ROR1 target binding molecules and uses thereof

Abstract

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

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present application relates to antibodies that specifically recognize ROR1, as well as methods for preparing and using the same. In addition, the present application also relates to antibody-drug conjugates (ADCs) that target ROR1 and compositions containing the molecules. In addition, the present invention also relates to therapeutic and diagnostic uses of these antibodies, antibody fragments and antibody-drug conjugates. [Background technology]

[0002] 2. Background of the Invention Antibody-drug conjugates (ADCs) are a class of drugs derived from traditional antibody therapy. They consist of a monoclonal antibody that targets a specific antigen, linked to a small molecule cytotoxic agent through a linker. This combination provides the targeted specificity of antibody drugs and the cytotoxic effect of traditional small molecules, making ADCs particularly suitable for the treatment of malignant tumors. Due to their excellent tumor-killing effect, ADCs have rapidly become the center of anticancer drug development. However, ADCs often have high toxicity and require high specificity for the target antigen. Ideally, targets for ADC development are antigens that are highly expressed in tumors but poorly or not expressed in healthy tissues. Target antigens should be membrane surface receptors upregulated on tumor cells that promote tumor growth or survival, and they should have intracellular uptake properties.

[0003] Both receptor tyrosine kinase-like orphan receptor 1 (ROR1, also known as receptor-related neurotrophin 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, the extracellular portion of which consists of one immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD domain and KRD domain), and the intracellular portion of which consists of one tyrosine kinase domain, two serine- or threonine-rich domains, and a proline-rich domain. ROR1 and ROR2 participate in non-canonical Wnt signaling by binding to the ligand Wnt5a through their FZD domains (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 inhibits apoptosis, promotes EGFR signaling, and induces epithelial-mesenchymal transition (EMT) (Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al (2008). Proc Natl Acad Sci USA 105:3047-3052; Yamaguchi T, Yanagisawa K, Sugiyama R, Hosono Y, Shimada Y, ArimaC, KatoS, TomidaS, Suzuki M,OsadaHet al (2012). Cancer Cell 21:348-361; Cui B, Zhang S, Chen L, Yu J, Widhopf GF, Fecteau JF, Rassenti LZ, Kipps TJ (2013). Cancer Res 73:3649-3660).

[0004] ROR1 is a conserved embryonic protein, whose expression gradually decreases with embryonic development, and is almost absent or only weakly expressed in most adult tissues. However, more and more literature has found that ROR1 is expressed in various cancer cells, such as B-cell chronic lymphocytic leukemia (CLL) and other hematological malignancies, renal cell carcinoma, colon cancer, and other specific 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 becomes an ideal drug target for cancer treatment.

[0005] Although some antibody drugs against ROR1 have been disclosed in the prior art, due to ROR1 as a pan-cancer tumor marker, there is still an urgent need to develop anti-ROR1 antibodies with high quality. Such antibodies can be used as a basis for developing antibody-based targeted therapy for cancers expressing ROR1, and can also be used as a diagnostic tool to detect ROR1 expression in ROR1-related diseases. In addition, considering the bright prospects shown by ADCs in the field of tumor treatment, there is still an urgent need for ADCs containing ROR1 with effective therapeutic effects, and the present invention addresses these needs. Summary of the Invention

[0006] In a first aspect, the present invention provides the following advantages: (1) binds to ROR1 and target cells expressing ROR1 with high affinity; (2) being able to enter cells via cellular uptake; (3) being suitable for constructing effective therapeutic antibody-drug conjugates; The present invention provides an antibody that targets ROR1, having the formula:

[0007] In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, including: 1) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 57 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 56; 2) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 55 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 54; 3) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 59 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 58; 4) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 61 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 60; 5) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 63 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 62; 6) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 65 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 64; 7) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 67 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 66; 8) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 68 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 66; 9) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region represented by SEQ ID NO: 70 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region represented by SEQ ID NO: 69; 10) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 72 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 71; 11) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region represented by SEQ ID NO: 74 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region represented by SEQ ID NO: 73; 12) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region represented by SEQ ID NO: 75 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region represented by SEQ ID NO: 79; 13) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region represented by SEQ ID NO: 76 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region represented by SEQ ID NO: 81; 14) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region represented by SEQ ID NO: 77 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region represented by SEQ ID NO: 82; 15) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region represented by SEQ ID NO: 78 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region represented by SEQ ID NO: 80; 16) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region represented by SEQ ID NO: 78 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region represented by SEQ ID NO: 81; 17) Three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 78 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 82; or 18) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region shown in SEQ ID NO: 84 and the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region shown in SEQ ID NO: 89.

[0008] In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, including: 1) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 28, 29 and 30, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 2) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 32 and 33, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 3) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 34, 32 and 33, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 4) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 35, 32 and 36, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 5) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 13 and 14, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 35, 32 and 37, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 6) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 15, 16 and 17, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 38, 32 and 33, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 7) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 18, 8 and 19, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 32 and 33, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 8) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 20, 21 and 12, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 32 and 33, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 9) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 22, 23 and 24, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 39, 40 and 41, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 10) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 25 and 26, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 35, 32 and 33, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 11) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 18, 25 and 27, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 35, 32 and 33, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 12) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 45 and 48, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 13) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 43, 46 and 48, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 14) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 44, 47 and 48, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 15) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 42 and 6, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 45 and 48, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 16) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 42 and 6, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 43, 46 and 48, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; 17) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 42 and 6, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 44, 47 and 48, or sequences containing one or more and less than three amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; or 18) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences containing one or more and up to 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 49, 50 and 30, or sequences containing one or more and up to 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to said sequences.

[0009] In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, comprising a heavy chain variable region comprising: 1) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:57, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:57, or consists of SEQ ID NO:57; 2) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:55, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:55, or consists of SEQ ID NO:55; 3) the heavy chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO:59, or consists of SEQ ID NO:59; 4) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:61, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:61, or consists of SEQ ID NO:61; 5) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:63, or consists of SEQ ID NO:63; 6) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:65, or consists of SEQ ID NO:65; 7) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:67, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:67, or consists of SEQ ID NO:67; 8) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:68, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:68, or consists of SEQ ID NO:68; 9) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:70, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:70, or consists of SEQ ID NO:70; 10) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:72, or consists of SEQ ID NO:72; 11) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:74, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:74, or consists of SEQ ID NO:74; 12) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:83, or consists of SEQ ID NO:83; 13) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:84, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84; 14) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:85, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:85, or consists of SEQ ID NO:85; 15) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 75, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 75, or consists of SEQ ID NO: 75; 16) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:76, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:76, or consists of SEQ ID NO:76; 17) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77; or 18) The heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:78, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:78, or consists of SEQ ID NO:78.

[0010] In one embodiment, the invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, comprising a light chain variable region comprising: 1) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:56, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:56, or consists of SEQ ID NO:56; 2) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:54, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:54, or consists of SEQ ID NO:54; 3) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:58, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:58, or consists of SEQ ID NO:58; 4) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:60, or consists of SEQ ID NO:60; 5) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:62, or consists of SEQ ID NO:62; 6) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:64, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:64, or consists of SEQ ID NO:64; 7) the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO:66, or consists of SEQ ID NO:66; 8) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:69, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:69, or consists of SEQ ID NO:69; 9) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:71, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:71, or consists of SEQ ID NO:71; 10) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:73, or consists of SEQ ID NO:73; 11) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:79, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:79, or consists of SEQ ID NO:79; 12) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:80, or consists of SEQ ID NO:80; 13) the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO:81, or consists of SEQ ID NO:81; 14) the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO:82, or consists of SEQ ID NO:82; 15) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:86, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:86, or consists of SEQ ID NO:86; 16) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:87, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:87, or consists of SEQ ID NO:87; 17) the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 88, or consists of SEQ ID NO: 88; or 18) The light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO:89, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:89, or consists of SEQ ID NO:89.

[0011] In another embodiment, the present invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, comprising a heavy chain variable region and a light chain variable region, wherein: 1) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:57, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:57, or consists of SEQ ID NO:57, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:56, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:56, or consists of SEQ ID NO:56; 2) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:55, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:55, or consists of SEQ ID NO:55, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:54, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:54, or consists of SEQ ID NO:54; 3) the heavy chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO:59, or consists of SEQ ID NO:59, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:58, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:58, or consists of SEQ ID NO:58; 4) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:61, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:61, or consists of SEQ ID NO:61, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:60, or consists of SEQ ID NO:60; 5) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:63, or consists of SEQ ID NO:63, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:62, or consists of SEQ ID NO:62; 6) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:65, or consists of SEQ ID NO:65, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:64, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:64, or consists of SEQ ID NO:64; 7) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:67, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:67, or consists of SEQ ID NO:67, and the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO:66, or consists of SEQ ID NO:66; 8) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:68, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:68, or consists of SEQ ID NO:68, and the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO:66, or consists of SEQ ID NO:66; 9) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:70, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:70, or consists of SEQ ID NO:70, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:69, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:69, or consists of SEQ ID NO:69; 10) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:72, or consists of SEQ ID NO:72, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:71, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:71, or consists of SEQ ID NO:71; 11) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:74, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:74, or consists of SEQ ID NO:74, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:73, or consists of SEQ ID NO:73; 12) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 75, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 75, or consists of SEQ ID NO: 75, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 79, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 79, or consists of SEQ ID NO: 79; 13) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 76, or consists of SEQ ID NO: 76, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 80, or consists of SEQ ID NO: 80; 14) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 76, or consists of SEQ ID NO: 76, and the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO: 81, or consists of SEQ ID NO: 81; 15) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 80, or consists of SEQ ID NO: 80; 16) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77, and the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO: 81, or consists of SEQ ID NO: 81; 17) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77, and the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO: 82, or consists of SEQ ID NO: 82; 18) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 78, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 78, or consists of SEQ ID NO: 78, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 80, or consists of SEQ ID NO: 80; 19) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 78, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 78, or consists of SEQ ID NO: 78, and the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO: 81, or consists of SEQ ID NO: 81; 20) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:78, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:78, or consists of SEQ ID NO:78, and the light chain variable region comprises an amino acid sequence set forth 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 to the amino acid sequence of SEQ ID NO:82, or consists of SEQ ID NO:82; 21) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 83, or consists of SEQ ID NO: 83, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 86, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 86, or consists of SEQ ID NO: 86; 22) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 83, or consists of SEQ ID NO: 83, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 87, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 87, or consists of SEQ ID NO: 87; 23) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:84, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:87, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:87, or consists of SEQ ID NO:87; 24) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:84, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:88, or consists of SEQ ID NO:88; 25) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO:84 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO:89 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:89, or consists of SEQ ID NO:89; 26) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 85, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 85, or consists of SEQ ID NO: 85, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 87, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 87, or consists of SEQ ID NO: 87; 27) the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 85, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 85, or consists of SEQ ID NO: 85, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 88, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 88, or consists of SEQ ID NO: 88; or 28) The heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 85 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 85, or consists of SEQ ID NO: 85, and the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 89 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 89, or consists of SEQ ID NO: 89.

[0012] In some embodiments, the above mentioned antibody or antigen-binding fragment thereof further comprises a heavy and / or light chain constant region sequence derived from a human antibody germline consensus sequence. The light chain constant region is preferably a human kappa or lambda chain constant region. The heavy chain constant region is a gamma, mu, alpha, delta, or epsilon chain. In some embodiments, the heavy chain constant region is preferably derived from a human IgG1, IgG2, IgG3, or IgG4 constant region sequence. In one embodiment, the light chain constant region comprises or consists of the sequence set forth in SEQ ID NO:53. In another embodiment, the heavy chain constant region comprises the sequence set forth in SEQ ID NO:52.

[0013] It will be understood that sequence variants of these constant region domains, for example containing one or more amino acid modifications, may also be used, where the amino acid positions are identified according to the EU Index System of Kabat et al. (1991).

[0014] In specific embodiments, the present invention provides anti-ROR1 antibodies and antigen-binding fragments thereof that specifically bind to ROR1, comprising a heavy chain and a light chain, wherein: 1) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:93, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:93, or consists of SEQ ID NO:93, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:92, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:92, or consists of SEQ ID NO:92; 2) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:91, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:91, or consists of SEQ ID NO:91, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:90, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:90, or consists of SEQ ID NO:90; 3) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:95, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:95, or consists of SEQ ID NO:95, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:94, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:94, or consists of SEQ ID NO:94; 4) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:97, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:97, or consists of SEQ ID NO:97, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:96, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:96, or consists of SEQ ID NO:96; 5) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:99, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:99, or consists of SEQ ID NO:99, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:98, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:98, or consists of SEQ ID NO:98; 6) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:101, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:101, or consists of SEQ ID NO:101, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:100, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:100, or consists of SEQ ID NO:100; 7) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 103, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 103, or consists of SEQ ID NO: 103, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 102, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 102, or consists of SEQ ID NO: 102; 8) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:104, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:104, or consists of SEQ ID NO:104, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:102, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:102, or consists of SEQ ID NO:102; 9) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 106, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 106, or consists of SEQ ID NO: 106, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 105, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 105, or consists of SEQ ID NO: 105; 10) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 108, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 108, or consists of SEQ ID NO: 108, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 107, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 107, or consists of SEQ ID NO: 107; 11) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:110, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:110, or consists of SEQ ID NO:110, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:109, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:109, or consists of SEQ ID NO:109; 12) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:111, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:111, or consists of SEQ ID NO:111, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:115, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:115, or consists of SEQ ID NO:115; 13) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:112, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:112, or consists of SEQ ID NO:112, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:116, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:116, or consists of SEQ ID NO:116; 14) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:112, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:112, or consists of SEQ ID NO:112, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:117, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:117, or consists of SEQ ID NO:117; 15) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:113, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:113, or consists of SEQ ID NO:113, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:116, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:116, or consists of SEQ ID NO:116; 16) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 113, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 113, or consists of SEQ ID NO: 113, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 117, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 117, or consists of SEQ ID NO: 117; 17) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:113, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:113, or consists of SEQ ID NO:113, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:118, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:118, or consists of SEQ ID NO:118; 18) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:114, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:114, or consists of SEQ ID NO:114, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:116, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:116, or consists of SEQ ID NO:116; 19) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:114, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:114, or consists of SEQ ID NO:114, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:117, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:117, or consists of SEQ ID NO:117; 20) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO:114, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:114, or consists of SEQ ID NO:114, and the light chain comprises an amino acid sequence set forth in SEQ ID NO:118, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:118, or consists of SEQ ID NO:118; 21) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 119, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 119, or consists of SEQ ID NO: 119, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 122, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 122, or consists of SEQ ID NO: 122; 22) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 119, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 119, or consists of SEQ ID NO: 119, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 123, or consists of SEQ ID NO: 123; 23) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 120, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 120, or consists of SEQ ID NO: 120, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 123, or consists of SEQ ID NO: 123; 24) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 120, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 120, or consists of SEQ ID NO: 120, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 124, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 124, or consists of SEQ ID NO: 124; 25) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 120, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 120, or consists of SEQ ID NO: 120, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 125, or consists of SEQ ID NO: 125; 26) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 121, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 121, or consists of SEQ ID NO: 121, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 123, or consists of SEQ ID NO: 123; 27) the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 121 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 121 or consists of SEQ ID NO: 121, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 124 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 124 or consists of SEQ ID NO: 124; or 28) The heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 121 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 121, or consists of SEQ ID NO: 121, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 125 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 125, or consists of SEQ ID NO: 125.

[0015] In certain embodiments of any of the above-described antibodies, the antibody is monoclonal. In certain embodiments of any of the above-described antibodies, the antibody is a full-length antibody. In one embodiment, the anti-ROR1 antibody of the invention is a complete antibody, such as an IgG1, IgG2, IgG3, IgG4 antibody, etc. In another embodiment, the anti-ROR1 antibody of the invention includes only the antigen-binding portion thereof, such as the following: Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragments.

[0016] In a second aspect, the present invention provides an antibody-drug conjugate targeting human ROR1, the antibody-drug conjugate having the following advantages: (1) It binds to target cells expressing ROR1 with high affinity; (2) being capable of entering cells and killing target cells through cellular uptake; in some embodiments, the ADCs of the invention have a high rate of cellular uptake; (3) treating, preventing, or ameliorating a disease associated with abnormal function or expression of ROR1 in a subject (e.g., cancer, such as a hematological malignancy, solid tumor, etc.), or treating, preventing, or ameliorating one or more symptoms of the disease; (4) reducing or inhibiting tumor growth or progression in a subject suffering from a tumor that expresses ROR1; (5) inducing regression (e.g., long-term regression) of ROR1-expressing tumors; (6) exert cytotoxic activity in cells expressing ROR1; has.

[0017] In one embodiment, the present application provides a conjugate comprising an anti-ROR1 antibody of the first aspect. In specific embodiments, the molecule capable of being conjugated to the anti-ROR1 antibody is, for example, a cytotoxic agent, an immunomodulatory agent, an imaging agent, a fluorescent protein, a molecular marker, a therapeutic protein, a biopolymer, and an oligonucleotide.

[0018] In one embodiment, the present invention provides an antibody-drug conjugate (ADC) comprising an anti-ROR1 antibody or antigen-binding fragment thereof according to the first aspect, and at least one therapeutically active substance or pharma- ceutical active ingredient having the structure Ab-(LD)n, where Ab is an antibody or antigen-binding fragment thereof that binds to ROR1 according to the first aspect of the invention; L is a linker; D is a therapeutically active substance or pharma-ceutical active ingredient, and n represents an integer from 1 to 20, such as 1, 2, 3, 4, 5, etc.

[0019] In one embodiment, the antibody-drug conjugate comprises multiple D moieties, which may be a combination of different therapeutic or pharma- ceutical active agents or a combination of the same therapeutic or pharma- ceutical active agents. In one embodiment, the antibody-drug conjugate has a drug / antibody ratio (DAR) of 1-20, such as a DAR value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In one embodiment, the DAR is the average DAR. In one embodiment, the average DAR ranges from 1-15, such as 1-10, 2-8, 2-6, or 3-5. In a specific embodiment, the average DAR is 3.7.

[0020] In specific embodiments, the therapeutically active agent or medicament is a cytotoxin, a plant toxin, a small molecule toxin, a radioisotope, a maytansine alkaloid, or the like. In a specific embodiment, the cytotoxin is 0101 (2-methylpropionyl-N-(3R,4S,5S)-3-methoxy-1-{(2S)-2-(1R,2R)-1-methoxy-2-methyl-3-oxy-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)-ethyl]amino}propylpyrrolidin-1-yl}-5-methyl-1-oxyheptan-4-yl]-N-methyl-L-valinamide), 8261 (2-methylpropionyl-N-(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenethyl]amino}-1-methoxy-2-methyl and auristatin derivatives thereof, such as auristatin F, auristatin PE, monomethylauristatin D (MMAD), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AFHPA). In another embodiment, the dolastatin and auristatin derivatives thereof are auristatin, dolastatin, MMAE, MMAF, auristatin F hydroxypropylamide, or auristatin F phenylenediamine.

[0021] In one embodiment, the cytotoxin is covalently linked to the anti-ROR1 antibody or antigen-binding fragment thereof via a linker in a non-site-selective or site-selective manner. In one embodiment, the linker is maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (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).

[0022] In one embodiment, the antibody-drug conjugate comprises an anti-ROR1 antibody or an antigen-binding fragment thereof according to the first aspect and a tubulin inhibitor (MMAE). In a further embodiment, the MMAE has the structure of the anti-ROR1 antibody MC-VC-PAB MMAE and is linked to the thiol group of a cysteine ​​on the anti-ROR1 antibody via a MC-VC-PAB linker. The IgG1 antibody has 16 pairs of cysteine ​​residues, which exist in the form of 12 pairs of intrachain disulfide bonds and 4 pairs of interchain disulfide bonds. The interchain disulfide bonds are solvent accessible and can be reduced by a reducing agent to form 8 sulfhydryl groups, which then become targets for conjugation (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015;17:339-51).

[0023] In a specific embodiment, the antibody-drug conjugate comprises or consists of the anti-ROR1 monoclonal antibody B62-H3L3 and MC-VC-PAB-MMAE. In a further embodiment, MMAE is attached to the thiol group of a cysteine ​​on B62-H3L3 via a MC-VC-PAB linker. In another specific embodiment, the antibody-drug conjugate comprises or consists of the anti-ROR1 monoclonal antibody B31-H3L3 and MC-VC-PAB-MMAE. In a further embodiment, MMAE is attached to the thiol group of a cysteine ​​on B31-H3L3 via a MC-VC-PAB linker.

[0024] 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 pharma- ceutical 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 according to the first aspect. In a fifth aspect, the present invention provides a vector comprising the polynucleotide molecule of the fourth aspect. In one embodiment, the vector is an expression vector.

[0025] In a sixth aspect, the present invention provides a host cell comprising the vector of the fifth aspect or the polynucleotide molecule of the fourth invention. In some embodiments, the host cell is a prokaryote, such as E. coli. In other embodiments, the host cell is a eukaryote, such as a HEK293 cell, a CHO cell, a yeast cell, or a plant cell.

[0026] 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, the method comprising administering to the subject a prophylactically or therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention, or a prophylactically or therapeutically effective amount of an antibody-drug conjugate of the present invention, or a prophylactically or therapeutically effective amount of a pharmaceutical composition of the present invention.

[0027] In one embodiment, the disease associated with the abnormal expression of ROR1 is cancer that highly expresses ROR1, such as chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer, gastric 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. In a specific embodiment, the breast cancer is triple-negative breast cancer.

[0028] In some embodiments, the ADC molecules or pharmaceutical compositions of the invention can also be administered in combination with one or more other therapies, such as therapeutic modalities and / or other therapeutic agents, for uses described herein, such as for treating or preventing an associated disease or condition mentioned herein.

[0029] In a specific embodiment, the present invention provides a method for killing a cell expressing ROR1 or inhibiting the proliferation of a cell expressing ROR1, the method comprising contacting the cell with an effective amount of an antibody or antigen-binding fragment thereof of the present invention, an effective amount of an antibody-drug conjugate of the present invention, or an effective amount of a pharmaceutical composition of the present invention.

[0030] In an eighth aspect, the present invention provides the use of an anti-ROR1 antibody, or an antigen-binding fragment thereof, in the preparation of an antibody-drug conjugate for preventing or treating cancer. The present invention also provides the use of an antibody-drug conjugate comprising an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of a medicament for preventing or treating cancer. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 shows the binding activity of each mouse antibody clone Fab obtained in the present application binding to huROR1-HEK293 cells as determined by FACS; FIG. 1A shows the binding activity of lysates of clones B31Fab, B32Fab, B34Fab, B39Fab, B62Fab, B74Fab, C38Fab, M71Fab and M78Fab binding to huROR1-HEK293 cells; FIG. 1B shows the binding activity of lysates of C42Fab and C77Fab binding to huROR1-HEK293 cells; NC represents the negative control, and MFI represents the median fluorescence intensity. [Diagram 2] Figures 2A to 2L show SEC-HPLC results of the antibodies of the present application; Figure 2A shows the SEC-HPLC result of B31; Figure 2B shows the SEC-HPLC result of B32; Figure 2C shows the SEC-HPLC result of B34; Figure 2D shows the SEC-HPLC result of B39; Figure 2E shows the SEC-HPLC result of B62; Figure 2F shows the SEC-HPLC result of B74; Figure 2G shows the SEC-HPLC result of C38; Figure 2H shows the SEC-HPLC result of M71; Figure 2I shows the SEC-HPLC result of M78; Figure 2J shows the SEC-HPLC result of C42; Figure 2K shows the SEC-HPLC result of C77; Figure 2L shows the SEC-HPLC result of 99961.1. [Diagram 3] Figures 3A-3B show the binding activity of the antibodies of the present application to the antigen protein huROR1-His, as determined by ELISA; Figure 3A shows the binding activity of B31, B32, B34, B39, B62, and B74 to the antigen protein huROR1-His; Figure 3B shows the binding activity of C38, C42, C77, M71, and M78 to the antigen protein huROR1-His. [Figure 4] Figures 4A-4B show the binding activity of the antibodies of the present application to A549 tumor cells as determined by FACS; Figure 4A shows the binding activity of B31, B32, B34, B39, B62 and B74 to A549 tumor cells; Figure 4B shows the binding activity of C38, C42, C77, M71 and M78 to A549 tumor cells. [Diagram 5] Figures 5A-5B show the activity of the antibodies of the present application binding to huROR1-HEK293 cells as determined by FACS; Figure 5A shows the activity of B31, B32, B34, B39, B62, and B74 binding to huROR1-HEK293 cells; Figure 5B shows the activity of C38, C42, C77, M71, and M78 binding to huROR1-HEK293 cells. [Figure 6] Figures 6A-6B show the activity of the antibodies of the present application in binding to the antigen protein MusROR1-His, as determined by ELISA; Figure 6A shows the activity of B31, B32, B34, B39, and B62 in binding to the antigen protein MusROR1-His; Figure 6B shows the activity of B74, C38, C42, C77, M71, and M78 in binding to the antigen protein MusROR1-His. [Figure 7] Figures 7A-7B show the activity of the antibodies of the present application in binding to the antigen protein huROR2-His, as determined by ELISA; Figure 7A shows the activity of B31, B32, B34, B39, and B62 in binding to the antigen protein huROR2-His; Figure 7B shows the activity of B74, C38, C42, C77, M71, and M78 in binding to the antigen protein huROR2-His. [Figure 8] Figures 8A and 8B show the intracellular uptake rates of the present antibodies in huROR1-HEK293 cells as determined by FACS; Figure 8A shows the intracellular uptake rates of B62 and C42 in huROR1-HEK293 cells; and Figure 8B shows the intracellular uptake rates of B31, B32, B34, B39, B74, C38, C77, M71, and M78 in huROR1-HEK293 cells. [Figure 9]Figures 9A to 9F show the intracellular uptake rates of the antibodies of the present application in huROR1-HEK293 cells, as determined by the Fab-Zap method; Figure 9A shows the intracellular uptake rates of B31 and B32 in huROR1-HEK293 cells; Figure 9B shows the intracellular uptake rates of B34 and B39 in huROR1-HEK293 cells; Figure 9C shows the intracellular uptake rates of B74 and C38 in huROR1-HEK293 cells; Figure 9D shows the intracellular uptake rates of C77 and M71 in huROR1-HEK293 cells; Figure 9E shows the intracellular uptake rate of M78 in huROR1-HEK293 cells; and Figure 9F shows the intracellular uptake rates of B62 and C42 in huROR1-HEK293 cells. [Figure 10] 10A-10E show the activity of humanized antibodies binding to the antigen protein huROR1-His, as determined by ELISA; FIG. 10A shows the activity of B31-H2L2, B31-H1L1, and B31-H2L3 binding to the antigen protein huROR1-His; FIG. 10B shows the activity of B31-H3L2, B31-H3L3, and B31-H3L4 binding to the antigen protein huROR1-His; FIG. 10C shows the activity of B31-H3L2, B31-H3L3, and B31-H3L4 binding to the antigen protein huROR1-His; FIG. 10D shows the activity of B62-H1L1, B62-H1L2, B62-H2L2, and B62-H2L3 binding to the antigen protein huROR1-His; FIG. 10E shows the activity of B62-H2L4, B62-H3L2, B62-H3L3, and B62-H3L4 binding to the antigen protein huROR1-His. [Figure 11]11A-11B show the activity of humanized antibody binding to A549 tumor cells as determined by FACS; FIG. 11A shows the activity of B31-H1L1, B31-H2L2, B31-H2L3, B31-H3L2, B31-H3L3, B31-H3L4, B31-H4L2, B31-H4L3, and B31-H4L4 binding to A549 tumor cells; FIG. 11B shows the activity of B62-H1L1, B62-H1L2, B62-H2L2, B62-H2L3, B62-H2L4, B62-H3L2, B62-H3L3, and B62-H3L4 binding to A549 tumor cells. [Figure 12] FIG. 12 shows the intracellular uptake rates of B31-H3L3 and B62-H3L3 in huROR1-HEK293 cells as determined by the Fab-Zap method. [Figure 13] Figures 13A-13B show the activity of the present ADCs binding to A549 and HT29 tumor cells as determined by FACS; Figure 13A shows the activity of B31-H3L3-MMAE and B62-H3L3-MMAE binding to A549 tumor cells; Figure 13B shows the activity of B31-H3L3-MMAE and B62-H3L3-MMAE binding to HT29 tumor cells. [Figure 14] Figures 14A-14D show the tumor cell killing rates of the present ADCs as detected by the MTS method; Figure 14A shows the effect of B31-H3L3-MMAE on killing of A549 tumor cells; Figure 14B shows the effect of B62-H3L3-MMAE on killing of A549 tumor cells; Figure 14C shows the effect of B31-H3L3-MMAE on killing of HT29 tumor cells; and Figure 14D shows the effect of B62-H3L3-MMAE on killing of HT29 tumor cells. [Figure 15]Figures 15A-15C show the effect of the present ADCs on killing of three types of tumor cells, Jeko-1, MDA-MB-468, and NCI-H1944, as determined by the CCK8 assay; Figure 15A shows the effect of B31-H3L3-MMAE and B62-H3L3-MMAE on killing of Jeko-1 cells; Figure 15B shows the effect of B31-H3L3-MMAE and B62-H3L3-MMAE on killing of Jeko-1 cells; Figure 15C shows the effect of B31-H3L3-MMAE and B62-H3L3-MMAE on killing of NCI-H1944 cells. [Figure 16] Figure 16 shows the antigen-dependent killing effect of the present ADC against huROR1-HEK293 cells, as measured by the CCK8 method. [Figure 17] Figures 17A-17B show the cellular uptake of the present ADCs in A549 and HT-29 tumor cells as determined by FACS; Figure 17A shows the cellular uptake of B31-H3L3-MMAE and B62-H3L3-MMAE in A549 tumor cells; Figure 17B shows the cellular uptake of B31-H3L3-MMAE and B62-H3L3-MMAE in HT-29 tumor cells. [Figure 18] Figures 18A-18C show the in vivo tumor suppressive effect of the subject ADCs in an HT-29 tumor mouse model; Figure 18A shows the change in tumor volume in mice in various experimental groups over the course of the experiment, with arrows indicating the time points of drug administration; Figure 18B shows the change in body weight in mice in various experimental groups over the course of the experiment; and Figure 18C shows the tumor weight in mice in various experimental groups after the completion of the experiment. [Figure 19] Figures 19A-19C show the in vivo tumor inhibitory effect of the present ADC in an A549 tumor mouse model; Figure 19A shows the change in tumor volume in mice in various experimental groups over the course of the experiment; Figure 19B shows the change in body weight in mice in various experimental groups over the course of the experiment; and Figure 19C shows the tumor weight in mice in various experimental groups after the completion of the experiment. [Figure 20]Figures 20A-20B show the in vivo tumor suppressive effect of the subject ADCs in an NCI-N87 tumor mouse model; Figure 20A shows the change in tumor volume in mice in various experimental groups over the course of the experiment, with arrows indicating the time points of drug administration; Figure 20B shows the change in body weight in mice in various experimental groups over the course of the experiment. [Figure 21] Figures 21A-21B show the in vivo tumor suppressive effect of the subject ADCs in an MDA-MB-231 tumor mouse model; Figure 21A shows the change in tumor volume in mice in various experimental groups over the course of the experiment, with arrows indicating the time points of drug administration; Figure 21B shows the change in body weight in mice in various experimental groups over the course of the experiment. [Figure 22] Figures 22A-22B show the in vivo tumor suppressive effect of the subject ADCs in an MDA-MB-468 tumor mouse model; Figure 22A shows the change in tumor volume in mice in various experimental groups over the course of the experiment, with arrows indicating the time points of drug administration; Figure 22B shows the change in body weight in mice in various experimental groups over the course of the experiment. [Diagram 23] Figures 23A-23B show the in vivo tumor inhibitory effect of the subject ADCs in a Jeko-1 tumor mouse model; Figure 23A shows the change in tumor volume in mice in various experimental groups over the course of the experiment, with arrows indicating the time points of drug administration; Figure 23B shows the change in body weight in mice in various experimental groups over the course of the experiment.

[0032] Detailed Description of the Invention Before describing the present invention in detail below, it is to be understood that the present invention is not limited to the particular methods and experimental conditions described herein, as such methods and conditions may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0033] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For purposes of the present invention, the following terms are defined below.

[0034] The term "about" when used in reference to numerical values ​​is meant to encompass numerical values ​​within a range from a lower limit of 10% less than the specified numerical value to an upper limit of 10% greater than the specified numerical value. The term "and / or", when used in connection with two or more options, should be understood to mean any one of those options, or any two or more of those options.

[0035] As used herein, the terms "comprising," "comprise," "including," or "include" mean the inclusion of the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. When the term "comprising" is used herein, unless otherwise specified, it also encompasses the situation of consisting of the recited elements, integers, or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass an antibody variable region consisting of that particular sequence.

[0036] The term "ROR1" refers to any recombinant or native Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), variants or homologs thereof, which, for example, maintain at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the activity of ROR1. The variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the native ROR1 protein in its entirety or a subsequence (e.g., 50, 100, 150 or 200 consecutive amino acids). In one embodiment, the ROR1 protein comprises the amino acid sequence of Uniprot ID: Q01973.

[0037] ROR1 is highly expressed in fetuses, and its expression level then decreases significantly in adulthood. However, the expression of ROR1 has been found to be significantly enhanced in various hematological malignancies and solid tumors. Hematological malignancies that highly express ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid hematological malignancies. Among solid tumors, cancers that express ROR1 include colon cancer, breast cancer, intestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, etc.

[0038] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, although in some cases may contain fewer chains, e.g., in camelids, naturally occurring antibodies may contain only heavy chains.

[0039] The term "anti-ROR1 antibody" refers to an antibody molecule that can specifically bind to ROR1 and inhibit ROR1 activity. The anti-ROR1 antibody can inhibit ROR1 activity relative to the absence of the ROR1 antibody, for example, by at least partially or completely preventing stimulation of ROR1, and can reduce, prevent or or delay activation of ROR1, or inactivate, blunt or downregulate ROR1 signaling, activity or amount. 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.

[0040] The term "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody and binds the antigen that 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 (e.g., bispecific antibodies) composed of antibody fragments.

[0041] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody binding to an antigen. Natural antibody heavy and light chain variable domains generally have a similar structure, in which each domain contains four conserved framework regions (FR) and three complementarity determining regions (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. page 91 (2007)). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0042] The terms "complementarity determining region" or "CDR region" or "CDR" are regions in an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to the antigen epitope. The CDRs of a variable domain are commonly referred to as CDR1, CDR2 and CDR3, and are numbered sequentially from the N-terminus. The precise amino acid sequence boundaries of each CDR of a given variable region can be determined, for example, by Chothia (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)), based on the three-dimensional structure of the antibody and the configuration of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), based on the variability of antibody sequences; AbM (University of Bath); Contact (University of London); the International ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ); and North American ImmunoGeneTics (http: / / anonymous), based on affinity propagation clustering using a large number of crystal structures. These can be determined by using any one or a combination of the various established antibody CDR designation schemes, including the CDR definitions.

[0043] Unless otherwise defined, the term "CDR" or "CDR sequence" encompasses CDR sequences determined by any of the methods described above. CDRs can also be determined based on a reference CDR sequence (eg, any of the exemplary CDRs of the invention) having the same AbM numbering position. In one embodiment, the CDRs of an antibody of the invention are arranged according to the AbM numbering scheme. Unless otherwise indicated, in the present invention, when referring to the positions of residues in antibody variable regions and CDRs (including heavy chain variable region residues), we refer to the AbM numbering system.

[0044] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) of a humanized antibody correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0045] The term "binding" or "specific binding" as used herein means that the binding interaction to the antigen is selective and can be distinguished from undesired or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin layer biomembrane interference assay, MSD assay, or surface plasmon resonance (SPR).

[0046] "Median Effective Concentration (EC 50 The term "maximum" refers to the concentration of a drug, antibody, or toxic agent that elicits a response that is 50% between baseline and maximum after a specified exposure time.

[0047] As used herein, the term "therapeutic agent" includes any substance effective to prevent or treat a tumor (e.g., a cancer), such as a chemotherapeutic agent, a cytokine, an angiogenesis inhibitor, a cytotoxic agent, another antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immunosuppressant).

[0048] The term "antibody-drug conjugate" or "ADC" refers to an antibody or antibody fragment covalently linked to a therapeutically active agent or active pharmaceutical ingredient such that the therapeutically active agent or active pharmaceutical ingredient is targeted to the antibody's binding target and exhibits a pharmacological function. The therapeutically active agent or active pharmaceutical ingredient may be a cytotoxin capable of killing cells, preferably cancer cells, targeted by the ADC. The covalent attachment of the therapeutically active agent, active pharmaceutical ingredient or cytotoxin is performed in a non-site-selective manner using a linker or in a site-selective manner.

[0049] The term "site-selective conjugation" refers to a method of specifically linking a therapeutic agent or active pharmaceutical ingredient to a specific site on an antibody. In one embodiment, conjugation is achieved through a linker.

[0050] The terms "cytotoxic agent" and "cytotoxin" are used interchangeably and refer to a substance that inhibits or disrupts cellular function and / or causes cell death or destruction. In one embodiment, cytotoxic agents can include, but are not limited to, bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin), small molecule toxins, radioisotopes, maytansine alkaloids, such as anthracyclines, camptothecins, combretastatins, dolastatins and their auristatin derivatives, duocarmycins, enediynes, geldanamycin, indolinobenzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterlins, spliceostatins, pladienolides, and calicheamicins.

[0051] Any antibody-drug conjugate of the present invention can be prepared by binding an antibody to dolastatin and its auristatin derivatives. Dolastatin and its auristatin derivatives are important cytotoxins used in antibody-drug conjugates (ADCs). They have antitumor and antifungal activity because they interfere with microtubule dynamics, cell division, etc. Modifications of the backbone, mainly of the terminal subunits: P1 (N-terminus) and P5 (C-terminus), have been widely reported in the literature. Modifications of the main peptide subunits also result in effective in vitro cytotoxicity of this type of substance. In one embodiment, dolastatins and their auristatin derivatives include, for example, 0101 (2-methylpropionyl-N-(3R,4S,5S)-3-methoxy-1-{(2S)-2-(1R,2R)-1-methoxy-2-methyl-3-oxy-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)-ethyl]amino}propylpyrrolidin-1-yl}-5-methyl-1-oxyheptan-4-yl]-N-methyl-L-valinamide), 8261 (2-methylpropionyl-N-(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenethyl]amino}-1-methoxy-2-methyl-3-oxy-3-{[(1S)-1-carboxyl-2-phenethyl]amino}-5-methyl-1-oxyheptan-4-yl]-N-methyl-L-valinamide), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AFHPA), and other auristatins, such as those described in U.S. Publication No. 20130129753.

[0052] Monomethylauristatin (MMAE), also known as demethyl-auristatin E, is a well-known member of the auristatin compound family. It has the following structural formula: [ka]

[0053] MMAE plays an effective inhibitory role in mitosis by suppressing tubulin polymerization. It cannot be used as a drug due to its cytotoxicity, but it is widely used to prepare antibody conjugates. MMAE is linked to a monoclonal antibody (MAB) via a linker to form MMAE-MAB. In general, MMAE-MAB targets tumor cells through the antibody, and the linker is cleaved after MMAE-MAB enters tumor cells, thus releasing MMAE, which can exert its cytotoxic effect and kill tumor cells.

[0054] The terms "linker" and "connector" are used interchangeably in this application and refer to a chemical moiety that covalently links an antibody to a therapeutically active agent or active pharmaceutical ingredient in an ADC. In one embodiment, the linker may comprise amino acid residues that connect the antibody to the payload. The amino acid residues may form a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. The amino acid residues include naturally occurring as well as unnatural amino acid analogs such as citrulline, or β-amino acids such as β-alanine, or ω-amino acids such as 4-aminobutyric acid.

[0055] According to their properties, linkers suitable for the present invention can be classified as cathepsin-degradable linkers, such as valine-citrulline (val-cit) linker, cBu-Cit linker and CX linker; non-cleavable linkers, such as SMCC linker or MD linker; 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.

[0056] The linker of the present invention may also be a combination of one or more linkers. For example, a linker that can be degraded by cathepsin may be combined with other types of linkers to form a new linker. Therefore, the "linker" according to the present invention includes a single type of linker or a combination of different types of linkers, as long as it can link the antibody of the present invention to a drug.

[0057] In specific embodiments, the linker may be, 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, maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p -aminobenzyloxycarbonyl (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), and N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

[0058] The terms "loading," "drug loading," or "payload" refer to the average number of available payloads per antibody in an ADC molecule (herein, "payload" is used interchangeably with "therapeutically active agent or active pharmaceutical ingredient"). Drug loading can range from 1 to 20 therapeutically active agents or active pharmaceutical ingredients per antibody. The term "drug / antibody ratio" or "DAR" refers to the ratio of therapeutically active agent or active pharmaceutical ingredient (D) conjugated to an antibody to the antibody. The ADCs described herein typically have a DAR of 1 to 20, and in certain embodiments, a DAR of 1 to 8, 2 to 8, 2 to 6, 2 to 5, 2 to 18, 4 to 16, 5 to 12, 6 to 10, 3 to 8, 4 to 6, 6 to 10, and 2 to 4. Representative DAR values ​​are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, and are usually expressed as a combination of the letter D and a number, where the number represents the numerical value of the DAR, for example, D2 represents the drug / antibody ratio, i.e., the DAR value is 2. In some embodiments, the DAR is an average DAR, i.e., characterized by a detection method (e.g., by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC). Quantitative DAR values ​​are also measured. The DAR may be limited by the number of binding sites on the antibody. For example, if the binding site is a cysteine ​​thiol, the antibody may have only one or more cysteine ​​thiol groups or only one or more sufficiently reactive thiol groups to which a linker unit may be attached.In some embodiments, the average DAR value of the conjugate of the present invention is 1 to 20, e.g., 2 to 18, 4 to 16, 5 to 12, 6 to 10, 2 to 8, 3 to 8, 2 to 6, 4 to 6, 6 to 10, etc., e.g., 1.0 to 8.0, 2.0 to 6.0, etc., e.g., 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, 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, and ranging between these two values ​​as the endpoints.

[0059] The term "treatment" refers to slowing, interrupting, blocking, mitigating, arresting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a disease, prevention of metastatic cancer, reduction in the rate of disease progression, improvement or alleviation of the disease state, and alleviation or improvement of prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or slow down the progression of a disease.

[0060] The term "prevention" includes inhibition of the appearance or progression of a disease or disorder, or a symptom of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for a preventive regimen. Typically, in the context of cancer, the term "prevention" refers to administration of a drug before signs or symptoms of cancer occur, particularly in subjects at risk of cancer.

[0061] The term "effective amount" refers to the amount or dose of the antibody or conjugate or composition of the present invention to produce the desired effect in a patient in need of treatment or prevention after administration to the patient in one or more doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by taking into account various factors such as, for example, the species of 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 profile of the administered preparation; the selected dosing regimen; and the use of any combination therapy.

[0062] A "therapeutically effective amount" refers to an amount that effectively achieves a desired therapeutic result at the required dosage and for the required period of time. A therapeutically effective amount of an antibody or antibody fragment, or a conjugate or composition thereof, can vary with various factors, such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody fragment, or a conjugate or composition thereof, are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and even more preferably at least about 80% or 90% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that is predictive of efficacy in human tumors.

[0063] The term "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic effect. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0064] The term "pharmaceutical composition" refers to a composition that allows the active ingredients contained therein to be present in a form that maintains their biological activity and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition will be administered.

[0065] II. Compositions of the Invention In some embodiments, the invention provides a composition, preferably a pharmaceutical composition, comprising any of the anti-ROR1 antibodies or ADC molecules thereof described herein. In one embodiment, the composition further comprises a pharma- ceutical acceptable adjuvant, such as a pharma- ceutical acceptable carrier, a pharma- ceutical acceptable excipient, including a buffer, known in the art. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises an anti-ROR1 antibody of the invention, or an ADC molecule thereof, in combination with one or more other therapeutic agents.

[0066] As used herein, "pharmaceutical acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, and the like. Also, regarding the use of pharma- ceutically acceptable adjuvants, see "Handbook of Pharmaceutical Excipients", Eighth Edition, RC Rowe, PJ Seskey and SC Owen, Pharmaceutical Press, London, Chicago.

[0067] The compositions of the present invention may exist in a variety of forms, including, for example, liquid, semi-solid, solid dosage forms, such as liquid solutions (e.g., injectable or infusible solutions), powders or suspensions, liposomes, or suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

[0068] For example, the route of administration of the compositions of the present invention may be oral, by intravenous injection, intraperitoneally, intracerebrally (intracerebroparenchyma), intracerebroventricularly, intramuscularly, intraocularly, intraarterially, intraportally or intralesionally; by sustained release system, or by implantation device, based on known methods. In certain embodiments, the compositions may be administered by bolus injection, by continuous infusion, or by implantation device.

[0069] The subject is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from or is at risk of suffering from a disease described herein (e.g., cancer). In certain embodiments, the subject is undergoing or has undergone other treatments, such as chemotherapy and / or radiation therapy. In some embodiments, the subject has previously undergone or is currently undergoing immunotherapy.

[0070] Medicaments containing the antibodies described herein can be prepared by mixing the anti-ROR1 antibody of the present invention or its ADC molecule, preferably in the form of a lyophilized preparation or aqueous solution, having the desired purity, with one or more optional pharma- ceutically acceptable adjuvants.

[0071] The pharmaceutical compositions or formulations of the present invention also contain two or more active ingredients as required for the particular indication to be treated, preferably those with complementary activities that do not adversely affect each other. It may also be desirable to provide other therapeutic agents, including, for example, chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immune checkpoint inhibitors or agonists). The active ingredients are preferably provided in combination in amounts effective for the intended purpose.

[0072] 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, e.g., films, or microcapsules.

[0073] III. Preparation of Antibodies and Antibody-Drug Conjugates of the Invention In one embodiment, the invention provides a method for preparing an anti-ROR1 antibody, the method comprising culturing a host cell comprising a nucleic acid encoding the anti-ROR1 antibody or an expression vector comprising the nucleic acid under conditions suitable for expression of the nucleic acid encoding the anti-ROR1 antibody, and, optionally, isolating the anti-ROR1 antibody. In certain embodiments, the method further comprises recovering the anti-ROR1 antibody from the host cell (or the host cell medium).

[0074] For recombinant production of the anti-ROR1 antibody of the invention, a nucleic acid encoding the anti-ROR1 antibody of the invention is first isolated and then inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid is amenable to isolation and sequencing by routine procedures, for example using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the anti-ROR1 antibody of the invention.

[0075] Anti-ROR1 antibodies produced as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art. The purity of the anti-ROR1 antibodies of the present invention can be determined by any one of several well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0076] Various methods for linking cytotoxic or other therapeutic agents to antibodies have been described in the prior art. For example, conjugation can occur via the amino groups of lysine side chains and the amino group at the N-terminus of the antibody, or via the carboxyl groups of aspartic acid, glutamic acid and the C-terminus, or via activated cysteine ​​sulfhydryl groups within the antibody. EXAMPLES

[0077] The present invention is further illustrated by the following examples; however, the examples are described in an illustrative rather than limiting manner, and it should be understood that various modifications may be made by those skilled in the art.

[0078] The present invention may be practiced by conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell physiology, unless expressly indicated to the contrary.

[0079] Example 1. Preparation and evaluation of raw materials 1.1 Preparation and evaluation of ROR1 control antibodies Preparation of ROR1 control antibody: In this application, anti-ROR1 antibody Cirmtuzumab is used as a positive control antibody. The gene of the target nuclear fragment was synthesized by General Biotech Co., Ltd. according to the sequence disclosed in WO / 2019 / 173843. The gene was then cloned into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination. The recombinant protein expression vector was transformed into E. coli DH5α, cultured overnight at 37°C, and the plasmid DNA was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The resulting expression vector (expressing Cirmtuzumab) was designated clone number 99961.1, and the expressed Cirmtuzumab was called 99961.1. The expression vector was transfected into 293 cells using ExpiFectamine™ CHO transfection reagent (Thermo Fisher, A29129). After 7 days, the cell culture supernatant was collected, centrifuged at 15,000 g for 10 min, and filtered through a 0.22 μm membrane. The antibodies in the supernatant were purified using a Protein A / G affinity chromatography column. The target antibodies were eluted with 100 mM glycine (pH 3.0) and then exchanged into PBS buffer using an ultrafiltration tube (Millipore, UFC901096).

[0080] Evaluation of ROR1 control antibody: The activity of the prepared positive control antibody 99961.1 (IgG1) was detected using the purchased huROR1-His antigen protein (Kaixia Biotechnology, ROR-HM401). The specific method was as follows: 96-well ELISA plate was coated with huROR1-His (2 μg / mL, 30 μL / well) overnight at 4°C; after washing three times, the plate was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing three times, the plate was added with control antibody 99961.1 gradient diluted with PBS and incubated at room temperature for 1 hour; after washing, the plate was added with secondary antibody anti-human IgG-κ+λ-HRP (Millipore, AP502P+AP506P) diluted with PBS (1:6000) and incubated at room temperature for 1 hour, then the plate was washed six times and TMB was added for color development for 5-20 minutes. After color development was completed, the data was read at OD450 using a microplate reader, and the data was processed and plotted using Graphpad Prism. The results showed that the expressed control antibody 99961.1 was able to bind to the ROR1 protein and had normal anti-ROR1 activity.

[0081] 1.2 Preparation and evaluation of antigen proteins Preparation of antigen proteins: His tag or human Fc (SEQ ID NO: 51) tag was added to the C-terminus of the sequences of human ROR1 protein huROR1ECD AA30-406 (Uniprot ID: Q01973), mouse ROR1 protein MusROR1ECD AA30-406 (Uniprot ID: Q9Z139), and human ROR2 protein huROR2ECD AA34-403 (Uniprot ID: Q01974) by genetic manipulation at the coding gene level. The resulting nucleic acid sequences were constructed into pcDNA3.4 vector, which was then transformed into Escherichia coli DH5α and cultured overnight at 37°C, and the plasmids were then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The resulting plasmid was transfected into HEK293 cells (ATCC® CRL-1573™) using ExpiFectamine™ 293 Transfection Kit (Gibco™, A14524). After 7 days of expression, the cell culture supernatant was collected, and the protein containing the Fc tag was subjected to affinity purification using COLUMN XK16 / 20 (Cytiva). After purification, the target protein was eluted with 100 mM glycine (pH=3.0), concentrated, and subjected to buffer exchange, and finally the antigen protein (huROR1-huFc) was obtained. The His-tagged protein was subjected to affinity purification using Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted using an imidazole gradient. The eluted proteins were each passed through an ultrafiltration concentration tube (Millipore, UFC901096) into PBS buffer, and finally, the antigen proteins (huROR1-His, MusROR1-His, huROR2-His) were obtained.

[0082] Antigen evaluation: The prepared antigens (huROR1-His, huROR1-huFc) were detected using the antibody 99961.1 (IgG1) obtained in Example 1.1, which had passed the quality inspection. The specific method was as follows: ELISA plates were coated overnight at 4°C with 2 μg / mL of huROR1-His and huROR1-huFc, respectively. The purchased antigen proteins huROR1-His and huROR1-huFc (Kaixia Biology, ROR-HM201) were used as positive controls. After washing three times, the plate was blocked with 5% skim milk prepared in PBS for 1 hour at room temperature; after washing three times, the plate was added with antibody 99961.1 gradient diluted in PBS and incubated at room temperature for 1 hour; after washing, the plate was added with secondary antibody anti-human IgG-κ+λ-HRP (Millipore, AP502P+AP506P) diluted in PBS (1:6000) and incubated at room temperature for 1 hour, then the plate was washed six times and TMB was added for 5-20 minutes for color development. After color development was completed, the data was read at OD450 using a microplate reader, and the data was processed and plotted using Graphpad prism. The results show that antibody 99961.1 binds to the in-house expressed antigens huROR1-His and huROR1-huFc with affinities comparable to those of the purchased ROR1 antigen protein.

[0083] Example 2. Construction and evaluation of cell lines overexpressing human ROR1 Construction of HEK293 cell line overexpressing human ROR1 (hereafter referred to as huROR1-HEK293): A nucleic acid sequence encoding full-length human ROR1 (Uniprot ID: Q01973) was constructed in pLVX-puro plasmid (Clontech, Cat#632164). The resulting plasmid was then electroporated into HEK293 cells (ATCC® CRL-1573™) using an electroporator (Invitrogen, Neon™ Transfection System, MP922947). After electroporation, the resulting cells were transferred into DMEM medium (Gibco, 11995065) containing 10% by volume FBS (Gibco, 15140-141) and without antibiotics, and then transferred to a 10×10 cm cell culture dish for 48 hours of culture. The cells were then transferred to a 10×10 cm cell culture dish for 48 hours of culture. 4 The cells were spread into 96-well cell culture plates at an average concentration of 100 cells / well, and puromycin was added to a final concentration of 2 μg / mL for screening pressure. After approximately 2 weeks, clonal cell lines were picked for identification.

[0084] Flow cytometry assay of huROR1-HEK293 cells: The cell line obtained above in logarithmic growth phase was digested and plated in a 96-well plate. After washing with FACS buffer (1×PBS buffer containing 2% FBS by volume), a gradient of primary antibody (99961.1) diluted in PBS was added and incubated at 4°C for 30 minutes. After washing, a prepared fluorescent secondary antibody anti-human IgG Fc (abcam, 98596) was added and incubated at 4°C for 30 minutes. Finally, the cells were detected by a flow cytometer (Beckman, CytoFLEXAOO-1-1102). The results show that huROR1-HEK293 cells with high expression of human ROR1 on the surface were obtained.

[0085] Example 3 Immunization of animals and construction of immune libraries 3.1 Immunization regimens Three Balb / C mice (Shanghai Lingchang Biotechnology Co., Ltd.) were cross-immunized with huROR1-huFc and huROR1-His antigens by subcutaneous and intraperitoneal injection once every two weeks for a total of four immunizations. One week after the fourth immunization, blood was collected from the mice for immune titer assay, and finally, huROR1-huFc was used for one booster immunization.

[0086] 3.2 Evaluation of serum antibody titers in mice after immunization ELISA plates were coated with 2 μg / mL huROR1-His and huROR1-huFc (30 μL / well) at 4° C. overnight. After washing three times, the plates were blocked with 5% skim milk prepared in PBS for 1 h at room temperature; after washing three times, mouse serum gradient diluted in PBS was added to the plates, and antibody 99961.1 was added as a positive control, and incubated for 1 h at room temperature; after washing, secondary antibody goat anti-mouse-lgG(1+2a+2b+3)-HRP (Jackson, 115-035-164) or goat anti-human κ+λ-HRP (Millipore, AP502P+AP506P) diluted in PBS was added to the plates, and incubated for 1 h at room temperature, then the plates were washed six times and TMB was added for 5-20 min for color development. After the color development was completed, the data was read at OD450 using a microplate reader, and the data was processed and plotted using Graphpad Prism. The results showed that the serum titers of all three mice met the criteria.

[0087] 3.3 Construction of antibody gene phage display library After immunization was completed, the spleen was removed from the mouse, and splenocytes were collected after crushing and filtration. 1 mL of TRIzol™ reagent (Thermo Fisher, 15596026) was added to dissolve the splenocytes, and total RNA was extracted by the phenol-chloroform method. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). The cDNA was then used as a PCR template to amplify the genes of the respective antibody light and heavy chain variable regions using specific primers of the mouse antibody sequence. The PCR products were digested with two enzymes NcoI and NotI to obtain antibody gene fragments, which were then inserted into a phage display vector and ligated with T4 ligase. The ligation products were recovered using a DNA recovery kit (OMEGA, D6492-02) and finally transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) using an Electroporator (Bio-Rad, MicroPulser). The electroporated bacteria were incubated in 2-YT (C + / K + The Fab sequences were spread onto solid plates (2-YT) and SS320 bacteria correctly transformed with the antibody plasmids were amplified and a phage display library containing the Fab sequences was obtained using VSCM13 helper phage (purchased from Stratagene).

[0088] Example 4 Screening of antibody gene phage display library 4.1 Cell-based screening of phage-displayed antibody gene libraries hROR1-HEK293T cells were cultured in T25 flasks. When the cell growth density approached 90%, the culture supernatant was removed, the cells were washed once with PBS (Yuanpei, B310KJ), then 2mL of 4% paraformaldehyde (Shenggong, E672002-0500) was added for fixation for 0.5 hours, and finally washed twice with PBS to be used as screening material. During screening, the phage display library was incubated with the fixed hROR1-HEK293T cells at room temperature for 1 hour, washed three times with 1×PBS, and then 2mL of glycine HCl (pH=2.0) was added and mixed gently for 10 minutes to elute the phages that specifically bound to human ROR1. The eluted supernatant was then used to infect log phase SS320 bacteria (Lucigen, 60512-1) for 30 minutes and then incubated for 1 hour at 37°C and 220 rpm. VSCM13 helper phage was added and incubated for 30 minutes and then incubated for 1 hour at 37°C and 220 rpm. After centrifugation, the cells were incubated for 1 hour at 37°C and 220 rpm. + / K + The phage were transferred to 2-YT medium. The final phage was used for the second round of screening. The screening was repeated several times, and 10 clones were randomly selected for sequence analysis in each round to evaluate the library. After three rounds of screening, the sequences of the library were significantly enriched.

[0089] 4.2 Immunotube-based and magnetic bead-based screening of antibody gene phage display libraries Immunotubes and magnetic beads were used to enrich for specific antibodies against antigens, and the two methods are complementary and validate each other.

[0090] Immunotube-based screening is a panning process, and consists of coating the surface of an immunotube with high adsorption capacity with antigen protein huROR1-His or huROR1-huFc, adding a phage display antibody library to the immunotube, incubating with the antigen protein adsorbed on the surface of the immunotube, washing, and eluting. After 2-4 rounds of panning, the specific monoclonal antibody Fab against the antigen is finally enriched. In this example, the monoclonal antibody Fab against human ROR1 is enriched after 3 rounds of panning. For the specific method, please refer to Example 2.4.2 of Patent CN112250763B.

[0091] Magnetic bead-based screening is a panning process and consists of labeling the antigen protein huROR1-His with biotin, binding the labeled antigen protein to streptavidin-linked magnetic beads, incubating the antigen-bound magnetic beads with the antibody gene phage display library, washing, and elution. Usually, specific monoclonal antibodies against antigens are enriched in large quantities by 3-4 rounds of panning. In this example, biotin-labeled huROR1-His was used for phage display library screening, and monoclonal antibody Fab against human ROR1 was first screened after 3 rounds of panning. For specific methods, please refer to Example 2.4.1 of Patent CN112250763B.

[0092] 4.3 Selection of single clones The phage pools eluted in each round were tested by ELISA to evaluate the enrichment effect, and 10 clones were randomly selected from the phage pools of each round of screening for sequence analysis. The ratio of enrichment effect and sequencing repeatability was comprehensively analyzed to select the proper round of single clone selection.

[0093] The antigen protein huROR1-His was used for ELISA monoclonal primary screening, and the antibody Fabs binding to huROR1-His obtained from the primary screening were prepared as Fab lysates, and then the huROR1-overexpressing HEK293 cells prepared in Example 2.1 were used for detection and assay by flow cytometry (FACS). A total of 11 antibody Fab molecules specifically binding to human ROR1 were screened, and the obtained 11 mouse antibody Fabs were named according to the corresponding clone numbers (B62, B31, B32, B74, B34, B39, C38, C77, C42, M71, and M78). The specific FACS results are shown in Figures 1A-1B. The CDR amino acid sequences of the obtained mouse antibody Fabs are shown in Table 1. The CDR sequences are determined using the AbM definition.

[0094] Table 1. Amino acid sequences of the CDR regions of mouse antibodies [Table 1-1] [Table 1-2]

[0095] Example 5. Antibody construction, expression, and purification 5.1 Plasmid construction The VH coding sequences of the screened monoclonal antibody Fab sequences B62, B31, B32, B74, B34, B39, C38, C77, C42, M71, and M78 were connected to the coding sequence of the human IgG1 heavy chain constant region (SEQ ID NO: 52) to obtain the heavy chain coding sequence of the chimeric antibody, and the VL coding sequence of the Fab sequence was connected to the coding sequence of the human light chain constant region (CL) kappa type (SEQ ID NO: 53) to obtain the light chain coding sequence of the chimeric antibody. The antibody heavy and light chain coding sequences were respectively inserted into the eukaryotic cell expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5α, and cultured overnight at 37°C. The plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free antibody plasmid for eukaryotic expression.

[0096] 5.2 Antibody expression and purification The full-length sequence of the antibody obtained above was expressed by the Expi CHO transient expression system (Thermo Fisher, A29133). The specific method was as follows: On the day of transfection, the CHO cell density was approximately 7 × 10 6 ~1×10 7 At this time, cells were cultured at 6 × 10 viable cells / mL and cell viability was >98%. 6 The final concentration was adjusted to cells / mL. The target plasmid was diluted in OptiPRO™ SFM pre-cooled to 4°C (1 μg of plasmid was added to 1 mL of culture medium), and ExpiFectamine™ CHO reagent was diluted in OptiPRO™ SFM. The two were mixed in equal amounts and gently mixed by pipetting to obtain the ExpiFectamine™ CHO / plasmid DNA mixture, which was incubated at room temperature for 1-5 minutes, slowly added to the prepared cell suspension with gentle shaking, and finally incubated at 37°C, 8% CO2 on a cell culture shaker.

[0097] 18-22 hours after transfection, ExpiCHO™ Enhancer Reagent and ExpiCHO™ Feed Reagent were added to the culture medium, and the flasks were placed on a shaker at 32°C with 5% CO2 and continued to be cultured. Five days after transfection, the same volume of ExpiCHO™ Feed Reagent was slowly added while gently mixing the cell suspension. Seven days after transfection, the cell culture supernatant in which the target protein was expressed was collected and centrifuged at 15,000g for 10 minutes. The resulting supernatant was subjected to affinity purification using MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1 M Tris HCl, and finally the resulting protein was placed into PBS buffer using an ultrafiltration concentration tube (Millipore, UFC901096).

[0098] Example 6 Physical and Chemical Properties of Antibodies In this example, the relative molecular weight and purity of the previously obtained antibodies were determined by SDS-PAGE and SEC-HPLC.

[0099] 6.1 Evaluation of antibodies by SDS-PAGE Preparation of non-reduced solutions: 1 μg of each obtained antibody and quality control product IPI (Ipilimumab) were added separately to 5×SDS loading buffer and 40 mM iodoacetamide, heated in a dry bath at 75° C. for 10 min, cooled to room temperature, and centrifuged at 12,000 rpm for 5 min to obtain the supernatant.

[0100] Preparation of reducing solution: 2 μg of each obtained antibody and quality control product IPI were added separately to 5×SDS loading buffer and 5 mM DTT, heated in a dry bath at 100°C for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min to obtain the supernatant, which was applied to a Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis, and protein bands were visualized by Coomassie brilliant blue staining.

[0101] Protein gels containing chromoprotein bands were scanned using an EPSON V550 color scanner (de-stained until the gel background was transparent), and the purity of reduced and non-reduced bands was calculated using ImageJ according to the peak area percentage method.

[0102] The results showed that the bands of each antibody in the non-reduced gel were around 150 kD, and the bands in the reduced gel were around 55 kD and 25 kD, which were consistent with the expected sizes. The purity of all antibodies obtained in this application detected by the reduced gel was more than 95% (Table 2).

[0103] 6.2 Evaluation of antibody monomer purity by SEC-HPLC Material preparation: 1. Mobile phase: 150 mmol / L phosphate buffer, pH 7.4; 2. Sample preparation: Each antibody and quality control product IPI were diluted to 0.5 mg / mL with mobile phase solution, respectively. Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph was used, the column was XBridge BEH (SEC 3.5 μm, 7.8 mm ID×30 cm), Waters flow rate was set to 0.8 mL / min, injection volume was 20 μL, and VWD detector wavelength was 280 nm and 214 nm. Blank solution, IPI quality control solution, and antibody sample solution were injected consecutively. The percentages of high molecular weight polymer, antibody monomer, and low molecular weight substances in the samples were calculated according to the area percentage method.

[0104] The results are shown in Figures 2A to 2K and Table 2. Except for antibody B74, the SEC monomer purity of the other antibodies was greater than 96%.

[0105] Table 2. Expression levels and physiochemical properties of the antibodies obtained in this application. [Table 2]

[0106] Example 7 Evaluation of antibody binding activity to antigen In this example, the binding profiles of 11 antibodies (B62, B31, B32, B34, B39, B74, C38, C42, C77, M71, and M78) against human ROR1 antigen protein huROR1-His were detected by ELISA assay, and the binding ability of the antibodies (B62, B31, B32, B34, B39, B74, C38, C42, C77, M71, and M78) against huROR1-HEK293 cells and A549 tumor cells overexpressing human ROR1 was also detected by FACS assay. A549 tumor cells are a human non-small cell lung cancer cell line overexpressing human ROR1.

[0107] 7.1 Evaluation of antibody binding ability to antigen protein huROR1-His by ELISA assay 96-well ELISA plates were coated with 2 μg / mL huROR1-His (30 μL / well) overnight at 4°C. The next day, the plates were washed three times with PBST and then blocked with 5% skim milk for 2 hours. After three washes with PBST, gradient dilutions of each antibody and positive control antibody 99961.1 (3.00000, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00046, 0.00005 μg / mL) were added and incubated for 1 hour. After three washes with PBST, secondary antibody goat anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the result of color development, 2M HCl was added to stop the reaction, and the data was read at OD450 using a microplate reader (Molecular Devices, SpecterMax190).

[0108] The results are shown in Figures 3A to 3B. All of the antibodies obtained in the present application had good affinity activity for the antigen protein huROR1-His, and the affinity of each antibody was equivalent to 99961.1.

[0109] 7.2 Evaluation of antibody binding ability to huROR1-HEK293 and A549 tumor cells by FACS In this example, the binding activity of the antibodies was evaluated using two types of cells, huROR1-HEK293 cells overexpressing human ROR1, and A549 tumor cells.

[0110] The specific method was as follows: huROR1-HEK293 cells or A549 cells in logarithmic growth phase were prepared into a single cell suspension and the density was adjusted to 1 × 10 6The cell concentration was adjusted to 100 μL / mL, and then 100 μL / well of cells was added to a 96-well round-bottom plate, centrifuged at 300 g at 4°C, and the supernatant was removed. Gradient dilutions of the antibodies obtained in this application and the positive control antibody 99961.1 were added to the corresponding wells, mixed thoroughly, and incubated at 4°C for 30 minutes. After incubation, the 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 in the dark at 4°C for 30 minutes. After washing three times, the cells were detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0111] The results are shown in the figures. For A549 cells (Figures 4A-4B), all the other antibodies, except C42, have superior affinity to that of the control antibody 99961.1. For huROR1-HEK293 cells (Figures 5A-5B), all the antibodies have affinity comparable to that of the control antibody 99961.1.

[0112] Example 8. Assessment of antibody cross-reactivity between species and allogroups In this example, the cross-reactivity of each antibody obtained in this application was tested between species and allogeneic groups. The cross-reactivity of the antibodies obtained in the present application between species and species groups was confirmed using the mouse ROR1 antigen protein MusROR1-His and the human ROR2 antigen protein huROR2-His prepared in Example 1.2.

[0113] 8.1 Assessment of antibody cross-reactivity between species A 96-well ELISA plate was coated with 2 μg / mL MusROR1-His (30 μL / well) overnight at 4°C. The next day, the plate was washed three times with PBST and then blocked with 5% skim milk for 2 hours. After three washes with PBST, gradient dilutions of each antibody and positive control antibody 99961.1 (1.00000, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, 0.00015, 0.00002 μg / mL) were added and incubated for 1 hour. After three washes with PBST, the secondary antibody goat anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. According to the result of color development, 2M HCl was added to stop the reaction, and the data was read at OD450 using a microplate reader (Molecular Devices, SpecterMax190).

[0114] The results are shown in Figures 6A-6B and Table 3. B62, B32, C38, and C42 bind to the antigen protein MusROR1-His and show good cross-reactivity with mice. Other antibodies and the positive control antibody 99961.1 do not bind to mouse antigen proteins. Therefore, the antibodies obtained in this application, B62, B32, C38, and C42, have a wide range of applications in mouse model-based experiments and tests.

[0115] 8.2 Evaluation of cross-reactivity of the present antibodies between allogeneic groups A 96-well ELISA plate was coated with 2 μg / mL huROR2-His (30 μL / well) overnight at 4°C. The next day, the plate was washed three times with PBST and then blocked with 5% skim milk for 2 hours. After washing three times with PBST, gradient dilutions of the antibodies and positive control antibody 99961.1 were added and incubated for 1 hour. After washing three times with PBST, secondary antibody goat anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After incubation, the plate was washed six times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the result of color development, 2M HCl was added to stop the reaction, and the data was read at OD450 using a microplate reader (Molecular Devices, SpecterMax190).

[0116] The results are shown in Figures 7A to 7B and Table 3. No antibodies bound to the antigen protein huROR2-His, indicating that the antibodies prepared in the present application specifically bound to human ROR1.

[0117] Table 3 Cross-reactivity of the antibodies obtained in this application among species and allogeneic groups [Table 3]

[0118] Example 9 Evaluation of intracellular uptake rate of antibody In this example, the intracellular uptake rate of the antibody obtained in this application was detected using two detection methods, FACS and Fab-Zap. The FACS-based intracellular uptake detection is to detect the antibody intracellular uptake rate in a short time by using supersaturated antibody-bound cells; and the Fab-Zap method reflects the long-term accumulation effect of the intracellular uptake rate by binding cells with different concentrations of Fab-Zap toxin-bound antibodies, and killing the target cells with the toxin that has entered the cells through intracellular uptake.

[0119] 9.1 Evaluation of intracellular uptake of the antibody of the present application by FACS assay Antibody dilution: Test antibodies were diluted in DMEM complete medium to a final concentration of 10.0000 μg / mL. Cell treatment: huROR1-HEK293 cells were digested and added to complete DMEM medium. After thorough mixing, the cells were counted and their viability was measured. 6 The cells were removed and added to a 1.5 mL centrifuge tube, centrifuged at 300 g for 5 minutes, the supernatant was discarded, 1 mL of pre-chilled DMEM medium was used for resuspension, centrifuged at 300 g for 5 minutes, and the supernatant was discarded.

[0120] Primary antibody incubation: 1000 μL of pre-chilled diluted antibody was removed and added to the centrifuge tube containing the cells to prepare an antibody-cell suspension, which was then added to the 96-well plate and incubated at 4°C. Extracellular secondary antibody incubation: The suspension in the 96-well plate was quickly transferred to a second 96-well plate. 180 μL of pre-chilled FACS buffer was added to each well of the second 96-well plate and washed twice. Then, 100 μL of diluted secondary antibody FITC-labeled anti-huFc or RPE-labeled anti-huFc (secondary antibody was diluted 1:150 using FACS buffer) was added to each well; incubated at 4° C. for 30 minutes.

[0121] Fixation of cells: After incubation, the supernatant was discarded by centrifugation, 180 μL of pre-chilled FACS buffer was added to each well, and the cells were washed twice. The supernatant was removed by centrifugation, and the cells were fixed with 100 μL of 4% paraformaldehyde in each well for 30 minutes at room temperature.

[0122] Disruption of cell membrane: After fixation, 180 μL of FACS buffer was added and washed twice. 100 μL of prewarmed 0.5% Triton X-100 was added to each well and permeabilized for 5 minutes at room temperature.

[0123] Intracellular secondary antibody incubation: After washing the 96-well plate, add 180 μL of pre-warmed permeabilization buffer (Invitrogen™ eBioscience™ 00-8333-56) to each well and wash twice. Add 100 μL of diluted secondary antibody RPE-labeled anti-huFc (diluted 1:150 using Perm buffer) to each well and incubate at room temperature for 60 minutes.

[0124] Fluorescence detection: After washing the 96-well plate, 100 μL of FACS buffer was used for resuspension and detection was performed by flow cytometry.

[0125] Two groups with different fluorescence settings were used in the examples to stain each sample. The first group was set as FITC+PE, i.e., first, FITC secondary antibody was used to stain extracellular primary antibody, and then, PE secondary antibody was used to stain intracellular primary antibody after membrane permeabilization. In this group, PE was the intracellular signal and FITC was the extracellular signal. The second group was set as PE+PE, i.e., first, PE secondary antibody was used to stain extracellular primary antibody, and then, PE secondary antibody was used to stain intracellular primary antibody after membrane permeabilization. In this group, PE was the sum of intracellular and extracellular signals. At the same time, both groups of samples were detected using FITC and PE channels, and the value of intracellular uptake rate was calculated as the detection value of PE channel. The specific formula is as follows: Intracellular uptake rate = Group 1 (FITC + PE) PE channel / Group 2 (PE + PE) PE channel × 100%

[0126] The results are shown in Figures 8A to 8B. The results of this example show that for all of the antibodies prepared in this application, the intracellular uptake rate is better than that of the control antibody 99961.1 in a short period of time (within 3 hours).

[0127] 9.2 Evaluation of intracellular uptake of antibodies using the Fab-Zap method The antibody internalization activity was detected in this experiment using the cytotoxicity of antibody-mediated Fab-ZAP internalization. Fab-ZAP is a Fab fragment linked to saporin, a ribosomal inhibitor that can inhibit protein synthesis and cause cell death. The Fab-ZAP used in this experiment is a Fab fragment that binds to the human Fc of a chimeric antibody. After incubation with the chimeric antibody, the Fab-ZAP creates a chimeric antibody that carries a toxin. When the chimeric antibody is internalized, the toxin enters the cell together with the chimeric antibody and causes cell death. Then, the activity of the cells is detected by MTS (Promega, G3580) to measure whether the antibody is internalized.

[0128] The detailed experimental procedure was as follows: First, Fab-Zap was diluted to 0.4 nM with DMEM complete medium, and then each antibody obtained in the present application and the positive control antibody were gradient-diluted with 0.4 nM Fab-ZAP (0.020000, 0.006667, 0.002222, 0.000741, 0.000247, 0.000082, 0.000027, 0.000009 μg / mL) to prepare antibody dilution solutions. huROR1-HEK293 cells in logarithmic growth phase were made into a single cell suspension and the density was adjusted to 6 × 10 6The antibody dilutions were adjusted to cells / mL and 50 μL was inoculated into each well of a 96-well plate. Then, the antibody dilutions were taken out and 50 μL per well was added to the cell culture plate, and the suspension was mixed thoroughly by pipetting. The cell culture plate was placed in a cell culture incubator at 37°C and incubated for 48 hours. After incubation, 7.5 μL of TritonX-100 solution was added to each well and mixed gently by tapping, and the cell culture plate was placed in a cell culture incubator at 37°C for 0.5 hours. Then, 20 μL of MTS was added to each well and incubated at 37°C for 1-4 hours. Finally, the cell culture plate was centrifuged at 1000 rpm for 5 minutes and the data was read by a microplate reader at the detection wavelength of A492.

[0129] The results are shown in Figures 9A to 9F, which show that the intracellular uptake effect of the antibody prepared in this application was equivalent to that of the control antibody under the antibody concentration conditions specified in this Example. Considering that cells only show a sensitive reaction to toxins above a certain threshold, it is difficult to widen the gap when there is a slight difference in the accumulated toxins.

[0130] Example 10 Evaluation of antibody affinity kinetics (Gator) In this example, the affinity of the antibodies obtained in the present application and the positive control antibody 99961.1 for the antigen protein huROR1-His was detected based on the Gator device.

[0131] First, Q buffer was prepared using PBS (10 mM, pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween20 (purchased from thermo) + 0.2% BSA (purchased from source culture), and the stock solution of the test antibody was diluted with the prepared Q buffer to a working solution with a final concentration of 30 nM; the stock solution of the antigen protein huROR1-His was prepared with Q buffer to a working solution with multiple dilutions (480, 240, 120, 60, 30, 15, 7.5 nM), and then the detection and analysis were carried out using the Gator instrument and its accompanying software by choosing to use the advanced kinetic experiment mode. The results are shown in Table 4.

[0132] The results show that, except for C42 antibody, which has an affinity comparable to that of the control antibody 99961.1, all other antibodies show affinities one or more orders of magnitude better than that of the control antibody 99961.1. Among them, B62 antibody has an affinity of 9.84E-10, which is about two orders of magnitude better than that of the control antibody 99961.1.

[0133] Table 4. Results of affinity kinetics test of the antibody obtained in this application [Table 4]

[0134] Example 11 Analysis of antibody affinity kinetics (Biacore) In this example, the affinity of the antibodies obtained in the present application and the positive control antibody 99961.1 to the antigen protein huROR1-His was detected based on a Biacore device.

[0135] Protein coupling: The huROR1-His protein prepared in Example 1.2 was diluted to 5.6 μg / mL with NaAc buffer, pH 5.0, the flow rate was set to 10 μL / min, the chip activation time was set to the initial value of 420 seconds using a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the antigen protein huROR1-His was immobilized to a level of about 75 RU using the coupling mode with a preset coupling amount, and the activated groups that were not bound to the test sample were blocked by ethanolamine.

[0136] 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 at 30 μL / min, the binding time was set at 120 s, and the dissociation time was set at 360 s. After 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.

[0137] Parameter fitting: Experiments were run over multiple cycles, with the response signal being the horizontal axis representing the analysis time and the vertical axis representing the response value. After double reference subtraction, the data obtained was fitted by the BIAcore T200 analysis software. The fitting model employed was the 1:1 Langmuir binding model, and affinity metrics such as binding dissociation constants were determined.

[0138] The results are shown in Table 5. Antibody B62, with a KD of 6.39E-11, had an affinity about two orders of magnitude better than that of the control antibody 99961.1 (the latter having a KD of 1.98E-9); in addition, the affinity of antibody B31 was comparable to that of the control antibody 99961.1.

[0139] Table 5. Results of affinity kinetics test of the antibody obtained in this application [Table 5]

[0140] Example 12 Epitope Classification by Affinity Kinetics The antibodies obtained in this application and the positive control antibody 99961.1 were divided into various epitope-based groups in this example by using affinity kinetic methods.

[0141] The detailed experimental procedure was as follows: First, prepare Q buffer by mixing PBS (10 mM, pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) with 0.02% Tween20 (purchased from Thermo) and 0.2% BSA (purchased from Source culture). Dilute the antibody stock solution to a final concentration of 100 nM with the prepared Q buffer. Similarly, dilute the antigen huROR1-His stock solution to a working solution of 50 nM with Q buffer. Then, detection and analysis were performed using the Gator instrument and its accompanying software with Tandem settings based on the Epitope Binning experiment mode.

[0142] The specific detection and analysis steps were as follows: Equilibration stage 1: the probe is equilibrated in Q buffer at 1000 rpm for 60 s; Antigen immobilization stage: after equilibration stage 1, the probe was immobilized in Q buffer containing 50 nM antigen at 400 rpm for 90 s; Equilibration time 2: after the antigen immobilization stage, the probe was equilibrated in Q buffer at 1000 rpm for 60 seconds; Antibody 1 binding stage: after equilibration stage 2 is completed, the probe is subjected to a binding reaction in Q buffer containing 100 nM antibody 1 at 1000 rpm for 180 seconds; Equilibration time 3: After the antibody 1 binding stage is completed, the probe is equilibrated with Q buffer at 1000 rpm for 30 seconds; Antibody 2 binding stage: after equilibration stage 3 is completed, the probe is subjected to a binding reaction in Q buffer containing 100 nM antibody 2 at 1000 rpm for 180 seconds; Regeneration phase: After one assay, ie, after equilibration stage 1 through antibody 2 binding stage is completed, the probe must be regenerated in Q buffer at 1000 rpm for 50 seconds before the next assay.

[0143] The epitope grouping results obtained for each antibody are shown in Table 6. The results show that the present antibodies and the control antibody 99961.1 are divided into two epitope-based groups, in which only C42 and the control antibody share the same binding epitope, and the remaining antibodies have epitopes different from that of the control antibody.

[0144] Table 6. Summary of epitope grouping results [Table 6]

[0145] Example 13 Humanization of Mouse Antibodies The VH and VL sequences of mouse antibodies B31 and B62 screened in Example 4 were separately compared with known human antibody databases, and the VH and VL sequences of human germline genes were found to have the highest homology to mouse VH and VL sequences, respectively. The framework regions (CDR and framework regions were defined by using AbM) of the corresponding VH and VL sequences of human germline genes were determined, and the complementarity determining region (CDR) sequences of the germline genes were replaced with the corresponding CDR sequences of mouse antibodies B31 and B62 of the present application. Then, with the help of computer prediction simulation, mouse amino acids in the framework regions of mouse antibodies B31 and B62 that have important effects on antigen binding were retained by back mutation. The amino acid sequences of the CDR regions of the humanized antibodies resulting from the humanization of mouse antibodies B31 and B62 are shown in Table 7. Various antibodies resulting from humanization of B31 and B62 were constructed, expressed and purified by the method of Example 5, and various antibody proteins resulting from humanization of B31 and B62 were identified by SDS-PAGE and SEC-HPLC. The results are shown in Table 8. The results show that each antibody showed a band of about 150 kD in non-reducing gel, and a band of about 55 kD and 25 kD in reducing gel, which were consistent with the expected size. The purity of all antibodies obtained in this application is more than 95% by reducing gel detection; the SEC monomer purity of all antibodies is more than 96%.

[0146] Table 7. Amino acid sequences of the CDR regions of humanized antibodies [Table 7-1] [Table 7-2]

[0147] Table 8. Physicochemical properties of humanized antibodies [Table 8]

[0148] Example 14 Evaluation of antigen affinity of humanized antibodies In this example, the affinity of the humanized antibody to the human ROR1 antigen protein huROR1-His was detected based on the ELISA method, and the affinity of the humanized antibody to A549 tumor cells was also detected based on the FACS assay. The specific test method is described in Example 7.

[0149] The ELISA results are shown in Figures 10A-10E. The humanized antibodies have affinities for the antigenic protein that are comparable to those of the corresponding parent molecules and the control antibody 99961.1.

[0150] The FACS results are shown in Figures 11A-11B. Except for B31-H3L4, B31-H4L3, B31-H4L2 and B31-H4L4, the other humanized antibodies have affinities for the antigenic proteins of A549 tumor cells comparable to those of the corresponding parent molecules and the control antibody 9996.1.

[0151] Example 15 Evaluation of intracellular uptake rate of humanized antibodies In this example, the humanized antibodies B31-H3L3 and B62-H3L3 of Example 13 were subjected to Fab-Zap-based intracellular uptake detection. The specific method was mentioned in Example 9. The results are shown in Figure 12. The intracellular uptake rates of B31-H3L3 and B62-H3L3 were comparable to that of the control antibody 99961.1.

[0152] Example 16 Preparation of ADC In this example, antibodies B62-H3L3, B31-H3L3 and control antibody 99961.1 were conjugated with the toxin MMAE (a tubulin inhibitor with anti-cancer activity) to construct antibody-drug conjugates. MMAE was connected to 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, thereby binding the antibody to MMAE to obtain the antibody-drug conjugate. IgG1 antibody has 16 pairs of cysteine ​​residues, which exist in the form of 12 pairs of intrachain disulfide bonds and 4 pairs of interchain disulfide bonds. The interchain disulfide bonds are solvent accessible and can be reduced by reducing agents to form eight sulfhydryl groups, which then become targets for conjugation (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015;17:339-51).

[0153] The specific preparation method was as follows: Antibodies B62-H3L3, B31-H3L3 and control antibody 99961.1 were removed from a -80°C refrigerator, thawed, and transferred separately to 15mL 30KD ultrafiltration centrifuge tubes, and coupling buffer (ingredients per liter: Na2HPO4·2H2O 6.86g, NaH2PO4·H2O 1.58g, purified water up to 1000g total, pH 7.4) was added to a total of 15mL. The solution was then centrifuged at 4500 rpm for approximately 30 minutes, concentrated to 2-3mL, and supplemented with dialysate (ingredients per liter: histidine 0.73g, histidine hydrochloride monohydrate 1.12g, purified water up to 1000g total, pH 6.0) to a final volume of 15mL. Dialysis was repeated 8-10 times to obtain antibody stock solutions, and antibody concentrations were measured after dialysis.

[0154] The thiol groups on the cysteines of the antibody were reduced by a reduction reaction system (formed by adding, in order, the antibody stock solution, 10 mM disulfide bond reducing agent TCEP stock solution (tris(2-carboxyethyl)phosphine hydrochloride stock solution, contents per 1 L: Na2HPO4·2H2O 6.86 g NaH2PO4·H2O 1.58 g, purified water up to a total of 1000 g), 10 mM DTPA stock solution (diethylenetriaminepentaacetic acid stock solution, contents per 1 L: DTPA 3.90 g, NaOH 1.20 g, purified water up to a total of 1000 g) and coupling buffer). The amount of each component added to the reduction reaction system so that the antibody concentration of the reduction reaction system was 5 mg / mL, the final concentration of DTPA was 1 mM, the molar ratio of TCEP to B62-H3L3 or B31-H3L3 was 2, and the molar ratio of TCEP to 9996.1 was 2.2 is shown in Table 9. After thorough mixing, the reduction reaction system was placed in a constant temperature shaker at 25° C. with a rotation speed of 400 rpm for 2 hours of reduction reaction.

[0155] MC-VC-PAB-MMAE was weighed and dissolved in DMSO to prepare 5 mM MC-VC-PAB-MMAE stock solution. After the reduction reaction was completed, the MC-VC-PAB-MMAE stock solution was added to the reduction reaction system in an ice-water bath in order, and the amount added to prepare the coupling reaction system was shown in Table 10. After mixing thoroughly, the coupling reaction system was placed in a constant temperature shaker at 400 rpm at 25° C. and coupled for 1 hour to obtain a solution containing ADC.

[0156] After the coupling reaction was completed, the solution containing ADC was centrifuged and filtered to obtain an ADC sample, which was transferred to a 15mL 30KD ultrafiltration centrifuge tube. Dialysis solution was added to 15mL, centrifuged at 4500rpm for 20 minutes to concentrate to 2-3mL, and then replenished with dialysate to 15mL again. Dialysis was repeated 8-10 times. The ADC sample after dialysis was subjected to SEC-HPLC detection, HIC-HPLC detection, concentration detection, free drug detection, etc. The results are shown in Table 11. The results show that ADC with a purity of more than 99% was obtained.

[0157] Table 9. Components of the reduction reaction system [Table 9]

[0158] Table 10. Amount of MC-VC-PAB-MMAE stock solution added [Table 10]

[0159] Table 11. ADC sample results [Table 11]

[0160] Example 17 Evaluation of antigen-binding activity of ADC In this example, the ability of the prepared ADCs (B62-H3L3-MMAE and B31-H3L3-MMAE) to bind to human ROR1 on tumor cells A549 and HT-29 (human colon cancer cells) was detected based on a FACS assay.

[0161] The specific method was as follows: A549 cells or HT-29 cells in the logarithmic growth phase were prepared into a single cell suspension and the density was adjusted to 1 × 10 6The cells were adjusted to 100 μL / well of cells were added to a 96-well round-bottom plate, then centrifuged at 300 g at 4° C. and the supernatant was removed. Gradient dilutions (1.0000, 0.3333, 0.1111, 0.0370, 0.0123, 0.0041, 0.0014, 0.0001 μg / mL) of the present antibodies (B62-H3L3 and B31-H3L3), the present ADCs (B62-H3L3-MMAE and B31-H3L3-MMAE), 99961.1, 99961.1-MMAE, and negative control were added to the corresponding wells, mixed thoroughly, and incubated at 4° C. for 30 minutes. After incubation, the 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 for 30 minutes at 4°C in the dark. After washing three times, the cells were detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0162] The results are shown in Figures 13A-13B: The results of binding to A549 cells are shown in Figure 13A. After coupling, the activity of B62-H3L3-MMAE and 99961.1-MMAE binding to A549 cells is slightly inferior to that of the corresponding naked antibodies, and the activity of B62-H3L3 and B62-H3L3-MMAE binding to A549 cells is superior to that of the corresponding positive control. The results of binding to HT-29 cells are shown in Figure 13B. The activity of the naked antibodies binding to HT-29 cells is equivalent to that of the corresponding ADC, and the activity of B62-H3L3 and B62-H3L3-MMAE binding to HT-29 cells is slightly superior to that of the corresponding positive control.

[0163] Example 18 Evaluation of tumor cell killing effect of ADC based on MTS assay In this example, A549 and HT-29 cells were used to detect the killing effects of the present and control ADCs.

[0164] The specific method was as follows: A549 cells or HT-29 cells in logarithmic growth phase were prepared into a single cell suspension, and the density of A549 was adjusted to 1 × 10 4 Adjust the density of HT-29 to 1.5 x 10 cells / mL. 4 The cells / mL were adjusted and 100 μL / well was added to a 96-well cell culture plate and incubated at 37° C., 5% CO2 for 12 hours. Gradient diluted ADC samples (2000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 1.953 nM) were then added and incubated at 37° C., 5% CO2 for 72 hours (A549 cells) or 96 hours (HT-29 cells). 40 μL of MTS (Promega, G3580) was then added to each well and incubated at 37° C. for 1 hour and data was read at OD492 using a microplate reader.

[0165] The results are shown in Figures 14A-14D: the killing effect of B31-H3L3-MMAE against two types of tumor cells, A549 (Figure 14A) and HT-29 (Figure 14C), is slightly superior to that of the control 99961.1-MMAE, whereas the killing effect of B62-H3L3-MMAE against A549 (Figure 14B) and HT-29 (Figure 14D) is slightly inferior to that of the control 99961.1-MMAE.

[0166] Example 19 Evaluation of tumor cell killing effect of ADC based on CCK8 assay In the examples, 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 effects of B31-H3L3-MMAE, B62-H3L3-MMAE, and control ADCs.

[0167] The specific method was as follows: Jeko-1 cells, MDA-MB-468 cells, or NCI-H1944 cells in the logarithmic growth phase were prepared into a single cell suspension, and the density of Jeko-1 was adjusted to 1 × 10 5 Adjust the density of MDA-MB-468 to 2 x 10 cells / mL.5 Adjust the cell density to 1.2 x 10 cells / mL for NCI-H1944. 5 The cells were adjusted to 1000 cells / mL and 90 μL / well of the single cell suspension was added to a 96-well cell culture plate and incubated at 37° C. and 5% CO2 for 12 hours. Gradient diluted ADC samples (500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, and 0.05 nM) were then added. The cells were incubated at 37° C. and 5% CO2 for 72 hours. Then, 10 μL of CCK8 (Bimake, B34304) was added to each well and incubated at 37° C. for 1 hour, and the data was read at OD450 using a microplate reader.

[0168] The experimental results are shown in Figures 15A to 15C. The killing effects of B31-H3L3-MMAE and B62-H3L3-MMAE against three tumor cells, Jeko-1 (FIG. 15A), MDA-MB-468 (FIG. 15B), and NCI-H1944 (FIG. 15C), were comparable to that of the control 99961.1-MMAE, where the EC50 of B31-H3L3-MMAE against Jeko-1, MDA-MB-468, and NCI-H1944 was 38.24 nM, 15.24 nM, and 117.3 nM, respectively, and the EC50 of B62-H3L3-MMAE against Jeko-1, MDA-MB-468, and NCI-H1944 was 50.14 nM, 28.64 nM, and 128.6 nM, respectively.

[0169] Example 20 Evaluation of antigen-dependent killing effect of ADC based on CCK8 assay In this example, huROR1-HEK293 cells were used to detect the antigen-dependent killing effect of B31-H3L3-MMAE and control ADC.

[0170] The specific method was as follows: huROR1-HEK293 cells in logarithmic growth phase were prepared into a single cell suspension, and the cell density was adjusted to 6 × 10 4The cells / mL were adjusted and 50 μL / well of the single cell suspension was added to a 96-well cell culture plate. After 24 hours of incubation at 37°C and 5% CO2, 50 μL of 100 μg / mL antibody B31-H3L3 or positive control antibody 99961.1 was added to each well. After 2 hours of incubation at 37°C, gradient dilutions of ADC B31-H3L3-MMAE or control ADC 99961.1-MMAE (42.6667, 21.3333, 10.6667, 5.3333) were added to the corresponding wells. After 96 hours of incubation at 37°C and 5% CO2, 30 μL of CCK8 (absin / Aibixin, abs50003) was added to each well and incubated at 37°C for 1-4 hours. The plates were then read at OD450 using a microplate reader. A system with only ADC but no antibody was used as a control.

[0171] The results are shown in Figure 16. The killing effect of the ADC against huROR1-HEK293 cells could be significantly antagonized by the corresponding antibodies, B31-H3L3 and 99961.1, demonstrating that the cell killing of the ADC prepared in this application was due to the binding of the antigen on the cell surface with the antibody attached to the ADC, and thus was antigen-dependent killing rather than non-specific killing by the attached toxin.

[0172] Example 21 Evaluation of cellular uptake efficiency of ADC In this example, A549 and HT-29 tumor cells were used to detect the intracellular uptake efficiency of the ADC obtained in this application based on FACS assay. For detailed experimental method, please refer to Example 9.

[0173] The experimental results showed that in A549 cells (Figure 17A), the intracellular uptake rate of B31-H3L3-MMAE was comparable to that of the control ADC 99961.1-MMA, the intracellular uptake rate of B62-H3L3-MMAE was slightly lower than that of the positive control ADC, and the intracellular uptake efficiency of the ADCs was all higher than that of the corresponding antibodies. In HT-29 cells (Figure 17B), the intracellular uptake rate of B31-H3L3-MMAE was slightly higher than that of the positive control ADC, the intracellular uptake rate of B62-H3L3-MMAE was slightly lower than that of the positive control ADC, and the intracellular uptake efficiency of the antibodies was mostly lower than that of the corresponding ADCs.

[0174] Example 22 Evaluation of the inhibitory effect of ADCs in a mouse model bearing HT-29 tumors In this example, the antitumor effects of two candidate ADCs (B31-H3L3-MMAE and B62-H3L3-MMAE) were examined in animals, 99961.1-MMAE was used as a positive control, and the tumor cells used were colon cancer cells HT-29 (BNCC337732).

[0175] The specific method was as follows: 6-8 week-old female Balb / C nude mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) weighing about 20 g were used, and 1 × 10 6 HT-29 cells were injected subcutaneously into each nude mouse alone. 3When the mice reached 10 mg / kg, they were randomly divided into cages into 9 groups (6 tumor-bearing nude mice per group), including a PBS negative control group, 5 candidate ADC groups (B31-H3L3-MMAE 0.4 mg / kg (mpk), B31-H3L3-MMAE 2 mg / kg, B31-H3L3-MMAE 10 mg / kg, B62-H3L3-MMAE 2 mg / kg, and B62-H3L3-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). Drugs were administered by tail vein injection twice a week, and tumor volumes were measured twice for a total of 8 doses / 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, the tumors were excised, and the tumor weights were measured. Data on tumor volume, tumor weight, and mouse body weight change were analyzed, and tumor growth inhibition was calculated. The results are shown in Figures 18A-18C and Table 12.

[0176] The experimental results show that there is no significant difference in the mouse weight of each group, and there is no significant change in the mouse weight of each group during treatment, indicating that the mice have good tolerance to the ADC (Figure 18B); B31-H3L3-MMAE, B62-H3L3-MMAE, and the control ADC 99961.1-MMAE all show significant tumor inhibition effects under high dose conditions (10mpk). Under high dose conditions, B31-H3L3-MMAE shows tumor inhibition effects equivalent to those of 99961.1-MMAE, and B62-H3L3-MMAE shows tumor growth inhibition that is slightly weaker than that of the control ADC 99961.1-MMAE. Under low dose conditions (2 mpk), the tumor growth inhibition of B62-H3L3-MMAE is significantly better than that of the control ADCs 99961.1-MMAE and B62-H3L3-MMAE (Figure 18A, Figure 18C, and Table 12).

[0177] Table 12. Tumor growth inhibition of ADCs in mice [Table 12]

[0178] Example 23 Evaluation of the inhibitory effect of ADCs in an A549 tumor-bearing mouse model In this example, the antitumor effects of two candidate ADCs (B31-H3L3-MMAE and B62-H3L3-MMAE) were detected in animals. The tumor cells used were non-small cell lung cancer cells A549 (Shanghai Cell Bank, Chinese Academy of Sciences, C2107019), and 99961.1-MMAE was used as a positive control.

[0179] The specific method was as follows: 6-8 week old female nude mice (Balb / C, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) weighing about 18-20 g were used, and 1 × 10 6 A549 cells were injected subcutaneously into the right dorsal surface of each nude mouse. 3 When the mice reached 10 mg / kg, they were randomly divided into cages. They were divided into a total of four groups (six tumor-bearing nude mice per group), including a PBS negative control group, two candidate ADC groups (B31-H3L3-MMAE 10 mpk (mg / kg) and B62-H3L3-MMAE 10 mpk), and one positive control ADC group (99961.1-MMAE 10 mpg). Drugs were administered via tail vein injection twice a week, and tumor volumes were measured twice for a total of six administrations / 3 weeks (BIW*3). Tumor volume (V) was calculated as follows: V=L×W 2 / 2 (wherein L is the longest tumor diameter and W is the shortest tumor diameter). After a specific observation period after the end of drug administration, the mice were euthanized, the tumors were excised, and the tumor weights were measured. The data on tumor volume, tumor weight, and mouse body weight change were analyzed, and the tumor growth inhibition was calculated. The results are shown in Figures 19A-19C and Table 13.

[0180] The experimental results show that there was no significant change in the mouse body weight of each group during treatment, indicating that the mice tolerated the ADC well (Figure 19B); B62-H3L3-MMAE showed an antitumor effect comparable to that of the positive control ADC at the same dose, and B31-H3L3-MMAE showed an antitumor effect superior to that of the positive control ADC (Figures 19A, 19C and Table 13).

[0181] Table 13. Tumor growth inhibition of ADCs in mice [Table 13]

[0182] Example 24 Evaluation of the inhibitory effect of ADCs in a mouse model bearing NCI-N87 tumors In this example, the antitumor effect of the candidate ADC (B31-H3L3-MMAE) was detected in animals. The tumor cells used were gastric cancer cells NCI-N87 (Shanghai Cell Bank of the Chinese Academy of Sciences, C2009021, P3), and 99961.1-MMAE was used as a positive control.

[0183] The specific method was as follows: 6-8 week old female nude mice (Balb / C, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) weighing about 18-20 g were used, and 1 × 10 6 NCI-N87 cells were injected subcutaneously into the right dorsal surface of each nude mouse. 3When the mice reached 10 mg / kg, they were randomly divided into cages. They were divided into a total of 5 groups (10 tumor-bearing nude mice per group), including a PBS negative control group, two candidate ADC groups (B31-H3L3-MMAE 5 mpk (mg / kg) and B31-H3L3-MMAE 10 mpk), and two positive control ADC groups (99961.1-MMAE 5 mpg and 99961.1-MMAE 10 mpg). Drugs were administered once a week via tail vein injection, and tumor volumes were measured twice for a total of 3 administrations / 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). Data on tumor volume and mouse weight change were analyzed, and tumor growth inhibition was calculated. The results are shown in Figures 20A-20B and Table 14.

[0184] The experimental results showed that there was no significant change in the mouse body weight of each group during treatment, indicating that the mice tolerated the ADC well (Figure 20B); compared with the PBS group, each dose group had a significant tumor inhibitory effect, and the 10 mpk B31-H3L3-MMAE molecule had a tumor inhibitory effect comparable to that of the 99961.1-MMAE molecule (Figure 20A and Table 14).

[0185] Table 14. Tumor growth inhibition of ADCs in mice [Table 14]

[0186] Example 25 Evaluation of the inhibitory effect of ADC in a mouse model bearing MDA-MB-231 tumors In this example, the antitumor effect of the candidate ADC (B31-H3L3-MMAE) was detected in animals. The tumor cells used were triple-negative breast cancer cells MDA-MB-231 (Shanghai Cell Bank of the Chinese Academy of Sciences, C2006040), and 99961.1-MMAE was used as a positive control.

[0187] The specific method was as follows: 6-8 week old female nude mice (NSG, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) weighing about 20-22 g were used, and 1 × 10 6 MDA-MB-231 cells were injected subcutaneously into the back of each nude mouse. 3 When the mice reached 10 mg / kg, they were randomly divided into cages. They were divided into a total of 5 groups (10 tumor-bearing nude mice per group), including a PBS negative control group, two candidate ADC groups (B31-H3L3-MMAE 5 mpk (mg / kg) and B31-H3L3-MMAE 10 mpk), and two positive control ADC groups (99961.1-MMAE 5 mpg and 99961.1-MMAE 10 mpg). Drugs were administered once a week via tail vein injection, and tumor volumes were measured twice for a total of 3 administrations / 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). Data on tumor volume and mouse weight change were analyzed, and tumor growth inhibition was calculated. The results are shown in Figures 21A-21B and Table 15.

[0188] The results show that there was no significant difference in the early stage of drug administration within each group of mice. The PBS group began to show weight loss on day 35, which was expected to be due to tumor overload. There was no significant change in mouse weight in the ADC group and the positive control group, indicating that the mice had good tolerance to the ADC (Figure 21B). Compared with the PBS group, the positive control and the high-dose test ADC group had considerable antitumor effects and showed significant effects. Under low-dose conditions, the test ADC (B31-H3L3-MMAE) shows a better antitumor effect than that of the positive control ADC. The low-dose group shows some tumor tissue rebound after withdrawal (Figure 21A and Table 15).

[0189] Table 15. Tumor growth inhibition of ADCs in mice [Table 15]

[0190] Example 26 Evaluation of the inhibitory effect of ADCs in a mouse model bearing MDA-MB-468 tumors In this example, the antitumor effect of the candidate ADC (B31-H3L3-MMAE) was detected in animals. The tumor cells used were triple-negative breast cancer cells MDA-MB-468 (Shanghai Cell Bank of the Chinese Academy of Sciences, TCHu136), and 99961.1-MMAE was used as a positive control.

[0191] The specific method was as follows: 6-8 week old female nude mice (NOD SCID, Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) weighing about 21-25 g were used, and 1 × 10 7 MDA-MB-468 cells were injected subcutaneously into the back of each nude mouse. 3 When the mice reached 100 mg / kg, they were randomly divided into cages into 5 groups (6 tumor-bearing nude mice in each group), including a PBS negative control group, 2 candidate ADC groups (B31-H3L3-MMAE 5mpk (mg / kg) (QW*3), B31-H3L3-MMAE 10mpk (QW*3)), and 2 positive control ADC groups (99961.1-MMAE 5mpk (QW*3), 99961.1-MMAE 10mpk (QW*3)); 2 groups (3 tumor-bearing nude mice in each group), including B31-H3L3-MMAE 10mpk (single dose) and 99961.1-MMAE 10mpk (single dose), and the administration methods were tail vein injection for a total of 3 doses / 3 weeks (BW*3) or a single dose. 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). The data on tumor volume and mouse weight change were analyzed, and tumor growth inhibition was calculated. The results are shown in Figures 22A-22B and Tables 16-17.

[0192] The experimental results showed that there was no significant change in the mouse weight of each group during the treatment period, indicating that the mice had good tolerance to ADC (Figure 22B); the tumor growth inhibition of the group administered 10mpk B31-H3L3-MMAE by QW*3 was slightly higher than that of the group administered 10mpk 99961.1-MMAE by QW*3 (Table 17), but all mice achieved complete remission (CR) about 18 days after administration; the tumor inhibition effect of the group administered 5mg / kg B31-H3L3-MMAE by QW*3 was superior to that of the group administered 5mg / kg 99961.1-MMAE by QW*3, and 4 / 6 mice achieved CR on the 25th day after administration. The single 10 mg / kg B31-H3L3-MMAE group showed superior tumor inhibition compared to the single 10 mg / kg 99961.1-MMAE group, with all mice achieving CR by day 18 after dosing, whereas the 99961.1-MMAE group showed no tumor recurrence by day 20 after dosing. In this experiment, no tumor recurrence was observed in any of the B31-H3L3-MMAE groups (Figure 22A and Tables 16-17).

[0193] Table 16. Tumor growth inhibition of ADCs in mice [Table 16]

[0194] Table 17. Complete response rate of ADC in mice [Table 17]

[0195] Example 27 Evaluation of the inhibitory effect of ADCs in a mouse model bearing Jeko-1 tumors In this example, the antitumor effect of the candidate ADC (B31-H3L3-MMAE) was detected in animals. The tumor cells used were mantle cell lymphoma Jeko-1 cells (ATCC, CRL-3006), and 99961.1-MMAE was used as a positive control.

[0196] The specific method was as follows: 6-8 week old female nude mice (BALB / c, Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) weighing about 21-25 g were used, and 1 × 10 7 Jeko-1 cells were injected subcutaneously into the back of each nude mouse. 3 When the mice reached 10 days of age, they were randomly assigned to cages. The PBS negative control group, four ADC groups (B31-H3L3-MMAE 2.5mpk (mg / kg) (QW*3), B31-H3L3-MMAE 10mpk (QW*3), B31-H3L3-MMAE 10mpk (single dose) and B31-H3L3-MMAE 2.5+3.5mpk (Q2W*2, i.e., 2.5mpk on day 15 and 3.5mpk on day 29, abbreviated as D15 2.5mpk+D29 3.5mpk) and four positive control ADC groups (99961.1-MMAE 2.5mpk (QW*3), 99961.1-MMAE 10mpk (QW*3), 99961.1-MMAE The tumor-bearing nude mice were divided into a total of 9 groups (7 mice per group), including 10mpk (single dose), and 99961.1-MMAE 2.5+3.5mpk (Q2W*2, i.e. 2.5mpk on day 15 and 3.5mpk on day 29, abbreviated as D15 2.5mpk+D29 3.5mpk), and the administration method was tail vein injection for a total of 3 doses / 3 weeks (BW*3) or single dose (single dose) or 2 doses / 3 weeks (Q2W*2). The 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). Data on tumor volume and mouse weight change were analyzed, and tumor growth inhibition was calculated. The results are shown in Figures 23A-23B and Tables 18-19.

[0197] The experimental results showed that there was no significant change in the body weight of mice in each group during treatment, indicating that the mice had good tolerance to the ADC (Figure 23B); Mice in the QW*3, B31-H3L3-MMAE-10mpk and 99961.1-MMAE-10mpk groups had comparable tumor growth inhibition rates TGI of about 96%, and no tumor recurrence was observed for 23 days after administration; B31-H3L3-MMAE-2.5mpk and 99961.1-MMAE-2.5mpk groups had comparable antitumor effects, with TGIs of 60.47% and 59.22%, respectively. In the single-dose groups, B31-H3L3-MMAE (10mpk) and 99961.1-MMAE (10mpk) groups had TGIs of 79.24% and 91.74%, respectively, and no tumor recurrence was observed for 23 days after administration. In the Q2W*2 dosing groups, the B31-H3L3-MMAE (2.5+3.5mpk) dosing group had a tumor inhibitory effect comparable to that of the 99961.1-MMAE (2.5+3.5mpk) group, with TGIs of 34.96% and 34.60%, respectively (Figure 23A and Tables 18-19).

[0198] Table 18. Tumor growth inhibition of ADCs in mice [Table 18]

[0199] Table 19. Tumor growth inhibition of ADCs in mice [Table 19]

[0200] Sequence Listing [Table 20-1]

Table 20-2

Table 20-3

Table 20-4

Table 20-5

Table 20-6

Table 20-7

Table 20-8

Table 20-9

Table 20-10

Table 20-11

Table 20-12

Table 20-13

Claims

1. Anti-ROR1 antibodies that specifically bind to ROR1 and their antigen-binding fragments, including the following: HCDR1, shown in the sequence of sequence number 4, The HCDR2 shown in sequence number 5, HCDR3, shown in sequence number 6, LCDR1, which is shown in sequence number 43, LCDR2, shown in the sequence of sequence number 46, and LCDR3 is shown in the sequence of sequence number 48.

2. It includes a heavy chain variable region and a light chain variable region, where: The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 77, or includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 77, and includes HCDR1, HCDR2, and HCDR3 shown in the sequences of SEQ ID NOs: 4, 5, and 6, or consists of SEQ ID NO: 77; the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 81, or includes an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 81, and includes LCDR1, LCDR2, and HCDR3 shown in the sequences of SEQ ID NOs: 43, 46, and 48, or consists of SEQ ID NO:

81. The anti-ROR1 antibody and its antigen-binding fragment according to claim 1.

3. An isolated anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, Fv, or (Fab')2 fragments.

4. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 3, wherein the Fv is scFv.

5. An isolated anti-ROR1 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a constant region sequence, wherein at least a portion of the constant region sequence is a human consensus constant region sequence.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 52, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

53.

7. Including heavy and light chains, here: The heavy chain contains an amino acid sequence shown in SEQ ID NO: 113, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 113, or consists of SEQ ID NO: 113, and the light chain contains an amino acid sequence shown in SEQ ID NO: 117, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 117, or consists of SEQ ID NO:

117. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

8. It has the structure Ab-(LD)n, where In the formula, Ab is the anti-ROR1 antibody or its antigen-binding fragment according to claim 1, L is a linker, D is a therapeutic active substance or pharmaceutically active ingredient, and n represents an integer from 1 to 20. The therapeutic active substance or pharmaceutically active ingredient is drastatin and its auristatin derivative. Antibody-drug conjugate.

9. The antibody-drug conjugate according to claim 8, wherein the therapeutic active substance or pharmaceutically active ingredient is 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), or auristatin F hydroxypropylamide (AFHPA).

10. The antibody-drug conjugate according to claim 9, wherein the aforementioned drastatin and its auristatin derivative are MMAE, MMAF, auristatin F hydroxypropylamide, or auristatin F phenylenediamine.

11. The linker, 1) Linkers that can be broken down by cathepsin: valine-citrulline (val-cit) linker, cBu-Cit linker and CX linker, 2) Linkers that cannot be cut: SMCC linker or MD linker, 3) Acid-sensitive linker, 4) Silicone structured linker, 5) Disulfide-carbamate linker, 6) MC-GGFG linker, 7) TRX linker, 8) Galactoside-containing linker, 9) Pyrophosphate linker, 10) Near-infrared sensitive linker, and 11) UV-sensitive linker: PC4AP An antibody-drug conjugate according to any one of claims 8 to 10, selected from the group consisting of the following.

12. The linker is maleimide-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimi An antibody-drug conjugate according to claim 11, selected from the group consisting of 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-(pyridine-2-yldithio)-propionate (SPDP).

13. The antibody-drug conjugate according to claim 12, wherein the linker is MC-VC-PAB, SMCC, or MC-GGFG.

14. The aforementioned Ab is as follows: HCDR1, shown in the sequence of sequence number 4, The HCDR2 shown in sequence number 5, The sequence of sequence number 6 shows HCDR3, LCDR1, shown in sequence number 43, LCDR2, shown in the sequence of sequence number 46, and LCDR3 shown in sequence number 48 including, The antibody-drug conjugate according to any one of claims 8 to 10.

15. The aforementioned Ab includes a heavy chain variable region and a light chain variable region, where: The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 77, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

81. The antibody-drug conjugate according to claim 14.

16. The aforementioned Ab is as follows: The amino acid sequence of the heavy chain shown in SEQ ID NO: 113, and the amino acid sequence of the light chain shown in SEQ ID NO: 117, including, The antibody-drug conjugate according to claim 15.

17. The antibody-drug conjugate according to claim 14, wherein the linker is MC-VC-PAB and the cytotoxin is MMAE.

18. below: (1) The anti-ROR1 antibody or antigen-binding fragment thereof as described in claim 1, or the antibody-drug conjugate as described in any one of claims 8 to 10; and (2) Carriers that are acceptable as pharmaceuticals, including, Pharmaceutical composition.

19. An isolated polynucleotide molecule encoding the anti-ROR1 antibody or its antigen-binding fragment as described in claim 1.

20. A vector comprising the polynucleotide molecule described in claim 19.

21. The vector according to claim 20, wherein the vector is an expression vector.

22. A host cell comprising a vector according to claim 20 or 21, or an isolated polynucleotide molecule encoding an anti-ROR1 antibody or its antigen-binding fragment according to claim 1.

23. Use of the anti-ROR1 antibody or its antigen-binding fragment according to claim 1 in the production of an antibody-drug conjugate for the prevention or treatment of cancer associated with high ROR1 expression.

24. Use of the anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 in the manufacture of a drug for preventing or treating a disease associated with high expression of ROR1.

25. Use of an antibody-drug conjugate according to any one of claims 8 to 10 in the manufacture of a drug for preventing or treating a disease associated with high expression of ROR1.

26. The use or method according to claim 23, wherein the cancer associated with high expression of ROR1 is chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer, breast cancer, neuroblastoma, lung cancer, gastric cancer, head and neck cancer, and melanoma.

27. A non-therapeutic method for killing cells expressing ROR1 or inhibiting the proliferation of cells expressing ROR1, wherein the cells An effective amount of the antibody or antigen-binding fragment thereof described in claim 1, or An effective amount of the antibody-drug conjugate described in any one of claims 8 to 10, or Effective amount, as follows: (1) The anti-ROR1 antibody or antigen-binding fragment thereof as described in claim 1, or the antibody-drug conjugate as described in any one of claims 8 to 10; and (2) Carriers that are acceptable as pharmaceuticals, A pharmaceutical composition containing A method that includes bringing into contact with it.

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