROR1 can inhibit apoptosis, enhance EGFR signaling, and induce epithelial-mesenchymal transition (EMT) (Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al(2008).Proc Natl Acad Sci USA 105:3047-3052;Yamaguchi T,Yanagisawa K,Sugiyama R,Hosono Y,Shimada Y,ArimaC,KatoS,TomidaS,Suzuki M,OsadaHet al(2012).Cancer Cell 21:348-361; Cui B, Zhang S, Chen L, Yu J, Widhopf GF, Fecteau J-F, Rassenti LZ, Kipps TJ (2013). Cancer Res 73: 3649-3660).

ROR1 is a conserved embryonic protein. Its expression gradually decreases with embryonic development and is almost non-existent or low-expressed in most adult tissues. However, more and more literature has found that ROR1 is expressed in a variety of cancer cells, such as B-cell chronic lymphocytes. Cellular leukemia (CLL) and other hematological malignancies, renal cell carcinoma, colon cancer and certain other cancer cell lines of breast cancer. In addition, ROR1 is It plays an important role in the progression of many hematological and solid malignancies. Therefore, as a cancer marker, ROR1 becomes an ideal drug target for cancer treatment.

Although the existing technology discloses several antibody drugs targeting ROR1, as a tumor marker for pan-cancer species, there is still an urgent need to develop high-quality anti-ROR1 antibodies, which can be used to develop antibody-based targeted therapies for ROR1-expressing cancers. basis and can also be used as a diagnostic tool to detect ROR1 expression in ROR1-related disorders. In addition, based on the good prospects shown by ADCs in the field of tumor treatment, there is still an urgent need for ROR1-containing ADCs with effective therapeutic effects, and the present invention meets these needs.

In a first aspect, the present invention provides antibodies targeting human ROR1, which have the following advantages:

(1) High affinity binding to human ROR1 and target cells expressing human ROR1;

(2) Has good mouse cross-activity;

(3) Able to enter cells through endocytosis;

(4) Antibody conjugate drugs suitable for constructing effective treatments;

The anti-ROR1 antibody provided by the present invention has a complete human sequence and is a human antibody. The anti-ROR1 antibodies of the invention are expected to be minimally immunogenic in human subjects, and the anti-ROR1 antibodies will be well tolerated by human subjects.

In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising:

1) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 52 and 3 included in the light chain variable region as shown in SEQ ID NO: 51 light chain CDRs (LCDR1, LCDR2, LCDR3);

2) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 54 and 3 included in the light chain variable region as shown in SEQ ID NO: 53 light chain CDRs (LCDR1, LCDR2, LCDR3);

3) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 56 and 3 included in the light chain variable region as shown in SEQ ID NO: 55 light chain CDRs (LCDR1, LCDR2, LCDR3);

4) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 58 and 3 included in the light chain variable region as shown in SEQ ID NO: 57 Light chain CDRs (LCDR1, LCDR2, LCDR3):

5) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 60 and the 3 included in the light chain variable region as shown in SEQ ID NO: 59 light chain CDRs (LCDR1, LCDR2, LCDR3);

6) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 62 and 3 included in the light chain variable region as shown in SEQ ID NO: 61 light chain CDRs (LCDR1, LCDR2, LCDR3);

7) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 64 and 3 included in the light chain variable region as shown in SEQ ID NO: 63 light chain CDRs (LCDR1, LCDR2, LCDR3);

8) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 66 and 3 included in the light chain variable region as shown in SEQ ID NO: 53 light chain CDRs (LCDR1, LCDR2, LCDR3);

9) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 68 and 3 included in the light chain variable region as shown in SEQ ID NO: 67 light chain CDRs (LCDR1, LCDR2, LCDR3);

10) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 70 and 3 contained in the light chain variable region as shown in SEQ ID NO: 69 light chain CDRs (LCDR1, LCDR2, LCDR3);

11) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 145 and 3 included in the light chain variable region as shown in SEQ ID NO: 144 light chain CDRs (LCDR1, LCDR2, LCDR3);

12) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 147 and the 3 included in the light chain variable region as shown in SEQ ID NO: 146 light chain CDRs (LCDR1, LCDR2, LCDR3);

13) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 141 and 3 included in the light chain variable region as shown in SEQ ID NO: 140 light chain CDRs (LCDR1, LCDR2, LCDR3);

14) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 143 and the 3 included in the light chain variable region as shown in SEQ ID NO: 142 light chain CDRs (LCDR1, LCDR2, LCDR3);

15) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 149 and the 3 included in the light chain variable region as shown in SEQ ID NO: 148 light chain CDRs (LCDR1, LCDR2, LCDR3);

16) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 161 and the 3 included in the light chain variable region as shown in SEQ ID NO: 160 light chain CDRs (LCDR1, LCDR2, LCDR3);

17) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 163 and the 3 included in the light chain variable region as shown in SEQ ID NO: 162 light chain CDRs (LCDR1, LCDR2, LCDR3);

18) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 165 and the 3 included in the light chain variable region as shown in SEQ ID NO: 164 light chain CDRs (LCDR1, LCDR2, LCDR3);

19) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 151 and the 3 contained in the light chain variable region as shown in SEQ ID NO: 150 light chain CDRs (LCDR1, LCDR2, LCDR3);

20) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 153 and the 3 included in the light chain variable region as shown in SEQ ID NO: 152 light chain CDRs (LCDR1, LCDR2, LCDR3);

21) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 155 and the 3 included in the light chain variable region as shown in SEQ ID NO: 154 light chain CDRs (LCDR1, LCDR2, LCDR3);

22) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 157 and the 3 included in the light chain variable region as shown in SEQ ID NO: 156 light chain CDRs (LCDR1, LCDR2, LCDR3);

23) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 52 and the 3 included in the light chain variable region as shown in SEQ ID NO: 158 light chain CDRs (LCDR1, LCDR2, LCDR3);

24) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 52 and the 3 contained in the light chain variable region as shown in SEQ ID NO: 159 light chain CDRs (LCDR1, LCDR2, LCDR3);

25) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 62 and the 3 included in the light chain variable region as shown in SEQ ID NO: 166 light chain CDRs (LCDR1, LCDR2, LCDR3);

26) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 168 and the 3 included in the light chain variable region as shown in SEQ ID NO: 167 light chain CDRs (LCDR1, LCDR2, LCDR3);

27) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 169 and the 3 included in the light chain variable region as shown in SEQ ID NO: 61 light chain CDRs (LCDR1, LCDR2, LCDR3).

In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising:

1) Contains the sequences shown in SEQ ID NO: 1, 2 and 3, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 19, 20 and 21, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

2) Contains the sequences shown in SEQ ID NO: 1, 2 and 6, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 22, 23 and 24, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

3) Contains the sequences shown in SEQ ID NO: 7, 8 and 9, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. The sequences of HCDR1, HCDR2, HCDR3; and as in SEQ ID NO: 25, 26 and 27 The sequence shown, or LCDR1, LCDR2, LCDR3 of a sequence containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence;

4) Contains the sequences shown in SEQ ID NO: 10, 11 and 12, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 28, 29 and 30, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

5) Contains the sequences shown in SEQ ID NO: 13, 14 and 15, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 31, 32 and 33, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

6) Contains the sequences shown in SEQ ID NO: 16, 17 and 18, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 34, 35 and 36, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

7) Contains the sequences shown in SEQ ID NO: 1, 2 and 37, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 19, 20 and 21, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, LCDR3 of sequences with sexual substitutions), deletions or insertions:

8) Contains the sequences shown in SEQ ID NO: 38, 39 and 12, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 28, 44 and 30, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

9) Contains the sequences shown in SEQ ID NO: 40, 41 and 15, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 31, 45 and 33, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

10) Contains the sequences shown in SEQ ID NO: 42, 43 and 18, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 34, 46 and 36, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

11) Contains the sequences shown in SEQ ID NO: 91, 92 and 93, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 101, 102 and 103, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

12) Contains the sequences shown in SEQ ID NO: 1, 2 and 94, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 104, 32 and 105, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

13) Contains the sequences shown in SEQ ID NO: 95, 96 and 97, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 106, 107 and 108, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

14) Contains the sequences shown in SEQ ID NO: 1, 2 and 98, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 109, 110 and 105, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

15) Contains the sequences shown in SEQ ID NO: 91, 99 and 100, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 111, 112 and 113, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

16) Contains the sequences shown in SEQ ID NO: 121, 122 and 3, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 19, 136 and 21, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

17) Contains the sequences shown in SEQ ID NO: 1, 123 and 3, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 19, 137 and 21, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

18) Contains the sequences shown in SEQ ID NO: 124, 2 and 3, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 19, 23 and 21, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

19) Contains the sequences shown in SEQ ID NO: 114, 115 and 12, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 28, 128 and 30, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

20) Comprising the sequences shown in SEQ ID NO: 116, 117 and 12, or containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 28, 129 and 30, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

21) Contains the sequences shown in SEQ ID NO: 118, 119 and 12, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 28, 130 and 30, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

22) Contains the sequences shown in SEQ ID NO: 16, 17 and 120, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 131, 35 and 132, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

23) Contains the sequences shown in SEQ ID NO: 16, 17 and 18, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of one or more and no more than 3 amino acids relative to the sequence. HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 34, 35 and 133, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

24) Contains the sequences shown in SEQ ID NO: 16, 17 and 18, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 134, 35 and 135, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

25) Comprising the sequences shown in SEQ ID NO: 13, 14 and 15, or containing one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 31, 138 and 33, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, and LCDR3 of sequences with sexual substitutions), deletions, or insertions;

26) Contains the sequences shown in SEQ ID NO: 125, 126 and 15, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 31, 139 and 33, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, LCDR3 of the sequence (sexual substitution), deletion or insertion; or

27) Contains the sequences shown in SEQ ID NO: 125, 127 and 15, or contains one or more amino acid substitutions (such as conservative substitutions), deletions or insertions of not more than 3 amino acids relative to the sequence HCDR1, HCDR2, HCDR3 of the sequences; and sequences as shown in SEQ ID NO: 31, 32 and 33, or amino acid substitutions containing one or more and no more than 3 amino acids relative to the sequence (e.g. conservative LCDR1, LCDR2, LCDR3 of the sequence (sexual substitution), deletion or insertion.

In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising heavy chain variable regions, wherein,

1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 52 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 52;

2) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 54 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 54;

3) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 56 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 56;

4) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 58, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 58. , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 58;

5) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 60 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 60;

6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 62 ,95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 62;

7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 64, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 64 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 64;

8) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 66, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 66 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 66;

9) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 68 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 68;

10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 70 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 70;

11) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 145, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 145 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 145;

12) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 147, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 147 ,95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 147;

13) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 141, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 141 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 141;

14) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 143, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 143 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 143;

15) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 149, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 149 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 149;

16) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 161, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 161 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 161;

17) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 163, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 163 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 163;

18) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 165, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 165 ,95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 165;

19) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 151, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 151 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 151;

20) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 153, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 153 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 153;

21) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 155, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 155 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 155;

22) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 157, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 157 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 157;

23) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 168, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 168 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 168; or

24) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 169, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 169 ,95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 169.

In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising light chain variable regions, wherein,

1) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 51 , an amino acid sequence of 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 51;

2) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 53, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 53 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 53;

3) The light chain variable region contains the amino acid sequence shown in SEQ ID NO: 55, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 55. , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 55;

4) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 57 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 57;

5) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 59, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 59 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 59;

6) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 61, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 61 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 61;

7) The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 63, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 63. , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 63;

8) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 67 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 67;

9) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 69, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 69 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 69;

10) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 144, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 144 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 144;

11) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 146, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 146 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 146;

12) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 140, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 140 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 140;

13) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 142, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 142 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 142;

14) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 148, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 148 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 148;

15) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 160, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 160 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 160;

16) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 162, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 162 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 162;

17) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 164, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 164 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 164;

18) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 150, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 150 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 150;

19) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 152, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 152 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 152;

20) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 154, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 154 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 154;

21) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 156, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 156 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 156;

22) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 158, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 158 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 158;

23) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 159, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 159 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 159;

24) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 166, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 166 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 166;

25) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 167, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 167 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 167.

In another embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, comprising a heavy chain variable region and a light chain variable region, wherein,

1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 52 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 52, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 51 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 51 acid sequence, or consisting of SEQ ID NO: 51;

2) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 54 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 54, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 53 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 53 acid sequence, or consisting of SEQ ID NO: 53;

3) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 56, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 56 ,95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 56, the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 55, or with The amino acid sequence of SEQ ID NO: 55 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or Composed of SEQ ID NO: 55;

4) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 58, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 58 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 58, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 57 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 57 acid sequence, or consisting of SEQ ID NO: 57;

5) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 60 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 60, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 59 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 59 acid sequence, or consisting of SEQ ID NO: 59;

6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 62 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 62, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 61 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 61 acid sequence, or consisting of SEQ ID NO: 61;

7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 64, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 64 ,95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 64, the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 63, or with The amino acid sequence of SEQ ID NO: 63 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or Composed of SEQ ID NO: 63;

8) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 66, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 66 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 66, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 53 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 53 acid sequence, or consisting of SEQ ID NO: 53;

9) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 68 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 68, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 67 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 67 acid sequence, or consisting of SEQ ID NO: 67;

10) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 70 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 70, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 69 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 69 acid sequence, or consisting of SEQ ID NO: 69;

11) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 145, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 145 ,95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 145, the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 144, or with The amino acid sequence of SEQ ID NO: 144 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or Composed of SEQ ID NO: 144;

12) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 147, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 147 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 147, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 146 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 146 acid sequence, or consisting of SEQ ID NO: 146;

13) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 141, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 141 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 141, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 140 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 140 acid sequence, or consisting of SEQ ID NO: 140;

14) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 143, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 143 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 143, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 142 sequence, or has at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 142;

15) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 149, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 149 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 149, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 148 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 148 acid sequence, or consisting of SEQ ID NO: 148;

16) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 161, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 161 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 161, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 160 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 160 acid sequence, or consisting of SEQ ID NO: 160;

17) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 163, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 163 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 163, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 162 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 162 acid sequence, or consisting of SEQ ID NO: 162;

18) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 165, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 165 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 165, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 164 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 164 acid sequence, or consisting of SEQ ID NO: 164;

19) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 151, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 151 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 151, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 150 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 150 acid sequence, or consisting of SEQ ID NO: 150;

20) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 153, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 153 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 153, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 152 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 152 acid sequence, or consisting of SEQ ID NO: 152;

21) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 155, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 155 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 155 Into, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 154, or has at least 90%, 91%, 92%, 93%, 94% with the amino acid sequence of SEQ ID NO: 154 , an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical, or consisting of SEQ ID NO: 154;

22) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 157, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 157 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 157, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 156 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 156 acid sequence, or consisting of SEQ ID NO: 156;

23) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 52 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 52, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 158 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 158 acid sequence, or consisting of SEQ ID NO: 158;

24) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52, or has at least 90%, 91%, 92%, 93%, or 94% of the amino acid sequence of SEQ ID NO: 52 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 52, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 159 sequence, or have at least 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 159;

25) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 62 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 62, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 166 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 166 acid sequence, or consisting of SEQ ID NO: 166;

26) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 168, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 168 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 168, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 167 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 167 acid sequence, or consisting of SEQ ID NO: 167; or

27) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 169, or has at least 90%, 91%, 92%, 93% or 94% of the amino acid sequence of SEQ ID NO: 169 , an amino acid sequence with 95%, 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 169, the light chain variable region comprising the amino acid set forth in SEQ ID NO: 61 sequence, or an amine group having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 61 acid sequence, or consisting of SEQ ID NO: 61.

In some embodiments, the above-described antibodies or antigen-binding fragments thereof further comprise heavy chain and/or light chain constant region sequences derived from human antibody germline consensus sequences. The light chain constant region is preferably human Source kappa or lambda chain constant region. The heavy chain constant region can be a gamma, mu, alpha, delta, or epsilon chain. In some embodiments, the heavy chain constant region is preferably from the constant region sequence of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the light chain constant region comprises or consists of the sequence set forth in SEQ ID NO: 49 or 50. In another embodiment, the heavy chain constant region comprises the Fc sequence set forth in SEQ ID NO:47. In yet another embodiment, the heavy chain constant region comprises or consists of the sequence set forth in SEQ ID NO: 48.

It will be appreciated that sequence variants of these constant region domains may also be used, for example containing one or more amino acid modifications, where the amino acid sites are identified according to the EU indexing system of Kabat et al. (1991).

In a specific embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind ROR1, comprising heavy chains and light chains, wherein,

1) The heavy chain includes the amino acid sequence shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72, the light chain comprising the amino acid sequence shown in SEQ ID NO: 71, or with SEQ ID The amino acid sequence of NO: 71 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 71 composition;

2) The heavy chain includes the amino acid sequence shown in SEQ ID NO: 74, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 74 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 74, the light chain comprising the amino acid sequence shown in SEQ ID NO: 73, or with SEQ ID NO. The amino acid sequence of NO: 73 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 73 composition;

3) The heavy chain contains the amino acid sequence shown in SEQ ID NO: 76, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 76 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 76, the light chain comprising the amino acid sequence shown in SEQ ID NO: 75, or with SEQ ID The amino acid sequence of NO: 75 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 75 composition;

4) The heavy chain includes the amino acid sequence shown in SEQ ID NO: 78, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 78 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 78, the light chain comprising the amino acid sequence shown in SEQ ID NO: 77, or with SEQ ID NO. The amino acid sequence of NO: 77 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 77 composition;

5) The heavy chain includes the amino acid sequence shown in SEQ ID NO: 80, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 80 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 80, the light chain comprising the amino acid sequence shown in SEQ ID NO: 79, or with SEQ ID NO. The amino acid sequence of NO: 79 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 79 composition;

6) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 82, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 82 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 82, the light chain comprising the amino acid sequence shown in SEQ ID NO: 81, or with SEQ ID NO. The amino acid sequence of NO: 81 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 81 composition;

7) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 84, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 84 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 84, the light chain comprising the amino acid sequence shown in SEQ ID NO: 83, or with SEQ ID NO. The amino acid sequence of NO: 83 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 83 composition;

8) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 86, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 86 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 86, the light chain comprising the amino acid sequence shown in SEQ ID NO: 73, or with SEQ ID NO. The amino acid sequence of NO: 73 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 73 composition;

9) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 88, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 88 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 88, the light chain comprising the amino acid sequence shown in SEQ ID NO: 87, or with SEQ ID The amino acid sequence of NO: 87 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 87 composition;

10) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 90, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 90 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 90, the light chain comprising the amino acid sequence shown in SEQ ID NO: 89, or with SEQ ID NO. The amino acid sequence of NO: 89 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 89 composition;

11) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 175, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 175 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 175, the light chain comprising the amino acid sequence shown in SEQ ID NO: 174, or with SEQ ID NO: 175 The amino acid sequence of NO: 174 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 174 composition;

12) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 177, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 177 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 177, the light chain comprising the amino acid sequence shown in SEQ ID NO: 176, or with SEQ ID NO: 177 The amino acid sequence of NO: 176 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 176 composition;

13) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 171, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 171 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 171, the light chain comprising the amino acid sequence shown in SEQ ID NO: 170, or with SEQ ID NO: 171 The amino acid sequence of NO: 170 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 170 composition;

14) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 173, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 173 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 173, the light chain comprising the amino acid sequence shown in SEQ ID NO: 172, or with SEQ ID NO: 173 The amino acid sequence of NO: 172 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 172 composition;

15) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 179, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 179 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 179, the light chain comprising the amino acid sequence shown in SEQ ID NO: 178, or with SEQ ID NO. The amino acid sequence of NO: 178 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 178 composition;

16) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 191, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 191 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 191, the light chain comprising the amino acid sequence shown in SEQ ID NO: 190, or with SEQ ID NO: 191 The amino acid sequence of NO: 190 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 190 composition;

17) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 193, or has at least 90%, 91%, 92%, 93%, 94%, or 95% of the amino acid sequence of SEQ ID NO: 193 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 193, the light chain comprising the amino acid sequence shown in SEQ ID NO: 192, or with SEQ ID NO. The amino acid sequence of NO: 192 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 192 composition;

18) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 195, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 195 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 195, the light chain comprising the amino acid sequence shown in SEQ ID NO: 194, or with SEQ ID NO. The amino acid sequence of NO: 194 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 194 composition;

19) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 181, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 181 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 181, the light chain comprising the amino acid sequence shown in SEQ ID NO: 180, or with SEQ ID The amino acid sequence of NO: 180 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 180 composition;

20) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 183, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 183 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 183, the light chain comprising the amino acid sequence shown in SEQ ID NO: 182, or with SEQ ID The amino acid sequence of NO: 182 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 182 composition;

21) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 185, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 185 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 185, the light chain comprising the amino acid sequence shown in SEQ ID NO: 184, or with SEQ ID NO. The amino acid sequence of NO: 184 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 184 composition;

22) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 187, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 187 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 187, the light chain comprising the amino acid sequence shown in SEQ ID NO: 186, or with SEQ ID NO. The amino acid sequence of NO: 186 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 186 composition;

23) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72, the light chain comprising the amino acid sequence shown in SEQ ID NO: 188, or with SEQ ID NO. The amino acid sequence of NO: 188 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 188 composition;

24) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 72, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 72 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 72, the light chain comprising the amino acid sequence shown in SEQ ID NO: 189, or with SEQ ID NO. The amino acid sequence of NO: 189 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 189 composition;

25) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 82, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 82 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 82, the light chain comprising an amino acid sequence as shown in SEQ ID NO: 196, or with SEQ ID NO. The amino acid sequence of NO: 196 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 196 composition;

26) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 198, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 198 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 198, the light chain comprising the amino acid sequence set forth in SEQ ID NO: 197, or with SEQ ID NO: 197 The amino acid sequence of NO: 197 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 197 composition; or

27) The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 199, or has at least 90%, 91%, 92%, 93%, 94%, 95% with the amino acid sequence of SEQ ID NO: 199 , an amino acid sequence with 96%, 97%, 98% or 99% identity, or consisting of SEQ ID NO: 199, the light chain comprising the amino acid sequence shown in SEQ ID NO: 81, or with SEQ ID NO. The amino acid sequence of NO: 81 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or is determined by SEQ. ID NO: 81 composition.

In certain embodiments of the antibody of any of the preceding, the antibody is monoclonal.

In certain embodiments of the antibody of any of the preceding, the antibody is a full-length antibody.

In one embodiment, the anti-ROR1 antibodies of the invention are intact antibodies, such as IgG1, IgG2, IgG3, IgG4 antibodies. In another embodiment, the anti-ROR1 antibodies of the invention encompass only the antigen-binding portion thereof, such as: Fab, Fab'-SH, Fv, scFv or (Fab') ₂ fragments.

In a second aspect, the present invention provides an antibody-conjugated drug targeting human ROR1. The antibody-conjugated drug has the following advantages:

(1) Binds to target cells expressing human ROR1 with high affinity;

(2) Able to enter cells through endocytosis and kill target cells; in some embodiments, the ADC of the present invention has high endocytosis efficiency;

(3) Treat, prevent, or improve conditions in subjects related to abnormal function or expression of ROR1 (e.g., cancer, such as blood cancers, solid tumors), or treat, prevent, or improve one or more symptoms of the disease;

(4) Reduce or inhibit tumor growth or progression in a subject having a ROR1-expressing tumor;

(5) induce regression (e.g., long-term regression) of ROR1-expressing tumors;

(6) Exert cytotoxic activity in cells expressing ROR1;

In one embodiment, the invention provides a conjugate comprising the anti-ROR1 antibody of the first aspect. In a specific embodiment, molecules that can be conjugated to anti-ROR1 antibodies are, for example, cytotoxic agents, immunomodulators, imaging agents, fluorescent proteins, molecular markers, therapeutic proteins, biopolymers, and oligonucleotides Acid etc.

In one embodiment, the invention provides an antibody drug conjugate (ADC), which comprises the anti-ROR1 antibody or antigen-binding fragment thereof according to the first aspect and at least one therapeutically active substance or pharmaceutically active ingredient, having Ab-( L-D) The structure of n, wherein Ab is the ROR1-binding antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; L is a linker; D is a therapeutically active substance or pharmaceutical active ingredient, and n represents an integer from 1 to 20 , such as 1, 2, 3, 4, 5....

In one embodiment, the antibody-drug conjugate contains multiple D components, and the multiple D components can be a combination of different therapeutically active substances or pharmaceutically active ingredients, or a combination of the same therapeutically active substance or pharmaceutically active ingredients. In one embodiment, the antibody drug conjugate has a drug/antibody ratio (DAR) of 1-20, such as a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14 or 15 DAR. In one embodiment, the DAR is the average DAR. In one embodiment, the average DAR is 1-15, such as 1-10, 2-8, 2-6, or 3-5. In a specific embodiment, the average DAR is 3.7.

In a specific embodiment, the therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a phytotoxin, a small molecule toxin, a radioactive isotope, a maytansinoid alkaloid, or the like. In a specific embodiment, the cytotoxin is dolastatin and its auristatin derivatives, such as 0101 (2-methylpropylamine acyl-N-[(3R,4S,5S )-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-benzene base-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N- Methyl-L-valamine), 8261(2-methylpropylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- {[(1S)-1-Carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxypropyl]pyrrolidine-1- Base}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) Aplysia toxin (Dolastatin) 10, Dolastatin ( Dolastatin) 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAE), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin Statin (auristatin) EFP (AEFP), auristatin (auristatin) F hydroxypropylamide (AFHPA). In another embodiment, the Aplysia toxin and its auristatin derivatives are auristatin, dolastatin, MMAE, MMAF, auristatin F-hydroxy Propylamide or auristatin F phenylenediamine.

In one embodiment, the cytotoxin is covalently linked to the anti-ROR1 antibody or antigen-binding fragment thereof via a linker in a non-site-specific manner or in a site-specific manner.

In one embodiment, the linker is selected from maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine Acid-Valine-Citrulline-p-Aminobenzyloxycarbonyl (AcLys-VC-PABC), Amino PEG6-Propanyl, and Maleimidecaproyl (mc), Maleimine Propanyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimide 4 -(2-Pyridylthio)valerate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-ethyl) amino benzoate (SIAB), N-succinimido-4-(2-pyridyl disulfide) butyrate (SPDB) ), N-succinimide 3-(2-pyridyldithio)-propionate (SPDP).

In one embodiment, the antibody drug conjugate comprises the anti-ROR1 antibody or antigen-binding fragment thereof described in the first aspect and a tubulin inhibitor (MMAE). In a further embodiment, MMAE binds cysteine on the anti-ROR1 antibody via a MC-VC-PAB linker. Thiol conjugation, with the structure of anti-ROR1 antibody-MC-VC-PAB-MMAE. IgG1 type antibodies have 16 pairs of cysteine residues, which exist in the form of 12 intrachain and 4 interchain disulfide bonds. The interchain disulfide bond has solvent accessibility and can be reduced by reducing agents to form eight sulfhydryl groups, which then become coupling targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J .2015;17:339-51).

In a specific embodiment, the antibody drug conjugate comprises or consists of anti-ROR1 monoclonal antibody N99 and MC-VC-PAB-MMAE. In a further embodiment, MMAE is conjugated to the sulfhydryl group of the cysteine on N99 via a MC-VC-PAB linker.

In another specific embodiment, the antibody drug conjugate comprises or consists of anti-ROR1 monoclonal antibody N147-135 and MC-VC-PAB-MMAE. In a further embodiment, MMAE is conjugated to the sulfhydryl group of the cysteine on N147-135 via a MC-VC-PAB linker.

In a third aspect, the present invention provides a pharmaceutical composition comprising (1) the antibody of the first aspect or its antigen-binding fragment or the antibody-conjugated drug of the second aspect, and (2) a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides an isolated polynucleotide molecule encoding any of the antibodies or antigen-binding fragments thereof described in the first aspect.

In a fifth aspect, the invention provides a vector comprising the nucleic acid molecule of the fourth aspect. In one embodiment, the vector is an expression vector.

In a sixth aspect, the present invention provides a host cell comprising the vector of the fifth aspect or the nucleic acid molecule of the fourth invention. In some embodiments, the host cell is prokaryotic, such as E. coli. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells.

In a seventh aspect, the present invention provides a method for preventing or treating diseases associated with abnormal expression of ROR1 in a subject in need thereof, comprising administering to the subject a prophylactically effective amount or a therapeutically effective amount of the antibody of the present invention or its antibody. An antigen-binding fragment, or a prophylactically effective or therapeutically effective dose of the antibody-conjugated drug of the present invention, or a prophylactically effective or therapeutically effective dose of the pharmaceutical composition of the present invention.

In one embodiment, the disease associated with abnormal expression of ROR1 is a cancer that highly expresses ROR1, such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer, Breast cancer, neuroblastoma, lung cancer, head and neck cancer, and melanoma. In a specific embodiment, wherein the lung cancer is non-small cell lung cancer, the breast cancer is triple negative breast cancer.

In some embodiments, the ADC molecules or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents, for the uses described herein, e.g., for prophylaxis and/or Treat related diseases or conditions mentioned in this article.

In a specific embodiment, the present invention provides a method for killing ROR1-expressing cells or inhibiting the growth of ROR1-expressing cells, comprising contacting the cells with an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, or an effective amount of the present invention. The antibody conjugate drug of the invention, or an effective amount of the pharmaceutical composition of the invention.

In an eighth aspect, the present invention provides the use of an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of an antibody-conjugated drug for preventing or treating cancer.

The present invention also provides the use of an antibody-conjugated drug comprising an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of a medicament for preventing or treating cancer.

Figures 1A to 1F show the binding activity of each antibody strain Fab obtained in the present invention to huROR1-HEK293 cells measured based on the FACS method; Figure 1A shows the binding activity of strains NW37 Fab and NW79 Fab to huROR1-HEK293 cells; Figure 1B shows the binding activity of strains N137 Fab, N174 Fab and N147 Fab to huROR1-HEK293 cells; Figure 1C shows the binding activity of strains N218 Fab and N204 Fab to huROR1-HEK293 cells; Figure 1D shows the binding activity of strain N99 Fab to huROR1-HEK293 Cell binding activity; Figure 1E shows the binding activity of strain N31 Fab to huROR1-HEK293 cells; Figure 1F shows the binding activity of strains N100 Fab and NW29 Fab to huROR1-HEK293 cells; NC represents the negative control, and MFI represents the median Count the fluorescence intensity.

Figures 2A to 2K show the SEC-HPLC results of the antibodies of the present invention; Figure 2A shows the SEC-HPLC results of N137; Figure 2B shows the SEC-HPLC results of N147; Figure 2C shows the SEC-HPLC results of N218; Figure Figure 2D shows the SEC-HPLC results for N31; Figure 2E shows the SEC-HPLC results for N99; Figure 2F shows the SEC-HPLC results for NW37; Figure 2G shows the SEC-HPLC results for N100; Figure 2H shows the SEC-HPLC results for N174 -HPLC results; Figure 2I shows the SEC-HPLC results for N204; Figure 2J shows the SEC-HPLC results for NW29; Figure 2K shows the SEC-HPLC results for NW79.

Figures 3A to 3F show the binding activity of the antibody of the present invention to the antigen protein huROR1-His measured based on the ELISA method; Figure 3A shows the binding activities of N100, N137 and N147 to the antigen protein huROR1-His; Figure 3B shows the binding activities of N204 and The binding activity of N218 to the antigen protein huROR1-His; Figure 3C shows the binding activity of N31 to the antigen protein huROR1-His; Figure 3D shows the binding activity of NW29, N99 and NW37 to the antigen protein huROR1-His; Figure 3E shows N174 Binding activity to the antigen protein huROR1-His; Figure 3F shows the binding activity of NW79 to the antigen protein huROR1-His.

Figure 4A to Figure 4D show the binding activity of the antibody of the present invention to A549 cells based on the FACS method; Figure 4A shows the binding activity of N100, N137, N174 and N147 to A549 cells; Figure 4B shows the binding activity of N204, N218 and N31 to A549 cells Binding activity of A549 cells; Figure 4C shows the binding activity of N99, NW29 and NW37 to A549 cells; Figure 4D shows the binding activity of NW79 to A549 cells.

Figures 5A to 5C show the binding activity of the antibody of the present invention to huROR1-HEK293 cells measured based on the FACS method; Figure 5A shows the binding activity of N100, N174, N204, N137, N147, N218 and N31 to huROR1-HEK293 cells; Figure 5B shows the binding activity of N99, NW29 and NW37 to huROR1-HEK293 cells; Figure 5C shows the binding activity of NW79 to huROR1-HEK293 cells.

Figures 6A to 6D show the binding activity of the antibody of the present invention to the antigenic protein MusROR1-His measured based on the ELISA method; Figure 6A shows the binding activity of N100, N174, N137 and N147 to the antigenic protein MusROR1-His; Figure 6B shows The binding activity of N204, N31 and N218 to the antigenic protein MusROR1-His; Figure 6C shows the binding activity of NW29, NW37 and N99 to the antigenic protein MusROR1-His; Figure 6D shows the binding activity of NW79 to the antigenic protein MusROR1-His.

Figures 7A to 7D show the binding activity of the antibody of the present invention to the antigen protein huROR2-His measured based on the ELISA method; Figure 7A shows that N100, N174, N137 and N147 do not bind to the antigen protein huROR2-His; Figure 7B shows N204 , N31 and N218 and the binding activity of the antigen protein huROR2-His; Figure 7C shows the binding activity of NW29, NW37 and N99 and the antigen protein huROR2-His; Figure 7D shows the binding activity of NW79 and the antigen protein huROR2-His.

Figure 8A and Figure 8B show the endocytosis efficiency of the antibody of the present invention on huROR1-HEK293 cells measured based on FACS method; Figure 8A shows N204, N147 and NW37 Endocytosis efficiency on huROR1-HEK293 cells; Figure 8B shows the endocytosis efficiency of N100, N174, NW29, NW79, N137, N218, N31, and N99 on huROR1-HEK293 cells.

Figures 9A to 9F show the endocytosis efficiency of the antibody of the present invention on huROR1-HEK293 cells based on the Fab-Zap method; Figure 9A shows the endocytosis efficiency of N31 and N99 on huROR1-HEK293 cells; Figure 9B shows The endocytosis efficiency of N174 and N218 on huROR1-HEK293 cells; Figure 9C shows the endocytosis efficiency of N147 on huROR1-HEK293 cells; Figure 9D shows the endocytosis efficiency of N100 and N137 on huROR1-HEK293 cells; Figure 9E The endocytosis efficiency of N204 and NW37 on huROR1-HEK293 cells is shown; Figure 9F shows the endocytosis efficiency of NW29 and NW79 on huROR1-HEK293 cells.

Figures 10A to 10D show the binding activity of antibodies to A549 cells after affinity maturation determined based on the FACS method; Figure 10A shows the binding activities of N31-6, N31-14, N31-18 and N31-56 to A549 cells; Figure 10A 10B shows the binding activity of N37-37, N37-99, N37-113 and N37-136 to A549 cells; Figure 10C shows the binding activity of N137-47, N137-53, N137-60 and N137-72 to A549 cells. ; Figure 10D shows the binding activity of N147-2, N147-86, N148-93 and N147-135 to A549 tumor cells.

Figure 11A to Figure 11E show the endocytosis efficiency of the antibody on huROR1-HEK293 cells after affinity maturation based on the Fab-Zap method; Figure 11A shows the endocytosis efficiency of N31-6 on huROR1-HEK293 cells; Figure 11B shows Figure 11C shows the endocytosis efficiency of NW37-37 on huROR1-HEK293 cells; Figure 11C shows the endocytosis efficiency of NW137-72 on huROR1-HEK293 cells; Figure 11D shows the endocytosis efficiency of N147-135 on huROR1-HEK293 cells ; Figure 11E shows the endocytosis efficiency of N147-2 on huROR1-HEK293 cells.

Figures 12A to 12D show the results of the binding of ADC of the present invention to A549 cells and HT-29 cells; Figure 12A shows the binding of N99-MMAE to A549 cells; Figure 12B shows the binding of N147-135-MMAE to A549 cells ; Figure 12C shows the binding of N99-MMAE to HT-29 cells; Figure 12D shows the binding of N147-135-MMAE to HT-29 cells.

Figure 13A and Figure 13B show the killing effect of the ADC of the present invention on two tumor cells, A549 and HT-29, based on the MTS method; Figure 13A shows the killing effect of N99-MMAE and N147-135-MMAE on A549 cells. ; Figure 13B shows the killing effect of N99-MMAE and N147-135-MMAE on HT-29 cells.

Figures 14A to 14F show the killing effect of the ADC of the present invention on three tumor cells Jeko-1, MDA-MB-468 and NCI-H1944 based on the CCK8 method; Figure 14A shows the killing effect of N99-MMAE on Jeko-1 cells The killing effect of N99-MMAE on MDA-MB-468 cells; Figure 14B shows the killing effect of N99-MMAE on MDA-MB-468 cells; Figure 14C shows the killing effect of N99-MMAE on NCI-H1944 cells; Figure 14D shows the killing effect of N147-135-MMAE The killing effect on Jeko-1 cells; Figure 14E shows the killing effect of N147-135-MMAE on MDA-MB-468 cells; Figure 14F shows the killing effect of N147-135-MMAE on NCI-H1944 cells.

Figure 15 shows the antigen-dependent killing effect of the ADC of the present invention on huROR1-HEK293 cells measured based on the CCK8 method.

Figures 16A to 16D show the endocytosis efficiency of the ADC of the present invention on two tumor cells, A549 and HT-29, based on FACS detection; Figure 16A shows the endocytosis efficiency of N99-MMAE on A549 cells, and Figure 16B shows Figure 16C shows the endocytosis efficiency of N147-135-MMAE on A549 cells; Figure 16C shows the endocytosis efficiency of N99-MMAE on HT-29 cells; Figure 16D shows the endocytosis efficiency of N147-135-MMAE on HT-29 cells. Swallowing efficiency.

Figures 17A to 17C show the tumor inhibitory effect of the ADC of the present invention in mice; Figure 17A shows the changes in tumor volume in mice in different experimental groups during the experimental period, and the arrows mark the time points of administration; Figure 17B shows Changes in body weight of mice in different experimental groups during the experimental period; Figure 17C shows the tumor weight in mice in different experimental groups after the end of the experiment.

Figures 18A to 18C show the tumor inhibitory effect of the ADC of the present invention in the mice of the A549 tumor model; Figure 18A shows the changes in tumor volume in the mice of different experimental groups during the experimental period, and the arrows mark the time points of administration; Figure 18B shows the changes in body weight of mice in different experimental groups during the experimental period; Figure 18C shows the tumor weight in mice in different experimental groups after the end of the experiment.

Figure 19A and Figure 19B show the tumor inhibitory effect of the ADC of the present invention in the mice of the NCI-N87 tumor model; Figure 19A shows the changes in tumor volume in the mice of different experimental groups during the experimental period, and the arrows mark the time of administration. Points; Figure 19B shows the body weight changes of mice in different experimental groups during the experimental period.

Figure 20A and Figure 20B show the tumor inhibitory effect of the ADC of the present invention in the mice of the MDA-MB-231 tumor model; Figure 20A shows the changes in tumor volume in the mice of different experimental groups during the experimental period, and the arrows mark administration. time points; Figure 20B shows the body weight changes of mice in different experimental groups during the experimental period.

Figure 21A and Figure 21B show the tumor inhibitory effect of the ADC of the present invention in the mice of the MDA-MB-468 tumor model; Figure 21A shows the changes in tumor volume in the mice of different experimental groups during the experimental period, and the arrows mark administration. time points; Figure 21B shows the body weight changes of mice in different experimental groups during the experimental period.

Figure 22A and Figure '22B show the tumor inhibitory effect of the ADC of the present invention in the mice of the Jeko-1 tumor model; Figure 22A shows the tumor suppression effect in the mice of different experimental groups during the experimental period. Tumor volume changes, arrows mark the time points of administration; Figure 22B shows the body weight changes of mice in different experimental groups during the experimental period.

Detailed description of the invention

Before the present invention is described in detail, it is to be understood that this invention is not limited to the specific methods and experimental conditions set forth in this specification, as such methods and conditions may vary. Additionally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

I.Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which they belong. For the purposes of the present invention, the following terms are defined below.

The term "about" when used in connection with a numerical value is meant to encompass a range of numerical values having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

The term "and/or" when used to connect two or more options shall be understood to mean any one of the options or any two or more of the options.

As used herein, the term "comprises" or "includes" means the inclusion of the stated element, integer or step, but not the exclusion of any other element, integer or step. When the term "comprises" or "includes" is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise indicated. For example, when reference is made to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass antibody variable regions that consist of that particular sequence.

The term "ROR1" refers to any recombinant or naturally occurring form of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), a variant or homologue thereof that maintains ROR1 activity. For example, at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity. The variant or homolog has at least 90%, 95%, 96%, 97% of the complete sequence or partial sequence (e.g., 50, 100, 150, or 200 consecutive amino acid portions) compared to the naturally occurring ROR1 protein. , 98%, 99% or 100% amino acid sequence identity. In one embodiment, the ROR1 protein includes the amino acid sequence of Uniprot ID: Q01973.

ROR1 is highly expressed in embryos, and then its expression levels decrease significantly in the adult stage. However, ROR1 expression was found to be significantly increased in a variety of hematological cancers and solid tumors. Blood cancers with high expression of ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL) and myeloid blood cancers. Among solid tumors, cancer types expressing ROR1 include colorectal cancer, intestinal cancer, lung cancer, pancreatic cancer, ovarian cancer and other cancers.

The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit The required antigen-binding activity can be obtained. A complete antibody will typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, for example naturally occurring antibodies in camels may contain only heavy chains.

The term "anti-ROR1 antibody" refers to an antibody molecule that specifically binds to ROR1 and is capable of inhibiting ROR1 activity. An anti-ROR1 antibody is capable of inhibiting ROR1 activity relative to the absence of ROR1 antibody, e.g., by at least partially or completely blocking stimulation of ROR1, reducing, preventing, or delaying the activation of ROR1, or inactivating, desensitizing, or downregulating ROR1. Signal transduction, activity or quantity. In some embodiments, the antibody can inhibit ROR1 activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control.

The term "antibody fragment" refers to a molecule that is distinct from an intact antibody, contains a portion of an intact antibody, and is capable of binding the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single chain antibodies (eg, scFv); single domain antibodies; bivalent or diabodies Specific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and multispecific antibodies formed from antibody fragments.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co. p. 91 (2007)). A single VH or VL domain is sufficient to confer antigen binding specificity.

A "complementarity determining region" or "CDR region" or "CDR" is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point") region. CDRs are mainly responsible for binding to antigenic epitopes. The CDRs in the variable domains are generally referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, including, for example, antibody-based three-dimensional Chothia structure and topology of CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 ( 1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) , Contact (University College London), the international ImMunoGeneTics database (IMGT) (http://imgt.cines.fr/), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

CDRs may also be determined based on having the same AbM numbering position as a reference CDR sequence (eg, any of the CDRs exemplified herein). In one embodiment, the CDRs of the antibodies of the invention are positioned according to the AbM numbering scheme.

Unless otherwise stated, in the present invention, when referring to residue positions in antibody variable regions and CDRs (including heavy chain variable region residues), reference is made to the numbering positions according to the AbM numbering system.

An "affinity matured" antibody refers to an antibody that has one or more changes in one or more CDRs of the antibody that results in an improved affinity of the antibody for the antigen compared to the parent antibody without these changes. Affinity matured antibodies can be generated by methods known in the art.

"Human antibody" or "fully human antibody" or "fully human antibody" are used interchangeably and refer to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody that Antibodies are produced by humans or human cells or are derived from non-human sources utilizing human antibody repertoires or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.

As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD measurement or surface plasmon resonance (SPR) measurement.

The term "half effective concentration ( _EC50 )" refers to the concentration of a drug, antibody, or agent that induces a response of 50% between baseline and maximum after a specified exposure time.

As used herein, the term "therapeutic agent" encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g. immunosuppressants).

The term "antibody drug conjugate" or "ADC" refers to an antibody or antibody fragment that is covalently coupled to a therapeutically active substance or active pharmaceutical ingredient, thereby targeting the therapeutically active substance or active pharmaceutical ingredient to the binding target of the antibody to express out of its pharmacological functions. The therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin capable of killing the cells targeted by the ADC, preferably cancer cells. Covalent attachment of therapeutically active substances, active pharmaceutical ingredients or cytotoxins can be carried out in a non-site-specific manner using linkers, or in a site-specific manner.

The term "site-specific conjugation" refers to the specific attachment of a therapeutically active substance or active pharmaceutical ingredient to a specific site on an antibody. In one embodiment, the coupling is accomplished via a linker.

The term "cytotoxic agent" may be used interchangeably with "cytotoxic" and in the present invention refers to a substance that inhibits or disrupts cellular function and/or causes cell death or destruction. In one embodiment, the cytotoxic agent may include, but is not limited to, bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., bacterin), small molecule toxins, radioactive isotopes, and the like, specifically, such as anthracycline ), camptothecin, combretastain, dolastatin and its auristatin derivatives, duocarmycin, enediyne, geldanamycin, indolino-benzodiazepine dimer, maytansine and its derivatives, puromycin, pyrrolocene Azepine dimer (pyrrolobenzodiazepine dimer), taxane (taxane), vinca alkaloid (vinca alkaloid), tubulysin (tubulysin), hemiasterlin (hemiasterlin), spliceostatin (spliceostatin), pladienolide and calicheamicin.

Any antibody-conjugated drug of the present invention can be prepared by conjugating dolastatin and its auristatin derivatives with antibodies. Dolastatin and its auristatin derivatives are important cytotoxins used in antibody-drug conjugates (ADCs). They interfere with microtubule dynamics, cell division, etc., and have anti-tumor and anti-fungal properties. active. The modification of its skeleton has been widely reported in the literature, mainly the modification of the terminal subunits: P1 (N-terminus) and P5 (C-terminus). The modification of the central peptide subunit also resulted in this type of substance having effective cytotoxicity in vitro. active. In one aspect, dolastatin and its auristatin derivatives can be, for example, 0101 (2-methylpropylamine acyl-N-[(3R,4S,5S)-3-methyl Oxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxy-3-{[(1S)-2-phenyl-1-( 1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L- Valamine), 8261(2-methylpropylamine-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S) -1-Carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxypropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl -1-Oxoheptan-4-yl]-N-methyl-L-valinamide) Dolastatin 10, Dolastatin 15, auristatin E, auristatin auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin (monomethyl auristatin) )F (MMAF), auristatin (auristatin) F phenylenediamine (AFP), auristatin (auristatin) EB (AEB), auristatin (auristatin) EFP (AEFP), auristatin ( auristatin) F hydroxypropylamide (AFHPA), and other auristatins (such as auristatin described in U.S. Publication No. 20130129753), etc.

Monomethyl auristatin (MMAE), desmethyl-auristatin E, is a well-known member of the auristatin compound family. Its structural formula is as follows:

MMAE plays an effective mitotic inhibitory effect by inhibiting tubulin polymerization. It cannot be used as a drug due to its cytotoxicity, but it is widely used to prepare antibody conjugates. MMAE is coupled to a monoclonal antibody (MAB) through a linker to form MMAE-MAB. Generally speaking, MMAE-MAB is targeted to tumor cells by antibodies, and the linker is cleaved after MMAE-MAB enters the tumor cells, thereby releasing MMAE, allowing it to exert a cytotoxic effect and kill tumor cells.

The terms "linker" and "linker" are used interchangeably in the present invention to refer to the chemical moiety that covalently links an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC. In one embodiment, the linker may comprise amino acid residues linking the antigen to the payload. Amino acid residues can form dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide, or dodecapeptide units. Amino acid residues include those naturally occurring as well as non-naturally occurring amino acid analogs, such as citrulline or beta-amino acids, such as beta-alanine, or omega-amino acids such as 4-amino- Butyric acid.

According to the classification of properties, the linkers suitable for the present invention can be cathepsin-degradable linkers, such as valine-citrulline (val-cit) linkers, cBu-Cit linkers and CX linkers; non-cleavable linkers such as SMCC linkers or MD Linkers; acid-sensitive linkers, silicone structure linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared sensitive linkers Connectors, UV-sensitive connectors such as PC4AP.

The linker of the present invention can also be a combination of one or more linkers. For example, a linker degraded by cathepsin can be combined with other types of linkers to form a new linker. Therefore, this invention The "linker" mentioned above covers a single type of linker, or a combination of different types of linkers, as long as it can couple the antibody of the present invention with the drug.

In specific embodiments, linkers include, but are not limited to, maleimino-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine Amino acid-Valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propyl, and maleimidecaproyl (mc), maleyl Aminopropyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimide 4-(2-Pyridylthio)valerate (SPP), N-succinimide 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) , N-succinimidyl (4-iodo-ethyl) amino benzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio)butyrate (SIAB) SPDB), N-succinimide 3-(2-pyridyldithio)-propionate (SPDP).

The term "load" or "drug load" or "payload" refers to the average number of payloads per antibody within the ADC molecule ("payload" is used interchangeably herein with "therapeutic active substance or active pharmaceutical ingredient"). Drug loads can range from 1-20 therapeutic actives or active pharmaceutical ingredients per antibody. The term "drug/antibody ratio" or "DAR" refers to the ratio of the therapeutically active substance or active pharmaceutical ingredient (D) coupled to the antibody to the antibody. The ADCs described herein generally have a DAR of 1-20, and in certain embodiments have a DAR of 1-8, 2-8, 2-6, 2-5, 2-18, 4-16, 5-12, 6 -DAR of 10, 3-8, 4-6, 6-10, and 2-4. Representative DAR values are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, usually expressed as a combination of the letter D and a number, where the number represents the numerical value of the DAR, For example, D2 represents the drug/antibody ratio with a DAR value of 2. In some embodiments, the DAR is the average DAR, that is, the product coupled herein as measured by a detection method (e.g., by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA assays, electrophoresis, and/or HPLC). The overall ratio of the small molecule drug part (D) of the Ab part to the Ab part. DARs may be limited by the number of attachment sites on the antibody. For example, in the link In the case of cysteine thiols, the antibody may have only one or a few cysteine thiol groups or may have only one or a few fully reactive thiol groups through which the thiol group can be attached connection unit). In some embodiments, the average DAR value of the conjugates of the invention is 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4 -6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 ,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4,4.1,4.2,4.3,4.4,4.5,4.6 ,4.7,4.8,4.9,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0,7.1 ,7.2,7.3,7.4,7.5,7.6,7.7,7.8.0,7.9,8,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9.0,9.1,9.2,9.3,9.4,9.5 , 9.6, 9.7, 9.8, 9.9, or 10.0, with two of these values as the endpoints.

The term "treat" means to slow, interrupt, arrest, alleviate, stop, reduce, or reverse the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the emergence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay disease progression or to slow the progression of disease.

The term "prevention" includes the inhibition of the occurrence or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventive regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

The term "effective amount" refers to an amount or dose of an antibody or conjugate or composition of the invention that produces a prognostic factor in a patient in need of treatment or prophylaxis upon administration to the patient in single or multiple doses. period effect. The effective amount can be readily determined by the attending physician, who is one of ordinary skill in the art, by considering factors such as: species of mammal; weight, age, and general health; the specific disease involved; and the extent or severity of the disease. properties; the individual patient's response; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at required doses and for required periods of time. The therapeutically effective amount of an antibody or antibody fragment, or conjugate or composition thereof, can vary depending on factors such as the disease state, the age, sex and weight of the individual and the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment, or conjugate or composition thereof, are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50% relative to an untreated subject , 60% or 70% and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (eg, cancer) can be evaluated in animal model systems that are predictive of efficacy in human tumors.

The term "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result, at required doses and for required periods of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.

The term "pharmaceutical composition" refers to a composition that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain additional substances that would be unacceptable toxicities to the subject to whom the composition is administered. Element.

II. Composition of the present invention

In some embodiments, the invention provides a composition comprising any anti-ROR1 antibody described herein, or an ADC molecule thereof, preferably the composition is a pharmaceutical composition. In a In one embodiment, the composition further includes pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. In one embodiment, a composition (eg, a pharmaceutical composition) includes an anti-ROR1 antibody of the invention, or an ADC molecule thereof, in combination with one or more other therapeutic agents.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

The compositions of the present invention may be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), powders or suspensions, liposomes, and suppositories. The preferred form will depend on the intended mode of administration and therapeutic use.

The administration route of the composition of the present invention is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebrally (intraparenchymal), intracerebroventricularly, intramuscularly, intraocularly, intraarterially, intraportally or Intralesional route; by sustained release system or by implanted device. In certain embodiments, the compositions may be administered by bolus injection or by continuous infusion or by an implanted device.

The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (eg, an individual suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject has a disease (eg, cancer) or is at risk of having a disease described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy treatments and/or radiation therapy. In some embodiments, the subject has previously received or is currently receiving immunotherapy.

Medicaments containing the antibodies described herein can be prepared by mixing the anti-ROR1 antibodies of the invention, or ADC molecules thereof, with the desired purity and one or more optional pharmaceutical excipients, preferably in a lyophilized formulation or form of aqueous solution.

The pharmaceutical compositions or preparations of the present invention may also contain more than one active ingredient required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immune modulators (eg, immune checkpoint inhibitors or agonists), and the like. The active ingredients are suitably present in combination in amounts effective for the intended use.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include a semipermeable matrix of a solid hydrophobic polymer containing the antibody in the form of a shaped article, such as a film or microcapsules.

III. Preparation of antibodies and antibody-drug conjugates of the present invention

In one embodiment, the invention provides a method for preparing an anti-ROR1 antibody, wherein the method comprises culturing a nucleic acid encoding an anti-ROR1 antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of a nucleic acid encoding the anti-ROR1 antibody. host cells, and optionally isolate the anti-ROR1 antibody. In a certain embodiment, the method further includes recovering an anti-ROR1 antibody from the host cell (or host cell culture medium).

In order to recombinantly produce the anti-ROR1 antibody of the present invention, the nucleic acid encoding the anti-ROR1 antibody of the present invention is first isolated, and the nucleic acid is inserted into a vector for further selection and/or expression in host cells. Such nucleic acids are readily isolated and sequenced using conventional procedures, for example by using oligonucleotide probes capable of specifically binding to nucleic acids encoding anti-ROR1 antibodies of the invention.

Anti-ROR1 antibodies of the invention prepared as described herein can be prepared by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion Chromatography and other purification. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those of ordinary skill in the art. The purity of the anti-ROR1 antibody of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, and the like.

The prior art describes a variety of methods for conjugating cytotoxic or other therapeutic agents to antibodies. For example, conjugation can be carried out in the antibody through the amine group of the lysine side chain and the amine group at the N-terminal end of the antibody, aspartic acid, glutamic acid and the carboxyl group at the C-terminal or activated cysteine thiol group. Combination reaction occurs.

Example

The following examples further illustrate the present invention, however, it should be understood that the examples are described by way of illustration rather than limitation, and that various modifications may be made by those skilled in the art.

Unless expressly stated to the contrary, the practice of the present invention will employ conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology within the skill in the art.

Example 1 Preparation and identification of raw materials

1.1 Preparation and identification of ROR1 control antibody

Preparation of ROR1 control antibody: The present invention uses the anti-ROR1 antibody Cirmtuzumab as a positive control antibody, and gene synthesis of the target fragment is performed by General Biotechnology Co., Ltd. according to the sequence disclosed in WO/2019/173843. Then, the eukaryotic expression vector pcDNA3.4 (Invitrogen) was constructed by homologous recombination. The constructed recombinant protein expression vectors were transformed into E. coli DH5α, cultured overnight at 37°C, and then endotoxin-free plasmid extraction reagents were used. cassette (OMEGA, D6950-01) for plasmid extraction, and the desired expression vector expressing cetolizumab was obtained. The strain number is 99961.1. The expressed cetolizumab will be referred to as 99961.1 in the following. The expression vector was transfected into 293 cells to express cetolizumab using ExpiFectamine ^TM CHO transfection kit (Thermo Fisher, A29129). Seven days after transfection, the cell culture supernatant was collected and centrifuged at 15000g for 10 min. After the supernatant was filtered through a 0.22 μm filter membrane, the antibodies in the supernatant were affinity purified using a Protein A/G affinity chromatography column. The antibody of interest was eluted with 100 mM glycinate (pH 3.0), and the eluted antibody was exchanged into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

ROR1 control antibody identification: Use the purchased huROR1-His antigen protein (Kaika Biotechnology, ROR-HM401) to detect the activity of the prepared positive control antibody 99961.1 (IgG1). The specific method is as follows: Coat huROR1-His on a 96-well ELISA plate (2μg/mL, 30μL/well), coated overnight at 4°C; after washing the plate three times, block with 5% skim milk in PBS for 1 hour at room temperature; after washing the plate three times, add control antibodies gradient diluted in PBS 99961.1 Incubate at room temperature for 1 hour; after washing the plate, add the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted in PBS (1:6000). After incubating at room temperature for 1 hour, wash the plate for 6 Add TMB at a time to develop color for 5-20 minutes. After the color development is terminated, read the data with a microplate reader OD450 and process the data for graphing with Graphpad prism. The results showed that the expressed control antibody 99961.1 could bind to ROR1 protein and had normal anti-ROR1 activity.

1.2 Preparation and identification of ROR1 antigen protein

Antigenic protein preparation: through genetic manipulation at the coding gene level, human ROR1 protein huROR1 ECD AA30-406 (Uniprot ID: Q01973), mouse ROR1 protein MusROR1 ECD AA30-406 (Uniprot ID: Q9Z139), and human ROR2 protein The sequence C-terminus of huROR2 ECD AA34-403 (Uniprot ID: Q01974) is added with a His tag. The obtained nucleic acid sequences were constructed into the pcDNA3.4 vector, then transformed into E. coli DH5α, cultured at 37°C overnight, and then the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The obtained plasmid was transiently transferred into HEK293 cells (ATCC® CRL-1573 ^TM ) using ExpiFectamine ^TM 293 transfection kit (Gibco ^TM , A14524). After expression for 7 days, the cell culture supernatant was collected and the cells containing His were The tagged protein was affinity purified using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was extracted with imidazole gradient. Each extracted protein was exchanged into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096), and finally the antigenic proteins (huROR1-His, MusROR1-His, huROR2-His) were obtained.

Antigen identification: Use the quality-tested antibody 99961.1 (IgG1) obtained in Example 1.1 to detect the prepared antigen (huROR1-His). The specific method is as follows: coat the Elisa plate with 2 μg/mL huROR1-His overnight at 4°C, and use the purchased antigen protein huROR1-His (Kaika Biotech ROR-HM201) as a positive control; after washing the plate three times, use 5 % skim milk for blocking at room temperature for 1 hour; after washing the plate three times, add antibody 99961.1 gradient diluted in PBS and incubate at room temperature for 1 hour; after washing the plate, add secondary antibody Anti-human-IgG- diluted in PBS (1:6000) Kappa+Lambda-HRP (Millipore, AP502P+AP506P) was incubated at room temperature for 1 hour, washed 6 times, then added TMB for color development for 5-20 minutes, terminated the color reaction, read the data using a microplate reader OD450, and processed the data using Graphpad prism. Figure. The results showed that the antibody 99961.1 could bind to the antigen huROR1-His constructed and expressed by the inventors of the present invention with an affinity comparable to that of the purchased ROR1 antigen protein.

Example 2 Construction and identification of cell lines overexpressing human ROR1

Construction of HEK293 cell line overexpressing human ROR1 (hereinafter referred to as huROR1-HEK293): The coding nucleic acid sequence of full-length human ROR1 (Uniprot ID: Q01973) was constructed into pLVX-puro plasmid (Clontech, Cat#632164). Then, the obtained plasmid was electrotransformed into HEK293 cells (ATCC® CRL-1573 ^TM ) by electroporation (Invitrogen, NeonTM Transfection System, MP922947). After electroporation, the obtained cells were transferred to DMEM medium (Gibco, 11995065) containing 10% volume percentage of FBS (Gibco, 15140-141) without antibiotics, and then the cells were transferred into 10 × 10 cm cells. Cultivate in a culture dish for 48 hours, then distribute the cells into a 96-well cell culture plate at an average density of 10 ⁴ cells/well, add puromycin at a final concentration of 2 μg/mL as the screening pressure, and pick them in about 2 weeks. The cell lines forming the strain were identified.

Flow cytometry identification of huROR1-HEK293 cells: Digest the above-mentioned cell lines in the logarithmic growth phase and plate them into a 96-well plate. After washing with FACS buffer (1×PBS buffer containing 2% FBS by volume), Add the primary antibody (99961.1) serially diluted in PBS and incubate at 4°C for 30 minutes; after washing, add the prepared fluorescent secondary antibody anti human IgG Fc (abcam, 98596), and incubate at 4°C for 30 minutes; finally, analyze by flow cytometry (Beckman , CytoFLEXAOO-1-1102) for detection. The test results showed that the huROR1-HEK293 cell line with high surface expression of human ROR1 was obtained.

Example 3 Construction and screening of human phage display recombinant antibody library

In this example, an antibody gene phage display library was constructed, and the antigen protein huROR1-His prepared in Example 1.2 and the huROR1-overexpressing cell huROR1-HEK293 prepared in Example 2 were used as screening antigens to screen the library, and obtained Multiple antibody molecules that specifically bind human ROR1.

3.1 Construction of human antibody gene library

Use Ficoll-Paque density gradient separation solution (purchased from GE, catalog number: 17144003S) to isolate peripheral blood mononuclear cells (PBMC) from normal human blood, and extract total RNA from the isolated PBMC cells by conventional methods. . The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa Company, catalog number: 6210A). Based on the sequence similarity of the heavy chain and light chain germline genes, degenerate primers were designed at the front end of the V region and the back end of the first constant region of the heavy chain and light chain respectively, and the heavy chain variable region gene fragment of the antibody was obtained after PCR. and light chain variable region gene segments. Fragments containing the light chain and heavy chain variable regions of the antibody were amplified by the fusion PCR method. The PCR product and the phage display vector were digested, recovered and ligated. The ligation product was purified using a recovery kit (Omega, catalog No.: D6492-02) recovery (Li Xiaolin, construction and preliminary screening of large-capacity non-immune human Fab phage antibody library, Master's thesis of "China Union Medical University", June 2007). Finally, it was transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) by electroporation (Bio-Rad, MicroPulser), and the transformed Escherichia coli SS320 liquid was coated on 2-YT solid with ampicillin resistance. Plate (solid plate is prepared from 1.5% trypsin, 1% yeast extract, 0.5% NaCl, 1.5% agar, based on mass volume g/mL). Through gradient dilution and plating, the library capacity was measured to be 3×10 ¹¹ cfu, that is, an antibody gene library of 3×10 ¹¹ antibody genes (refer to Example 2.2 in patent CN112250763B for the library capacity calculation method). The VSCM13 helper phage (purchased from Stratagene) was used to package it, and an antibody gene phage display library was obtained (for the preparation of the antibody gene phage display library, refer to Example 2.3 in patent CN112250763B).

3.2 Screening of antibody gene phage display library

3.2.1 Magnetic bead method for screening antibody gene phage display library

Magnetic bead screening is based on labeling the antigen protein huROR1-His with biotin and then binding it to magnetic beads coupled with streptavidin. The antigen-bound magnetic beads and the antibody gene phage display library are incubated and washed. and the extraction process. It usually undergoes 3-4 rounds of panning, whereby specific monoclonal antibodies against the antigen can be enriched in large quantities. In this example, biotin-labeled huROR1-His was used for phage display library screening, and after three rounds of panning, monoclonal antibody Fab against human ROR1 was initially screened. For specific methods, refer to Example 2.4.1 in patent CN112250763B. .

3.2.2 Immunotube method to screen antibody gene phage display library

The principle of immune tube screening is to coat the antigen protein huROR1-His on the surface of an immune tube with high adsorption capacity, add the phage display antibody library to the immune tube and incubate and wash it with the antigen protein adsorbed on the surface of the immune tube. After 2-4 rounds of panning, the specific monoclonal antibody Fab against the antigen is finally enriched. In this example, monoclonal antibody Fab against human ROR1 was enriched after three rounds of panning. For specific methods, refer to Example 2.4.2 in patent CN112250763B.

3.3 Selection of individual plants

The phage pool extracted in each round was tested by ELISA to evaluate the enrichment effect, and 10 strains were randomly selected from the phage pool screened in each round for sequence analysis, and a comprehensive analysis was conducted based on the enrichment effect and the repeatability ratio of the measured sequences. , choose the appropriate round for individual plant selection.

ELISA single strain primary screening uses the antigen protein huROR1-His, and the huROR1-His-binding antibody Fab obtained in the primary screening is prepared into Fab lysate, and then the overexpressed cell huROR1-HEK293 prepared in Example 2.1 is used for flow cytometric analysis. (FACS) was used for detection and review, and a total of 11 antibody Fab molecules that specifically bind to human ROR1 were screened. The 11 antibody Fab strains obtained were named according to the corresponding strain numbers (N99, N218, N31, N137, N147, N100, N204, N174, NW29, NW79 and NW37), the specific FACS results are shown in Figure 1A to Figure 1F. The amino acid sequence of the CDR region of the obtained antibody Fab is shown in Table 1. The CDR sequence was determined using the method of AbM defining CDR.

Example 4 Antibody construction, expression and purification

4.1 Plasmid construction

The VH coding sequence in the Fab sequences of individual strains N99, N218, N31, N137, N147, N100, N204, N174, NW29, NW79 and NW37 obtained through screening was combined with the heavy chain constant region of human IgG1 (SEQ ID NO: 48) The coding sequence of the human antibody is connected to obtain the heavy chain coding sequence of the fully human antibody, and the VL coding sequence in the Fab sequence is combined with the Kappa type (SEQ ID NO: 49) or Lambda type (SEQ ID NO: CL) of the human light chain constant region (CL). The coding sequence of 50) was ligated to obtain the light chain coding sequence of a fully human antibody. The coding sequences of the antibody heavy chain and light chain were inserted into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen) respectively, transformed into E. coli DH5α, and cultured at 37°C overnight. Use endotoxin-free plasmid extraction kit (OMEGA, D6950-01) for plasmid extraction to obtain endotoxin-free antibody plasmid for eukaryotic expression.

4.2 Expression and purification of antibodies

The full-length antibody sequence obtained above was expressed by the ExpiCHO transient expression system (Thermo Fisher, A29133). The specific method is as follows: on the day of transfection, confirm that the CHO cell density is about 7×10 ⁶ to 1×10 ⁷ viable cells/mL. The cell survival rate is >98%. At this time, use fresh ExpiCHO expression medium pre-warmed at 37°C to adjust the cells to a final concentration of 6×10 ⁶ cells/mL. Dilute the target plasmid with OptiPRO ^TM SFM pre-cooled at 4°C (add 1 μg of plasmid to 1 mL of the culture medium), and at the same time dilute the ExpiFectamine ^TM CHO reagent with OptiPRO ^TM SFM. Mix the two in equal volumes and mix gently by pipetting to prepare ExpiFectamine. ^TM CHO/plasmid DNA mixture was incubated at room temperature for 1-5 minutes, slowly added to the prepared cell suspension while shaking gently, and finally placed in a cell culture shaker at 37°C and 8% _CO2. Cultivate.

18-22 hours after transfection, add ExpiCHO ^TM Enhancer reagent and ExpiCHO ^TM Feed reagent to the cell culture medium, and place the shake flask on a 32°C shaker and 5% CO ₂ to continue culturing. On day 5 after transfection, add the same volume of ExpiCHO ^TM Feed reagent and mix the cell suspension gently while adding slowly. Seven days after transfection, the cell culture supernatant expressing the target antibody protein was collected, centrifuged at 15,000g for 10 minutes, and the supernatant was affinity purified with MabSelect SuRe LX (GE, 17547403), and then eluted with 100mM sodium acetate (pH 3.0) The target antibody protein was then neutralized with 1M Tris-HCl, and finally the resulting antibody protein was exchanged into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

Example 5 Detection of physical and chemical properties of antibodies

In this example, SDS-PAGE and SEC-HPLC were used to detect the relative molecular weight and purity of the candidate antibodies.

5.1 Antibody SDS-PAGE identification

Preparation of non-reducing solution: Add 1 μg of each obtained antibody and quality control product IPI (Ipilimumab) to 5×SDS loading buffer and 40mM iodoacetamide, heat in a dry bath at 75°C for 10 minutes, and cool After reaching room temperature, centrifuge at 12,000 rpm for 5 minutes to collect the supernatant.

Preparation of reducing solution: Add 2 μg of each obtained antibody and quality control product IPI to 5×SDS loading buffer and 5mM DTT, heat in a dry bath at 100°C for 10 minutes, cool to room temperature, and centrifuge at 12,000 rpm for 5 minutes to collect the supernatant. The supernatant was added to Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and Coomassie brilliant blue staining was used to develop the protein bands.

Use EPSON V550 color scanner to scan the protein gel with colored protein bands (decolorize with destaining solution until the gel background is transparent), and use ImageJ to calculate the purity of the reduced and non-reduced bands according to the peak area normalization method.

The test results show that the bands of each antibody on non-reducing gel are around 150kD, and the bands on reducing gel are around 55kD and 25kD, which are in line with the expected size. The purity of all candidate antibodies tested by reducing gel was greater than 95% (Table 2).

5.2 SEC-HPLC identification of antibody monomer purity

Material preparation: 1. Mobile phase: 150mmol/L phosphate buffer, pH 7.4; 2. Sample preparation: Dilute each antibody and quality control IPI to 0.5 with mobile phase solution. mg/mL. Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph, the chromatographic column is XBridge BEH (SEC 3.5μm, 7.8mm I.D.×30cm), the Waters flow rate is set to 0.8mL/min, the injection volume is 20μL, and the VWD detector wavelength is 280nm and 214nm. Inject blank solution, IPI quality control solution and antibody sample solution in sequence. Calculate the percentage of high molecular polymers, antibody monomers and low molecular substances in the sample according to the area normalization method.

The results are shown in Figure 2A to Figure 2K and Table 2. Except for N204 and NW79, whose monomer purity is around 95%, the monomer purity of other antibodies is greater than 98%.

Example 6 Detection of Antigen Binding Activity of Antibodies

In this example, the expressed antibodies (N99, N218, N147, N137, N31, NW37, N100, N204, N174, NW29 and NW79) were detected based on the ELISA method The binding to human ROR1 antigen protein huROR1-His was also tested based on the FACS method between the expressed antibodies (N99, N218, N147, N137, N31, NW37, N100, N204, N174, NW29 and NW79) and human ROR1 overexpressing cells. Binding ability of huROR1-HEK293 and A549 cells. A549 cells are a human non-small cell lung cancer cell line that overexpress huROR1.

6.1 Detection of antibody binding ability to antigenic protein huROR1-His based on ELISA

Coat a 96-well ELISA plate (30 μL/well) with 2 μg/mL huROR1-His and incubate at 4°C overnight. The next day, wash the well plate three times with PBST and then block it with 5% skim milk for 2 hours. After washing the plate three times with PBST, add gradient dilution (3.00000, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00046, 0.00005μg/mL ) of each antibody and the positive control antibody 99961.1 and incubated for 1 h. After washing with PBST three times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed 6 times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in Figures 3A to 3F. All the antibodies obtained in the present invention have good binding ability to the antigen protein huROR1-His. The binding ability of each antibody to the antigen protein is equivalent to the positive control antibody 99961.1.

6.2 Detection of antibody binding ability to huROR1-HEK293 and A549 cells based on FACS

In this example, human ROR1 overexpressing cells huROR1-HEK293 and A549 cells were used to evaluate the binding activity of the antibody.

The specific method is as follows: huROR1-HEK293 cells or A549 cells in the logarithmic growth phase are prepared into a single cell suspension, the density is adjusted to 1×10 ⁶ cells/mL, and 100 μL per well is added to a 96-well round bottom plate at 4°C, Centrifuge at 300g and remove supernatant. Add various gradient dilutions (3.000000, 0.300000, 0.100000, 0.033333, 0.011111, 0.003704, 0.001235, 0.000123μg/mL) of each antibody and positive control antibody 99961.1 to the corresponding wells, mix and incubate at 4°C for 30 minutes. Wash the incubated cell mixture three times, add 100 μL of secondary antibody Goat F(ab')2 Anti-Human IgG-Fc (abcam, ab98596) diluted 1:300, incubate for 30 minutes at 4°C in the dark, and wash three times. Detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

The results are shown in Figure 4A to Figure 4D and Figure 5A to Figure 5C. On A549 cells, the affinity of NW29, N147 and N99 to human ROR1 is better than that of the positive control antibody 99961.1, and the affinity of the remaining antibodies to human ROR1 is slightly weaker than that of the positive control antibody. 99961.1; on huROR1-HEK293 cells, the affinity of N137 and N31 to human ROR1 is better than that of positive control antibody 99961.1, and the affinities of other antibodies are equivalent to or weaker than positive control antibody 99961.1.

Example 7 Antibody species and homogeneous cross-activity detection

In this example, the cross-reactivity of each antibody obtained in the present invention in terms of species and congeners was tested. The mouse ROR1 antigen protein MusROR1-His and the human ROR2 antigen protein huROR2-His prepared in Example 1.2 were used for measurement.

7.1 Species cross-reactivity detection of antibodies of the present invention

Coat a 96-well ELISA plate (30 μL/well) with 2 μg/mL MusROR1-His and incubate at 4°C overnight. The next day, wash the well plate three times with PBST and then block it with 5% skim milk for 2 hours. After washing the plate three times with PBST, add gradient dilution (1.00000, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, 0.00015, 0.00002 μg/mL ) of each antibody and the positive control antibody 99961.1 and incubated for 1 h. After washing with PBST three times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed 6 times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, add 2M HCl The reaction was terminated and the absorbance was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in Figures 6A to 6D and Table 3. All antibodies obtained in the present invention can bind to the mouse antigen protein MusROR1-His, showing good mouse cross-activity. The positive control antibody 99961.1 does not bind to mouse antigen proteins. Therefore, the antibodies obtained in the present invention are widely used in experiments and tests based on mouse models.

7.2 Detection of homologous cross-reactivity of antibodies of the present invention

Coat a 96-well ELISA plate (30 μL/well) with 2 μg/mL huROR2-His and incubate at 4°C overnight. The next day, the well plate was washed three times with PBST and blocked with 5% skim milk for 2 hours. After washing the plate three times with PBST, gradient dilutions of the candidate antibody and positive control antibody 99961.1 were added and incubated for 1 hour. After washing with PBST three times, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After the incubation was completed, the plate was washed 6 times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the absorbance was read at OD450 by a microplate reader (Molecular Devices, SpecterMax 190).

The results are shown in Figure 7A to Figure 7D and Table 3. N204, NW29, NW79, N218, N31 and N99 can bind to the antigen protein huROR2-His, while the remaining antibodies do not bind to the antigen protein huROR2-His.

Example 8 Detection of endocytosis efficiency of antibodies of the present invention

In this example, two detection methods, FACS and Fab-Zap, were used to identify the endocytosis efficiency of the antibodies obtained in the present invention. The FACS endocytosis test uses supersaturated antibodies to bind to cells to detect the antibody endocytosis efficiency in a short period of time; the Fab-Zap method uses antibodies that bind saporin toxins at different concentrations to bind to cells and bring the toxins into the cells through endocytosis. To kill target cells, this method reflects the long-term cumulative effect of endocytosis efficiency.

8.1 FACS method to detect antibody endocytosis efficiency

Antibody dilution: Dilute the test antibody with DMEM complete medium to a final concentration of 10.0000 μg/mL.

Treatment of cells: Digest huROR1-HEK293 cells and add DMEM complete medium. After mixing the cells thoroughly, count the cells and determine their viability. Add 10 ⁶ cells to a 1.5mL centrifuge tube, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend in 1mL of pre-cooled DMEM culture medium, centrifuge at 300g for 5 minutes, discard the supernatant.

Primary antibody incubation: Take 1000 μL of the pre-cooled above-mentioned diluted antibody and add it to a centrifuge tube with existing cells to prepare an antibody cell suspension. Add the antibody cell suspension to a 96-well plate and incubate at 4°C.

Extracellular secondary antibody incubation: Quickly transfer the suspension in the 96-well plate to a second 96-well plate. Add 180 μL of pre-cooled FACS buffer to each well of the second 96-well plate and wash twice. Then add 100 μL of diluted secondary antibody FITC-labeled rabit-anit-huFc or RPE-labeled rabit-anit-huFc to each well (the secondary antibody is diluted 1:150 in FACS buffer); incubate at 4°C for 30 minutes.

Cell fixation: After incubation, centrifuge and discard the supernatant, add 180 μL pre-cooled FACS buffer to each well and wash the cells twice. Centrifuge to remove the supernatant, and fix the cells with 100 μL of 4% paraformaldehyde at room temperature for 30 min in each well.

Broken cell membrane: After fixation, add 180 μL FACS buffer and wash twice. Add 100 μL of preheated 0.5% Triton X-100 to each well and punch at room temperature for 5 min.

Intracellular secondary antibody incubation: After washing the 96-well plate, add 180 μL of preheated Permeabilization Buffer (Invitrogen ^TM eBioscience ^TM , 00-8333-56) to each well and wash twice. Add 100 μL of diluted secondary antibody RPE-labeled rabit-anit-huFc to each well (diluted 1:150 using Permeabilization buffer). Incubate at room temperature for 60 minutes.

Detect fluorescence: After washing the 96-well plate, resuspend it in 100 μL FACS buffer and detect it by flow cytometry.

This experiment uses two sets of different fluorescence settings to stain each sample. The first set is FITC+PE, that is, the FITC secondary antibody is first used to identify the extracellular primary antibody, and after the membrane is broken, the PE secondary antibody is used to identify the intracellular primary antibody. In this group, PE is the intracellular signal and FITC is the extracellular signal; the second group is set to PE+PE, that is, the PE secondary antibody is first used to recognize the extracellular primary antibody, and after the membrane is broken, the PE secondary antibody is used to recognize the intracellular primary antibody. This group of PE is the sum of intracellular and extracellular signals. At the same time, these two groups of samples are tested using FITC and PE channels. The calculated endocytosis rate values are based on the PE channel detection values. The specific formula is: endocytosis rate = one group (FITC+PE) PE channel value/two groups (PE +PE)PE channel value×100%.

The results are shown in Figure 8A and Figure 8B. The results of this example show that the endocytosis efficiency of N147, N100, NW37, N99 and N31 is better than that of the control antibody 99961.1 in a shorter period of time (within 3 hours). N174, N218, N204 and NW29 is equivalent to control antibody 99961.1.

8.2 Fab-Zap method to detect antibody endocytosis efficiency

This experiment detects the endocytic activity of the antibody through antibody-mediated Fab-ZAP endocytosis cytotoxicity. Fab-ZAP is a Fab fragment connected to saporin (saponin), a ribosome inhibitor that can inhibit protein synthesis and cause cell death. The Fab-ZAP used in this experiment is a Fab fragment that can bind to human antibodies. After incubation of Fab-ZAP and human antibodies, the human antibodies are loaded with toxins. When the human antibodies are endocytosed, the toxins follow the antibodies. It enters the cells, causes cell death, and then uses MTS (Promega, G3580) to detect the activity of the cells to detect whether the antibody is internalized.

The specific experimental method is as follows: first, Fab-ZAP is diluted to 0.4nM with DMEM complete culture medium, and then gradient diluted with 0.4nM Fab-ZAP (0.400000, 0.080000, 0.026667, 0.008889, 0.002963, 0.000988, 0.000329, 0.000066μg/mL). The obtained antibodies and positive control antibodies were prepared into antibody diluents. Prepare a single cell suspension from huROR1-HEK293 cells in the logarithmic growth phase, adjust the density to 6×10 ⁶ cells/mL, inoculate 50 μL per well into a 96-well plate, then take the aforementioned antibody dilution and add 50 μL per well. In the cell culture plate, mix thoroughly by pipetting. Place the cell culture plate into a 37°C cell culture incubator and incubate for 48 hours. After the incubation, add 7.5 μL TritonX-100 solution to each well, mix gently, and place the cell culture plate in a 37°C cell culture incubator for 0.5 hours. Then add 20 μL MTS to each well and incubate at 37°C for 1-4 hours. Finally, centrifuge the cell culture plate at 1000 rpm for 5 minutes, read the data with a microplate reader, and detect wavelength A492.

The results are shown in Figures 9A to 9F, indicating that under the antibody concentration conditions defined in this example, the endocytosis effect of the antibody prepared in the present invention is equivalent to that of the control antibody. Considering that cells are sensitive to toxins only when they are above a certain threshold, it is difficult to widen the gap when the difference in accumulated toxins is not large.

Example 9 Antibody affinity kinetics (Biacore) analysis

In this example, Biacore equipment was used to detect the affinity of the antibody obtained in the present invention and the positive control antibody 99961.1 with the antigen protein huROR1-His.

Coupling of protein: The huROR1-His protein produced in Example 1.2 was diluted to 5.6 μg/mL with NaAc buffer at pH 5.0, and the flow rate was set to 10 μL/min. The chip was activated with a mixture of EDC and N-hydroxysuccinimide (NHS) at the default value of 420s, and the antigen protein huROR1-His was immobilized to about 75RU using the coupling mode with the preset coupling amount. level, use ethanolamine to block the activated groups that are not bound to the test product.

Sample test conditions: Use PBS buffer (pH7.4) containing 0.05% Tween-20 as the running buffer, use the running buffer as the control test sample, set a series of antibody concentrations (4nM, 20nM), and set the flow rate during sample analysis to 30μL/min, binding time is 120s, dissociation time is 360s. After the dissociation is completed, 10mM Gly-HCl (pH2.0) is used for regeneration for 20 seconds to completely remove the antibody bound to the ligand.

Parameter fitting: The experiment adopts multi-cycle operation, with the response signal taking the analysis time as the abscissa and the response value as the ordinate. After double-reference deduction, the obtained data were fitted by BIAcore T200 analysis software. The fitting model used was a 1:1 Langmuir binding model to determine its binding and dissociation constants and other affinity indicators.

The results are shown in Table 4. The affinity of N147 is 2 orders of magnitude better than that of the control antibody 99961.1. The KD of N147 reaches 6.62E-11, while the KD of the control antibody 99961.1 is 1.98E-9. The affinity of the remaining antibodies is the same as that of the control antibody 99961.1. The affinity is quite similar.

Example 10 Antibody affinity kinetics (Gator) detection

In this example, Gator equipment was used to detect the affinity of the antibody obtained in the present invention and the positive control antibody 99961.1 with the antigen protein huROR1-His.

The specific test method is as follows: First, PBS (10mM pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from Yuanpei) is configured into Q buffer. , use the configured Q buffer buffer to dilute the storage solution of the antibody to be tested to a working solution with a final concentration of 30nM; use Q buffer to configure the antigen protein huROR1-His storage solution into multiple dilutions (480, 240, 120, 60, 30, 15, 7.5nM) working solution, and then use the Gator instrument and its supporting software to select the Advanced Kinetics experimental mode for detection and analysis. The test results are shown in Table 5. The affinities of N100, NW29 and NW79 are higher than the affinity of the control antibody 99961.1, among which the KD of N100 reaches By 8.85E-9, the KD of NW29 reaches 6.60E-9, the KD of NW79 reaches 6.94E-8, while the KD of control antibody 99961.1 is 1.35E-8, and the affinity of the remaining antibodies is equivalent to that of control antibody 99961.1.

Example 11 Affinity Kinetics Epitope Grouping

This example uses the affinity dynamics method to group the epitopes of the antibodies obtained in the present invention and the positive control antibody 99961.1.

The specific test method is as follows: First, PBS (10mM pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from Yuanpei) is configured into Q buffer. , use the configured Q buffer buffer to dilute the storage solution of the antibody to be tested to a working solution with a final concentration of 100nM; use Q buffer to configure the antigen protein huROR1-His storage solution into a 50nM working solution, and then use the Gator instrument and its The accompanying software performs detection and analysis based on the Tandem settings of the Epitope Binning experimental mode. The results thus obtained for each antibody epitope grouping are shown in Table 6, where each group of antibodies has the same epitope.

Example 12 Antibody affinity maturation

In this example, antibodies N147, N31, N137 and NW37 were subjected to affinity maturation transformation to improve antibody affinity and other biological activities. Affinity maturation transformation is based on M13 phage display technology, using codon-based primers (during the primer synthesis process, a single codon is composed of NNK) to introduce CDR region mutations, and construct four phage display libraries: Library 1 and Library 2 are single-point combination mutations. (i.e., each CDR contains only one mutation site), library 1 is a CDRL1+CDRL3+CDRH3 combination mutation, library 2 is a CDRL2+CDRH1+CDRH2 combination mutation; library 3 and library 4 are double-point saturation mutations, and library 3 is A double-point saturation mutation of CDRL3, and library 4 is a double-point saturation mutation of CDRH3.

The specific library construction method: first synthesize primers containing point mutations (Jinweizhi Biotechnology Co., Ltd.); secondly, use the coding sequences of the antibodies to be modified (also called maternal antibodies) N147, N31, N137 and NW37 as PCR amplification templates. , amplify the sequence containing mutations in the CDR region, combine the fragments containing different CDR mutations by bridging PCR, and then connect the point mutation antibody to the In the phage display vector, the antibody sequence with the mutation site was finally transferred into E. coli SS320 by electroporation. The detailed procedures for library volume calculation, phage library preparation and library screening are described in the previous embodiments. N147 affinity After force maturation, N147-135 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 66, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 53), N147-2 (the heavy chain variable region amino acid sequence is shown in SEQ ID NO: 53), The amino acid sequence of the variable region is shown in SEQ ID NO: 161, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 160), N147-86 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO :163, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 162) and N147-93 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 165, the amino acid sequence of the light chain variable region The sequence is shown in SEQ ID NO: 164); N31-6 is obtained after N31 affinity maturation (the amino acid of the heavy chain variable region is shown in SEQ ID NO: 64, and the amino acid of the light chain variable region is shown in SEQ ID NO: 63 ), N31-14 (the amino acid of the heavy chain variable region is shown in SEQ ID NO: 151, and the amino acid of the light chain variable region is shown in SEQ ID NO: 150), N31-18 (the amino acid of the heavy chain variable region is shown in SEQ ID NO: 150) The acid is shown in SEQ ID NO: 153, the light chain variable region amino acid is shown in SEQ ID NO: 152) and N31-56 (the heavy chain variable region amino acid is shown in SEQ ID NO: 155, the light chain variable The amino acid of the region is shown in SEQ ID NO: 154); N137 affinity matured to obtain N137-72 (the amino acid of the heavy chain variable region is shown in SEQ ID NO: 68, and the amino acid of the light chain variable region is shown in SEQ ID NO : 67), N137-47 (the heavy chain variable region amino acid is shown in SEQ ID NO: 157, the light chain variable region amino acid is shown in SEQ ID NO: 156), N137-53 (heavy chain variable region amino acid is shown in SEQ ID NO: 157) The amino acid is shown in SEQ ID NO: 52, the amino acid of the light chain variable region is shown in SEQ ID NO: 158), N137-60 (the amino acid of the heavy chain variable region is shown in SEQ ID NO: 52, the light chain The amino acids of the variable region are shown in SEQ ID NO: 159); NW37-37 is obtained after affinity maturation of NW37 (the amino acids of the heavy chain variable region are shown in SEQ ID NO: 70, and the amino acids of the light chain variable region are shown in SEQ ID NO: 69), NW37-99 (the amino acid of the heavy chain variable region is shown in SEQ ID NO: 62, and the amino acid of the light chain variable region is shown in SEQ ID NO: 166), NW37-113 (the amino acid of the heavy chain variable region is shown in SEQ ID NO: 62) The variable region amino acids are shown in SEQ ID NO: 168, the light chain variable region amino acids are shown in SEQ ID NO: 167) and NW37-136 (the heavy chain variable region amino acids are shown in SEQ ID NO: 169 , the light chain variable region amino acid is shown in SEQ ID NO: 61); the CDR region amino acid sequence of the antibody after affinity maturation modification is shown in Table 7. Express and purify by the methods disclosed in the previous embodiments The corresponding antibody protein obtained. The molecular weight and purity of the antibody protein were determined by SDS PAGE. The test results showed that after affinity maturation, the non-reducing gel band of the antibody was around 150kD, and the reducing gel band was around 55kD and 25kD, which were in line with the expected size. The purity of all affinity matured antibodies tested by reducing gel was greater than 95%.

Example 13 Antigen affinity detection of antibodies after affinity maturation

In this example, the affinity ability of the antibody to A549 cells after affinity maturation was detected based on the FACS method. Please refer to Example 6 for specific detection methods. The test results are shown in Figure 10A to Figure 10D. The results show that after affinity maturation, the affinity of the antibody to A549 cells has increased. Improvement, including N31-6, N31-14, N31-18, N31-56, NW37-37, NW37-99, NW37-113, NW37-136, N137-47, N137-53, N137-60 and N137-72 The affinities of N147-135, N147-2, N147-86 and N147-93 are all significantly better than those of the corresponding parent antibody molecules N31, NW37 and N137; the affinities of N147-135, N147-2, N147-86 and N147-93 are all better than those of the corresponding parent molecule N147 and better than the control antibody 99961.1.

Example 14 Antibody affinity kinetics (Biacore) analysis after affinity maturation

In this example, the affinity of 16 affinity matured antibodies and parent molecules to the human ROR1 antigen protein huROR1-His was detected based on Biacore equipment. For specific experimental methods, refer to Example 9. The results are shown in Table 8. The KD of NW37-37 is 1 order of magnitude higher than that of the parent antibody; the affinities of the other affinity matured antibodies with the human ROR1 antigen protein huROR1-His are also better than those of the parent antibody. This molecule.

Example 15 Detection of antibody endocytosis efficiency after affinity maturation

In this example, the affinity matured antibodies N147-135, N31-6, N137-72, N147-2 and NW37-37 in Example 11 were subjected to Fab-ZAP endocytosis detection. For the specific method, refer to Example 8.2. The results As shown in Figures 11A to 11E, within the antibody concentration range defined in this example, the endocytosis efficiency of the affinity matured antibody was comparable to that of the control antibody.

Example 16 ADC preparation

In this example, the antibodies N99 and N147-135 and the control antibody 99961.1 were coupled to the toxin MMAE (a tubulin inhibitor with anti-cancer activity) to construct an antibody-conjugated drug. MMAE was connected to the linker MC-VC-PAB to form MC. -VC-PAB-MMAE, and is covalently connected to the sulfhydryl group on the cysteine of the antibody through a linker, thereby conjugating the antibody with MMAE to prepare an antibody-conjugated drug. IgG1 type antibodies have 16 pairs of cysteine residues, which exist in the form of 12 intrachain and 4 interchain disulfide bonds. The interchain disulfide bond has solvent accessibility and can be reduced by reducing agents to form eight sulfhydryl groups, which then become coupling targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J .2015;17:339-51).

The specific preparation method is as follows:

Take out the antibodies N99, N147-135 and control antibody 99961.1 from the -80°C refrigerator, transfer them to 15mL 30KD ultrafiltration centrifuge tubes after melting, and add coupling buffer (content per 1L: Na ₂ HPO _{4.2H 2} O 6.86g, NaH ₂ PO _{4.H 2} O 1.58g, purified water (dilute to 1000g, pH7.4) to 15mL, centrifuge at 4500rpm for about 30min, concentrate to 2-3mL, re-add dialysate (content per 1L: group 0.73g of amino acid, 1.12g of histidine hydrochloride monohydrate, purified water to 1000g, pH 6.0) to 15mL, dialyze repeatedly 8-10 times to obtain the antibody stock solution, and detect the antibody concentration after dialysis.

Add the antibody stock solution and 10mM disulfide bond reducing agent TCEP mother solution (tris(2-carboxyethyl)phosphine hydrochloride mother solution) in sequence. Contents per 1L: Na ₂ HPO ₄ .2H ₂ O 6.86g, NaH ₂ PO ₄ .H ₂ O 1.58g), 10mM DTPA stock solution (diethylenetriaminepentaacetic acid stock solution, content per 1L: DTPA 3.90g, NaOH 1.20g) and coupling buffer constitute a reduction reaction system to reduce the sulfhydryl group on the cysteine of the antibody . The amounts of each component added in the reduction reaction system are shown in Table 9, so that the final concentration of the antibody in the reduction reaction system is 5mg/mL (N99 and 999611) or 4mg/mL (N147-135), the final concentration of DTPA is 1mM, TCEP and antibody The molar ratio is 2.2 (N99 and 99961.1) or 2.7 (N147-135). After thorough mixing, the reduction reaction system was placed in a constant temperature mixer at 25°C, 400 rpm, and the reduction reaction was carried out for 2 hours.

Weigh MC-VC-PAB-MMAE, dissolve it in DMSO, and prepare a 5mM MC-VC-PAB-MMAE stock solution. After the reduction reaction is completed, MC-VC-PAB-MMAE mother liquor is sequentially added to the reduction reaction system in an ice-water bath in an amount as shown in Table 10 to prepare a coupling reaction system. After thorough mixing, the coupling reaction system was placed in a constant temperature mixer at 25°C, 400 rpm, and the coupling reaction was carried out for 1 hour to obtain a solution containing ADC.

After coupling, the solution containing ADC was centrifuged and filtered to obtain the ADC sample and transferred to a 15 mL 30KD ultrafiltration centrifuge tube. Add dialysate to 15mL, 4500 Centrifuge at rpm for 20 minutes, concentrate to 2-3mL, add dialysate again to 15mL, and repeat dialysis 8-10 times. Conduct SEC-HPLC detection, HIC-HPLC detection, concentration detection, free drug detection, etc. on the dialyzed ADC samples. See Table 11 for test results. The test results showed that an ADC with a purity of more than 99% was obtained.

Example 17 Detection of Antigen Binding Activity of ADC

In this example, the binding ability of the prepared antibody-conjugated drugs N99-MMAE and N147-135-MMAE to human ROR1 on tumor cells A549 and HT-29 (human colon cancer cells) was tested based on the FACS method.

The specific method is as follows: A549 cells or HT-29 cells in the logarithmic growth phase are prepared into a single cell suspension, the density is adjusted to 1×10 ⁶ cells/mL, and 100 μL per well is added to a 96-well round bottom plate at 4°C, Centrifuge at 300 g and remove supernatant. Add gradient dilutions (1.0000, 0.3333, 0.1111, 0.0370, 0.0123, 0.0041, 0.0014, 0.0001μg/mL) of N99, N99-MMAE, N147-135, N147-135-MMAE, 99961.1, 99961.1-MMAE and For negative control, mix well and incubate at 4°C for 30 minutes. Wash the incubated cell mixture three times, add 100 μL of secondary antibody Goat F(ab') ₂ Anti-Human IgG-Fc (abcam, ab98596) diluted 1:300, incubate for 30 minutes at 4°C in the dark, and wash three times. Detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

The results are shown in Figure 12: The A549 cell results show (Figure 12A and Figure 12B) that the binding activities of N99-MMAE and control 99961.1-MMA to A549 cells are comparable to those of the corresponding parent antibodies. However, the binding activities of N99 and N99-MMAE to A549 cells The binding activity of N147-135-MMAE, N147-135 and A549 cells is equivalent to that of the control antibody 99961.1; the HT-29 cell results show (Figure 12C and 12D), N99 -The binding activity of MMAE and control 99961.1-MMA to HT-29 cells is equivalent to that of the corresponding parent antibody, and the binding activity of N99-MMAE to HT-29 cells is equivalent to that of control 99961.1-MMAE; N147-135-MMAE, N147-135 Binding activity to HT-29 cells is comparable to control antibody 99961.1.

Example 18 Detection of ADC tumor cell killing effect based on MTS method

In this example, A549 and HT-29 cells were used to detect the killing of tumor cells by the candidate ADC and the control ADC respectively.

The specific method is as follows: A549 cells or HT-29 cells in the logarithmic growth phase are prepared into a single cell suspension. The density of A549 is adjusted to 1×10 ⁴ cells/mL, and the density of HT-29 is adjusted to 1.5×10 ⁴ cells/mL. , add 100 μL per well to a 96-well cell culture plate, culture at 37°C, 5% CO ₂ for 12 hours. Then add gradient diluted (2000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 1.953nM) ADC samples, 37°C, 5% CO ₂ , and culture for 72h (A549 cells) or 96h (HT-29 cells) . Then 40 μl MTS (Promega, G3580) was added to each well, and after incubation at 37°C for 1 hour, the data was read at OD492 by a microplate reader.

The results are shown in Figure 13A and Figure 13B: The killing effect of N99-MMAE on two tumor cells, A549 (Figure 13A) and HT-29 (Figure 13B), was better than that of the control 99961.1-MMAE. The IC ₅₀ on HT-29 are 11.07nM and 5.952nM respectively; the killing effect of N147-135-MMAE on HT-29 tumor cells is better than that of the control 99961.1-MMAE, and the killing effect on A549 tumor cells is equivalent to that of the control ADC.

Example 19 Detection of ADC tumor cell killing effect based on CCK8 method

In this example, Jeko-1 cells (human mantle cell lymphoma cells), MDA-MB-468 cells (human breast cancer cells) and NCI-H1944 cells (human lung cancer cells) were used to detect the effect of N99-MMAE and control ADC on tumors. Cell killing.

The specific method is as follows: Jeko-1 cells, MDA-MB-468 cells or NCI-H1944 cells in the logarithmic growth phase are prepared into a single cell suspension, the Jeko-1 density is adjusted to 1×10 ⁵ cells/mL, and MDA-MB The density of -468 was adjusted to 2×10 ⁵ cells/mL, and the density of NCI-H1944 was adjusted to 1.2×10 ⁵ cells/mL. Add 90 μL per well to a 96-well cell culture plate at 37°C, 5% CO ₂ , Incubate for 12h. Then add ADC sample, dilute the drug concentration gradient to 500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, 0.05nM, 37°C, 5% CO ₂ and incubate for 72 hours. Then 10 μl of CCK8 (Bimake, B34304) was added to each well, and after incubation at 37°C for 1 hour, the data was read at OD450 by a microplate reader.

The results are shown in Figure 14: The killing effect of N99-MMAE on three tumor cells, Jeko-1 (Figure 14A), MDA-MB-468 (Figure 14B) and NCI-H1944 (Figure 14C), is better than the control ADC 99961.1 -MMAE, among which the IC ₅₀ of N99-MMAE on Jeko-1, MDA-MB-468 and NCI-H1944 are 5.188nM, 5.126nM and 48.62nM respectively; N147-135-MMAE on three tumor cells Jeko-1( The killing effect on MDA-MB-468 (Figure 14E) and NCI-H1944 (Figure 14F) was comparable to that of the control ADC 99961.1-MMAE (Figure 14D), where N147-135-MMAE was effective on Jeko-1, MDA-MB-468 and The IC ₅₀ on NCI-H1944 are 52.98nM, 16.17nM and 113.4nM respectively.

Example 20 Detection of ADC antigen-dependent killing effect based on CCK8 method

In this example, huROR1-HEK293 cells were used to detect the antigen-dependent killing effects of the ADC obtained in the present invention and the control ADC.

The specific method is as follows: prepare huROR1-HEK293 cells in the logarithmic growth phase into a single cell suspension, adjust the cell density to 6×10 ⁴ cells/mL, add 50 μL per well to a 96-well cell culture plate, 37°C, 5 % CO ₂ , after 24 hours of culture, add 50 μL of 100 μg/mL antibodies (N99 and N147-135) and positive control antibody 99961.1 to each well. After incubation at 37°C for 2 hours, add concentration gradient dilutions (85.3333, 42.6667, 21.3333, 10.6667 , 5.3333, 2.6667nM) corresponding ADCs (N99-MMAE and N147-135-MMAE) and positive control ADC 99961.1-MMAE, 37°C, 5% CO ₂ , after culturing for 96h, add 30μL CCK8 (absin/Ai) to each well Bixin, abs50003), after incubation at 37°C for 1-4 hours, read the plate at OD450 with a microplate reader. The system without adding antibody and only adding ADC was used as the control group.

The results are shown in Figure 15A and Figure 15B. The competition between antibodies N99, N147-135 and 99961.1 can significantly inhibit the killing effect of their corresponding ADCs on huROR1-HEK293 cells, proving that the killing of cells by the ADC prepared in the present invention depends on the connection between The binding of the antibody on the ADC to the antigen on the cell surface results in antigen-dependent killing rather than non-specific killing of the linked toxin.

Example 21 ADC endocytosis efficiency detection

In this example, A549 and HT-29 tumor cells were used to detect the endocytosis efficiency of the ADC obtained by the present invention based on the FACS method. Please refer to Example 8 for specific experimental methods.

Experimental results show that on A549 cells (Figure 16A and Figure 16B), the endocytosis efficiency of N99-MMAE is significantly higher than that of the control 9996.1-MMAE, and the endocytosis efficiency of ADC is higher than that of its corresponding parent antibody, N147-135- The endocytosis efficiency of MMAE was also significantly higher than the control 99961.1-MMAE; on HT-29 cells (Figure 16C and Figure 16D), the endocytosis efficiency of N99-MMAE was also significantly higher than the control 9996.1-MMAE, and the endocytosis efficiency of ADC are higher than their corresponding parent antibodies; the endocytosis efficiency of N147-135-MMAE is slightly weaker than that of the positive control.

Example 22 Detection of efficacy of ADC in inhibiting HT-29 mouse tumor-bearing model

This example tested the tumor inhibitory effect of two candidate ADCs (N99-MMAE and N147-135-MMAE) in animals. The tumor cells used were colon cancer cells HT-29 (BNCC337732), and 99961.1-MMAE was used as a positive control. .

The specific method is as follows: Use female nude mice (Balb/C Beijing Vitong Lever Experimental Animal Technology Co., Ltd.) that are 6-8 weeks old and weigh about 20g. Each nude mouse is subcutaneously injected with 1×10 ⁶ HT-29 unilaterally. The cells were randomly grouped into cages when the tumor-bearing volume reached about 100 mm ³ . There were 6 tumor-bearing nude mice in each group, a total of 9 groups, including a PBS negative control group, 5 ADC groups (N99-MMAE 0.4mg/kg (mpk), N99-MMAE 2mg/kg, N99-MMAE 10mg/kg, N147-135-MMAE 2mg/kg and N147-135-MMAE 10mg/kg) and 3 positive control ADC groups (99961.1-MMAE 0.4mg/kg, 99961.1-MMAE 2mg/kg and 99961.1-MMAE 10mg/kg), giving The drug is administered by tail vein injection, twice a week and the tumor volume is measured twice, for a total of 8 times/4 weeks (BIW*4). Tumor volume (V) is calculated as: V=L×W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The mice were euthanized 1 week after the end of the administration, and the tumors were removed and their weights were measured. The tumor volume, tumor weight and mouse weight change data were analyzed to calculate the tumor inhibition rate. The results are shown in Figures 17A to 17C and Table 12.

The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well (Figure 17B); N99-MMAE, M147-135MMAE and control 99961.1-MMAE were all at high doses (10mpk) Under the conditions of 100%, significantly better than the positive control 99961.1-MMAE (Figure 17A, Figure 17C and Table 12).

Example 23 Detection of efficacy of ADC in inhibiting A549 mouse tumor-bearing model

This example tested the tumor inhibitory effect of two candidate ADCs (N99-MMAE and N147-135-MMAE) in animals. The tumor cells used were non-small cell lung cancer cells A549 (Shanghai Cell Bank, Chinese Academy of Sciences, C2107019), with 99961.1 -MMAE as positive control.

The specific method is as follows: Use 6-8 weeks old female nude mice weighing 18-20g (Balb/C Beijing Vitong Lihua Experimental Animal Technology Co., Ltd.), and unilaterally inject 1×10 ⁶ mice subcutaneously on the right back of each nude mouse. A549 cells were randomly grouped into cages when the tumor-bearing volume reached about 100 mm ³ . There were 6 tumor-bearing nude mice in each group, a total of 7 groups, including a PBS negative control group, 4 ADC groups (N99-MMAE 5mpk (mg/kg), N99-MMAE 10mpk , N147-135-MMAE 5mpk and N147- 135-MMAE 10mp k ) and 2 positive control ADC groups (99961.1-MMAE 5mpk and 99961.1-MMAE 10mp k ). The administration method was tail vein injection. The drugs were administered twice a week and the tumor volume was measured twice. A total of Take medicine 6 times/3 weeks (BIW*3). Tumor volume (V) is calculated as: V=L×W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). After administration, the mice were observed for a certain period of time before being euthanized, and the tumors were removed and their weights were measured. The tumor volume, tumor weight and mouse weight change data were analyzed to calculate the tumor inhibition rate. The results are shown in Figures 18A to 18C and Table 13.

The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well (Figure 18B); N99-MMAE showed significant changes under high-dose (10mpk) and low-dose (5mpk) conditions. The anti-tumor effect is close to that of the positive control ADC 99961.1-MMAE under low-dose conditions. M147-135MMAE also showed a certain anti-tumor effect under high-dose conditions (Figure 18A and Table 13) ; However, some tumor tissue rebounded after the 41st day in both the high-dose group and the low-dose group.

Example 24 Detection of efficacy of ADC in inhibiting NCI-N87 mouse tumor-bearing model

This example tested the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals. The tumor cells used were gastric cancer cells NCI-N87 (Shanghai Cell Bank, Chinese Academy of Sciences, C2009021, P3), and 99961.1-MMAE was used as Positive control.

The specific method is as follows: Use female nude mice (Balb/C Beijing Vitong Lever Experimental Animal Technology Co., Ltd.) that are 6-8 weeks old and weigh 18-20g. Each nude mouse is subcutaneously injected with 1×10 ⁶ NCI- on the back. N87 cells were randomly grouped into cages when the tumor-bearing volume reached about 100 mm ³ . There were 10 tumor-bearing nude mice in each group, with a total of 5 groups, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mp k ) and 2 positive controls ADC group (99961.1-MMAE 5mpk and 99961.1-MMAE 10mp k ), the administration method is microvenous injection, once a week and the tumor volume is measured twice, a total of 3 times/3 weeks (QW*3) . Tumor volume (V) is calculated as: V=L×W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The tumor volume and mouse weight change data were analyzed to calculate the tumor inhibition rate. The results are shown in Figure 19A and Figure 19B and Table 14.

The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well (Figure 19B); compared with the PBS group, each dose group had significant tumor inhibitory effects, N147-135 -MMAE molecule and 99961.1-MMAE molecule have similar tumor inhibitory effects at the same dose of 10mpk and 5mpk, and there is no significant difference at the same dose (Figure 19A and Table 14).

Example 25 ADC inhibits the efficacy of MDA-MB-231 mouse tumor-bearing model

This example tested the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals. The tumor cells used were triple-negative breast cancer cells MDA-MB-231 (Shanghai Cell Bank, Chinese Academy of Sciences, C2006040), with 99961.1 -MMAE as positive control.

The specific method is as follows: Use 6-8 weeks old female nude mice weighing 20-22g (NSG Beijing Vitong Lever Experimental Animal Technology Co., Ltd.), and subcutaneously inject 1×10 ⁶ MDA-MB-231 on the back of each nude mouse. The cells were randomly grouped into cages when the tumor-bearing volume reached about 100 mm ³ . There were 10 tumor-bearing nude mice in each group, with a total of 5 groups, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mp k ) and 2 positive controls ADC group (99961.1-MMAE 5mpk and 99961.1-MMAE 10mp k ), the administration method is tail vein injection, once a week and the tumor volume is measured twice, a total of 3 times/3 weeks (QW*3) . Tumor volume (V) is calculated as: V=L×W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The tumor volume and mouse weight change data were analyzed to calculate the tumor inhibition rate. The results are shown in Figure 20A and Figure 20B and Table 15.

The test results showed that there was no significant difference in the early stage of administration of mice in each group. The PBS group began to lose weight on the 35th day, and the ADC molecule combination positive control ADC group had no significant change in the weight of mice, indicating the mice's tolerance to ADC. Good (Figure 20B); compared with the PBS group, the high-dose group of the positive control molecule and the candidate ADC molecule had similar significant tumor inhibitory effects, and both molecules had obvious dose correlation. The low-dose group had a certain effect after drug withdrawal. Tissue tumor rebound (Figure 20A and Table 15).

Example 26 ADC inhibits the efficacy of MDA-MB-468 mouse tumor-bearing model

This example tested the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals. The tumor cells used were triple-negative breast cancer cells MDA-MB-468 (Shanghai Cell Bank, Chinese Academy of Sciences, TCHu136), with 99961.1 -MMAE as positive control.

The specific method is as follows: Use 6-8 weeks old female nude mice weighing 21-25g (NOD SCID Zhejiang Vitong Lihua Experimental Animal Technology Co., Ltd.), and subcutaneously inject 1×10 ⁷ MDA-MB- on the back of each nude mouse. 468 cells were randomly divided into cages when the tumor-bearing volume reached about 100 mm ³ . There were 6 tumor-bearing nude mice in each group, a total of 5 groups, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mp k ) and 2 positive controls ADC group (99961.1-MMAE 5mpk and 99961.1-MMAE 10mp k ), the administration method is tail vein injection, once a week and the tumor volume is measured twice a week, a total of 3 times/3 weeks (QW* 3). Tumor volume (V) is calculated as: V=L×W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The tumor volume and mouse weight change data were analyzed to calculate the tumor inhibition rate. The results are shown in Figure 21A and Figure 21B and Tables 16-17.

The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well (Figure 21B); in the multiple administration group, the 10 mg/kg dose group N147-135-MMAE (first All patients were completely relieved on the 14th day after the first dose), and its anti-tumor effect was better than that of 99961.1-MMAE (all patients were completely relieved on the 18th day after the first dose); the 5 mg/kg dose group N147-135-MMAE was the first All achieved complete remission on the 20th day after the first dose, and the tumor inhibitory effect was better than that of the same dose group 99961.1-MMAE (4 out of 6 mice achieved complete remission on the 25th day after the first dose) (Figure 21A and Table 16 -17).

Example 27 ADC efficacy test in inhibiting Jeko-1 mouse tumor-bearing model

This example tested the tumor inhibitory effect of a candidate ADC (N147-135-MMAE) in animals. The tumor cells used were mantle cell lymphoma cells Jeko-1 (ATCC, CRL-3006), and 99961.1-MMAE was used as Positive control.

The specific method is as follows: use female nude mice (BALB/c Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) that are 6-8 weeks old and weigh 21-25g, and subcutaneously inject 1×10 ⁷ Jeko-1 on the back of each nude mouse. The cells were randomly grouped into cages when the tumor-bearing volume reached about 100 mm ³ . There were 7 tumor-bearing nude mice in each group, a total of 9 groups, including a PBS negative control group, 4 ADC groups (N147-135-MMAE 2mpk (mg/kg) (QW*3), N147-135-MMAE 8mp k ( QW*3), N147-135-MMAE 8mpk (single dose,) and N147-135-MMAE 2+3mpk (Q2W*2 means 2mpk administered on day 15 and 3mpk administered on day 29, abbreviated as D15 2mpk+ D29 3mpk)) and 4 positive control ADC groups (99961.1-MMAE 2.5mpk (QW*3), 99961.1-MMAE 10mpk (QW*3), 99961.1-MMAE 10mpk (single dose) and 99961.1-MMAE 2.5+3.5 mpk (Q2W*2, that is, 2.5mpk is administered on the 15th day and 3.5mpk is administered on the 29th day, abbreviated as D15 2.5mpk+D29 3.5mpk)). The administration method is tail vein injection, and it is administered 3 times in total. /3 weeks (QW*3) or single dose (single dose) or 2 times /3 weeks (Q2W*2). Tumor volume (V) is calculated as: V=L×W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The tumor volume and mouse weight change data were analyzed to calculate the tumor inhibition rate. The results are shown in Figure 22A and Figure 22B and Tables 18-19.

The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well (Figure 22B); QW*3 high-dose group, N147-135-MMAE (8mpk) and 99961.1 -The tumor growth inhibition rate TGI of mice in the MMAE (10mpk) dose group was about 96%, and the tumor inhibition rate was comparable. The tumor did not recur 23 days after administration. In the single dose group, the tumor inhibitory effect of N147-135-MMAE (8mpk) was slightly worse than that of 99961.1-MMAE (10mpk), with TGIs of 76% and 93% respectively. In the 99961.1-MMAE (10mpk) group, The tumor did not recur 23 days after administration, but tumor volume began to rebound in the N147-135-MMAE (8mpk) group 16 days after administration. In the Q2W*2 dosage group, the tumor inhibitory effect of N147-135-MMAE (2+3mpk) was slightly better than that of the 99961.1-MMAE (2.5+3.5mpk) dosage group, with TGIs of 47% and 32% respectively (Figure 22A and Table 18 -19).

sequence list

TW202330620A_112102385_SEQL.xml

Claims (29)

An anti-ROR1 antibody or antigen-binding fragment thereof that specifically binds ROR1, comprising: 1) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 52 and 3 included in the light chain variable region as shown in SEQ ID NO: 51 light chain CDRs (LCDR1, LCDR2, LCDR3); 2) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 54 and 3 included in the light chain variable region as shown in SEQ ID NO: 53 light chain CDRs (LCDR1, LCDR2, LCDR3); 3) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 56 and 3 included in the light chain variable region as shown in SEQ ID NO: 55 light chain CDRs (LCDR1, LCDR2, LCDR3); 4) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 58 and 3 included in the light chain variable region as shown in SEQ ID NO: 57 light chain CDRs (LCDR1, LCDR2, LCDR3); 5) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 60 and the 3 included in the light chain variable region as shown in SEQ ID NO: 59 light chain CDRs (LCDR1, LCDR2, LCDR3); 6) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 62 and 3 included in the light chain variable region as shown in SEQ ID NO: 61 light chain CDRs (LCDR1, LCDR2, LCDR3); 7) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 64 and 3 included in the light chain variable region as shown in SEQ ID NO: 63 light chain CDRs (LCDR1, LCDR2, LCDR3); 8) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 66 and 3 included in the light chain variable region as shown in SEQ ID NO: 53 light chain CDRs (LCDR1, LCDR2, LCDR3); 9) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 68 and 3 included in the light chain variable region as shown in SEQ ID NO: 67 light chain CDRs (LCDR1, LCDR2, LCDR3); 10) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 70 and 3 contained in the light chain variable region as shown in SEQ ID NO: 69 light chain CDRs (LCDR1, LCDR2, LCDR3); 11) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 145 and 3 included in the light chain variable region as shown in SEQ ID NO: 144 light chain CDRs (LCDR1, LCDR2, LCDR3); 12) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 147 and the 3 included in the light chain variable region as shown in SEQ ID NO: 146 light chain CDRs (LCDR1, LCDR2, LCDR3); 13) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 141 and 3 included in the light chain variable region as shown in SEQ ID NO: 140 light chain CDRs (LCDR1, LCDR2, LCDR3); 14) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 143 and the 3 included in the light chain variable region as shown in SEQ ID NO: 142 light chain CDRs (LCDR1, LCDR2, LCDR3); 15) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 149 and the 3 included in the light chain variable region as shown in SEQ ID NO: 148 light chain CDRs (LCDR1, LCDR2, LCDR3); 16) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 161 and the 3 included in the light chain variable region as shown in SEQ ID NO: 160 light chain CDRs (LCDR1, LCDR2, LCDR3); 17) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 163 and the 3 included in the light chain variable region as shown in SEQ ID NO: 162 light chain CDRs (LCDR1, LCDR2, LCDR3); 18) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 165 and the 3 included in the light chain variable region as shown in SEQ ID NO: 164 light chain CDRs (LCDR1, LCDR2, LCDR3); 19) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 151 and the 3 contained in the light chain variable region as shown in SEQ ID NO: 150 light chain CDRs (LCDR1, LCDR2, LCDR3); 20) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 153 and the 3 included in the light chain variable region as shown in SEQ ID NO: 152 light chain CDRs (LCDR1, LCDR2, LCDR3); 21) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 155 and the 3 included in the light chain variable region as shown in SEQ ID NO: 154 light chain CDRs (LCDR1, LCDR2, LCDR3); 22) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 157 and the 3 included in the light chain variable region as shown in SEQ ID NO: 156 light chain CDRs (LCDR1, LCDR2, LCDR3); 23) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 52 and the 3 included in the light chain variable region as shown in SEQ ID NO: 158 light chain CDRs (LCDR1, LCDR2, LCDR3); 24) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 52 and the 3 contained in the light chain variable region as shown in SEQ ID NO: 159 light chain CDRs (LCDR1, LCDR2, LCDR3); 25) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 62 and the 3 included in the light chain variable region as shown in SEQ ID NO: 166 light chain CDRs (LCDR1, LCDR2, LCDR3); 26) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 168 and the 3 included in the light chain variable region as shown in SEQ ID NO: 167 A light chain CDR (LCDR1, LCDR2, LCDR3); or 27) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) included in the heavy chain variable region as shown in SEQ ID NO: 169 and the 3 included in the light chain variable region as shown in SEQ ID NO: 61 light chain CDRs (LCDR1, LCDR2, LCDR3). An anti-ROR1 antibody or antigen-binding fragment thereof that specifically binds ROR1, comprising: 1) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 1, 2, and 3; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 19, 20, and 21; 2) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 1, 2, and 6; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 22, 23, and 24; 3) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 7, 8, and 9; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 25, 26, and 27; 4) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 10, 11, and 12; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 28, 29, and 30; 5) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 13, 14, and 15; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 31, 32, and 33; 6) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 16, 17, and 18; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 34, 35, and 36; 7) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 1, 2, and 37; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 19, 20, and 21; 8) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 38, 39, and 12; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 28, 44, and 30; 9) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 40, 41, and 15; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 31, 45, and 33; or 11) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 91, 92, and 93; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 101, 102, and 103; 12) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 1, 2, and 94; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 104, 32, and 105; 13) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 95, 96, and 97; and LCDR1, LCDR2, and LCDR3 containing the sequences shown in SEQ ID NO: 106, 107, and 108; 14) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 1, 2, and 98; and LCDR1, LCDR2, and LCDR3 containing the sequences shown in SEQ ID NO: 109, 110, and 105; 15) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 91, 99, and 100; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 111, 112, and 113; 16) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 121, 122, and 3; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 19, 136, and 21; 17) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 1, 123, and 3; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 19, 137, and 21; 18) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 124, 2, and 3; and LCDR1, LCDR2, and LCDR3 containing the sequences shown in SEQ ID NO: 19, 23, and 21; 19) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 114, 115, and 12; and LCDR1, LCDR2, and LCDR3 containing the sequences shown in SEQ ID NO: 28, 128, and 30; 20) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 116, 117, and 12; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 28, 129, and 30; 21) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 118, 119, and 12; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 28, 130, and 30; 22) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 16, 17, and 120; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 131, 35, and 132; 23) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 16, 17, and 18; and LCDR1, LCDR2, and LCDR3 having the sequences shown in SEQ ID NO: 34, 35, and 133; 24) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 16, 17, and 18; and LCDR1, LCDR2, and LCDR3 containing the sequences shown in SEQ ID NO: 134, 35, and 135; 25) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO: 13, 14 and 15; and LCDR1, LCDR2, LCDR3 having the sequences shown in SEQ ID NO: 31, 138 and 33; or 27) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO: 125, 127 and 15; and LCDR1, LCDR2, LCDR3 having the sequences shown in SEQ ID NO: 31, 32 and 33. The anti-ROR1 antibody or antigen-binding fragment thereof as described in claim 1 or 2, which includes a heavy chain variable region and a light chain variable region, wherein, 1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence determined by SEQ ID NO: 52. Composed of NO: 52, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 51, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 51, or Composed of SEQ ID NO: 51; 2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 54, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence determined by SEQ ID NO: 54. NO: 54 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 53, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 53, or Composed of SEQ ID NO: 53; 3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 56, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence determined by SEQ ID NO: 56. NO: 56 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 55, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 55, or Composed of SEQ ID NO: 55; 4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence determined by SEQ ID NO: 58. NO: 58 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 57, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 57, or Composed of SEQ ID NO: 57; 5) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 60, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence determined by SEQ ID NO: 60. ID NO: 60, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 59, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 59, or consisting of SEQ ID NO: 59; 6) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence determined by SEQ ID NO: 62. NO: 62 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 61, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 61, or Composed of SEQ ID NO: 61; 7) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 64, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence determined by SEQ ID NO: 64. NO: 64 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 63, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 63, or Composed of SEQ ID NO: 63; 8) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 66, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence determined by SEQ ID NO: 66. NO: 66 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 53, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 53, or Composed of SEQ ID NO: 53; 9) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 68, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence determined by SEQ ID NO: 68. NO: 68 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 67, or Composed of SEQ ID NO: 67; 10) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence determined by SEQ ID NO: 70. NO: 70, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 69, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 69, or Consisting of SEQ ID NO: 69; or 11) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 145, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence determined by SEQ ID NO: 145. NO: 145 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 144, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 144, or Composed of SEQ ID NO: 144; 12) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 147, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence determined by SEQ ID NO: 147. NO: 147 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 146, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 146, or Composed of SEQ ID NO: 146; 13) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 141, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence determined by SEQ ID NO: 141. NO: 141 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 140, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 140, or Composed of SEQ ID NO: 140; 14) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 143, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence determined by SEQ ID NO: 143. Composed of NO: 143, the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 142 A sequence, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 142, or consisting of SEQ ID NO: 142; 15) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 149, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 149, or an amino acid sequence determined by SEQ ID NO: 149. NO: 149 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 148, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 148, or Composed of SEQ ID NO: 148; 16) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 161, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 161, or an amino acid sequence determined by SEQ ID NO: 161. NO: 161 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 160, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 160, or Composed of SEQ ID NO: 160; 17) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 163, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 163, or an amino acid sequence determined by SEQ ID NO: 163. NO: 163 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 162, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 162, or Composed of SEQ ID NO: 162; 18) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 165, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence determined by SEQ ID NO: 165. NO: 165 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 164, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 164, or Composed of SEQ ID NO: 164; 19) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 151, or an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 151, or is determined by SEQ ID NO: 151. ID NO: 151, the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 150, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 150, Or consisting of SEQ ID NO: 150; 20) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 153, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 153, or an amino acid sequence determined by SEQ ID NO: 153. NO: 153 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 152, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 152, or Composed of SEQ ID NO: 152; 21) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 155, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence determined by SEQ ID NO: 155. NO: 155 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 154, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 154, or Composed of SEQ ID NO: 154; 22) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 157, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 157, or an amino acid sequence determined by SEQ ID NO: 157. NO: 157 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 156, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 156, or Composed of SEQ ID NO: 156; 23) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence determined by SEQ ID NO: 52. Composed of NO: 52, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 158, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 158, or Composed of SEQ ID NO: 158; 24) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence determined by SEQ ID NO: 52. Composed of NO: 52, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 159, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 159, or Composed of SEQ ID NO: 159; 25) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence determined by SEQ ID NO: 62. NO: 62 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 166, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 166, or Composed of SEQ ID NO: 166; 26) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 168, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 168, or an amino acid sequence determined by SEQ ID NO: 168. NO: 168 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 167, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 167, or Consisting of SEQ ID NO: 167; or 27) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 169, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 169, or an amino acid sequence determined by SEQ ID NO: 169. NO: 169 consists of the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 61, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 61, or Consists of SEQ ID NO:61. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is a human monoclonal antibody. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antigen-binding fragment is selected from Fab, Fab'-SH, Fv (such as scFv) or (Fab')2 fragment. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which comprises a constant region sequence, wherein at least part of the constant region sequence is a human consensus constant region sequence. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the heavy chain constant region of the antibody includes the Fc sequence shown in SEQ ID NO: 47 or includes the Fc sequence shown in SEQ ID NO: 48 The amino acid sequence of the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which includes a heavy chain and a light chain, wherein, 1) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence consisting of SEQ ID NO: 72 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 71, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 71, or consisting of SEQ ID NO: 71 composition; 2) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 74, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence consisting of SEQ ID NO: 74 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 73, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 73, or consisting of SEQ ID NO: 73 composition; 3) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 76, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence consisting of SEQ ID NO: 76 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 75, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 75, or consisting of SEQ ID NO: 75 composition; 4) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 78, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 78, or an amino acid sequence consisting of SEQ ID NO: 78 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 77, or consisting of SEQ ID NO: 77 composition; 5) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 80, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence consisting of SEQ ID NO: 80 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 79, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 79, or consisting of SEQ ID NO: 79 composition; 6) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 82, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence consisting of SEQ ID NO: 82 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 81, or consisting of SEQ ID NO: 81 composition; 7) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 84, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence consisting of SEQ ID NO: 84 Composed, the light chain includes the amino acid sequence shown in SEQ ID NO: 83, or is the same as SEQ ID NO: 83 The amino acid sequence of NO: 83 has an amino acid sequence that is at least 90% identical, or consists of SEQ ID NO: 83; 8) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 86, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence consisting of SEQ ID NO: 86 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 73, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 73, or consisting of SEQ ID NO: 73 composition; 9) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 88, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence consisting of SEQ ID NO: 88 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 87, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 87, or consisting of SEQ ID NO: 87 composition; 10) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 90, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 90, or an amino acid sequence consisting of SEQ ID NO: 90 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 89, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 89, or consisting of SEQ ID NO: 89 composition; 11) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 175, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 175, or an amino acid sequence consisting of SEQ ID NO: 175 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 174, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 174, or consisting of SEQ ID NO: 174 composition; 12) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 177, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 177, or an amino acid sequence consisting of SEQ ID NO: 177 NO: 177 consists of the light chain comprising the amino acid sequence shown in SEQ ID NO: 176, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 176, or consisting of SEQ ID NO: 177. NO: 176 composition; 13) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 171, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 171, or an amino acid sequence consisting of SEQ ID NO: 171 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 170, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 170, or consisting of SEQ ID NO: 170 composition; 14) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 173, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 173, or an amino acid sequence consisting of SEQ ID NO: 173 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 172, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 172, or consisting of SEQ ID NO: 172 composition; 15) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 179, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 179, or an amino acid sequence consisting of SEQ ID NO: 179 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 178, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 178, or consisting of SEQ ID NO: 178 composition; 16) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 191, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 191, or an amino acid sequence consisting of SEQ ID NO: 191 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 190, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 190, or consisting of SEQ ID NO: 190 composition; 17) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 193, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence consisting of SEQ ID NO: 193 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 192, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 192, or consisting of SEQ ID NO: 192 composition; 18) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 195, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 195, or an amino acid sequence consisting of SEQ ID NO: 195 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 194, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 194, or consisting of SEQ ID NO: 194 composition; 19) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 181, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence consisting of SEQ ID NO: 181 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 180, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 180, or consisting of SEQ ID NO: 180 composition; 20) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 183, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 183, or an amino acid sequence consisting of SEQ ID NO: 183 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 182, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 182, or consisting of SEQ ID NO: 182 composition; 21) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 185, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 185, or an amino acid sequence consisting of SEQ ID NO: 185 Composed of, the light chain includes the amino acid sequence shown in SEQ ID NO: 184, or with The amino acid sequence of SEQ ID NO: 184 has at least 90% identity to the amino acid sequence, or consists of SEQ ID NO: 184; 22) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 187, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 187, or an amino acid sequence consisting of SEQ ID NO: 187 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 186, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 186, or consisting of SEQ ID NO: 186 composition; 23) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence consisting of SEQ ID NO: 72 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 188, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 188, or consisting of SEQ ID NO: 188 composition; 24) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 72, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence consisting of SEQ ID NO: 72 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 189, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 189, or consisting of SEQ ID NO: 189 composition; 25) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 82, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence consisting of SEQ ID NO: 82 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 196, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 196, or consisting of SEQ ID NO: 196 composition; 26) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 198, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 198, or an amino acid sequence consisting of SEQ ID NO: 198. NO: 198 consists of the light chain comprising the amino acid sequence shown in SEQ ID NO: 197, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 197, or consisting of SEQ ID NO: 197. NO: 197 composition; or 27) The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 199, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 199, or an amino acid sequence consisting of SEQ ID NO: 199 Composed of, the light chain comprises the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 81, or consisting of SEQ ID NO: 81 composition. An antibody-conjugated drug with an Ab-(L-D)n structure, wherein Ab is an anti-ROR1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8, L is a linker, and D is a therapeutically active substance Or pharmaceutical active ingredient, n represents an integer from 1 to 20. The antibody-conjugated drug according to claim 9, wherein the therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a plant toxin, a small molecule toxin, or a radioactive isotope. The antibody-conjugated drug as described in claim 9 or 10, wherein the therapeutically active substance or pharmaceutical active ingredient is Dolastatin and its auristatin derivatives, such as Dolastatin 10. Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin Statin (monomethyl auristatin) E (MMAE), monomethyl auristatin (monomethyl auristatin) F (MMAF), auristatin (auristatin) F phenylenediamine (AFP), auristatin (auristatin) EB ( AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AF HPA). The antibody-conjugated drug according to any one of claims 9 to 11, wherein the Dolastatin and its auristatin derivatives are MMAE, MMAF, auristatin F hydroxypropylamide or auristatin F phenylenediamine. The antibody-conjugated drug according to any one of claims 9 to 12, wherein the linker is a cathepsin-degradable linker (such as valine-citrulline (val-cit) linker, cBu-Cit linker and CX connectors), non-breakable connectors (such as SMCC connectors or MD connectors), acid-sensitive connectors, silicone structure connectors, disulfide-carbamate connectors, MC-GGFG connectors, TRX connectors, galactocontaining connectors Glycoside linkers, pyrophosphate linkers, near-infrared-sensitive linkers, and UV-sensitive linkers (such as PC4AP). The antibody-conjugated drug according to any one of claims 9 to 13, wherein the linker is selected from the group consisting of maleimide-hexyl-valine-citrulline-p-aminobenzyloxy ( mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG6-propyl, and maleyl Aminocaproic acid (mc), maleiminopropyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxy Carbonyl (PAB), N-succinimide 4-(2-pyridylthio)valerate (SPP), N-succinimide 4-(N-maleiminomethyl)- Cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl-4-( 2-Pyridyldithio)butyrate (SPDB), N-succinimide 3-(2-pyridyldithio)-propionate (SPDP). The antibody-conjugated drug according to any one of claims 9 to 14, wherein the linker is MC-VC-PAB, SMCC or MC-GGFG. The antibody conjugate drug according to any one of claims 9 to 15, wherein the Ab includes: or (2) HCDR1, HCDR2, and HCDR3 of the sequences shown in SEQ ID NO: 1, 2, and 37 and LCDR1, LCDR2, and LCDR3 of the sequences shown in SEQ ID NO: 19, 20, and 21. The antibody drug conjugate according to any one of claims 9 to 16, wherein the Ab comprises a heavy chain variable region and a light chain variable region, wherein (1) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 60, the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 59, or (2) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 66, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 53. The antibody conjugate drug according to any one of claims 9 to 17, wherein the Ab includes: (1) The heavy chain of the amino acid sequence shown in SEQ ID NO: 80 and the light chain of the amino acid sequence shown in SEQ ID NO: 79; or (2) The heavy chain of the amino acid sequence shown in SEQ ID NO: 86 and the light chain of the amino acid sequence shown in SEQ ID NO: 73. The antibody-conjugated drug according to any one of claims 16 to 18, wherein the linker is MC-VC-PAB and the cytotoxin is MMAE. A pharmaceutical composition containing: (1) The anti-ROR1 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, or the antibody conjugate drug as described in any one of claims 9 to 19, and; (2) Pharmaceutically acceptable carrier. An isolated polynucleotide molecule encoding an anti-ROR1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8. A vector comprising the nucleic acid molecule as described in claim 21. Preferably, the vector is an expression vector. A host cell comprising the vector according to claim 22 or the nucleic acid molecule according to claim 21. The use of an anti-ROR1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8 in the preparation of an antibody-conjugated drug for preventing or treating cancer, or in the preparation of an antibody-conjugated drug for preventing or treating abnormalities related to ROR1 Use in medicines related to expression-related diseases. The use of an antibody-conjugated drug as described in any one of claims 9 to 19 in the preparation of a drug for preventing or treating diseases related to abnormal expression of ROR1. A method of killing ROR1-expressing cells or inhibiting the growth of ROR1-expressing cells, comprising contacting the cells with an effective amount of the anti-ROR1 antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, or an effective amount of The antibody conjugate drug as described in any one of claims 9 to 19, or an effective amount of the pharmaceutical composition as described in claim 20. A method for preventing or treating a disease associated with abnormal expression of ROR1 in a subject in need, comprising administering to the subject a prophylactically effective amount or a therapeutically effective amount of the antibody described in any one of claims 1 to 8. ROR1 antibody or antigen-binding fragment thereof, or a prophylactically effective amount, or A therapeutically effective amount of the antibody-conjugated drug as described in any one of claims 9 to 19, or a prophylactically effective amount or a therapeutically effective amount of the pharmaceutical composition as described in claim 20. The use as described in claim 25 or the method as described in claim 27, wherein the disease associated with abnormal expression of ROR1 is cancer that highly expresses ROR1. The use or method as described in claim 28, wherein the cancer with high expression of ROR1 is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer, breast cancer , neuroblastoma, lung cancer, stomach cancer, head and neck cancer, and melanoma.

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