CN118574845A - ROR 1-targeted antibody or antigen binding fragment thereof and application thereof - Google Patents

Abstract

The present application relates to antibodies specifically recognizing ROR1, methods of preparation and uses thereof. The application also relates to an antibody conjugated drug (ADC) targeting ROR1 and compositions containing the same. Furthermore, the application relates to therapeutic and diagnostic uses of these antibodies, antibody fragments and antibody-conjugated drugs.

Description

An antibody or antigen-binding fragment targeting ROR1 and its application Technical Field

The present application relates to antibodies that specifically recognize ROR1, methods for preparing the same, and uses thereof. The present application also relates to antibody-drug conjugates (ADCs) targeting ROR1 and compositions containing the same. In addition, the present invention also relates to therapeutic and diagnostic uses of these antibodies, antibody fragments, and antibody-drug conjugates.

Background Art

Antibody-Drug Conjugates (ADC) are derivative drugs of traditional antibody drugs. They are made by coupling monoclonal antibody drugs targeting specific antigens and small molecule cytotoxic drugs through linkers. They have both the specific targeting of antibody drugs and the killing effect of traditional small molecule drugs. Their main indications are malignant tumors. ADC has quickly become a hot spot in the development of anti-tumor drugs due to its excellent tumor killing effect. However, the toxicity of ADC is usually high, so the specificity of the target antigen is required to be high. The targets suitable for the development of ADC are generally highly expressed in tumors and low or almost not expressed in healthy tissues; the antigen is a surface receptor expressed upregulated by tumor cells, which 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-related 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. They are composed of an immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD domain and KRD domain) extracellularly, and a tyrosine kinase domain, two serine- or threonine-rich domains and a proline-rich domain intracellularly. ROR1 and ROR2 participate in the non-canonical Wnt signaling pathway by binding to the ligand Wnt5a through the FZD domain (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 cell 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, Arima C, Kato S, Tomida S, Suzuki M, Osada Het 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 absent or lowly 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 lymphocytic leukemia (CLL) and other hematological malignancies, renal cell carcinoma, colon cancer, and some other cancer cell lines of breast cancer. In addition, ROR1 plays an important role in the progression of many hematological and solid malignancies. Therefore, as a cancer marker, ROR1 has become an ideal drug target for cancer treatment.

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

Summary of the invention

In a first aspect, the present invention provides an antibody targeting human ROR1, which has the following advantages:

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

(2) Have good mouse cross-reactivity;

(3) Ability to enter cells via endocytosis;

(4) Suitable for constructing effective therapeutic antibody-drug conjugates;

The anti-ROR1 antibody provided by the present invention has a completely human sequence and is a human antibody. It is expected that the anti-ROR1 antibody of the present invention has minimal immunogenicity to human subjects, and human subjects will have good tolerance to the anti-ROR1 antibody.

In one embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising:

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

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

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

4) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:58 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:57;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In one embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising:

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

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

3) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 25, 26 and 27, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

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

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

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

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

8) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO:38, 39 and 12, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NO:28, 44 and 30, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

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

10) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 42, 43 and 18, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 34, 46 and 36, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

11) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 91, 92 and 93, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 101, 102 and 103, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

12) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 1, 2 and 94, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 104, 32 and 105, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

13) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 95, 96 and 97, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 106, 107 and 108, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

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

15) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 91, 99 and 100, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 111, 112 and 113, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

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

17) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 1, 123 and 3, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 19, 137 and 21, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

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

19) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 114, 115 and 12, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 28, 128 and 30, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

20) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 116, 117 and 12, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 28, 129 and 30, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

21) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 118, 119 and 12, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 28, 130 and 30, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

22) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 16, 17 and 120, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 131, 35 and 132, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

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

24) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 16, 17 and 18, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 134, 35 and 135, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

25) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NO: 13, 14 and 15, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NO: 31, 138 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

26) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 125, 126 and 15, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 139 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to said sequences; or

27) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 125, 127 and 15, or sequences containing one or more and no more than 3 amino acids of amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 32 and 33, or sequences containing one or more and no more than 3 amino acids of amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to the sequences.

In one embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising a heavy chain variable region, wherein:

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

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

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

4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:58, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:58, or consists of SEQ ID NO:58;

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

6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:62, or consists of SEQ ID NO:62;

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

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

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

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

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

12) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 147, or consists of SEQ ID NO: 147;

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

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

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

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

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

18) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 165, or consists of SEQ ID NO: 165;

19) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 151, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 151, or consists of SEQ ID NO: 151;

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

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

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

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

24) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 169, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 169, or consists of SEQ ID NO: 169.

In one embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising a light chain variable region, wherein:

1) the light chain variable region comprises the amino acid sequence of SEQ ID NO:51, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:51, or consists of SEQ ID NO:51;

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

3) the light chain variable region comprises the amino acid sequence of SEQ ID NO:55, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:55, or consists of SEQ ID NO:55;

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

5) The light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 59, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 59, or consists of SEQ ID NO: 59;

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

7) the light chain variable region comprises the amino acid sequence of SEQ ID NO:63, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:63, or consists of SEQ ID NO:63;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In another embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds 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 of SEQ ID NO:52, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:52, or consists of SEQ ID NO:52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:51, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:51, or consists of SEQ ID NO:51;

2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:54, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:54, or consists of SEQ ID NO:54, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:53, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:53, or consists of SEQ ID NO:53;

3) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:56, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:56, or consists of SEQ ID NO:56, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:55, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:55, or consists of SEQ ID NO:55;

4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:58, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:58, or consists of SEQ ID NO:58, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:57, or consists of SEQ ID NO:57;

5) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:60, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:60, or consists of SEQ ID NO:60, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:59, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:59, or consists of SEQ ID NO:59;

6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:62, or consists of SEQ ID NO:62, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:61, or consists of SEQ ID NO:61;

7) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:64, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:64, or consists of SEQ ID NO:64, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:63, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:63, or consists 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 66, or consists of SEQ ID NO: 66, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 53, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 53, or consists of SEQ ID NO: 53;

9) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:68, or consists of SEQ ID NO:68, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:67, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:67, or consists of SEQ ID NO:67;

10) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:70, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:70, or consists of SEQ ID NO:70, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:69, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:69, or consists of SEQ ID NO:69;

11) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:145, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:145, or consists of SEQ ID NO:145, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:144, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:144, or consists of SEQ ID NO:144;

12) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 147, or consists of SEQ ID NO: 147, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 146, or consists of SEQ ID NO: 146;

13) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 141, or consists of SEQ ID NO: 141, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 140, or consists of SEQ ID NO: 140;

14) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 143, or consists of SEQ ID NO: 143, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 142, or consists of SEQ ID NO: 142;

15) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 149, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 149, or consists of SEQ ID NO: 149, and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 148, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 148, or consists of SEQ ID NO: 148;

16) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 161, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 161, or consists of SEQ ID NO: 161, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 160, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 160, or consists of SEQ ID NO: 160;

17) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 163, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 163, or consists of SEQ ID NO: 163, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 162, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 162, or consists of SEQ ID NO: 162;

18) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 165, or consists of SEQ ID NO: 165, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 164, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 164, or consists of SEQ ID NO: 164;

19) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 151, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 151, or consists of SEQ ID NO: 151, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 150, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 150, or consists of SEQ ID NO: 150;

20) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 153, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 153, or consists of SEQ ID NO: 153, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 152, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 152, or consists of SEQ ID NO: 152;

21) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:155, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:155, or consists of SEQ ID NO:155, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:154, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:154, or consists of SEQ ID NO:154;

22) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 157, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 157, or consists of SEQ ID NO: 157, and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 156, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 156, or consists of SEQ ID NO: 156;

23) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:52, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:52, or consists of SEQ ID NO:52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:158, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:158, or consists of SEQ ID NO:158;

24) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:52, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:52, or consists of SEQ ID NO:52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:159, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:159, or consists of SEQ ID NO:159;

25) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:62, or consists of SEQ ID NO:62, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:166, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:166, or consists of SEQ ID NO:166;

26) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 168, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 168, or consists of SEQ ID NO: 168, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 167, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 167, or consists of SEQ ID NO: 167; or

27) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 169, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 169, or consists of SEQ ID NO: 169, and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 61, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 61, or consists of SEQ ID NO: 61.

In some embodiments, the above-mentioned antibodies or antigen-binding fragments thereof further comprise heavy chain and/or light chain constant region sequences from human antibody germline consensus sequences. The light chain constant region is preferably a human κ or λ chain constant region. The heavy chain constant region may be a γ, μ, α, δ, or ε chain. In some embodiments, the heavy chain constant region is preferably a constant region sequence from human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the light chain constant region comprises the sequence shown in SEQ ID NO: 49 or 50, or consists of the sequence. In another embodiment, the heavy chain constant region comprises the Fc sequence shown in SEQ ID NO: 47. In another embodiment, the heavy chain constant region comprises the sequence shown in SEQ ID NO: 48, or consists of the sequence.

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

In a specific embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising a heavy chain and a light chain, wherein:

1) the heavy chain comprises the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:72, or consists of SEQ ID NO:72, and the light chain comprises the amino acid sequence of SEQ ID NO:71, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:71, or consists of SEQ ID NO:71;

2) the heavy chain comprises the amino acid sequence of SEQ ID NO:74, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:74, or consists of SEQ ID NO:74, and the light chain comprises the amino acid sequence of SEQ ID NO:73, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:73, or consists of SEQ ID NO:73;

3) the heavy chain comprises the amino acid sequence of SEQ ID NO:76, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:76, or consists of SEQ ID NO:76, and the light chain comprises the amino acid sequence of SEQ ID NO:75, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:75, or consists of SEQ ID NO:75;

4) the heavy chain comprises the amino acid sequence of SEQ ID NO:78, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:78, or consists of SEQ ID NO:78, and the light chain comprises the amino acid sequence of SEQ ID NO:77, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:77, or consists of SEQ ID NO:77;

5) the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:80, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:80, or consists of SEQ ID NO:80, and the light chain comprises the amino acid sequence as set forth in SEQ ID NO:79, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:79, or consists of SEQ ID NO:79;

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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 82, or consists of SEQ ID NO: 82, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 81, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 81, or consists of SEQ ID NO: 81;

7) the heavy chain comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84, and the light chain comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:83, or consists of SEQ ID NO:83;

8) the heavy chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO:86, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:86, and the light chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO:73, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:73;

9) the heavy chain comprises the amino acid sequence of SEQ ID NO:88, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:88, or consists of SEQ ID NO:88, and the light chain comprises the amino acid sequence of SEQ ID NO:87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:87, or consists of SEQ ID NO:87;

10) the heavy chain comprises the amino acid sequence of SEQ ID NO:90, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:90, or consists of SEQ ID NO:90, and the light chain comprises the amino acid sequence of SEQ ID NO:89, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:89, or consists of SEQ ID NO:89;

11) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 175, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 175, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 174, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 174;

12) the heavy chain comprises the amino acid sequence of SEQ ID NO: 177, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 177, or consists of SEQ ID NO: 177, and the light chain comprises the amino acid sequence of SEQ ID NO: 176, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 176, or consists of SEQ ID NO: 176;

13) The heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 171, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 171, or consists of SEQ ID NO: 171, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 170, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 170, or consists of SEQ ID NO: 170;

14) the heavy chain comprises the amino acid sequence of SEQ ID NO: 173, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 173, or consists of SEQ ID NO: 173, and the light chain comprises the amino acid sequence of SEQ ID NO: 172, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 172, or consists of SEQ ID NO: 172;

15) the heavy chain comprises the amino acid sequence of SEQ ID NO: 179, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 179, or consists of SEQ ID NO: 179, and the light chain comprises the amino acid sequence of SEQ ID NO: 178, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 178, or consists of SEQ ID NO: 178;

16) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 191, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 191, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 190;

17) the heavy chain comprises the amino acid sequence of SEQ ID NO: 193, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 193, or consists of SEQ ID NO: 193, and the light chain comprises the amino acid sequence of SEQ ID NO: 192, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 192, or consists of SEQ ID NO: 192;

18) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 195, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 195, and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 194, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 194;

19) the heavy chain comprises the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 181, or consists of SEQ ID NO: 181, and the light chain comprises the amino acid sequence of SEQ ID NO: 180, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 180, or consists of SEQ ID NO: 180;

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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 183, or consists of SEQ ID NO: 183, and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 182, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 182, or consists of SEQ ID NO: 182;

21) the heavy chain comprises the amino acid sequence of SEQ ID NO:185, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:185, or consists of SEQ ID NO:185, and the light chain comprises the amino acid sequence of SEQ ID NO:184, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:184, or consists of SEQ ID NO:184;

22) the heavy chain comprises the amino acid sequence of SEQ ID NO: 187, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 187, or consists of SEQ ID NO: 187, and the light chain comprises the amino acid sequence of SEQ ID NO: 186, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 186, or consists of SEQ ID NO: 186;

23) the heavy chain comprises the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:72, or consists of SEQ ID NO:72, and the light chain comprises the amino acid sequence of SEQ ID NO:188, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:188, or consists of SEQ ID NO:188;

24) the heavy chain comprises the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:72, or consists of SEQ ID NO:72, and the light chain comprises the amino acid sequence of SEQ ID NO:189, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:189, or consists of SEQ ID NO:189;

25) the heavy chain comprises the amino acid sequence of SEQ ID NO:82, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:82, or consists of SEQ ID NO:82, and the light chain comprises the amino acid sequence of SEQ ID NO:196, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:196, or consists of SEQ ID NO:196;

26) the heavy chain comprises the amino acid sequence of SEQ ID NO: 198, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 198, or consists of SEQ ID NO: 198, and the light chain comprises the amino acid sequence of SEQ ID NO: 197, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 197, or consists of SEQ ID NO: 197; or

27) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 199, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 199, or consists of SEQ ID NO: 199, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 81, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 81, or consists of SEQ ID NO: 81.

In certain embodiments of any of the foregoing antibodies, the antibody is monoclonal.

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

In one embodiment, the anti-ROR1 antibody of the present invention is a complete antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the anti-ROR1 antibody of the present invention only encompasses its antigen binding portion, such as: Fab, Fab'-SH, Fv, scFv, or (Fab') ₂ fragment.

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

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

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

(3) treating, preventing, or ameliorating a condition (e.g., cancer, such as blood cancer, solid tumor) associated with abnormal function or expression of ROR1 in a subject, or treating, preventing, or ameliorating one or more symptoms of the disease;

(4) reducing or inhibiting tumor growth or progression in a subject having a tumor expressing ROR1;

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

(6) exert cytotoxic activity in cells expressing ROR1;

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

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

In one embodiment, the antibody-drug conjugate comprises a plurality of D components, and the plurality of D components can be a combination of different therapeutically active substances or pharmaceutically active ingredients, or a combination of the same therapeutically active substances or pharmaceutically active ingredients. In one embodiment, the antibody-drug conjugate has a drug/antibody ratio (DAR) of 1-20, for example, a DAR of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In one embodiment, the DAR is an average DAR. In one embodiment, the average DAR is 1-15, for example, 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 plant toxin, a small molecule toxin, a radioactive isotope, a maytansinoid alkaloid, etc. In a specific embodiment, the cytotoxin is dolastatin and its auristatin derivatives, such as 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5 -methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide), 8261 (2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide) Dolastatin 10. Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AFHPA). In another embodiment, the dolastatin toxin and its auristatin derivatives are auristatin, dolastatin, MMAE, MMAF, auristatin F hydroxypropylamide or auristatin F phenylenediamine.

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

In one embodiment, the linker is selected from maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-amino benzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

In one embodiment, the antibody-drug conjugate comprises the anti-ROR1 antibody or its antigen-binding fragment described in the first aspect and a microtubule inhibitor (MMAE). In a further embodiment, MMAE is conjugated to the thiol group of cysteine on the anti-ROR1 antibody through a MC-VC-PAB linker, having a structure of anti-ROR1 antibody-MC-VC-PAB-MMAE. IgG1 type antibodies have 16 pairs of cysteine residues, of which 12 are present in the form of intrachain and 4 interchain disulfide bonds. The interchain disulfide bonds are solvent accessible and can be reduced by a reducing agent to form eight thiol groups, thereby becoming a coupling target (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 thiol group of 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 thiol group of 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 or antigen-binding fragment thereof of the first aspect or the antibody-drug conjugate of the second aspect, and (2) a pharmaceutically acceptable carrier.

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

In a fifth aspect, the present 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 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 Escherichia 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 a disease associated with abnormal expression of ROR1 in a subject in need thereof, comprising administering to the subject a preventively or therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention, or a preventively or therapeutically effective amount of an antibody-drug conjugate of the present invention, or a preventively or therapeutically effective amount of a 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 lymphocytic 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, the lung cancer is non-small cell lung cancer and the breast cancer is triple negative breast cancer.

In some embodiments, the ADC molecules or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and/or other therapeutic agents, for the uses described herein, such as for preventing and/or treating the relevant diseases or disorders mentioned herein.

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

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

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figures 2A-2K show the SEC-HPLC results of the antibodies of the present application; 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 2D shows the SEC-HPLC results of N31; Figure 2E shows the SEC-HPLC results of N99; Figure 2F shows the SEC-HPLC results of NW37; Figure 2G shows the SEC-HPLC results of N100; Figure 2H shows the SEC-HPLC results of N174; Figure 2I shows the SEC-HPLC results of N204; Figure 2J shows the SEC-HPLC results of NW29; Figure 2K shows the SEC-HPLC results of NW79.

Figures 3A-3F show the binding activity of the antibodies of the present application with the antigen protein huROR1-His determined based on the ELISA method; Figure 3A shows the binding activity of N100, N137 and N147 with the antigen protein huROR1-His; Figure 3B shows the binding activity of N204 and N218 with the antigen protein huROR1-His; Figure 3C shows the binding activity of N31 with the antigen protein huROR1-His; Figure 3D shows the binding activity of NW29, N99 and NW37 with the antigen protein huROR1-His; Figure 3E shows the binding activity of N174 with the antigen protein huROR1-His; Figure 3F shows the binding activity of NW79 with the antigen protein huROR1-His.

Figures 4A-4D show the binding activity of the antibodies of the present application to A549 cells determined 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; 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-5C show the binding activity of the antibodies of the present application to huROR1-HEK293 cells determined 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-6D show the binding activity of the antibodies of the present application with the antigen protein MusROR1-His determined based on the ELISA method; Figure 6A shows the binding activity of N100, N174, N137 and N147 with the antigen protein MusROR1-His; Figure 6B shows the binding activity of N204, N31 and N218 with the antigen protein MusROR1-His; Figure 6C shows the binding activity of NW29, NW37 and N99 with the antigen protein MusROR1-His; Figure 6D shows the binding activity of NW79 with the antigen protein MusROR1-His.

Figures 7A-7D show the binding activity of the antibodies of the present application with the antigen protein huROR2-His determined 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 the binding activity of N204, N31 and N218 with the antigen protein huROR2-His; Figure 7C shows the binding activity of NW29, NW37 and N99 with the antigen protein huROR2-His; Figure 7D shows the binding activity of NW79 with the antigen protein huROR2-His.

Figures 8A-8B show the endocytosis efficiency of the antibodies of the present application in huROR1-HEK293 cells determined based on the FACS method; Figure 8A shows the endocytosis efficiency of N204, N147 and NW37 in huROR1-HEK293 cells; Figure 8B shows the endocytosis efficiency of N100, N174, NW29, NW79, N137, N218, N31 and N99 in huROR1-HEK293 cells.

Figures 9A-9F show the endocytosis efficiency of the antibodies of the present application in huROR1-HEK293 cells determined based on the Fab-Zap method; Figure 9A shows the endocytosis efficiency of N31 and N99 in huROR1-HEK293 cells; Figure 9B shows the endocytosis efficiency of N174 and N218 in huROR1-HEK293 cells; Figure 9C shows the endocytosis efficiency of N147 in huROR1-HEK293 cells; Figure 9D shows the endocytosis efficiency of N100 and N137 in huROR1-HEK293 cells; Figure 9E shows the endocytosis efficiency of N204 and NW37 in huROR1-HEK293 cells; Figure 9F shows the endocytosis efficiency of NW29 and NW79 in huROR1-HEK293 cells.

Figures 10A-10D show the binding activity of affinity matured antibodies to A549 cells determined based on the FACS method; Figure 10A shows the binding activity of N31-6, N31-14, N31-18 and N31-56 to A549 cells; Figure 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.

Figures 11A-11E show the endocytosis efficiency of affinity matured antibodies in huROR1-HEK293 cells determined based on the Fab-Zap method; Figure 11A shows the endocytosis efficiency of N31-6 in huROR1-HEK293 cells; Figure 11B shows the endocytosis efficiency of NW37-37 in huROR1-HEK293 cells; Figure 11C shows the endocytosis efficiency of NW137-72 in huROR1-HEK293 cells; Figure 11D shows the endocytosis efficiency of N147-135 in huROR1-HEK293 cells; Figure 11E shows the endocytosis efficiency of N147-2 in huROR1-HEK293 cells.

Figures 12A-12D show the results of the binding of the ADC of the present application 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.

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

Figures 14A-14F show the killing effect of the ADC of the present application on three tumor cells, Jeko-1, MDA-MB-468 and NCI-H1944, detected based on the CCK8 method; Figure 14A shows the killing effect of N99-MMAE on Jeko-1 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 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.

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

Figures 16A-16D show the endocytosis efficiency of the ADC of the present application on two tumor cells A549 and HT-29 detected based on the FACS method; Figure 16A shows the endocytosis efficiency of N99-MMAE on A549 cells, and Figure 16B 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.

Figures 17A-17C show the tumor-suppressing effect of the ADC of the present application in mice; Figure 17A shows the changes in tumor volume in mice of different experimental groups during the experimental period, and arrows indicate the time points of drug administration; Figure 17B shows the changes in body weight of mice of different experimental groups during the experimental period; Figure 17C shows the tumor weight of mice of different experimental groups after the end of the experiment.

Figures 18A-18C show the tumor-suppressing effect of the ADC of the present application in mice with an A549 tumor model; Figure 18A shows the changes in tumor volume in mice in different experimental groups during the experimental period, with arrows marking the time points of drug 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 of mice in different experimental groups after the end of the experiment.

Figures 19A-19B show the tumor-suppressing effect of the ADC of the present application in mice with the NCI-N87 tumor model; Figure 19A shows the changes in tumor volume in mice in different experimental groups during the experimental period, with arrows marking the time points of drug administration; Figure 19B shows the changes in body weight of mice in different experimental groups during the experimental period.

Figures 20A-20B show the tumor-suppressing effect of the ADC of the present application in mice with an MDA-MB-231 tumor model; Figure 20A shows the changes in tumor volume in mice in different experimental groups during the experimental period, with arrows marking the time points of drug administration; Figure 20B shows the changes in body weight of mice in different experimental groups during the experimental period.

Figures 21A-21B show the tumor-suppressing effect of the ADC of the present application in mice with an MDA-MB-468 tumor model; Figure 21A shows the changes in tumor volume in mice in different experimental groups during the experimental period, with arrows marking the time points of drug administration; Figure 21B shows the changes in body weight of mice in different experimental groups during the experimental period.

Figures 22A-22B show the tumor-suppressing effect of the ADC of the present application in mice with a Jeko-1 tumor model; Figure 22A shows the changes in tumor volume in mice in different experimental groups during the experimental period, with arrows marking the time points of drug administration; Figure 22B shows the changes in body weight of mice in different experimental groups during the experimental period.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be changed. In addition, the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting.

I. Definitions

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

The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range 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 optional items, should be understood to mean any one of the optional items or any two or more of the optional items.

As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise indicated, the situation consisting of the stated elements, integers or steps is also covered. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

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

ROR1 is highly expressed in the embryo, and then its expression level decreases significantly in the adult stage. However, it was found that the expression of ROR1 was significantly increased in a variety of blood cancers and solid tumors. Blood cancers with high expression of ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL) and myeloid blood cancers. In solid tumors, cancer types that express ROR1 include colorectal cancer, breast cancer, intestinal cancer, lung cancer, pancreatic cancer, ovarian cancer and many other cancers.

The term "antibody" is used in the broadest sense herein 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, as long as they exhibit the desired antigen-binding activity. A complete antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in certain cases, e.g., antibodies naturally occurring in camels may comprise 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. Relative to the absence of the ROR1 antibody, the anti-ROR1 antibody is capable of inhibiting ROR1 activity, for example, by at least partially or completely blocking the stimulation of ROR1, reducing, preventing or delaying the activation of ROR1, or inactivating, desensitizing or downregulating the signal transduction, activity or amount of ROR1. 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 different from an intact antibody, which comprises a portion of an intact antibody and is capable of binding to 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 (e.g., scFv); single-domain antibodies; bivalent or bispecific 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 chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co. 91 pages (2007)). A single VH or VL domain is sufficient to confer antigen binding specificity.

"Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. CDRs in a variable domain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially 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 a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the 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), 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 specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

CDRs can also be identified 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 indicated, in the present invention, when referring to residue positions in antibody variable regions and CDRs (including heavy chain variable region residues), the numbered positions are those according to the AbM numbering system.

An "affinity matured" antibody is one that has one or more changes in one or more CDRs of the antibody that result in an improvement in the 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 humanized antibody" are used interchangeably and refer to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody encoding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

As used herein, the term "binding" or "specific binding" means that the binding effect is selective for the antigen and can be distinguished from unwanted 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 thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

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

The term "therapeutic agent" as used herein encompasses any substance 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 to which a therapeutically active substance or active pharmaceutical ingredient is covalently coupled, so that the therapeutically active substance or active pharmaceutical ingredient is targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin that can kill cells (preferably cancer cells) targeted by the ADC. The covalent attachment of the therapeutically active substance, active pharmaceutical ingredient or cytotoxin may be performed in a non-site-specific manner using a linker, or in a site-specific manner.

The term "site-specific conjugation" refers to a method of specifically conjugating a therapeutically active substance or active pharmaceutical ingredient to a specific site of an antibody. In one embodiment, the conjugation is accomplished with the aid of a linker.

The term "cytotoxic agent" can be used interchangeably with "cytotoxin", and in the present invention refers to a substance that inhibits or destroys cell 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., ricin), small molecule toxins, radioactive isotopes, etc., specifically, anthracyclines, camptothecins, combretastatins, dolastatins and their auristatin derivatives, duocarmycins, enediynes, geldanamycins, indolino-benzodiazepine dimers, maytansine and its derivatives, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, and the like. alkaloid, tubulysin, hemiasterlin, spliceostatin, pladienolide, and calicheamicin.

Any antibody-drug conjugate 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), which interfere with microtubule dynamics, cell division, etc., and have anti-tumor and antifungal activities. 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), and the modification of the central peptide subunit also leads to the effective cytotoxic activity of such substances in vitro. In one aspect, dolastatin and its auristatin derivatives can be, for example, 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1 -oxoheptane-4-yl]-N-methyl-L-valinamide), 8261 (2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide) Dolastatin 10. Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AFHPA), and other auristatins (e.g., auristatins described in U.S. Publication No. 20130129753), etc.

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

MMAE plays an effective role in inhibiting mitosis by inhibiting tubulin polymerization. Due to its cytotoxicity, it cannot be used as a drug, 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 targets tumor cells through antibodies, and the linker is cleaved after MMAE-MAB enters the tumor cells, thereby releasing MMAE, which exerts its cytotoxic effect and kills tumor cells.

The terms "linker" and "connector" are used interchangeably in this application and refer to a chemical module that covalently connects an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC. In one embodiment, the linker may comprise amino acid residues that connect the antigen to the payload. The amino acid residues may form dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptides. The amino acid residues include naturally occurring ones and non-naturally occurring amino acid analogs, such as citrulline or β-amino acids, such as β-alanine, or ω-amino acids such as 4-amino-butyric acid.

According to the property classification, the linkers suitable for the present invention can be linkers degradable by tissue proteases, 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-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, ultraviolet-sensitive linkers 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, the "linker" described in the present invention 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 to the drug.

In a specific embodiment, the linker includes, but is not limited to, maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-amino The present invention also contains the following compounds: N-succinimidyl benzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), and N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

The term "load" or "drug load" or "effective load" refers to the average effective load number per antibody in the ADC molecule ("effective load" can be used interchangeably with "therapeutically active substance or active pharmaceutical ingredient" in this article). The drug load can range from 1-20 therapeutically active substances 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 ADC described herein generally has a DAR of 1-20, and in certain specific embodiments has a DAR of 1-8, 2-8, 2-6, 2-5, 2-18, 4-16, 5-12, 6-10, 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, wherein the number represents the numerical value of the DAR, for example, D2 represents a drug/antibody ratio with a DAR value of 2. In some embodiments, the DAR is the average DAR, i.e., the overall ratio of the small molecule drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in the product as measured by a detection method (e.g., by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay, electrophoresis and/or HPLC). The DAR may be limited by the number of attachment sites on the antibody. For example, in the case where the attachment site is a cysteine thiol, the antibody may have only one or a few cysteine thiol groups or may have only one or a few sufficiently reactive thiol groups through which the linking unit can be attached. In some embodiments, the average DAR value of the conjugate 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, or a range with two of these values as endpoints.

The term "treat" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay disease development or to slow the progression of a disease.

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

The term "effective amount" refers to the amount or dosage of the antibody or conjugate or composition of the invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of the mammal; body weight, age and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; 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 outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition can vary according to a variety of factors such as disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to stimulate 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 its conjugate or composition are outweighed by the therapeutically beneficial effects. Relative to untreated subjects, 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%, 60% or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

The term "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

II. Compositions of the Invention

In some embodiments, the present invention provides a composition comprising any anti-ROR1 antibody described herein, or its ADC molecule, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient, such as a pharmaceutical carrier, a pharmaceutical excipient, including a buffer as known in the art. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises an anti-ROR1 antibody of the present invention, or its ADC molecule, and a combination of 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", Eighth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends 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, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional route; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implant device.

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

A medicament comprising the antibody described herein can be prepared by mixing the anti-ROR1 antibody of the present invention, or its ADC molecule, having a desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized preparation or an aqueous solution.

The pharmaceutical composition or preparation of the present invention may also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists), etc. The active ingredient is suitably combined in an amount effective for the intended use.

Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films, or microcapsules.

III. Preparation of Antibodies and Antibody-Drug Conjugates of the Invention

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

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 inserted into a vector for further cloning and/or expression in a host cell. Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to nucleic acids encoding the anti-ROR1 antibody of the present invention.

The anti-ROR1 antibodies of the present invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art. The purity of the anti-ROR1 antibodies 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, etc.

The prior art describes a variety of methods for coupling cytotoxic agents or other therapeutic agents to antibodies. For example, conjugation can occur in antibodies via the amino group of a lysine side chain and the amino group at the N-terminus of the antibody, the carboxyl group at the C-terminus of aspartic acid, glutamic acid, or an activated cysteine sulfhydryl group.

Example

The present invention is further illustrated by the following examples, however, it should be understood that the examples are described in an illustrative rather than a limiting manner and that various modifications may be made by those skilled in the art.

The practice of the present invention will employ, unless explicitly indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, which are within the skill of the art.

Example 1 Preparation and identification of raw materials

1.1 Preparation and identification of ROR1 control antibodies

Preparation of ROR1 control antibody: This application uses the anti-ROR1 antibody Cirmtuzumab as a positive control antibody, and the gene synthesis of the target fragment is performed by General Biotechnology Co., Ltd. according to the sequence disclosed in WO/2019/173843. Then, it is constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination, and the constructed recombinant protein expression vector is transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the plasmid is extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain the desired expression vector expressing cirmtuzumab, with a clone number of 99961.1. The expressed cirmtuzumab is referred to as 99961.1 below. The expression vector was transfected into 293 cells to express cimetuzumab by ExpiFectamine ^TM CHO transfection kit (Thermo Fisher, A29129). After 7 days of transfection, the cell culture supernatant was collected and centrifuged at 15000g for 10 min. The obtained supernatant was filtered through a 0.22 μm filter membrane, and the antibody in the supernatant was affinity purified using a Protein A/G affinity chromatography column. The target antibody was eluted with 100 mM glycine (pH 3.0), and the eluted antibody was exchanged into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).

Identification of ROR1 control antibody: The activity of the prepared positive control antibody 99961.1 (IgG1) was detected with the purchased huROR1-His antigen protein (Kaixia Biotechnology, ROR-HM401). The specific method is as follows: huROR1-His (2 μg/mL, 30 μL/well) was coated on a 96-well ELISA plate at 4°C overnight; after washing the plate 3 times, it was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing the plate 3 times, the control antibody 99961.1 diluted with PBS was added and incubated at room temperature for 1 hour; after washing the plate, the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted with PBS (1:6000) was added and incubated at room temperature for 1 hour, the plate was washed 6 times, TMB was added for color development for 5-20 minutes, and the data was read by the OD450 of the microplate reader after the color development was stopped, and the data was processed and plotted using Graphpad prism. The results showed that the expressed control antibody 99961.1 could bind to the ROR1 protein and had normal anti-ROR1 activity.

1.2 Preparation and identification of ROR1 antigen protein

Preparation of antigen protein: Through genetic manipulation at the coding gene level, His tags were added to the C-terminus of the sequences of human ROR1 protein huROR1 ECD AA30-406 (Uniprot ID: Q01973), mouse ROR1 protein MusROR1 ECD AA30-406 (Uniprot ID: Q9Z139), and human ROR2 protein huROR2 ECD AA34-403 (Uniprot ID: Q01974). The obtained nucleic acid sequences were constructed into pcDNA3.4 vectors, and then transformed into Escherichia coli DH5α, cultured at 37°C overnight, and then the plasmids were extracted using an endotoxin ^- free plasmid extraction kit ( ^OMEGA , D6950-01). The obtained plasmids were transiently transfected into HEK293 cells ( CRL-1573 ^TM ), after 7 days of expression, the cell culture supernatant was collected, and the protein containing the His tag was affinity purified using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with an imidazole gradient. The eluted proteins were exchanged into PBS buffer through ultrafiltration concentration tubes (Millipore, UFC901096), and finally the antigen proteins (huROR1-His, MusROR1-His, huROR2-His) were obtained.

Antigen identification: The prepared antigen (huROR1-His) was detected with the antibody 99961.1 (IgG1) obtained in Example 1.1. The specific method is as follows: 2 μg/mL huROR1-His was used to coat the Elisa plate at 4°C overnight, and the purchased antigen protein huROR1-His (Kaixia Bio ROR-HM201) was used as a positive control; after washing the plate 3 times, it was blocked with 5% skim milk prepared with PBS at room temperature for 1 hour; after washing the plate 3 times, the antibody 99961.1 diluted with PBS was added and incubated at room temperature for 1 hour; after washing the plate, the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted with PBS (1:6000) was added and incubated at room temperature for 1 hour, the plate was washed 6 times and then TMB was added for color development for 5-20 minutes, the color development reaction was terminated, the data was read using an OD450 microplate reader, and the data was processed using Graphpad prism for plotting. The results showed that antibody 99961.1 could bind to the antigen huROR1-His constructed and expressed by the inventors of the present application 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 electroporated into HEK293 cells ( CRL-1573 ^TM ). After electroporation, the obtained cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) by volume and without antibiotics, and then transferred to 10×10 cm cell culture dishes for 48 hours, and then the cells were distributed to 96-well cell culture plates at an average density of 10 ⁴ cells/well, and puromycin with a final concentration of 2 μg/mL was added as a screening pressure. After about 2 weeks, the cell lines that formed clones were picked for identification.

Flow cytometry identification of huROR1-HEK293 cells: digest the above cell lines in the logarithmic growth phase and plate them into 96-well plates. After washing with FACS buffer (1×PBS buffer containing 2% FBS by volume), add primary antibody (99961.1) diluted with PBS gradient and incubate at 4℃ for 30min; after washing, add prepared fluorescent secondary antibody anti human IgG Fc (abcam, 98596) and incubate at 4℃ for 30min; finally, detect by flow cytometer (Beckman, CytoFLEXAOO-1-1102). Test results Show that huROR1-HEK293 cell lines with high surface expression of human ROR1 were 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 library was screened using the antigen protein huROR1-His prepared in Example 1.2 and the huROR1-HEK293 cells overexpressing huROR1 prepared in Example 2 as screening antigens to obtain multiple antibody molecules that specifically bind to human ROR1.

3.1 Construction of human antibody gene library

Ficoll-Paque density gradient separation solution (purchased from GE, catalog number: 17144003S) was used to separate the peripheral blood mononuclear cells (PBMC) of normal human blood, and total RNA was extracted from the isolated PBMC cells by conventional methods. The extracted total RNA was reversely transcribed into cDNA using a reverse transcription kit (purchased from TaKaRa, 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 of the heavy chain and light chain and the rear end of the first constant region, and the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody were obtained after PCR. The fragments containing the light chain and heavy chain variable regions of the antibody were amplified by the fusion PCR method, and the PCR product and the phage display vector were digested, recovered and connected. The connection product was recovered by a recovery kit (Omega, catalog number: D6492-02) (Li Xiaolin, Construction and preliminary screening of a large-capacity non-immune humanized Fab phage antibody library, Master's thesis of Peking Union Medical College, June 2007). Finally, it was transformed into competent Escherichia coli SS320 (Lucigen, MC1061 F) by an electroporator (Bio-Rad, MicroPulser), and the transformed Escherichia coli SS320 bacterial solution was coated on a 2-YT solid plate with ampicillin resistance (solid plate is made of 1.5% tryptone, 1% yeast extract, 0.5% NaCl, 1.5% agar, and is prepared by mass volume g/mL). By gradient dilution plating, the library capacity was measured to be 3×10 ¹¹ cfu, i.e., an antibody gene library of 3×10 ¹¹ antibody genes (the library capacity calculation method refers to Example 2.2 in patent CN112250763B). VSCM13 helper phage (purchased from Stratagene) was used to package it to obtain an antibody gene phage display library (the preparation of the antibody gene phage display library refers to Example 2.3 in patent CN112250763B).

3.2 Screening of antibody gene phage display library

3.2.1 Screening of antibody gene phage display library by magnetic bead method

The magnetic bead screening is based on the process of incubating, washing and eluting the antigen protein huROR1-His with magnetic beads coupled with streptavidin after biotin labeling, and then the antigen-bound magnetic beads and the antibody gene phage display library are incubated, washed and eluted. Usually 3-4 rounds of panning are carried out, so that specific monoclonal antibodies against the antigen can be enriched in large quantities. In this embodiment, biotin-labeled huROR1-His is used for phage display library screening, and after 3 rounds of panning, the monoclonal antibody Fab against human ROR1 is initially screened. For specific methods, refer to Example 2.4.1 in patent CN112250763B.

3.2.2 Screening of antibody gene phage display library by immunotube method

The principle of the immunotube screening is to coat the antigen protein huROR1-His on the surface of an immunotube with high adsorption capacity, add the phage display antibody library to the immunotube and perform a panning process of incubation, washing and elution with the antigen protein adsorbed on the surface of the immunotube, and finally enrich the specific monoclonal antibody Fab against the antigen after 2-4 rounds of panning. In this example, the monoclonal antibody Fab against human ROR1 was enriched after 3 rounds of panning. The specific method is referred to Example 2.4.2 in patent CN112250763B.

3.3 Selection of monoclones

The phage pool eluted in each round was tested by ELISA to evaluate the enrichment effect, and 10 clones were randomly selected from the phage pool in each round of screening for sequence analysis. The enrichment effect and the repeatability ratio of the measured sequences were comprehensively analyzed to select the appropriate round for single clone selection.

The ELISA monoclonal primary screening used the antigen protein huROR1-His, and the antibody Fab binding to huROR1-His obtained in the primary screening was prepared into Fab lysate, and then the overexpression cell huROR1-HEK293 prepared in Example 2.1 was used for detection and verification by flow cytometry (FACS). A total of 11 antibody Fab molecules that specifically bind to human ROR1 were screened, and the 11 obtained antibody Fabs were named according to the corresponding clone numbers (N99, N218, N31, N137, N147, N100, N204, N174, NW29, NW79 and NW37). The specific FACS results are shown in Figures 1A-1F. The amino acid sequence of the CDR region of the obtained antibody Fab is shown in Table 1, and the CDR sequence is determined by using the AbM definition method.

Table 1. Amino acid sequences of CDR regions of 11 antibodies

Example 4 Antibody Construction, Expression and Purification

4.1 Plasmid construction

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

4.2 Antibody expression and purification

The full-length sequence of the antibody obtained above was expressed by the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, the CHO cell density was confirmed to be about 7×10 ⁶ to 1×10 ⁷ viable cells/mL, and the cell survival rate was >98%. At this time, the cells were adjusted to a final concentration of 6×10 ⁶ cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C. The target plasmid was diluted with OptiPRO ^™ SFM pre-cooled at 4°C (1 μg of plasmid was added to 1 mL of the culture medium), and the ExpiFectamine ^™ CHO reagent was diluted with OptiPRO ^™ SFM at the same time, and then the two were mixed in equal volumes and gently pipetted to mix to prepare ExpiFectamine ^™ CHO/plasmid DNA mixed solution, incubated at room temperature for 1-5 minutes, slowly added to the prepared cell suspension and gently shaken at the same time, and finally placed in a cell culture shaker and cultured at 37°C and 8% CO ₂ .

18-22h after transfection, add ExpiCHO ^™ Enhancer reagent and ExpiCHO ^™ Feed reagent to the cell culture medium, and place the shake flask in a 32°C shaker and 5% CO ₂ for continued culture. On the 5th day after transfection, add the same volume of ExpiCHO ^™ Feed reagent, and gently mix the cell suspension while slowly adding it. 7 days after transfection, collect the cell culture supernatant expressing the target antibody protein, centrifuge at 15000g for 10min, and the resulting supernatant is affinity purified with MabSelect SuRe LX (GE, 17547403), and then the target antibody protein is eluted with 100mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl, and finally the resulting antibody protein is exchanged into PBS buffer by ultrafiltration concentration tube (Millipore, UFC901096).

Example 5 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: 1 μg of each obtained antibody and quality control product IPI (ipilimumab) were added to 5×SDS loading buffer and 40 mM iodoacetamide, heated in a 75°C dry bath for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min to obtain the supernatant.

Preparation of reducing solution: 2 μg of each obtained antibody and quality control product IPI were added to 5×SDS loading buffer and 5 mM DTT, heated in a 100°C dry bath for 10 min, cooled to room temperature, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The supernatant was added to Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and the protein bands were visualized by Coomassie brilliant blue staining.

The protein gel with chromogenic protein bands was scanned using EPSON V550 color scanner (decolorization was performed until the gel background was transparent), and the purity of the reduced and non-reduced bands was calculated using ImageJ according to the peak area normalization method.

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

5.2 SEC-HPLC Identification of Monomeric Purity of Antibodies

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

The results are shown in Figures 2A-2K and Table 2. Except for N204 and NW79 whose monomer purities were around 95%, the monomer purities of other antibodies were all greater than 98%.

Table 2 Expression levels and physicochemical properties of candidate antibodies

Example 6 Antigen Binding Activity Detection of Antibodies

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

6.1 ELISA-based detection of antibody binding ability to antigen protein huROR1-His

96-well ELISA plates (30 μL/well) were coated with 2 μg/mL huROR1-His at 4°C overnight. The next day, the plates were washed 3 times with PBST and blocked with 5% skim milk for 2 hours. After washing the plates 3 times with PBST, gradient dilutions (3.00000, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00046, 0.00005 μg/mL) of each antibody and positive control antibody 99961.1 were added and incubated for 1 hour. After washing 3 times with PBST, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After incubation, the plates were 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 using a microplate reader (Molecular Devices, SpecterMax 190).

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

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

In this example, the binding activity of the antibody was evaluated using two types of cells, huROR1-HEK293 cells overexpressing human ROR1 and A549 cells.

The specific method is as follows: huROR1-HEK293 cells or A549 cells in the logarithmic growth phase were prepared into a single cell suspension, the density was adjusted to 1×10 ⁶ cells/mL, and 100 μL was added to each well of a 96-well round-bottom plate, centrifuged at 4°C and 300g, and the supernatant was removed. Each antibody with each gradient dilution (3.000000, 0.300000, 0.100000, 0.033333, 0.011111, 0.003704, 0.001235, 0.000123 μg/mL) and the positive control antibody 99961.1 were added to the corresponding wells, mixed and incubated at 4°C for 30 minutes. The incubated cell mixture was washed three times and then 100 μL of 1:300 diluted secondary antibody Goat F(ab')2 Anti-Human IgG-Fc (abcam, ab98596) was added. The mixture was incubated at 4°C in the dark for 30 min. After washing three times, the cells were detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

The results are shown in Figures 4A-4D and 5A-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 other 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 the positive control antibody 99961.1, and the affinity of the other antibodies is equivalent to or weaker than that of the positive control antibody 99961.1.

Example 7 Antibody species and homologous cross-reactivity detection

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

7.1 Species Cross-Reactivity Testing of Antibodies in the Application

Coat a 96-well ELISA plate (30 μL/well) with 2 μg/mL MusROR1-His at 4°C overnight. The next day, wash the plate 3 times with PBST and block with 5% skim milk for 2 hours. Wash the plate 3 times with PBST, add gradient dilutions (1.00000, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, 0.00015, 0.00002 μg/mL) of each antibody and positive control antibody 99961.1 and incubate for 1 hour. After washing 3 times with PBST, add the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) and incubate for 1 hour. After incubation, wash the plate 6 times with PBST and add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the absorbance was read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

As shown in Figures 6A-6D and Table 3, all antibodies obtained in this application can bind to the mouse antigen protein MusROR1-His, showing good mouse cross-reactivity. The positive control antibody 99961.1 does not bind to the mouse antigen protein. Therefore, the antibodies obtained in this application have a wide range of uses in experiments and tests based on mouse models.

7.2 Detection of cross-reactivity of antibodies of the present application

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

As shown in Figures 7A-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.

Table 3 Cross-species and homologous cross-activity of candidate antibodies

Example 8 Detection of antibody endocytosis efficiency of the present application

In this example, the endocytosis efficiency of the antibody obtained in this application was identified by using two detection methods, FACS and Fab-Zap. The FACS method endocytosis detection uses supersaturated antibodies to bind cells and detects the antibody endocytosis efficiency in a short period of time; the Fab-Zap method uses antibodies bound to saporin toxins at different concentrations to bind cells and bring the toxins into the target cells through endocytosis to kill them. This method reflects the long-term cumulative effect of endocytosis efficiency.

8.1 FACS assay to detect antibody internalization efficiency

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

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

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

Incubation with extracellular secondary antibody: 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 (diluted 1:150 with FACS buffer) to each well; incubate at 4°C for 30 min.

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

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

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 (diluted 1:150 with Permeabilization buffer) to each well. Incubate at room temperature for 60 min.

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 different fluorescence settings to stain each sample. The first group is set to FITC+PE, that is, FITC secondary antibody is used to identify extracellular primary antibody first, and PE secondary antibody is used to identify intracellular primary antibody after membrane permeabilization. In this group, PE is the intracellular signal and FITC is the extracellular signal; the second group is set to PE+PE, that is, PE secondary antibody is used to identify extracellular primary antibody first, and PE secondary antibody is used to identify intracellular primary antibody after membrane permeabilization. In this group, PE is the sum of intracellular and extracellular signals. At the same time, both groups of samples are detected using FITC and PE channels, and the calculation value of endocytosis rate is the calculation value of PE channel detection. 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 Figures 8A-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), and that of N174, N218, N204 and NW29 is comparable to that of the control antibody 99961.1.

8.2 Fab-Zap method to detect antibody internalization efficiency

This experiment detects the endocytic activity of antibodies through the cytotoxicity of antibody-mediated Fab-ZAP endocytosis. Fab-ZAP is a Fab fragment connected to saporin, which is 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 with human antibodies, Fab-ZAP and human antibodies carry toxins to the human antibodies. When the human antibodies are internalized, the toxins enter the cells along with the antibodies, causing cell death. Then, MTS (Promega, G3580) is used to detect the activity of the cells to detect whether the antibodies are internalized.

The specific experimental method is as follows: First, dilute Fab-ZAP to 0.4nM with DMEM complete medium, and then use 0.4nM Fab-ZAP gradient dilution (0.400000, 0.080000, 0.026667, 0.008889, 0.002963, 0.000988, 0.000329, 0.000066μg/mL) to prepare antibody dilution solution. Prepare single cell suspension of huROR1-HEK293 cells in logarithmic growth phase, adjust the density to 6×10 ⁶ cells/mL, and inoculate 50μL per well into a 96-well plate, then take the aforementioned antibody dilution solution, add 50μL per well into the cell culture plate, and mix thoroughly by blowing. Place the cell culture plate in a 37°C cell culture incubator and incubate for 48 hours. After the incubation, add 7.5 μL TritonX-100 solution to each well, tap gently to mix, 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 an ELISA reader, and detect the wavelength A492.

The results are shown in Figures 9A-9F, indicating that under the antibody concentration conditions specified in this example, the endocytosis effect of the antibody prepared in this application is comparable to that of the control antibody. Considering that cells are sensitive to toxins only when they react above a certain threshold, it is difficult to increase the difference when the accumulated toxins are not much different.

Example 9 Antibody Affinity Kinetics (Biacore) Analysis

In this example, the affinity of the antibody obtained in the present application and the positive control antibody 99961.1 to the antigen protein huROR1-His was detected based on the Biacore device.

Protein coupling: The huROR1-His protein produced in Example 1.2 was diluted to 5.6 μg/mL with NaAc buffer at pH 5.0, the flow rate was set to 10 μL/min, the chip activation time was set to the default value of 420 s with a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the antigen protein was fixed using the coupling mode with a preset coupling amount huROR1-His to a level of about 75 RU, and the activation groups that were not bound to the test sample were blocked with ethanolamine.

Sample test conditions: PBS buffer (pH 7.4) containing 0.05% Tween-20 was used as the running buffer, and the running buffer was used as the control test sample. A series of antibody concentrations (4 nM, 20 nM) were set. During sample analysis, the flow rate was set to 30 μL/min, the binding time was 120 s, and the dissociation time was 360 s. After the dissociation was completed, 10 mM Gly-HCl (pH 2.0) was used for regeneration for 20 s to completely remove the antibody bound to the ligand.

Parameter fitting: The experiment was run in multiple cycles, with the response signal taking the analysis time as the horizontal axis and the response value as the vertical axis. After double reference subtraction, the obtained data were fitted by BIAcore T200 analysis software. The fitting model used was the 1:1 Langmuir binding model to determine affinity indicators such as the binding dissociation constant.

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, where the KD of N147 reaches 6.62E-11, while the KD of the control antibody 99961.1 is 1.98E-9. The affinities of the remaining antibodies are comparable to that of the control antibody 99961.1.

Table 4 KD values of antibodies

Example 10 Antibody Affinity Kinetics (Gator) Detection

In this example, the affinity of the antibody obtained in the present application and the positive control antibody 99961.1 to the antigen protein huROR1-His was detected based on the Gator device.

The specific experimental 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 source culture) is configured into Q buffer buffer, and the storage solution of the antibody to be tested is diluted to a working solution with a final concentration of 30nM with the configured Q buffer buffer; the antigen protein huROR1-His storage solution is configured with Q buffer into a working solution with multiple dilutions (480, 240, 120, 60, 30, 15, 7.5nM), and then the Gator instrument and its supporting software are used 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 that of the control antibody 99961.1, among which the KD of N100 reaches 8.85E-9, the KD of NW29 reaches 6.60E-9, and the KD of NW79 reaches 6.94E-8, while the KD of the control antibody 99961.1 is 1.35E-8. The affinities of the remaining antibodies are comparable to that of the control antibody 99961.1.

Table 5 KD values of antibodies

Example 11 Affinity Kinetics Epitope Grouping

This example uses the affinity kinetics method to group the epitopes of the antibodies obtained in this application 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 source culture) is configured into Q buffer buffer, and the storage solution of the antibody to be tested is diluted to a working solution with a final concentration of 100nM with the configured Q buffer buffer; the antigen protein huROR1-His storage solution is configured into a 50nM working solution with Q buffer, and then the Gator instrument and its supporting software are used to detect and analyze based on the Tandem setting of the Epitope Binning experimental mode. The results of the epitope grouping of each antibody obtained are shown in Table 6, wherein each group of antibodies has the same epitope.

Table 6 Summary of epitope grouping results

Example 12 Antibody Affinity Maturation

In this example, affinity maturation modification was performed on antibodies N147, N31, N137 and NW37 to improve antibody affinity and other biological activities. Affinity maturation modification is based on M13 phage display technology, using codon-based primers (in the process of primer synthesis, a single codon is composed of NNK) to introduce mutations in the CDR region, 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 CDRL1+CDRL3+CDRH3 combination mutation, Library 2 is CDRL2+CDRH1+CDRH2 combination mutation; Library 3 and Library 4 are double-point saturation mutations, 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 is as follows: first, primers containing point mutations are synthesized (Golden Wisdom Biotechnology Co., Ltd.); secondly, the coding sequences of the antibodies to be modified (also called parent antibodies) N147, N31, N137 and NW37 are used as PCR amplification templates to amplify the CDR region containing mutation sequences, and the fragments containing different CDR mutations are combined by the bridge PCR method, and then the point mutation antibodies are connected to the phage display vector by double enzyme digestion (HindⅢ and NotⅠ) and double sticky end connection, and finally the antibody sequence with mutation sites is transferred into Escherichia coli SS320 by electroporation. The library capacity calculation, phage library preparation and library screening operation process are detailed in the above examples. After affinity maturation of N147, 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 amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 161, and 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, and 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, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 164) were obtained; after affinity maturation of N31, N31-6 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 64, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 63), N31-14 (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 151, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: NO: 150), N31-18 (heavy chain variable region amino acids are shown in SEQ ID NO: 153, light chain variable region amino acids are shown in SEQ ID NO: 152) and N31-56 (heavy chain variable region amino acids are shown in SEQ ID NO: 155, light chain variable region amino acids are shown in SEQ ID NO: 154); N137 affinity maturation obtained N137-72 (heavy chain variable region amino acids are shown in SEQ ID NO: 68, light chain variable region amino acids are shown in SEQ ID NO: 67), N137-47 (heavy chain variable region amino acids are shown in SEQ ID NO: 157, light chain variable region amino acids are shown in SEQ ID NO: 156), N137-53 (heavy chain variable region amino acids are shown in SEQ ID NO: 52, light chain variable region amino acids are shown in SEQ ID NO: 158), N137-60 (heavy chain variable region amino acids are shown in SEQ ID NO: 52, light chain variable region amino acids are shown in SEQ ID NO: NO:159); after affinity maturation of NW37, NW37-37 (amino acids in the heavy chain variable region are shown in SEQ ID NO:70, and amino acids in the light chain variable region are shown in SEQ ID NO:69), NW37-99 (amino acids in the heavy chain variable region are shown in SEQ ID NO:62, and amino acids in the light chain variable region are shown in SEQ ID NO:166), NW37-113 (amino acids in the heavy chain variable region are shown in SEQ ID NO:168, and amino acids in the light chain variable region are shown in SEQ ID NO:167) and NW37-136 (amino acids in the heavy chain variable region are shown in SEQ ID NO:169, and amino acids in the light chain variable region are shown in SEQ ID NO:61) were obtained; the amino acid sequences of the CDR regions of the antibodies after affinity maturation are shown in Table 7. The corresponding antibody proteins were expressed and purified by the methods disclosed in the aforementioned embodiments. The molecular weight and purity of the antibody protein were identified by SDS PAGE. The test results showed that the band of the non-reducing gel of the affinity matured antibody was around 150kD, and the band of the reducing gel was around 55kD and 25kD, which was in line with the expected size. The purity of all affinity matured antibodies detected by reducing gel was greater than 95%.

Table 7 Amino acid sequences of CDR regions of affinity matured antibodies (using AbM definition)

Example 13 Antigen affinity detection of antibodies after affinity maturation

In this embodiment, the affinity of the antibody after affinity maturation and A549 cells was detected based on the FACS method. For specific detection methods, see Example 6. The test results are shown in Figures 10A-10D, and the results show that the affinity of the antibody after affinity maturation and A549 cells is improved, among which 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 have significantly better affinity than their corresponding parent antibody molecules N31, NW37 and N137; N147-135, N147-2, N147-86 and N147-93 have better affinity than their 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 the parent molecule to the human ROR1 antigen protein huROR1-His was tested based on the Biacore device. The specific experimental method is shown in Example 9, and 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 affinity of the remaining affinity matured antibodies to the human ROR1 antigen protein huROR1-His is also better than that of the parent molecule.

Table 8 KD values of antibodies after affinity maturation

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. The specific method is shown in Example 8.2. The results are shown in Figures 11A-11E. Within the antibody concentration range specified in this example, the endocytosis efficiency of the affinity matured antibodies is equivalent to that of the control antibodies.

Example 16 ADC Preparation

In this embodiment, antibodies N99 and N147-135 and control antibody 99961.1 were coupled with toxin MMAE (a tubulin inhibitor with anticancer activity) to construct antibody-drug conjugates. MMAE was connected with the linker MC-VC-PAB to form MC-VC-PAB-MMAE, and the linker was covalently linked to the sulfhydryl group on the cysteine of the antibody, so that the antibody was conjugated with MMAE to obtain an antibody-drug conjugate. IgG1 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 a reducing agent to form eight sulfhydryl groups, which then become the coupling target (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:

Antibodies N99, N147-135 and control antibody 99961.1 were taken out of the -80°C refrigerator, thawed and transferred to 15 mL 30KD ultrafiltration centrifuge tubes respectively, and coupling buffer (content per 1L: Na ₂ HPO ₄ ·2H ₂ O 6.86 g, NaH ₂ PO ₄ ·H ₂ O 1.58 g, purified water to 1000 g, pH 7.4) was added to 15 mL, centrifuged at 4500 rpm for about 30 min, concentrated to 2-3 mL, and dialysate (content per 1L: histidine 0.73 g, histidine hydrochloride monohydrate 1.12 g, purified water to 1000 g, pH 6.0) was added again to 15 mL, dialyzed repeatedly 8-10 times to obtain antibody stock solution, and the antibody concentration after dialysis was detected.

The antibody stock solution, 10 mM disulfide bond reducing agent TCEP mother solution (tris(2-carboxyethyl)phosphine hydrochloride mother solution, content per 1L: Na ₂ HPO ₄ ·2H ₂ O 6.86 g, NaH ₂ PO ₄ ·H ₂ O 1.58 g), 10 mM DTPA mother solution (diethylenetriaminepentaacetic acid mother solution, content per 1L: DTPA 3.90 g, NaOH 1.20 g) and coupling buffer were added in sequence to form a reduction reaction system to reduce the sulfhydryl group on the cysteine of the antibody. The amount of each component added in the reduction reaction system is shown in Table 9, so that the final antibody concentration in the reduction reaction system is 5 mg/mL (N99 and 999611) or 4 mg/mL (N147-135), the final DTPA concentration is 1 mM, and the molar ratio of TCEP to antibody 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 and 400 rpm for reduction reaction for 2 h.

Weigh MC-VC-PAB-MMAE, dissolve it in DMSO, and prepare a 5mM MC-VC-PAB-MMAE mother solution. After the reduction reaction is completed, add the MC-VC-PAB-MMAE mother solution to the reduction reaction system in an ice-water bath in sequence, and the addition amount is shown in Table 10 to prepare a coupling reaction system. After sufficient mixing, place the coupling reaction system in a 25°C constant temperature mixer, 400 rpm, and perform the coupling reaction for 1 hour to obtain a solution containing ADC.

After the coupling is completed, the solution containing ADC is centrifuged and filtered to obtain an ADC sample, which is transferred to a 15mL 30KD ultrafiltration centrifuge tube. The dialysate is added to 15mL, centrifuged at 4500rpm for 20min, concentrated to 2-3mL, and the dialysate is added again to 15mL, and the dialysate is repeated 8-10 times. The dialyzed ADC sample is subjected to SEC-HPLC detection, HIC-HPLC detection, concentration detection, free drug detection, etc. The test results are shown in Table 11. The test results show that an ADC with a purity of more than 99% is obtained.

Table 9 Reduction reaction system composition

Table 10 MC-VC-PAB-MMAE mother liquor addition amount

Table 11 ADC sample test results

Example 17 Antigen Binding Activity Detection of ADC

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

The specific method is as follows: A549 cells or HT-29 cells in the logarithmic growth phase were prepared into a single cell suspension, the density was adjusted to 1×10 ⁶ cells/mL, and 100 μL was added to each well of a 96-well round-bottom plate, centrifuged at 4°C and 300g, and the supernatant was removed. N99, N99-MMAE, N147-135, N147-135-MMAE, 99961.1, 99961.1-MMAE and negative control were added to the corresponding wells with gradient dilutions (1.0000, 0.3333, 0.1111, 0.0370, 0.0123, 0.0041, 0.0014, 0.0001 μg/mL), mixed and incubated at 4°C for 30 min. The incubated cell mixture was washed three times and then 100 μL of 1:300 diluted secondary antibody Goat F(ab') ₂ Anti-Human IgG-Fc (abcam, ab98596) was added, incubated at 4°C in the dark for 30 min, washed three times and detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

The results are shown in Figure 12: The results of A549 cells show (Figure 12A and Figure 12B), the binding activity of N99-MMAE and the control 99961.1-MMA to A549 cells is comparable to that of the corresponding parent antibody, but the binding activity of N99 and N99-MMAE to A549 cells is better than that of the corresponding control 99961.1 and 99961.1-MMAE; the binding activity of N147-135-MMAE and N147-135 to A549 cells is comparable to that of the control The results of HT-29 cells showed (Figures 12C and 12D) that the binding activity of N99-MMAE and the control 99961.1-MMA to HT-29 cells was equivalent to that of the corresponding parent antibody, and the binding activity of N99-MMAE to HT-29 cells was equivalent to that of the control 99961.1-MMAE; the binding activity of N147-135-MMAE and N147-135 to HT-29 cells was equivalent to that of the 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 candidate ADC and control ADC.

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, and 100 μL per well is added to a 96-well cell culture plate, and cultured at 37°C, 5% CO ₂ for 12 hours. Then, a gradient dilution (2000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 1.953 nM) of ADC samples is added, and cultured at 37°C, 5% CO ₂ for 72 hours (A549 cells) or 96 hours (HT-29 cells). Then, 40 μL of MTS (Promega, G3580) is added to each well, and after incubation at 37°C for 1 hour, the data is read at OD492 by an ELISA reader.

The results are shown in Figures 13A-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, among which the IC50 of _N99 -MMAE on A549 and HT-29 were 11.07nM and 5.952nM, respectively; the killing effect of N147-135-MMAE on HT-29 tumor cells was better than that of the control 99961.1-MMAE, and the killing effect on A549 tumor cells was 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 killing of tumor cells by N99-MMAE and control ADC.

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 density of Jeko-1 is adjusted to 1×10 ⁵ cells/mL, the density of MDA-MB-468 is adjusted to 2×10 ⁵ cells/mL, and the density of NCI-H1944 is adjusted to 1.2×10 ⁵ cells/mL, and 90 μL per well is added to a 96-well cell culture plate, and cultured at 37°C, 5% CO ₂ for 12 hours. Then, ADC samples are added, and the drug concentration is gradiently diluted to 500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, 0.05 nM, and cultured at 37°C, 5% CO ₂ for 72 hours. Then 10 μl of CCK8 (Bimake, B34304) was added to each well, incubated at 37°C for 1 h, and the data were read at OD450 using a microplate reader.

The results are shown in Figure 14: The killing effects of N99-MMAE on three tumor cells Jeko-1 (Figure 14A), MDA-MB-468 (Figure 14B) and NCI-H1944 (Figure 14C) were better than those of the control ADC 99961.1-MMAE, among which the IC ₅₀ of N99-MMAE on Jeko-1, MDA-MB-468 and NCI-H1944 were 5.188nM, 5.126nM and 48.62nM, respectively; the killing effects of N147-135-MMAE on three tumor cells Jeko-1 (Figure 14D), MDA-MB-468 (Figure 14E) and NCI-H1944 (Figure 14F) were comparable to those of the control ADC The _IC50 of N147-135-MMAE on Jeko-1, MDA-MB-468 and NCI-H1944 were 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 application and the control ADC.

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

The results are shown in Figures 15A-15B. The competition of antibodies N99, N147-135 and 99961.1 can significantly inhibit the killing effect of their corresponding ADCs on huROR1-HEK293 cells, proving that the cell killing of the ADC prepared in the present application depends on the binding of the antibody connected to the ADC with the antigen on the cell surface, and is therefore antigen-dependent killing rather than non-specific killing by the connected 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 in this application based on the FACS method. For specific experimental methods, see Example 8.

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

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

This example detects the tumor-suppressing effects of two candidate ADCs (N99-MMAE and N147-135-MMAE) in animals. The tumor cells used are colon cancer cells HT-29 (BNCC337732), and 99961.1-MMAE is used as a positive control.

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

The experimental results showed that there was no significant change in the body weight of mice in each group during the treatment, indicating that the mice had good tolerance to ADC (Figure 17B); N99-MMAE, M147-135MMAE and the control 99961.1-MMAE all showed significant tumor inhibition effects under high-dose conditions (10mpk). Under high-dose conditions, the tumor inhibition effects of N99-MMAE and 99961.1-MMAE were equivalent, and the tumor inhibition rate of N147-135-MMAE almost reached 100% on the 41st day, which was significantly better than the positive control 99961.1-MMAE (Figure 17A, Figure 17C and Table 12).

Table 12 Tumor inhibition rate of ADC in mice

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

This example detects the tumor inhibition effects of two candidate ADCs (N99-MMAE and N147-135-MMAE) in animals. The tumor cells used are non-small cell lung cancer cells A549 (Shanghai Cell Bank, Chinese Academy of Sciences, C2107019), and 99961.1-MMAE is used as a positive control.

The specific method is as follows: 6-8 week old female nude mice (Balb/C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) weighing 18-20 g were used, and 1×10 ⁶ A549 cells were injected subcutaneously unilaterally on the right back of each nude mouse. When the tumor volume reached about 100 mm ³ , they were randomly divided into groups and cages. There were 6 tumor-bearing nude mice in each group, and a total of 7 groups, including a PBS negative control group, 4 ADC groups (N99-MMAE 5mpk (mg/kg), N99-MMAE 10mp k , 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, and the administration was 2 times a week and the tumor volume was measured 2 times, for a total of 6 times/3 weeks (BIW*3). Tumor volume (V) was calculated as follows: V = L × W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). After a certain period of observation after administration, the mice were euthanized, and the tumors were removed and the tumor weight was measured. The tumor volume, tumor weight and mouse body weight change data were analyzed to calculate the tumor inhibition rate. The results are shown in Figures 18A-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, indicating that the mice had good tolerance to ADC (Figure 18B); N99-MMAE showed similar tumor inhibition effects under high-dose (10mpk) and low-dose (5mpk) conditions, and the tumor inhibition effect was better than the positive control ADC 99961.1-MMAE under low-dose conditions. M147-135MMAE also showed a certain tumor inhibition effect under high-dose conditions (Figure 18A and Table 13); However, some tumor tissue rebound occurred in both the high-dose group and the low-dose group after the 41st day.

Table 13 Tumor inhibition rate of ADC in mice

Example 24 ADC efficacy test on NCI-N87 mouse tumor model

This example detects the tumor inhibition effect of a candidate ADC (N147-135-MMAE) in animals. The tumor cells used are gastric cancer cells NCI-N87 (Shanghai Cell Bank of the Chinese Academy of Sciences, C2009021, P3), and 99961.1-MMAE is used as a positive control.

The specific method is as follows: 6-8 week old female nude mice (Balb/C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) weighing 18-20g were used, and 1×10 ⁶ NCI-N87 cells were injected subcutaneously on the back of each nude mouse. When the tumor volume reached about 100 mm ³ , they were randomly divided into groups and cages. There were 10 tumor-bearing nude mice in each group, and there were 5 groups in total, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) and N147-135-MMAE 10mpk) and 2 positive control ADC groups (99961.1-MMAE 5mpk and 99961.1-MMAE 10mpk). The drug was administered by microvenous injection, and the drug was administered once a week and the tumor volume was measured twice, for a total of 3 times/3 weeks (QW*3). Tumor volume (V) was calculated as follows: V = L x W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). Tumor volume and mouse body weight change data were analyzed to calculate tumor inhibition rate. The results are shown in FIGS. 19A-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 had good tolerance to ADC (Figure 19B); compared with the PBS group, each dose group had a significant tumor inhibition effect, and the tumor inhibition effects of N147-135-MMAE molecule and 99961.1-MMAE molecule were comparable at the same dose of 10mpk and 5mpk, and there was no significant difference at the same dose (Figure 19A and Table 14).

Table 14 Tumor inhibition rate of ADC in mice

Example 25 Detection of the efficacy of ADC in inhibiting MDA-MB-231 mouse tumor model

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

The specific method is as follows: 6-8 week old female nude mice weighing 20-22 g (NSG Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used, and 1×10 ⁶ MDA-MB-231 cells were subcutaneously injected into the back of each nude mouse. When the tumor volume reached about 100 mm ³ , they were randomly divided into groups and cages. There were 10 tumor-bearing nude mice in each group, and there were 5 groups in total, including a PBS negative control group, 2 ADC groups (N147-135-MMAE 5mpk (mg/kg) 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, and the administration was once a week and the tumor volume was measured twice, for a total of 3 times/3 weeks (QW*3). Tumor volume (V) was calculated as follows: V = L x W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). Tumor volume and mouse body weight change data were analyzed to calculate tumor inhibition rate. The results are shown in FIGS. 20A-20B and Table 15.

The test results showed that there was no significant difference in the early stage of drug administration among the mice in each group. The PBS group began to lose weight on the 35th day, and the weight of the mice in the ADC molecule combination positive control ADC group did not change significantly, indicating that the mice had good tolerance to ADC (Figure 20B). Compared with the PBS group, the high-dose groups of the positive control molecule and the candidate ADC molecule had similar significant tumor inhibition effects, and both molecules had obvious dose correlation. The low-dose group had a certain tissue tumor rebound after drug withdrawal (Figure 20A and Table 15).

Table 15 Tumor inhibition rate of ADC in mice

Example 26 Detection of the efficacy of ADC in inhibiting MDA-MB-468 mouse tumor model

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

The specific method is as follows: 6-8 week old female nude mice (NOD SCID Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) weighing 21-25 g were used, and 1×10 ⁷ MDA-MB-468 cells were subcutaneously injected into the back of each nude mouse. When the tumor volume reached about 100 mm ³ , they were randomly divided into groups and cages. There were 6 tumor-bearing nude mice in each group, and 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 control ADC groups (99961.1-MMAE 5mpk and 99961.1-MMAE 10mp k ), and the administration method was tail vein injection, once a week, and the tumor volume was measured twice a week, for a total of 3 times/3 weeks (QW*3). Tumor volume (V) was calculated as follows: V = L x W ² /2 (where L is the longest tumor diameter and W is the shortest tumor diameter). Tumor volume and mouse body weight change data were analyzed to calculate tumor inhibition rate. The results are shown in Figures 21A-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 had good tolerance to ADC (Figure 21B); in the multiple-dose group, the 10 mg/kg dose group N147-135-MMAE (all mice were completely relieved on the 14th day after the first dose), and its tumor inhibition effect was better than that of 99961.1-MMAE (all mice were completely relieved on the 18th day after the first dose); the 5 mg/kg dose group N147-135-MMAE was completely relieved on the 20th day after the first dose, and the tumor inhibition effect was better than that of the same dose group 99961.1-MMAE (4 of 6 mice achieved complete remission on the 25th day after the first dose) (Figure 21A and Tables 16-17).

Table 16 Tumor inhibition rate of ADC in mice

Table 17 Complete remission rate of ADC in mice

Example 27 Detection of the efficacy of ADC in inhibiting Jeko-1 mouse tumor model

This example detects the tumor-suppressing effect of a candidate ADC (N147-135-MMAE) in animals. The tumor cells used are mantle cell lymphoma cells Jeko-1 (ATCC, CRL-3006), and 99961.1-MMAE is used as a positive control.

The specific method is as follows: 6-8 week old female nude mice (BALB/c Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) weighing 21-25 g were used, and 1×10 ⁷ Jeko-1 cells were injected subcutaneously on the back of each nude mouse. When the tumor volume reached about 100 mm ³ , the mice were randomly divided into groups and cages. There were 7 tumor-bearing nude mice in each group, for 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 8mp k (single dose), and N147-135-MMAE 2+3mpk (Q2W*2, i.e., 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 10mp k (QW*3), 99961.1-MMAE 10mp k (single dose), and 99961.1-MMAE 2.5+3.5 mpk (Q2W*2, i.e., 2.5 mpk on day 15 and 3.5 mpk on day 29, abbreviated as D15 2.5 mpk + D29 3.5 mpk)), the administration method is tail vein injection, a total of 3 times/3 weeks (QW*3) or single dose (single dose) or 2 times/3 weeks (Q2W*2). Tumor volume (V) calculation method: V = L × W ² /2 (where L is the longest tumor diameter, W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed to calculate the tumor inhibition rate. The results are shown in Figures 22A-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 had good tolerance to ADC (Figure 22B); the tumor growth inhibition rate TGI of mice in the QW*3 high-dose group, N147-135-MMAE (8mpk) and 99961.1-MMAE (10mpk) dose groups was about 96%, and the tumor inhibition rate was equivalent. The tumor did not recur 23 days after administration. In the single dose group, the tumor inhibition effect of N147-135-MMAE (8mpk) was slightly inferior to that of 99961.1-MMAE (10mpk), with TGIs of 76% and 93%, respectively. The tumor did not recur 23 days after administration in the 99961.1-MMAE (10mpk) group, but the tumor volume of the N147-135-MMAE (8mpk) group began to rebound 16 days after administration. In the Q2W*2 dosing group, the tumor inhibition 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 ( FIG. 22A and Tables 18-19 ).

Table 18 Tumor inhibition rate of ADC in mice after QW administration

Table 19 Tumor inhibition rate of ADC in mice after single dose administration and Q2W administration

Sequence Listing

Claims (29)

1. An anti-ROR 1 antibody that specifically binds ROR1, and antigen binding fragments thereof, comprising:

   1) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 52 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 51;

   2) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 54 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 53;

   3) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 56 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 55;

   4) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 58 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 57;

   5) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 60 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 59;

   6) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 62 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 61;

   7) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 64 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 63;

   8) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 66 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 53;

   9) 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 68 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 67;

   103 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 70 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 69;

   113 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 145 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 144;

   123 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 147 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 146;

   13 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 141 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 140;

   143 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 143 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 142;

   153 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 149 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 148;

   163 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 161 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 160;

   17 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 163 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 162;

   183 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 165 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 164;

   193 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 151 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 150;

   20 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 153 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 152;

   213 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 155 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 154;

   22 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 157 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 156;

   23 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 52 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 158;

   243 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 52 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 159;

   25 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 62 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 166;

   26 3 heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region as shown in SEQ ID No. 168 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region as shown in SEQ ID No. 167; or (b)

   27 3 Heavy chain CDRs (HCDR 1, HCDR2, HCDR 3) comprised by the heavy chain variable region shown in SEQ ID NO. 169 and 3 light chain CDRs (LCDR 1, LCDR2, LCDR 3) comprised by the light chain variable region shown in SEQ ID NO. 61.
2. An anti-ROR 1 antibody that specifically binds ROR1, and antigen binding fragments thereof, comprising:

   1) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 1,2 and 3; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 19, 20 and 21;

   2) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 1,2 and 6; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS.22, 23 and 24;

   3) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 7, 8 and 9; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 25, 26 and 27;

   4) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 10, 11 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 28, 29 and 30;

   5) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 13, 14 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS.31, 32 and 33;

   6) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 16, 17 and 18; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 34, 35 and 36;

   7) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 1,2 and 37; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 19, 20 and 21;

   8) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 38, 39 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 28, 44 and 30;

   9) HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 40, 41 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS.31, 45 and 33;

   10 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 42, 43 and 18; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 34, 46 and 36; or (b)

   11 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 91, 92 and 93; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 101, 102 and 103;

   12 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 1,2 and 94; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 104, 32 and 105;

   13 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 95, 96 and 97; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 106, 107 and 108;

   14 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 1,2 and 98; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 109, 110 and 105;

   15 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 91, 99 and 100; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 111, 112 and 113;

   16 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 121, 122 and 3; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 19, 136 and 21;

   17 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 1, 123 and 3; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 19, 137 and 21;

   18 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 124, 2 and 3; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 19, 23 and 21;

   19 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 114, 115 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 28, 128 and 30;

   20 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 116, 117 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 28, 129 and 30;

   21 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 118, 119 and 12; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 28, 130 and 30;

   22 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 16, 17 and 120; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS.131, 35 and 132;

   23 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 16, 17 and 18; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 34, 35 and 133;

   24 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 16, 17 and 18; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS 134, 35 and 135;

   25 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 13, 14 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS.31, 138 and 33;

   26 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 125, 126 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS.31, 139 and 33; or (b)

   27 HCDR1, HCDR2, HCDR3 comprising the sequences as shown in SEQ ID NOs 125, 127 and 15; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOS.31, 32 and 33.
3. The anti-ROR 1 antibody and antigen binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein:

   1) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:52, or an amino acid sequence as set forth in SEQ ID NO:52, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:52, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:51, or an amino acid sequence as set forth in SEQ ID NO:51, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 51;

   2) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:54, or an amino acid sequence as set forth in SEQ ID NO:54, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:54, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:53, or an amino acid sequence as set forth in SEQ ID NO:53, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53;

   3) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:56, or an amino acid sequence as set forth in SEQ ID NO:56, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:56, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:55, or an amino acid sequence as set forth in SEQ ID NO:55, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 55;

   4) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:58, or an amino acid sequence as set forth in SEQ ID NO:58, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:58, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:57, or an amino acid sequence as set forth in SEQ ID NO:57, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 57;

   5) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:60, or an amino acid sequence as set forth in SEQ ID NO:60, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:60, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:59, or an amino acid sequence as set forth in SEQ ID NO:59, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 59;

   6) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:62, or an amino acid sequence as set forth in SEQ ID NO:62, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:62, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:61, or an amino acid sequence as set forth in SEQ ID NO:61, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61;

   7) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:64, or an amino acid sequence as set forth in SEQ ID NO:64, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:64, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:63, or an amino acid sequence as set forth in SEQ ID NO:63, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 63;

   8) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:66, or an amino acid sequence as set forth in SEQ ID NO:66, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:66, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:53, or an amino acid sequence as set forth in SEQ ID NO:53, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 53;

   9) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:68, or an amino acid sequence as set forth in SEQ ID NO:68, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:68, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:67, or an amino acid sequence as set forth in SEQ ID NO:67, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 67;

   10 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:70, or an amino acid sequence as set forth in SEQ ID NO:70, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:70, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:69, or an amino acid sequence as set forth in SEQ ID NO:69, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 69; or (b)

   11 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:145, or an amino acid sequence as set forth in SEQ ID NO:145, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:145, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:144, or an amino acid sequence as set forth in SEQ ID NO:144, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 144;

   12 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:147, or an amino acid sequence as set forth in SEQ ID NO:147 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:147, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:146, or an amino acid sequence as set forth in SEQ ID NO:146, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 146;

   13 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:141, or an amino acid sequence as set forth in SEQ ID NO:141, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:141, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:140, or an amino acid sequence as set forth in SEQ ID NO:140, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 140;

   14 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:143, or an amino acid sequence as set forth in SEQ ID NO:143, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:143 comprising a light chain variable region as set forth in SEQ ID NO:142, or an amino acid sequence as set forth in SEQ ID NO:142, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 142;

   15 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:149, or an amino acid sequence as set forth in SEQ ID NO:149, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:149 comprising a light chain variable region as set forth in SEQ ID NO:148, or an amino acid sequence as set forth in SEQ ID NO:148, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:148, a composition;

   16 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:161, or an amino acid sequence as set forth in SEQ ID NO:161, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:161, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:160, or an amino acid sequence as set forth in SEQ ID NO:160, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:160, a composition;

   17 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:163, or an amino acid sequence as set forth in SEQ ID NO:163, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:163, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:162, or an amino acid sequence as set forth in SEQ ID NO:162, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 162;

   18 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:165, or an amino acid sequence as set forth in SEQ ID NO:165, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:165 comprising a light chain variable region as set forth in SEQ ID NO:164, or an amino acid sequence as set forth in SEQ ID NO:164, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 164;

   19 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:151, or an amino acid sequence as set forth in SEQ ID NO:151, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:151, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:150, or an amino acid sequence as set forth in SEQ ID NO:150, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 150;

   20 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:153, or an amino acid sequence as set forth in SEQ ID NO:153, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:153, said light chain variable region comprising a light chain variable region as set forth in SEQ ID NO:152, or an amino acid sequence as set forth in SEQ ID NO:152, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 152;

   21 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:155, or an amino acid sequence as set forth in SEQ ID NO:155, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:155, comprising a light chain variable region as set forth in SEQ ID NO:154, or an amino acid sequence as set forth in SEQ ID NO:154, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 154;

   22 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:157, or an amino acid sequence as set forth in SEQ ID NO:157, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:157, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:156, or an amino acid sequence as set forth in SEQ ID NO:156, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:156, a composition;

   23 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:52, or an amino acid sequence as set forth in SEQ ID NO:52, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:52, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:158, or an amino acid sequence as set forth in SEQ ID NO:158, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:158 is composed of;

   24 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:52, or an amino acid sequence as set forth in SEQ ID NO:52, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:52, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:159, or an amino acid sequence as set forth in SEQ ID NO:159, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 159;

   25 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:62, or an amino acid sequence as set forth in SEQ ID NO:62, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:62, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:166, or an amino acid sequence as set forth in SEQ ID NO:166, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 166;

   26 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:168, or an amino acid sequence as set forth in SEQ ID NO:168, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:168, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:167, or an amino acid sequence as set forth in SEQ ID NO:167, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 167; or (b)

   27 The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:169, or an amino acid sequence as set forth in SEQ ID NO:169, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:169 comprising a light chain variable region as set forth in SEQ ID NO:61, or an amino acid sequence as set forth in SEQ ID NO:61, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 61.
4. The antibody and antigen-binding fragment thereof that specifically bind ROR1 of any one of claims 1 to 3, wherein the antibody is a human monoclonal antibody.
5. The isolated anti-ROR 1 antibody or antigen binding fragment thereof of any one of claims 1 to 4, wherein the antigen binding fragment is selected from Fab, fab '-SH, fv (e.g. scFv) or (Fab') 2 fragments.
6. The isolated anti-ROR 1 monoclonal antibody or antigen binding fragment thereof according to any one of claims 1 to 5, comprising a constant region sequence, wherein at least a portion of the constant region sequence is a human consensus constant region sequence.
7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the heavy chain constant region of the antibody comprises the Fc sequence set forth in SEQ ID No. 47 or comprises the amino acid sequence set forth in SEQ ID No. 48 and the light chain constant region comprises the amino acid sequence set forth in SEQ ID No. 49 or SEQ ID No. 50.
8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, comprising a heavy chain and a light chain, wherein:

   1) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:72, or an amino acid sequence as set forth in SEQ ID NO:72, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:72, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:71, or an amino acid sequence as set forth in SEQ ID NO:71, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 71;

   2) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:74, or an amino acid sequence as set forth in SEQ ID NO:74, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:74, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:73, or an amino acid sequence as set forth in SEQ ID NO:73, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73;

   3) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:76, or an amino acid sequence as set forth in SEQ ID NO:76, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:76, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:75, or an amino acid sequence set forth in SEQ ID NO:75, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 75;

   4) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:78, or an amino acid sequence as set forth in SEQ ID NO:78, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:78, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:77, or an amino acid sequence as set forth in SEQ ID NO:77, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 77;

   5) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:80, or an amino acid sequence as set forth in SEQ ID NO:80, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:80, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:79, or an amino acid sequence as set forth in SEQ ID NO:79, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 79;

   6) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:82, or an amino acid sequence as set forth in SEQ ID NO:82, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:82, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:81, or an amino acid sequence as set forth in SEQ ID NO:81, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 81;

   7) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:84, or an amino acid sequence as set forth in SEQ ID NO:84, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:84, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:83, or an amino acid sequence as set forth in SEQ ID NO:83, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 83;

   8) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:86, or an amino acid sequence as set forth in SEQ ID NO:86, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:86, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:73, or an amino acid sequence as set forth in SEQ ID NO:73, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 73;

   9) The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:88, or an amino acid sequence as set forth in SEQ ID NO:88, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:88, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:87, or an amino acid sequence as set forth in SEQ ID NO:87, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 87;

   10 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:90, or an amino acid sequence as set forth in SEQ ID NO:90, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:90, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:89, or an amino acid sequence as set forth in SEQ ID NO:89, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 89;

   11 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:175, or an amino acid sequence as set forth in SEQ ID NO:175, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:175, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:174, or an amino acid sequence as set forth in SEQ ID NO:174, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 174;

   12 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:177, or an amino acid sequence as set forth in SEQ ID NO:177, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:177, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:176, or an amino acid sequence as set forth in SEQ ID NO:176, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 176;

   13 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:171, or an amino acid sequence as set forth in SEQ ID NO:171, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:171, said light chain comprising the amino acid sequence set forth in SEQ ID NO:170, or an amino acid sequence as set forth in SEQ ID NO:170, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 170;

   14 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:173, or an amino acid sequence as set forth in SEQ ID NO:173, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:173, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:172, or an amino acid sequence as set forth in SEQ ID NO:172, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 172;

   15 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:179, or an amino acid sequence as set forth in SEQ ID NO:179, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:179, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:178, or an amino acid sequence as set forth in SEQ ID NO:178, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 178;

   16 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:191, or an amino acid sequence as set forth in SEQ ID NO:191, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:191, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:190, or an amino acid sequence as set forth in SEQ ID NO:190, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 190;

   17 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:193, or an amino acid sequence corresponding to SEQ ID NO:193, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:193, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:192, or an amino acid sequence as set forth in SEQ ID NO:192, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:192, a composition;

   18 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:195, or an amino acid sequence as set forth in SEQ ID NO:195, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:195, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:194, or an amino acid sequence as set forth in SEQ ID NO:194, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:194, composition;

   19 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:181, or an amino acid sequence as set forth in SEQ ID NO:181, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:181, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:180, or an amino acid sequence as set forth in SEQ ID NO:180, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:180 parts;

   20 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:183, or an amino acid sequence as set forth in SEQ ID NO:183, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:183, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:182, or an amino acid sequence as set forth in SEQ ID NO:182, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:182, a composition;

   21 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:185, or an amino acid sequence as set forth in SEQ ID NO:185, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:185, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:184, or an amino acid sequence set forth in SEQ ID NO:184, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 184;

   22 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:187, or an amino acid sequence as set forth in SEQ ID NO:187, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:187, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:186, or an amino acid sequence as set forth in SEQ ID NO:186, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:186, the composition;

   23 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:72, or an amino acid sequence as set forth in SEQ ID NO:72, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:72, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:188, or an amino acid sequence as set forth in SEQ ID NO:188, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 188;

   24 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:72, or an amino acid sequence as set forth in SEQ ID NO:72, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:72, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:189, or an amino acid sequence as set forth in SEQ ID NO:189 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 189;

   25 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:82, or an amino acid sequence as set forth in SEQ ID NO:82, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:82, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:196, or an amino acid sequence as set forth in SEQ ID NO:196 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:196, composition;

   26 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:198, or an amino acid sequence as set forth in SEQ ID NO:198, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:198, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:197, or an amino acid sequence as set forth in SEQ ID NO:197, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 197; or (b)

   27 The heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:199, or an amino acid sequence as set forth in SEQ ID NO:199, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:199, said light chain comprising the amino acid sequence as set forth in SEQ ID NO:81, or an amino acid sequence as set forth in SEQ ID NO:81, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 81.
9. An antibody-conjugated drug having an Ab- (L-D) n structure, wherein Ab is the antibody or antigen-binding fragment thereof of ROR1 of any one of claims 1-8, L is a linker, D is a therapeutically active substance or pharmaceutically active ingredient, and n represents an integer of 1-20.
10. The antibody-conjugated drug of claim 9, wherein the therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a phytotoxin, a small molecule toxin, a radioisotope.
11. The antibody-conjugated drug according to claim 9 or 10, wherein the therapeutically active substance or pharmaceutical active ingredient is Dolastatin (Dolastatin) and its auristatin derivatives, such as Dolastatin 10、Dolastatin 15、auristatin E、auristatin PE、monomethyl auristatin D(MMAD)、monomethyl auristatin E(MMAE)、monomethyl auristatin F(MMAF)、auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropyl amide (AF HPA).
12. The antibody-conjugated drug of any one of claims 9-11, wherein the Dolastatin (Dolastatin) and its auristatin derivatives are MMAE, MMAF, auristatin F hydroxypropyl amide, or auristatin F phenylenediamine.
13. The antibody-conjugated drug of any of claims 9-12, wherein the linker is a cathepsin-degradable linker (e.g., valine-citrulline (val-Cit) linker, cBu-Cit linker, and CX linker), a non-cleavable linker (e.g., SMCC linker or MD linker), an acid-sensitive linker, a silicone-structured linker, disulfide-carbamate linker, MC-GGFG linker, TRX linker, a galactoside-containing linker, a pyrophosphate linker, a near infrared sensitive linker, a uv sensitive linker (e.g., PC4 AP).
14. The antibody-conjugated drug of any of claims 9-13, wherein said linker is selected from the group consisting of maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-VC-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino PEG 6-propionyl, and maleimido caproic acid (mc), maleimido Propionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimido 4- (2-pyridylthio) pentanoate (SPP), N-succinimido 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC), N-succinimido (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimido-4- (2-pyridyldithio) butyrate (SPDB), N-succinimido-3- (2-pyridylthio) -propionate (SPP).
15. The antibody-conjugated drug of any of claims 9-14, wherein said linker is MC-VC-PAB, SMCC or MC-GGFG.
16. The antibody-conjugated drug of any of claims 9-15, wherein said Ab comprises:

    (1) HCDR1, HCDR2, HCDR3, as shown in SEQ ID NO 7, 8 and 9, and LCDR1, LCDR2, LCDR3, as shown in SEQ ID NO 25, 26 and 27, or

    (2) HCDR1, HCDR2, HCDR3 having the sequences shown in SEQ ID nos. 1,2 and 37 and LCDR1, LCDR2, LCDR3 having the sequences shown in SEQ ID nos. 19, 20 and 21.
17. The antibody-conjugated drug of any of claims 9-16, wherein said Ab comprises a heavy chain variable region and a light chain variable region, wherein

    (1) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:60, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:59, or

    (2) The heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:66, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 53.
18. The antibody-conjugated drug of any of claims 9-17, wherein said Ab comprises:

    (1) As set forth in SEQ ID NO:80 and a heavy chain of the amino acid sequence shown as SEQ ID NO:79, and a light chain of the amino acid sequence shown in seq id no; or (b)

    (2) As set forth in SEQ ID NO:86 and a heavy chain of the amino acid sequence shown as SEQ ID NO:73, and a light chain of the amino acid sequence shown in seq id no.
19. The antibody-conjugated drug of any of claims 16-18, wherein said linker is MC-VC-PAB and said cytotoxin is MMAE.
20. A pharmaceutical composition comprising:

    (1) The anti-ROR 1 antibody or antigen binding fragment thereof of any one of claims 1-8, or the antibody-conjugated drug of any one of claims 9-19, and;

    (2) A pharmaceutically acceptable carrier.
21. An isolated polynucleotide molecule encoding the anti-ROR 1 antibody or antigen binding fragment thereof of any one of claims 1-8.
22. A vector comprising the nucleic acid molecule of claim 21, preferably the vector is an expression vector.
23. A host cell comprising the vector of claim 22 or the nucleic acid molecule of claim 21.
24. Use of an anti-ROR 1 antibody or antigen binding fragment thereof of any one of claims 1-8 in the manufacture of an antibody-coupled medicament for the prevention or treatment of cancer, or in the manufacture of a medicament for the prevention or treatment of a disease associated with abnormal expression of ROR 1.
25. Use of an antibody-conjugated drug according to any one of claims 9-19 in the manufacture of a medicament for the prevention or treatment of a disease associated with aberrant expression of ROR 1.
26. A method of killing a ROR1 expressing cell or inhibiting the growth of a ROR1 expressing cell, comprising contacting the cell with an effective amount of the antibody or antigen binding fragment thereof of any one of claims 1-8, or an effective amount of the antibody-conjugated drug of any one of claims 9-19, or an effective amount of the pharmaceutical composition of claim 20.
27. A method of preventing or treating a disease associated with abnormal expression of ROR1 in a subject in need thereof, comprising administering to the subject a prophylactically or therapeutically effective amount of the antibody or antigen binding fragment thereof of any one of claims 1-8, or a prophylactically or therapeutically effective amount of the antibody-conjugated drug of any one of claims 9-19, or a prophylactically or therapeutically effective amount of the pharmaceutical composition of claim 20.
28. The use of claim 25 or the method of claim 27, wherein the disease associated with aberrant expression of ROR1 is a cancer that highly expresses ROR 1.
29. The use or method of claim 28, wherein the cancer that highly expresses ROR1 is Chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon carcinoma, breast carcinoma, neuroblastoma, lung carcinoma, gastric carcinoma, head and neck carcinoma, and melanoma.