Binding molecule targeting ROR1 and application thereof

Abstract

The invention relates to an antibody capable of specifically recognizing ROR1 as well as a preparation method and application of the antibody. The present application also relates to antibody-conjugated drugs (ADCs) targeting ROR1 and compositions containing the molecules. In addition, the invention also relates to therapeutic and diagnostic uses of these antibodies, antibody fragments and antibody-conjugated drugs.

Description

[0001] This application is a divisional application of Chinese application 202380025939.0, whose application date is March 3, 2023 and whose invention name is “A binding molecule targeting ROR1 and its application”. Technical Field

[0002] The present application relates to antibodies that specifically recognize ROR1, and methods for preparing and using the same. In addition, the present application also relates to antibody-drug conjugates (ADCs) targeting ROR1 and compositions containing the same. In addition, the present invention also relates to therapeutic and diagnostic uses of these antibodies, antibody fragments, and antibody-drug conjugates. Background Art

[0003] Antibody-Drug Conjugates (ADC) are derivative drugs of traditional antibody drugs. They are made by coupling monoclonal antibody drugs targeting specific antigens and small molecule cytotoxic drugs through linkers. They have both the specific targeting of antibody drugs and the killing effect of traditional small molecule drugs. Their main indications are malignant tumors. ADC has quickly become a hot spot in the development of anti-tumor drugs due to its excellent tumor killing effect. However, the toxicity of ADC is usually high, so the specificity of the target antigen is required to be high. The targets suitable for the development of ADC are generally highly expressed in tumors and low or almost not expressed in healthy tissues; the antigen is a surface receptor expressed upregulated by tumor cells, which can promote tumor growth or survival, and the target antigen should have internalization properties.

[0004] Receptor tyrosine kinase-like orphan receptor 1 (ROR1, also known as receptor-associated neurotrophic tyrosine kinase 1, NTRKR1) and receptor tyrosine kinase-like orphan receptor 2 (ROR2) are single-pass transmembrane proteins belonging to the receptor tyrosine kinase (RTK) family. They are composed of an immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD domain and KRD domain) extracellularly, and a tyrosine kinase domain, two serine- or threonine-rich domains and a proline-rich domain intracellularly. ROR1 and ROR2 participate in the non-canonical Wnt signaling pathway by binding to the ligand Wnt5a through the FZD domain (Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara B, Koshida I, Suzuki K, Yamada G, Schwabe GC et al (2003). Genes Cells 8:645–654; Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al (2008). Proc Natl Acad Sci USA 105:3047–3052; Paganoni S, Bernstein J, Ferreira A (2010). Neuroscience 165:1261–1274).ROR1 can inhibit cell apoptosis, enhance EGFR signaling, and induce epithelial-mesenchymal transition (EMT) (Fukuda T, Chen L, Endo T, Tang L, Lu D, Castro JE, Widhopf GF II, Rassenti LZ, Cantwell MJ, Prussak CE et al (2008). Proc Natl Acad Sci USA 105:3047–3052; Yamaguchi T, Yanagisawa K, Sugiyama R, Hosono Y, Shimada Y, Arima C, Kato S, Tomida S, Suzuki M, Osada Het al (2012). Cancer Cell 21:348–361; Cui B, Zhang S, Chen L, Yu J, Widhopf GF, Fecteau JF, Rassenti LZ, Kipps TJ (2013). Cancer Res 73:3649–3660).

[0005] ROR1 is a conserved embryonic protein, whose expression gradually decreases with embryonic development and is almost absent or lowly expressed in most adult tissues. However, more and more literature has found that ROR1 is expressed in a variety of cancer cells, such as B-cell chronic lymphocytic leukemia (CLL) and other hematological malignancies, renal cell carcinoma, colon cancer and some other cancer cell lines of breast cancer. In addition, ROR1 plays an important role in the progression of many hematological and solid malignancies. Therefore, as a cancer marker, ROR1 has become an ideal drug target for cancer treatment.

[0006] Although the prior art discloses several antibody drugs targeting ROR1, as a pan-cancer tumor marker, there is still an urgent need to develop high-quality anti-ROR1 antibodies, which can be used as the basis for developing antibody-based targeted therapies for cancers expressing ROR1, and can also be used as a diagnostic tool to detect ROR1 expression in ROR1-related diseases. In addition, based on the good prospects shown by ADC in the field of tumor treatment, there is still an urgent need for ADCs containing ROR1 with effective therapeutic effects, and the present invention meets these needs. Summary of the invention

[0007] In a first aspect, the present invention provides an antibody targeting ROR1, which has the following advantages:

[0008] (1) High affinity binding to ROR1 and target cells expressing ROR1;

[0009] (2) Ability to enter cells via endocytosis;

[0010] (3) Suitable for constructing effective therapeutic antibody-drug conjugates.

[0011] In one embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising:

[0012] 1) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 57 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 56;

[0013] 2) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:55 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:54;

[0014] 3) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:59 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:58;

[0015] 4) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:61 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:60;

[0016] 5) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 63 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 62;

[0017] 6) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 65 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 64;

[0018] 7) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 67 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 66;

[0019] 8) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:68 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:66;

[0020] 9) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 70 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 69;

[0021] 10) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 72 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 71;

[0022] 11) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 74 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 73;

[0023] 12) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 75 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 79;

[0024] 13) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 76 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 81;

[0025] 14) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 77 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 82;

[0026] 15) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 78 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 80;

[0027] 16) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 78 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 81;

[0028] 17) three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 78 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO: 82; or

[0029] 18) The three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO:84 and the three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:89.

[0030] In one embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising:

[0031] 1) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 28, 29 and 30, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0032] 2) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 32 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0033] 3) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 34, 32 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0034] 4) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 35, 32 and 36, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0035] 5) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 13 and 14, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 35, 32 and 37, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0036] 6) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 15, 16 and 17, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 38, 32 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0037] 7) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 18, 8 and 19, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 32 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0038] 8) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 20, 21 and 12, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 32 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0039] 9) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 22, 23 and 24, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 39, 40 and 41, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0040] 10) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 25 and 26, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 35, 32 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0041] 11) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 18, 25 and 27, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 35, 32 and 33, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0042] 12) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 45 and 48, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0043] 13) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 43, 46 and 48, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0044] 14) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 44, 47 and 48, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0045] 15) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 42 and 6, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 31, 45 and 48, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0046] 16) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 42 and 6, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 43, 46 and 48, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to the sequences;

[0047] 17) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 4, 42 and 6, or sequences containing one or more and no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to 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 no more than 3 amino acid substitutions (e.g., conservative substitutions), deletions or insertions of amino acids relative to said sequences; or

[0048] 18) HCDR1, HCDR2, HCDR3 comprising the sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences containing one or more and no more than 3 amino acids in amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to the sequences; and LCDR1, LCDR2, LCDR3 comprising the sequences shown in SEQ ID NOs: 49, 50 and 30, or sequences containing one or more and no more than 3 amino acids in amino acid substitutions (e.g., conservative substitutions), deletions or insertions relative to the sequences.

[0049] In one embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising a heavy chain variable region, wherein:

[0050] 1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:57, or consists of SEQ ID NO:57;

[0051] 2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:55, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:55, or consists of SEQ ID NO:55;

[0052] 3) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:59, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:59, or consists of SEQ ID NO:59;

[0053] 4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:61, or consists of SEQ ID NO:61;

[0054] 5) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:63, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:63, or consists of SEQ ID NO:63;

[0055] 6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:65, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:65, or consists of SEQ ID NO:65;

[0056] 7) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:67, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:67, or consists of SEQ ID NO:67;

[0057] 8) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:68, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:68, or consists of SEQ ID NO:68;

[0058] 9) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:70, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:70, or consists of SEQ ID NO:70;

[0059] 10) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:72, or consists of SEQ ID NO:72;

[0060] 11) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:74, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:74, or consists of SEQ ID NO:74;

[0061] 12) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:83, or consists of SEQ ID NO:83;

[0062] 13) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84;

[0063] 14) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:85, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:85, or consists of SEQ ID NO:85;

[0064] 15) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:75, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:75, or consists of SEQ ID NO:75;

[0065] 16) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:76, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:76, or consists of SEQ ID NO:76;

[0066] 17) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77; or

[0067] 18) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:78, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:78, or consists of SEQ ID NO:78.

[0068] In one embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising a light chain variable region, wherein:

[0069] 1) the light chain variable region comprises the amino acid sequence of SEQ ID NO:56, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:56, or consists of SEQ ID NO:56;

[0070] 2) the light chain variable region comprises the amino acid sequence of SEQ ID NO:54, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:54, or consists of SEQ ID NO:54;

[0071] 3) the light chain variable region comprises the amino acid sequence of SEQ ID NO:58, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:58, or consists of SEQ ID NO:58;

[0072] 4) the light chain variable region comprises the amino acid sequence of SEQ ID NO:60, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:60, or consists of SEQ ID NO:60;

[0073] 5) the light chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:62, or consists of SEQ ID NO:62;

[0074] 6) the light chain variable region comprises the amino acid sequence of SEQ ID NO:64, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:64, or consists of SEQ ID NO:64;

[0075] 7) the light chain variable region comprises the amino acid sequence of SEQ ID NO:66, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:66, or consists of SEQ ID NO:66;

[0076] 8) the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:69, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:69, or consists of SEQ ID NO:69;

[0077] 9) the light chain variable region comprises the amino acid sequence of SEQ ID NO:71, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:71, or consists of SEQ ID NO:71;

[0078] 10) the light chain variable region comprises the amino acid sequence of SEQ ID NO:73, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:73, or consists of SEQ ID NO:73;

[0079] 11) the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:79, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:79, or consists of SEQ ID NO:79;

[0080] 12) the light chain variable region comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:80, or consists of SEQ ID NO:80;

[0081] 13) the light chain variable region comprises the amino acid sequence of SEQ ID NO:81, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:81, or consists of SEQ ID NO:81;

[0082] 14) the light chain variable region comprises the amino acid sequence of SEQ ID NO:82, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:82, or consists of SEQ ID NO:82;

[0083] 15) the light chain variable region comprises the amino acid sequence of SEQ ID NO:86, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:86, or consists of SEQ ID NO:86;

[0084] 16) the light chain variable region comprises the amino acid sequence of SEQ ID NO:87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:87, or consists of SEQ ID NO:87;

[0085] 17) the light chain variable region comprises the amino acid sequence of SEQ ID NO:88, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:88, or consists of SEQ ID NO:88; or

[0086] 18) The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:89, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:89, or consists of SEQ ID NO:89.

[0087] In another embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising a heavy chain variable region and a light chain variable region, wherein:

[0088] 1) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:57, or consists of SEQ ID NO:57, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:56, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:56, or consists of SEQ ID NO:56;

[0089] 2) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:55, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:55, or consists of SEQ ID NO:55, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:54, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:54, or consists of SEQ ID NO:54;

[0090] 3) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:59, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:59, or consists of SEQ ID NO:59, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:58, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:58, or consists of SEQ ID NO:58;

[0091] 4) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:61, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:61, or consists of SEQ ID NO:61, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:60, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:60, or consists of SEQ ID NO:60;

[0092] 5) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:63, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:63, or consists of SEQ ID NO:63, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:62, or consists of SEQ ID NO:62;

[0093] 6) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:65, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:65, or consists of SEQ ID NO:65, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:64, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:64, or consists of SEQ ID NO:64;

[0094] 7) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:67, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:67, or consists of SEQ ID NO:67, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:66, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:66, or consists of SEQ ID NO:66;

[0095] 8) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:68, or consists of SEQ ID NO:68, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:66, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:66, or consists of SEQ ID NO:66;

[0096] 9) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:70, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:70, or consists of SEQ ID NO:70, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:69, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:69, or consists of SEQ ID NO:69;

[0097] 10) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:72, or consists of SEQ ID NO:72, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:71, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:71, or consists of SEQ ID NO:71;

[0098] 11) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:74, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:74, or consists of SEQ ID NO:74, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:73, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:73, or consists of SEQ ID NO:73;

[0099] 12) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:75, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:75, or consists of SEQ ID NO:75, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:79, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:79, or consists of SEQ ID NO:79;

[0100] 13) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:76, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:76, or consists of SEQ ID NO:76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:80, or consists of SEQ ID NO:80;

[0101] 14) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:76, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:76, or consists of SEQ ID NO:76, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:81, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:81, or consists of SEQ ID NO:81;

[0102] 15) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:77, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:77, or consists of SEQ ID NO:77, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:80, or consists of SEQ ID NO:80;

[0103] 16) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:77, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:77, or consists of SEQ ID NO:77, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:81, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:81, or consists of SEQ ID NO:81;

[0104] 17) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:77, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:77, or consists of SEQ ID NO:77, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:82, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:82, or consists of SEQ ID NO:82;

[0105] 18) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:78, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:78, or consists of SEQ ID NO:78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:80, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:80, or consists of SEQ ID NO:80;

[0106] 19) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:78, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:78, or consists of SEQ ID NO:78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:81, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:81, or consists of SEQ ID NO:81;

[0107] 20) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:78, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:78, or consists of SEQ ID NO:78, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:82, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:82, or consists of SEQ ID NO:82;

[0108] 21) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:83, or consists of SEQ ID NO:83, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:86, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:86, or consists of SEQ ID NO:86;

[0109] 22) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:83, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:83, or consists of SEQ ID NO:83, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:87, or consists of SEQ ID NO:87;

[0110] 23) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:87, or consists of SEQ ID NO:87;

[0111] 24) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:88, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:88, or consists of SEQ ID NO:88;

[0112] 25) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:84, or consists of SEQ ID NO:84, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:89, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:89, or consists of SEQ ID NO:89;

[0113] 26) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:85, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:85, or consists of SEQ ID NO:85, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:87, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:87, or consists of SEQ ID NO:87;

[0114] 27) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:85, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:85, or consists of SEQ ID NO:85, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:88, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:88, or consists of SEQ ID NO:88; or

[0115] 28) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:85, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:85, or consists of SEQ ID NO:85, and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:89, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:89, or consists of SEQ ID NO:89.

[0116] In some embodiments, the above-mentioned antibodies or antigen-binding fragments thereof further comprise heavy chain and / or light chain constant region sequences from human antibody germline consensus sequences. The light chain constant region is preferably a human κ or λ chain constant region. The heavy chain constant region may be a γ, μ, α, δ, or ε chain. In some embodiments, the heavy chain constant region is preferably derived from the constant region sequence of human IgG1, IgG2, IgG3, or IgG4. In one embodiment, the light chain constant region comprises the sequence shown in SEQ ID NO: 53, or consists of the sequence. In another embodiment, the heavy chain constant region comprises the sequence shown in SEQ ID NO: 52.

[0117] It will be appreciated that sequence variants of these constant region domains may also be used, for example comprising one or more amino acid modifications, wherein amino acid positions are identified according to the EU index system of Kabat et al. (1991).

[0118] In a specific embodiment, the present invention provides an anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising a heavy chain and a light chain, wherein:

[0119] 1) the heavy chain comprises the amino acid sequence of SEQ ID NO:93, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:93, or consists of SEQ ID NO:93, and the light chain comprises the amino acid sequence of SEQ ID NO:92, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:92, or consists of SEQ ID NO:92;

[0120] 2) the heavy chain comprises the amino acid sequence of SEQ ID NO:91, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:91, or consists of SEQ ID NO:91, and the light chain comprises the amino acid sequence of SEQ ID NO:90, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:90, or consists of SEQ ID NO:90;

[0121] 3) the heavy chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO:95, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:95, and the light chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO:94, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:94;

[0122] 4) the heavy chain comprises the amino acid sequence of SEQ ID NO:97, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:97, or consists of SEQ ID NO:97, and the light chain comprises the amino acid sequence of SEQ ID NO:96, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:96, or consists of SEQ ID NO:96;

[0123] 5) the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:99, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:99, or consists of SEQ ID NO:99, and the light chain comprises the amino acid sequence as set forth in SEQ ID NO:98, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:98, or consists of SEQ ID NO:98;

[0124] 6) the heavy chain comprises the amino acid sequence of SEQ ID NO:101, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:101, or consists of SEQ ID NO:101, and the light chain comprises the amino acid sequence of SEQ ID NO:100, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:100, or consists of SEQ ID NO:100;

[0125] 7) the heavy chain comprises the amino acid sequence of SEQ ID NO:103, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:103, or consists of SEQ ID NO:103, and the light chain comprises the amino acid sequence of SEQ ID NO:102, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:102, or consists of SEQ ID NO:102;

[0126] 8) the heavy chain comprises the amino acid sequence of SEQ ID NO:104, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:104, or consists of SEQ ID NO:104, and the light chain comprises the amino acid sequence of SEQ ID NO:102, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:102, or consists of SEQ ID NO:102;

[0127] 9) the heavy chain comprises the amino acid sequence of SEQ ID NO:106, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:106, or consists of SEQ ID NO:106, and the light chain comprises the amino acid sequence of SEQ ID NO:105, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:105, or consists of SEQ ID NO:105;

[0128] 10) the heavy chain comprises the amino acid sequence of SEQ ID NO:108, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:108, or consists of SEQ ID NO:108, and the light chain comprises the amino acid sequence of SEQ ID NO:107, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:107, or consists of SEQ ID NO:107;

[0129] 11) the heavy chain comprises the amino acid sequence of SEQ ID NO:110, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:110, or consists of SEQ ID NO:110, and the light chain comprises the amino acid sequence of SEQ ID NO:109, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:109, or consists of SEQ ID NO:109;

[0130] 12) the heavy chain comprises the amino acid sequence of SEQ ID NO:111, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:111, or consists of SEQ ID NO:111, and the light chain comprises the amino acid sequence of SEQ ID NO:115, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:115, or consists of SEQ ID NO:115;

[0131] 13) the heavy chain comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 112, or consists of SEQ ID NO: 112, and the light chain comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 116, or consists of SEQ ID NO: 116;

[0132] 14) the heavy chain comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 112, or consists of SEQ ID NO: 112, and the light chain comprises the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 117, or consists of SEQ ID NO: 117;

[0133] 15) the heavy chain comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 113, or consists of SEQ ID NO: 113, and the light chain comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 116, or consists of SEQ ID NO: 116;

[0134] 16) the heavy chain comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 113, or consists of SEQ ID NO: 113, and the light chain comprises the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 117, or consists of SEQ ID NO: 117;

[0135] 17) the heavy chain comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 113, or consists of SEQ ID NO: 113, and the light chain comprises the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 118, or consists of SEQ ID NO: 118;

[0136] 18) the heavy chain comprises the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 114, or consists of SEQ ID NO: 114, and the light chain comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 116, or consists of SEQ ID NO: 116;

[0137] 19) the heavy chain comprises the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 114, or consists of SEQ ID NO: 114, and the light chain comprises the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 117, or consists of SEQ ID NO: 117;

[0138] 20) the heavy chain comprises the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 114, or consists of SEQ ID NO: 114, and the light chain comprises the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 118, or consists of SEQ ID NO: 118;

[0139] 21) the heavy chain comprises the amino acid sequence of SEQ ID NO:119, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:119, or consists of SEQ ID NO:119, and the light chain comprises the amino acid sequence of SEQ ID NO:122, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:122, or consists of SEQ ID NO:122;

[0140] 22) the heavy chain comprises the amino acid sequence of SEQ ID NO:119, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:119, or consists of SEQ ID NO:119, and the light chain comprises the amino acid sequence of SEQ ID NO:123, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:123, or consists of SEQ ID NO:123;

[0141] 23) the heavy chain comprises the amino acid sequence of SEQ ID NO:120, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:120, or consists of SEQ ID NO:120, and the light chain comprises the amino acid sequence of SEQ ID NO:123, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:123, or consists of SEQ ID NO:123;

[0142] 24) the heavy chain comprises the amino acid sequence of SEQ ID NO:120, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:120, or consists of SEQ ID NO:120, and the light chain comprises the amino acid sequence of SEQ ID NO:124, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:124, or consists of SEQ ID NO:124;

[0143] 25) the heavy chain comprises the amino acid sequence of SEQ ID NO:120, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:120, or consists of SEQ ID NO:120, and the light chain comprises the amino acid sequence of SEQ ID NO:125, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:125, or consists of SEQ ID NO:125;

[0144] 26) the heavy chain comprises the amino acid sequence of SEQ ID NO:121, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:121, or consists of SEQ ID NO:121, and the light chain comprises the amino acid sequence of SEQ ID NO:123, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:123, or consists of SEQ ID NO:123;

[0145] 27) the heavy chain comprises the amino acid sequence of SEQ ID NO:121, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:121, or consists of SEQ ID NO:121, and the light chain comprises the amino acid sequence of SEQ ID NO:124, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:124, or consists of SEQ ID NO:124; or

[0146] 28) The heavy chain comprises the amino acid sequence shown in SEQ ID NO:121, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:121, or consists of SEQ ID NO:121, and the light chain comprises the amino acid sequence shown in SEQ ID NO:125, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:125, or consists of SEQ ID NO:125.

[0147] In certain embodiments of any of the foregoing antibodies, the antibody is monoclonal.

[0148] In certain embodiments of the antibody of any of the foregoing, the antibody is a full length antibody.

[0149] In one embodiment, the anti-ROR1 antibody of the present invention is a complete antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the anti-ROR1 antibody of the present invention only encompasses its antigen binding portion, such as: Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment.

[0150] In a second aspect, the present invention provides an antibody-drug conjugate targeting human ROR1, wherein the antibody-drug conjugate has the following advantages:

[0151] (1) Binds to target cells expressing human ROR1 with high affinity;

[0152] (2) Ability to enter cells through endocytosis and kill target cells; in some embodiments, the ADC of the present invention has high endocytosis efficiency;

[0153] (3) treating, preventing, or ameliorating a condition (e.g., cancer, such as blood cancer, solid tumor) associated with abnormal function or expression of ROR1 in a subject, or treating, preventing, or ameliorating one or more symptoms of the disease;

[0154] (4) reducing or inhibiting tumor growth or progression in a subject having a tumor expressing ROR1;

[0155] (5) inducing regression (e.g., long-term regression) of tumors expressing ROR1;

[0156] (6) exert cytotoxic activity in cells expressing ROR1;

[0157] In one embodiment, the present application provides a conjugate comprising the anti-ROR1 antibody of the first aspect. In a specific embodiment, the molecules that can be conjugated to the anti-ROR1 antibody are, for example, cytotoxic agents, immunomodulators, imaging agents, fluorescent proteins, molecular markers, therapeutic proteins, biopolymers, and oligonucleotides.

[0158] In one embodiment, the present invention provides an antibody-drug conjugate (ADC), comprising the anti-ROR1 antibody or antigen-binding fragment thereof as described in the first aspect and at least one therapeutically active substance or pharmaceutically active ingredient, and having a structure of Ab-(LD)n, wherein: Ab is the antibody or antigen-binding fragment thereof that binds to ROR1 as described in the first aspect of the present invention; L is a linker; D is a therapeutically active substance or pharmaceutically active ingredient, and n represents an integer of 1-20, such as 1, 2, 3, 4, 5...

[0159] In one embodiment, the antibody-drug conjugate comprises a plurality of D components, and the plurality of D components can be a combination of different therapeutically active substances or pharmaceutically active ingredients, or a combination of the same therapeutically active substances or pharmaceutically active ingredients. In one embodiment, the antibody-drug conjugate has a drug / antibody ratio (DAR) of 1-20, for example, a DAR of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In one embodiment, the DAR is an average DAR. In one embodiment, the average DAR is 1-15, for example 1-10, 2-8, 2-6 or 3-5. In a specific embodiment, the average DAR is 3.7.

[0160] In a specific embodiment, the therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a plant toxin, a small molecule toxin, a radioactive isotope, a maytansinoid alkaloid, etc. In a specific embodiment, the cytotoxin is dolastatin and its auristatin derivatives, such as 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5 ... -methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide), 8261 (2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide) Dolastatin 10, Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AFHPA). In another embodiment, the dolastatin toxin and its auristatin derivatives are auristatin, dolastatin, MMAE, MMAF, auristatin F hydroxypropylamide or auristatin F phenylenediamine.

[0161] In one embodiment, the cytotoxin is covalently linked to the anti-ROR1 antibody or antigen-binding fragment thereof in a non-site-specific manner or in a site-specific manner via a linker.

[0162] In one embodiment, the linker is selected from maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-amino benzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

[0163] In one embodiment, the antibody-drug conjugate comprises the anti-ROR1 antibody or its antigen-binding fragment described in the first aspect and a microtubule inhibitor (MMAE). In a further embodiment, MMAE is conjugated to the thiol group of cysteine ​​on the anti-ROR1 antibody through a MC-VC-PAB linker, having a structure of anti-ROR1 antibody-MC-VC-PAB-MMAE. IgG1 type antibodies have 16 pairs of cysteine ​​residues, of which 12 are present in the form of intrachain and 4 interchain disulfide bonds. The interchain disulfide bonds are solvent accessible and can be reduced by a reducing agent to form eight thiol groups, thereby becoming a coupling target (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015; 17: 339-51).

[0164] 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 conjugated to the thiol group of cysteine ​​on B62-H3L3 via a MC-VC-PAB linker.

[0165] In another specific embodiment, the antibody-drug conjugate comprises or consists of anti-ROR1 monoclonal antibody B31-H3L3 and MC-VC-PAB-MMAE. In a further embodiment, MMAE is conjugated to the thiol group of cysteine ​​on B31-H3L3 via a MC-VC-PAB linker.

[0166] In a third aspect, the present invention provides a pharmaceutical composition comprising (1) the antibody or antigen-binding fragment thereof of the first aspect or the antibody-drug conjugate of the second aspect, and (2) a pharmaceutically acceptable carrier.

[0167] In a fourth aspect, the present invention provides an isolated polynucleotide molecule encoding any one of the antibodies or antigen-binding fragments thereof described in the first aspect.

[0168] In a fifth aspect, the present invention provides a vector comprising the nucleic acid molecule of the fourth aspect. In one embodiment, the vector is an expression vector.

[0169] In a sixth aspect, the present invention provides a host cell comprising the vector of the fifth aspect or the nucleic acid molecule of the fourth invention. In some embodiments, the host cell is prokaryotic, such as Escherichia coli. In other embodiments, the host cell is eukaryotic, such as HEK293 cells, CHO cells, yeast cells, or plant cells.

[0170] In a seventh aspect, the present invention provides a method for preventing or treating a disease associated with abnormal expression of ROR1 in a subject in need thereof, comprising administering to the subject a preventively or therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention, or a preventively or therapeutically effective amount of an antibody-drug conjugate of the present invention, or a preventively or therapeutically effective amount of a pharmaceutical composition of the present invention.

[0171] In one embodiment, the disease associated with abnormal expression of ROR1 is a cancer that highly expresses ROR1, such as chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer, 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.

[0172] In some embodiments, the ADC molecules or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, such as for preventing and / or treating the relevant diseases or disorders mentioned herein.

[0173] In a specific embodiment, the present invention provides a method for killing cells expressing ROR1 or inhibiting the growth of cells expressing ROR1, comprising contacting the cells with an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, or an effective amount of an antibody-drug conjugate of the present invention, or an effective amount of a pharmaceutical composition of the present invention.

[0174] In an eighth aspect, the present invention provides use of an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of an antibody-drug conjugate for preventing or treating cancer.

[0175] The present invention also provides use of an antibody-drug conjugate comprising an anti-ROR1 antibody or an antigen-binding fragment thereof in the preparation of a drug for preventing or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0176] FIG1 shows the binding activity of each mouse antibody clone Fab obtained in the present application to huROR1-HEK293 cells determined based on the FACS method; Figure 1A The binding activity of clone B31 Fab, B32 Fab, B34 Fab, B39 Fab, B62 Fab, B74 Fab, C38 Fab, M71 Fab, and M78 Fab lysates to huROR1-HEK293 cells is shown; Figure 1B The binding activity of C42 Fab and C77 Fab lysates to huROR1-HEK293 cells is shown; NC indicates negative control, and MFI indicates median fluorescence intensity.

[0177] Figure 2A-2L The SEC-HPLC results of the antibodies of the present application are shown; Figure 2A SEC-HPLC results of B31 are shown; Figure 2B SEC-HPLC results of B32 are shown; Figure 2C SEC-HPLC results of B34 are shown; Figure 2D SEC-HPLC results of B39 are shown; Figure 2E SEC-HPLC results of B62 are shown; Figure 2F SEC-HPLC results of B74 are shown; Figure 2G SEC-HPLC results of C38 are shown; Figure 2H SEC-HPLC results of M71 are shown; Fig.2I SEC-HPLC results of M78 are shown; Figure 2J SEC-HPLC results of C42 are shown; Figure 2K SEC-HPLC results of C77 are shown; Figure 2L The SEC-HPLC result of 99961.1 is shown.

[0178] Figure 3A-3B The binding activity of the antibody of the present application and the antigen protein huROR1-His determined by ELISA method is shown; Figure 3A The binding activities of B31, B32, B34, B39, B62, and B74 to the antigen protein huROR1-His were shown; Figure 3B The binding activities of C38, C42, C77, M71 and M78 to the antigen protein huROR1-His are shown.

[0179] Figures 4A-4B The binding activity of the antibody of the present application to A549 tumor cells determined by FACS method is shown; Figure 4A The binding activity of B31, B32, B34, B39, B62, and B74 to A549 tumor cells was shown; Figure 4B The binding activity of C38, C42, C77, M71 and M78 to A549 tumor cells is shown.

[0180] Figure 5A-5B The binding activity of the antibody of the present application to huROR1-HEK293 cells determined by FACS method is shown; Figure 5A The binding activities of B31, B32, B34, B39, B62, and B74 to huROR1-HEK293 cells are shown; Figure 5B The binding activities of C38, C42, C77, M71 and M78 to huROR1-HEK293 cells are shown.

[0181] Figure 6A-6B The binding activity of the antibody of the present application to the antigen protein MusROR1-His determined by ELISA method is shown; Fig. 6A The binding activities of B31, B32, B34, B39, and B62 with the antigen protein MusROR1-His were shown; Figure 6B The binding activities of B74, C38, C42, C77, M71 and M78 to the antigen protein MusROR1-His are shown.

[0182] Figure 7A-7B The binding activity of the antibody of the present application to the antigen protein huROR2-His determined by ELISA method is shown; Fig. 7A The binding activities of B31, B32, B34, B39, and B62 with the antigen protein huROR2-His were shown; Figure 7B The binding activities of B74, C38, C42, C77, M71 and M78 to the antigen protein huROR2-His are shown.

[0183] Figures 8A-8B The internalization efficiency of the antibody of the present application on huROR1-HEK293 cells was shown based on FACS determination; Fig. 8A The internalization efficiency of B62 and C42 on huROR1-HEK293 cells is shown; Figure 8BThe internalization efficiency of B31, B32, B34, B39, B74, C38, C77, M71, and M78 on huROR1-HEK293 cells is shown.

[0184] Figures 9A-9F The endocytosis efficiency of the antibody of the present application in huROR1-HEK293 cells was shown based on the Fab-Zap method; Fig. 9A The internalization efficiency of B31 and B32 on huROR1-HEK293 cells is shown; Fig. 9B The internalization efficiency of B34 and B39 on huROR1-HEK293 cells is shown; Fig. 9C The internalization efficiency of B74 and C38 on huROR1-HEK293 cells is shown; Fig.9D The internalization efficiency of C77 and M71 on huROR1-HEK293 cells is shown; Fig.9E The internalization efficiency of M78 on huROR1-HEK293 cells is shown; Fig.9F The internalization efficiency of B62 and C42 on huROR1-HEK293 cells is shown.

[0185] Figures 10A-10E The binding activity of humanized antibodies to the antigen protein huROR1-His determined based on the ELISA method is shown; Fig. 10A The binding activities of B31-H2L2, B31-H1L1, and B31-H2L3 with the antigen protein huROR1-His were shown; Fig. 10B The binding activities of B31-H3L2, B31-H3L3, and B31-H3L4 to the antigen protein huROR1-His were shown; Fig. 10C The binding activities of B31-H4L2, B31-H4L3, and B31-H4L4 to the antigen protein huROR1-His were shown; Fig. 10D The binding activities of B62-H1L1, B62-H1L2, B62-H2L2, and B62-H2L3 to the antigen protein huROR1-His were shown; Fig. 10E The binding activities of B62-H2L4, B62-H3L2, B62-H3L3 and B62-H3L4 to the antigen protein huROR1-His are shown.

[0186] Figures 11A-11B The binding activity of humanized antibodies to A549 tumor cells determined based on FACS method is shown; Fig.11AThe binding activity of B31-H1L1, B31-H2L2, B31-H2L3, B31-H3L2, B31-H3L3, B31-H3L4, B31-H4L2, B31-H4L3, and B31-H4L4 to A549 tumor cells was shown; Fig. 11B The binding activity of B62-H1L1, B62-H1L2, B62-H2L2, B62-H2L3, B62-H2L4, B62-H3L2, B62-H3L3 and B62-H3L4 to A549 tumor cells is shown.

[0187] Fig.12 The internalization efficiency of B31-H3L3 and B62-H3L3 in huROR1-HEK293 cells determined based on the Fab-Zap method is shown.

[0188] Figures 13A-13B The binding activity of the ADC of the present application to A549 and HT29 tumor cells determined based on the FACS method is shown; Fig.13A The binding activity of B31-H3L3-MMAE and B62-H3L3-MMAE to A549 tumor cells is shown; Fig. 13B The binding activity of B31-H3L3-MMAE and B62-H3L3-MMAE to HT29 tumor cells is shown.

[0189] Figures 14A-14D The tumor cell killing effect of the ADC of the present application detected based on the MTS method is shown; Fig.14A The killing effect of B31-H3L3-MMAE on A549 tumor cells was shown; Fig. 14B The killing effect of B62-H3L3-MMAE on A549 tumor cells was shown; Fig. 14C The killing effect of B31-H3L3-MMAE on HT29 tumor cells was shown; Fig.14D The killing effect of B62-H3L3-MMAE on HT29 tumor cells is shown.

[0190] Figures 15A-15C The killing effect of the ADC of the present application on three tumor cells, Jeko-1, MDA-MB-468 and NCI-H1944, determined based on the CCK8 method is shown; Fig.15A The killing effects of B31-H3L3-MMAE and B62-H3L3-MMAE on Jeko-1 cells are shown; Fig. 15B The killing effects of B31-H3L3-MMAE and B62-H3L3-MMAE on Jeko-1 cells are shown; Fig. 15CThe killing effects of B31-H3L3-MMAE and B62-H3L3-MMAE on NCI-H1944 cells are shown.

[0191] Fig.16 The antigen-dependent killing effect of the ADC of the present application on huROR1-HEK293 cells determined based on the CCK8 method is shown.

[0192] Figures 17A-17B The internalization efficiency of the ADC of the present application on A549 and HT-29 tumor cells determined based on the FACS method is shown; Fig.17A The internalization efficiency of B31-H3L3-MMAE and B62-H3L3-MMAE on A549 tumor cells is shown; Fig. 17B The internalization efficiency of B31-H3L3-MMAE and B62-H3L3-MMAE on HT-29 tumor cells is shown.

[0193] Figures 18A-18C The results show that the ADC of the present application has an anti-tumor effect in mice with HT-29 tumor model; Fig.18A The figure shows the changes in tumor volume in mice of different experimental groups during the experimental period, and the arrows indicate the time points of drug administration; Fig.18B The weight changes of mice in different experimental groups during the experimental period are shown; Fig. 18C The tumor weights in mice of different experimental groups after the experiment are shown.

[0194] Figures 19A-19C The results show that the ADC of the present application has an anti-tumor effect in mice with A549 tumor model; Fig.19A The changes in tumor volume in mice of different experimental groups during the experimental period are shown; Fig.19B The weight changes of mice in different experimental groups during the experimental period are shown; Fig.19C The tumor weights in mice of different experimental groups after the experiment are shown.

[0195] Figures 20A-20B The results show that the ADC of the present application has an anti-tumor effect in mice with the NCI-N87 tumor model; Fig. 20A The figure shows the changes in tumor volume in mice of different experimental groups during the experimental period, and the arrows indicate the time points of drug administration; Fig. 20B The body weight changes of mice in different experimental groups during the experimental period are shown.

[0196] Figures 21A-21B The results show that the ADC of the present application has an anti-tumor effect in mice with MDA-MB-231 tumor model; Fig.21A The figure shows the changes in tumor volume in mice of different experimental groups during the experimental period, and the arrows indicate the time points of drug administration; Fig.21BThe body weight changes of mice in different experimental groups during the experimental period are shown.

[0197] Figures 22A-22B The results show that the ADC of the present application has an anti-tumor effect in mice with MDA-MB-468 tumor model; Fig.22A The figure shows the changes in tumor volume in mice of different experimental groups during the experimental period, and the arrows indicate the time points of drug administration; Fig. 22B The body weight changes of mice in different experimental groups during the experimental period are shown.

[0198] Figures 23A-23B The results show that the ADC of the present application has an anti-tumor effect in mice with Jeko-1 tumor model; Fig.23A The figure shows the changes in tumor volume in mice of different experimental groups during the experimental period, and the arrows indicate the time points of drug administration; Fig. 23B The body weight changes of mice in different experimental groups during the experimental period are shown. DETAILED DESCRIPTION OF THE INVENTION

[0200] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be changed. In addition, the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting.

[0201] I. Definitions

[0202] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0203] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0204] The term “and / or”, when used to connect two or more optional items, should be understood to mean any one of the optional items or any two or more of the optional items.

[0205] As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

[0206] The term "ROR1" refers to any recombinant or naturally occurring form of receptor tyrosine kinase-like orphan receptor 1 (ROR1), a variant or homolog thereof, which maintains, for example, at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the activity of ROR1. The variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity in the entire sequence or partial sequence (e.g., 50, 100, 150 or 200 consecutive amino acid portions) compared to the naturally occurring ROR1 protein. In one embodiment, the ROR1 protein includes the amino acid sequence of Uniprot ID: Q01973.

[0207] ROR1 is highly expressed in the embryo, and then its expression level decreases significantly in the adult stage. However, it was found that the expression of ROR1 was significantly increased in a variety of blood cancers and solid tumors. Blood cancers that highly express ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL) and myeloid blood cancers. In solid tumors, cancer types that express ROR1 include colorectal cancer, breast cancer, intestinal cancer, lung cancer, pancreatic cancer, ovarian cancer and many other cancers.

[0208] The term "antibody" is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen-binding activity. A complete antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but may comprise fewer chains in certain cases, e.g., antibodies naturally occurring in camels may comprise only heavy chains.

[0209] The term "anti-ROR1 antibody" refers to an antibody molecule that specifically binds to ROR1 and is capable of inhibiting ROR1 activity. Relative to the absence of the ROR1 antibody, the anti-ROR1 antibody is capable of inhibiting ROR1 activity, for example, by at least partially or completely blocking the stimulation of ROR1, reducing, preventing or delaying the activation of ROR1, or inactivating, desensitizing or downregulating the signal transduction, activity or amount of ROR1. In some embodiments, the antibody can inhibit ROR1 activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control.

[0210] The term "antibody fragment" refers to a molecule different from an intact antibody, which comprises a portion of an intact antibody and is capable of binding to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or fragments thereof; camelid antibodies (heavy chain antibodies); and multispecific antibodies (e.g., bispecific antibodies) formed from antibody fragments.

[0211] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. 91 pages (2007)). A single VH or VL domain is sufficient to confer antigen binding specificity.

[0212] "Complementarity determining region" or "CDR region" or "CDR" is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. CDRs in the variable domain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0213] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

[0214] CDRs can also be identified based on having the same AbM numbering position as a reference CDR sequence (eg, any of the CDRs exemplified herein). In one embodiment, the CDRs of the antibodies of the invention are positioned according to the AbM numbering scheme.

[0215] Unless otherwise indicated, in the present invention, when referring to residue positions in antibody variable regions and CDRs (including heavy chain variable region residues), the numbered positions are those according to the AbM numbering system.

[0216] "Humanized antibody" refers to a chimeric antibody comprising 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) in the humanized antibody correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. The humanized antibody optionally may comprise 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.

[0217] As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0218] The term “median effective concentration (EC 50 )” refers to the concentration of a drug, antibody or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time.

[0219] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0220] The term "antibody-drug conjugate" or "ADC" refers to an antibody or antibody fragment to which a therapeutically active substance or active pharmaceutical ingredient is covalently coupled, so that the therapeutically active substance or active pharmaceutical ingredient is targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient may be a cytotoxin that can kill cells (preferably cancer cells) targeted by the ADC. The covalent attachment of the therapeutically active substance, active pharmaceutical ingredient or cytotoxin may be performed in a non-site-specific manner using a linker, or in a site-specific manner.

[0221] The term "site-specific conjugation" refers to a method of specifically conjugating a therapeutically active substance or active pharmaceutical ingredient to a specific site of an antibody. In one embodiment, the conjugation is accomplished with the aid of a linker.

[0222] The term "cytotoxic agent" can be used interchangeably with "cytotoxin" and refers to a substance that inhibits or destroys cell function and / or causes cell death or destruction in the present invention. In one embodiment, the cytotoxic agent may include, but is not limited to, bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin), small molecule toxins, radioactive isotopes, maytansinoids, etc., specifically, anthracyclines, camptothecins, combretastatins, dolastatins and their auristatin derivatives, duocarmycins, enediynes, geldanamycins, indolino-benzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, etc. alkaloid, tubulysin, hemiasterlin, spliceostatin, pladienolide, and calicheamicin.

[0223] Any antibody-drug conjugate of the present invention can be prepared by coupling dolastatin and its auristatin derivatives with antibodies. Dolastatin and its auristatin derivatives are important cytotoxins used in antibody-drug conjugates (ADCs), which interfere with microtubule dynamics, cell division, etc., and have anti-tumor and antifungal activities. The modification of its skeleton has been widely reported in the literature, mainly the modification of the terminal subunits: P1 (N-terminus) and P5 (C-terminus). The modification of the central peptide subunit also leads to the effective cytotoxic activity of such substances in vitro. In one aspect, dolastatin and its auristatin derivatives can be, for example, 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1- oxoheptane-4-yl]-N-methyl-L-valinamide), 8261 (2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide), Dolastatin 10. Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethylauristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AF HPA), and other auristatins (e.g., auristatins described in U.S. Publication No. 20130129753), etc.

[0224] Monomethyl auristatin (MMAE), also known as demethyl-auristatin E, is a well-known member of the auristatin compound family. Its structural formula is as follows:

[0225]

[0226] MMAE plays an effective role in inhibiting mitosis by inhibiting tubulin polymerization. Due to its cytotoxicity, it cannot be used as a drug, but it is widely used to prepare antibody conjugates. MMAE is coupled to a monoclonal antibody (MAB) through a linker to form MMAE-MAB. Generally speaking, MMAE-MAB targets tumor cells through antibodies, and the linker is cleaved after MMAE-MAB enters the tumor cells, thereby releasing MMAE, which exerts its cytotoxic effect and kills tumor cells.

[0227] The terms "linker" and "connector" are used interchangeably in this application and refer to a chemical module that covalently connects an antibody to a therapeutically active substance or active pharmaceutical ingredient in an ADC. In one embodiment, the linker may comprise amino acid residues that connect the antigen to the payload. The amino acid residues may form dipeptides, tripeptides, tetrapeptides, pentapeptides, hexapeptides, heptapeptides, octapeptides, nonapeptides, decapeptides, undecapeptides, or dodecapeptides. The amino acid residues include naturally occurring ones and non-naturally occurring amino acid analogs, such as citrulline or β-amino acids, such as β-alanine, or ω-amino acids such as 4-amino-butyric acid.

[0228] According to the property classification, the linkers suitable for the present invention can be linkers degradable by tissue proteases, such as valine-citrulline (val-cit) linkers, cBu-Cit linkers and CX linkers; non-cleavable linkers such as SMCC linkers or MD linkers; acid-sensitive linkers, silicone-structured linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared-sensitive linkers, ultraviolet-sensitive linkers such as PC4AP.

[0229] The linker of the present invention can also be a combination of one or more linkers, for example, a linker degraded by cathepsin can be combined with other types of linkers to form a new linker. Therefore, the "linker" described in the present invention covers a single type of linker, or a combination of different types of linkers, as long as it can couple the antibody of the present invention to the drug.

[0230] In a specific embodiment, the linker includes, but is not limited to, maleimido-hexanoyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-amino The present invention also contains the following compounds: N-succinimidyl benzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), and N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

[0231] The term "load" or "drug load" or "effective load" refers to the average effective load number per antibody in the ADC molecule ("effective load" can be used interchangeably with "therapeutically active substance or active pharmaceutical ingredient" in this article). The drug load can range from 1-20 therapeutically active substances or active pharmaceutical ingredients per antibody. The term "drug / antibody ratio" or "DAR" refers to the ratio of the therapeutically active substance or active pharmaceutical ingredient (D) coupled to the antibody to the antibody. The ADC described herein generally has a DAR of 1-20, and in certain specific embodiments has a DAR of 1-8, 2-8, 2-6, 2-5, 2-18, 4-16, 5-12, 6-10, 3-8, 4-6, 6-10, and 2-4. Representative DAR values ​​are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, usually expressed as a combination of the letter D and a number, wherein the number represents the numerical value of the DAR, for example, D2 represents a drug / antibody ratio with a DAR value of 2. In some embodiments, the DAR is an average DAR, i.e., the average DAR measured by the detection method (e.g., by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC). Quantitative DAR values ​​can also be determined. The DAR may be limited by the number of attachment sites on the antibody. For example, in the case where the attachment site is a cysteine ​​thiol, the antibody may have only one or a few cysteine ​​thiol groups or may have only one or a few sufficiently reactive thiol groups (through which the thiol group can be attached). In some embodiments, the average DAR value of the conjugate of the invention is 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2. 2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6. 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, or a range having two of these values ​​as endpoints.

[0232] The term "treat" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay disease development or to slow the progression of a disease.

[0233] The term "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0234] The term "effective amount" refers to the amount or dosage of an antibody or conjugate or composition of the invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses. The effective amount can be readily determined by the attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of the mammal; body weight, age and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0235] The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition can vary according to a variety of factors such as disease state, age, sex and weight of the individual and the ability of the antibody or antibody portion to stimulate the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition are outweighed by the therapeutically beneficial effects. Relative to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors.

[0236] The term "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0237] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0238] II. Compositions of the Invention

[0239] In some embodiments, the present invention provides a composition comprising any anti-ROR1 antibody described herein, or its ADC molecule, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient, such as a pharmaceutical carrier, a pharmaceutical excipient, including a buffer, as known in the art. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises an anti-ROR1 antibody of the present invention, or its ADC molecule, and a combination of one or more other therapeutic agents.

[0240] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0241] For the use of pharmaceutical excipients and their uses, see also "Handbook of Pharmaceutical Excipients", Eighth Edition, RC Rowe, PJ Seskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0242] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0243] The administration route of the composition of the present invention is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional route; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implant device.

[0244] The subject can be a mammal, e.g., a primate, preferably, a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or having a risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., cancer) or has a risk of suffering from a disease described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and / or radiotherapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

[0245] A medicament comprising the antibody described herein can be prepared by mixing the anti-ROR1 antibody of the present invention, or its ADC molecule, having a desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized preparation or an aqueous solution.

[0246] The pharmaceutical composition or preparation of the present invention may also include more than one active ingredient, which is required for the specific indication to be treated, preferably with those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutics, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (such as immune checkpoint inhibitors or agonists), etc. The active ingredient is suitably combined in an amount effective for the intended use.

[0247] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films, or microcapsules.

[0248] III. Preparation of Antibodies and Antibody-Drug Conjugates of the Invention

[0249] In one embodiment, the present invention provides a method for preparing an anti-ROR1 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an anti-ROR1 antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the anti-ROR1 antibody, and optionally isolating the anti-ROR1 antibody. In a certain embodiment, the method further comprises recovering the anti-ROR1 antibody from the host cell (or host cell culture medium).

[0250] In order to recombinantly produce the anti-ROR1 antibody of the present invention, the nucleic acid encoding the anti-ROR1 antibody of the present invention is first isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to nucleic acids encoding the anti-ROR1 antibody of the present invention.

[0251] The anti-ROR1 antibodies of the present invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art. The purity of the anti-ROR1 antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0252] The prior art describes a variety of methods for coupling cytotoxic agents or other therapeutic agents to antibodies. For example, conjugation can be carried out in the antibody through the amino group of the lysine side chain and the amino group at the N-terminus of the antibody, the carboxyl group at the aspartic acid, glutamic acid and C-terminus, or the activated cysteine ​​sulfhydryl group. Example

[0253] The present invention is further illustrated by the following examples, however, it should be understood that the examples are described in an illustrative rather than a limiting manner and that various modifications may be made by those skilled in the art.

[0254] The practice of the present invention will employ, unless explicitly indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, which are within the skill of the art.

[0255] Example 1 Preparation and identification of raw materials

[0256] 1.1 Preparation and identification of ROR1 control antibodies

[0257] Preparation of ROR1 control antibody: This application uses the anti-ROR1 antibody Cirmtuzumab as a positive control antibody, and the gene synthesis of the target fragment is performed by General Biotechnology Co., Ltd. according to the sequence disclosed in WO / 2019 / 173843. Then, it was constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination, and the constructed recombinant protein expression vector was transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the endotoxin-free plasmid extraction kit (OMEGA, D6950-01) was used for plasmid extraction to obtain the desired expression vector expressing cirmtuzumab, clone number 99961.1, and the expressed cirmtuzumab will be referred to as 99961.1 below. Through ExpiFectamine TMThe expression vector was transfected into 293 cells to express cimetuzumab using a CHO transfection kit (Thermo Fisher, A29129). Seven days after transfection, the cell culture supernatant was collected and centrifuged at 15,000 g for 10 min. The resulting supernatant was filtered through a 0.22 μm filter membrane and then affinity purified using a Protein A / G affinity chromatography column. The target antibody was eluted with 100 mM glycine (pH 3.0), and the eluted antibody was exchanged into PBS buffer using an ultrafiltration concentrator (Millipore, UFC901096).

[0258] Identification of ROR1 control antibody: The activity of the prepared positive control antibody 99961.1 (IgG1) was detected with the purchased huROR1-His antigen protein (Kaixia Biotechnology, ROR-HM401). The specific method is as follows: huROR1-His (2 μg / mL, 30 μL / well) was coated on a 96-well ELISA plate at 4°C overnight; after washing the plate 3 times, it was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing the plate 3 times, the control antibody 99961.1 diluted in gradient with PBS was added and incubated at room temperature for 1 hour; after washing the plate, the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted with PBS (1:6000) was added and incubated at room temperature for 1 hour, the plate was washed 6 times, TMB was added for color development for 5-20 minutes, and the data was read by the OD450 of the microplate reader after the color development was stopped, and the data was processed and plotted using Graphpad prism. The results showed that the expressed control antibody 99961.1 could bind to the ROR1 protein and had normal anti-ROR1 activity.

[0259] 1.2 Antigen protein preparation and identification

[0260] Preparation of antigen protein: Through genetic manipulation at the coding gene level, His tag or human Fc (SEQ ID NO: 51) tag was added to the C-terminus of the sequence of human ROR1 protein huROR1 ECDAA30-406 (Uniprot ID: Q01973), mouse ROR1 protein MusROR1 ECD AA30-406 (Uniprot ID: Q9Z139), and human ROR2 protein huROR2 ECD AA34-403 (Uniprot ID: Q01974). The obtained nucleic acid sequence was constructed into the pcDNA3.4 vector, and then transformed into Escherichia coli DH5α, cultured at 37°C overnight, and then the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01). The obtained plasmid was stained with ExpiFectamine TM293 transfection kit (Gibco TM , A14524) were transiently transfected into HEK293 cells ( CRL-1573 TM ), after 7 days of expression, the cell culture supernatant was collected, and the protein containing the Fc tag was affinity purified by COLUMN XK16 / 20 (Cytiva). After purification, the target protein was eluted with 100mM glycine salt (pH=3.0), concentrated and replaced with buffer, and finally the antigen protein (huROR1-huFc) was obtained. The protein containing the His tag was affinity purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then the target protein was eluted with an imidazole gradient. The eluted proteins were exchanged into PBS buffer by ultrafiltration concentration tubes (Millipore, UFC901096), and finally the antigen proteins (huROR1-His, MusROR1-His, huROR2-His) were obtained.

[0261] Antigen identification: The prepared antigens (huROR1-His, huROR1-huFc) were detected using the antibody 99961.1 (IgG1) obtained in Example 1.1 and qualified for quality inspection. The specific method is as follows: 2 μg / mL huROR1-His and huROR1-huFc were used to coat the Elisa plate at 4°C overnight, and the purchased antigen proteins huROR1-His and huROR1-huFc (Kaixia Bio ROR-HM201) were used as positive controls; after washing the plate 3 times, it was blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; after washing the plate 3 times, the antibody 99961.1 diluted in gradient with PBS was added and incubated at room temperature for 1 hour; after washing the plate, the secondary antibody Anti-human-IgG-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted with PBS (1:6000) was added and incubated at room temperature for 1 hour, the plate was washed 6 times and then TMB was added for color development for 5-20 minutes, the color development reaction was terminated, the data was read using an enzyme reader OD450, and the data was processed and plotted using Graphpad prism. The results showed that antibody 99961.1 could bind to the antigens huROR1-His and huROR1-huFc constructed and expressed by the inventors of the present application with an affinity comparable to that of the purchased ROR1 antigen protein.

[0262] Example 2 Construction and identification of cell lines overexpressing human ROR1

[0263] Construction of HEK293 cell line overexpressing human ROR1 (hereinafter referred to as huROR1-HEK293): The coding nucleic acid sequence of full-length human ROR1 (Uniprot ID: Q01973) was constructed into pLVX-puro plasmid (Clontech, Cat#632164). Then, the obtained plasmid was electroporated by Invitrogen, Neon TM Transfection System, MP922947) and electroporated into HEK293 cells ( CRL-1573 TM After electroporation, the cells were transferred to DMEM medium (Gibco, 11995065) containing 10% FBS (Gibco, 15140-141) and without antibiotics, and then transferred to 10×10 cm cell culture dishes for 48 hours. 4 The cells were dispensed into 96-well cell culture plates at a density of 10 cells / well, and puromycin with a final concentration of 2 μg / mL was added as a screening pressure. After about 2 weeks, the cell lines that formed clones were picked for identification.

[0264] Flow cytometry identification of huROR1-HEK293 cells: digest the above cell lines in the logarithmic growth phase and plate them into 96-well plates, wash them with FACS buffer (1×PBS buffer containing 2% FBS by volume), add primary antibody (99961.1) diluted with PBS gradient and incubate at 4°C for 30 minutes; after washing, add the prepared fluorescent secondary antibody anti human IgGFc (abcam, 98596), incubate at 4°C for 30 minutes; finally, detect by flow cytometer (Beckman, CytoFLEXAOO-1-1102). The test results show that the huROR1-HEK293 cell line with high surface expression of human ROR1 was obtained.

[0265] Example 3 Animal immunization and immune library construction

[0266] 3.1 Immunization regimen

[0267] Three Balb / C mice (Shanghai Lingchang Biotechnology Co., Ltd.) were cross-immunized with huROR1-huFc and huROR1-His antigens by subcutaneous injection and intraperitoneal injection, once every two weeks, for a total of four immunizations. One week after the fourth immunization, the mice were blooded for immune titer detection, and finally, huROR1-huFc was used for booster immunization.

[0268] 3.2 Detection of serum antibody titers in mice after immunization

[0269] Elisa plates were coated with 2 μg / mL huROR1-His and huROR1-huFc at 4°C overnight (30 μL / well), washed 3 times, and blocked with 5% skim milk prepared in PBS at room temperature for 1 hour; washed 3 times, mouse serum diluted with PBS was added, and antibody 99961.1 was added as a positive control, and incubated at room temperature for 1 hour; after washing, the secondary antibody Goat-anti-mouse-lgG (1+2a+2b+3)-HRP (Jackson, 115-035-164) or Goat-anti-human-Kappa+Lambda-HRP (Millipore, AP502P+AP506P) diluted in PBS was added and incubated at room temperature for 1 hour, washed 6 times, TMB was added for color development for 5-20 minutes, and the OD450 data was read by an enzyme reader after terminating the color development reaction, and the data was processed by Graphpad prism for plotting. The results showed that the titers of the sera of the three mice were all up to standard.

[0270] 3.3 Construction of phage display antibody gene library

[0271] After immunization, the spleen of the mouse was taken, and the spleen cells were collected by grinding and filtering, and 1 mL of TRIzol was added. TM Reagent (Thermo Fisher, 15596026) was used to lyse spleen cells, and total RNA was extracted by the phenol-chloroform method. The extracted RNA was reverse transcribed into cDNA by a reverse transcription kit (TaKaRa, 6210A). After that, cDNA was used as a PCR template, and specific primers of the mouse antibody sequence were used to amplify the light chain and heavy chain variable region genes of the antibody respectively. The PCR product was double-digested by NcoI and NotI to obtain the antibody gene fragment, which was inserted into the phage display vector, connected by T4 ligase, and the ligation product was recovered by a DNA recovery kit (Omega, D6492-02), and finally transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) by an electroporator (Bio-Rad, MicroPulser), and the electroporated bacteria were coated on 2-YT (C + / K + 2-YT) solid plates were used to amplify the SS320 bacteria that had been correctly transformed with the antibody plasmid, and VSCM13 helper phage (purchased from Stratagene) was used to package them to obtain a phage display library containing Fab sequences.

[0272] Example 4 Screening of phage display antibody gene library

[0273] 4.1 Cell-based screening of phage-displayed antibody gene libraries

[0274] hROR1-HEK293T cells were cultured in T25 culture flasks. When the cell growth density was close to 90%, the culture supernatant was removed and washed once with PBS (Source Culture, B310KJ), then 2 mL of 4% paraformaldehyde (Sangong, E672002-0500) was added for fixation for 0.5 hours, and finally washed twice with PBS and used as the screening raw material. During screening, the phage display library was incubated with fixed hROR1-HEK293T cells at room temperature for 1 hour, washed three times with 1×PBS, and then 2 mL of glycine-HCl (pH=2.0) was added and gently mixed for 10 minutes to elute the phage that specifically binds to human ROR1. The eluted supernatant was then infected with logarithmic phase SS320 bacteria (Lucigen, 60512-1), allowed to stand for 30 minutes, and then cultured at 37°C, 220 rpm for 1 hour. VSCM13 helper phage was then added, allowed to stand for 30 minutes, and continued to be cultured at 37°C, 220 rpm for 1 hour, centrifuged and replaced to C + / K + The final phages were used for the second round of screening in 2-YT medium. 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 in the library were significantly enriched.

[0275] 4.2 Screening of phage display antibody gene libraries by immunotube and magnetic bead methods

[0276] The immunotube method and magnetic bead method were used to enrich specific antibodies against antigens, and the two methods complemented and verified each other.

[0277] The immunotube screening is to coat the antigen protein huROR1-His or huROR1-huFc on the surface of an immunotube with high adsorption capacity, add the phage display antibody library to the immunotube and incubate, wash and elute with the antigen protein adsorbed on the surface of the immunotube, undergo 2-4 rounds of panning, and finally enrich the specific monoclonal antibody Fab against the antigen. In this embodiment, the monoclonal antibody Fab against human ROR1 was enriched after 3 rounds of panning. The specific method refers to Example 2.4.2 in patent CN112250763B.

[0278] Magnetic bead screening is a panning process in which the antigen protein huROR1-His is biotin-labeled and then combined with magnetic beads coupled with streptavidin, and the magnetic beads bound to the antigen and the antibody gene phage display library are incubated, washed and eluted. Usually 3-4 rounds of panning are performed, so that specific monoclonal antibodies against the antigen can be enriched in large quantities. In this embodiment, biotin-labeled huROR1-His is used for phage display library screening, and after 3 rounds of panning, the monoclonal antibody Fab against human ROR1 is initially screened. For specific methods, refer to Example 2.4.1 in patent CN112250763B.

[0279] 4.3 Selection of single clones

[0280] The phage pool eluted in each round was tested by ELISA to evaluate the enrichment effect, and 10 clones were randomly selected from the phage pool in each round of screening for sequence analysis. The enrichment effect and the repeatability ratio of the measured sequences were comprehensively analyzed to select the appropriate round for single clone selection.

[0281] The ELISA monoclonal primary screening used the antigen protein huROR1-His, and the antibody Fab binding to huROR1-His obtained in the primary screening was prepared into Fab lysate, and then the overexpression cells huROR1-HEK293 prepared in Example 2.1 were used for detection and verification by flow cytometry (FACS). A total of 11 antibody Fab molecules that specifically bind to human ROR1 were screened, and the 11 mouse antibody Fabs obtained 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 Figure 1A-1B The CDR amino acid sequence of the obtained mouse antibody Fab is shown in Table 1. The CDR sequence was determined using the AbM definition method.

[0282] Table 1 Amino acid sequences of CDR regions of mouse antibodies

[0283]

[0284]

[0285]

[0286] Example 5 Antibody Construction, Expression and Purification

[0287] 5.1 Plasmid construction

[0288] The VH coding sequence in the Fab sequence of the monoclonal B62, B31, B32, B74, B34, B39, C38, C77, C42, M71 and M78 obtained by screening was connected with the coding sequence of the heavy chain constant region (SEQ ID NO: 52) of human IgG1 to obtain the heavy chain coding sequence of the chimeric antibody, and the VL coding sequence in the Fab sequence was connected with the coding sequence of the Kappa type (SEQ ID NO: 53) of the human light chain constant region (CL) to obtain the light chain coding sequence of the chimeric antibody. The coding sequences of the antibody heavy chain and light chain were respectively inserted into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5α, and cultured at 37°C overnight. The plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain an endotoxin-free antibody plasmid for eukaryotic expression.

[0289] 5.2 Antibody Expression and Purification

[0290] The full-length sequence of the antibody obtained above was expressed by the Expi CHO transient expression system (Thermo Fisher, A29133). The specific method is as follows: on the day of transfection, the CHO cell density was confirmed to be 7×10 6 Up to 1×10 7 The cell viability was >98%. Fresh ExpiCHO expression medium pre-warmed at 37°C was used to adjust the cells to a final concentration of 6×10 6 cells / mL. Use OptiPRO pre-cooled at 4°C TM The target plasmid was diluted in SFM (1 μg of plasmid was added to 1 mL of the culture medium) and OptiPRO TM Dilute ExpiFectamine in SFM TM CHO reagent, and then mix the two in equal volumes and gently pipette to mix to prepare ExpiFectamine TM The CHO / plasmid DNA mixture was incubated at room temperature for 1-5 minutes, slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker and cultured at 37°C and 8% CO2.

[0291] 18-22 hours after transfection, add ExpiCHO TM Enhancer Reagents and ExpiCHO TM Feed reagent, and place the shake flask in a 32°C shaker with 5% CO2 to continue culturing. On the 5th day after transfection, add the same volume of ExpiCHO TMFeed reagent, slowly add and gently mix the cell suspension. 7 days after transfection, collect the cell culture supernatant expressing the target protein, centrifuge at 15000g for 10min, and use MabSelect SuRe LX (GE, 17547403) for affinity purification, then elute the target protein with 100mM sodium acetate (pH3.0), then neutralize with 1M Tris-HCl, and finally exchange the obtained protein into PBS buffer through ultrafiltration concentration tube (Millipore, UFC901096).

[0292] Example 6 Physical and chemical properties of antibodies

[0293] In this example, SDS-PAGE and SEC-HPLC were used to detect the relative molecular weight and purity of the antibodies obtained above.

[0294] 6.1 Antibody SDS-PAGE Identification

[0295] Preparation of non-reducing solution: 1 μg of each obtained antibody and quality control product IPI (ipilimumab) were added to 5×SDS loading buffer and 40 mM iodoacetamide, heated in a 75°C dry bath for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min to obtain the supernatant.

[0296] Preparation of reducing solution: 2 μg of each obtained antibody and quality control product IPI were added to 5×SDS loading buffer and 5 mM DTT, heated in a 100°C dry bath for 10 min, cooled to room temperature, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The supernatant was added to Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis and the protein bands were visualized by Coomassie Brilliant Blue staining.

[0297] The protein gel with chromogenic protein bands was scanned using EPSON V550 color scanner (decolorization was performed until the gel background was transparent), and the purity of the reduced and non-reduced bands was calculated using ImageJ according to the peak area normalization method.

[0298] The test results show that the bands of each antibody in non-reduced gel are around 150kD, and the bands of reduced gel are around 55kD and 25kD, which are in line with the expected size. The purity of all antibodies obtained in this application detected by reduced gel is greater than 95% (Table 2).

[0299] 6.2 SEC-HPLC Identification of Monomeric Purity of Antibodies

[0300] Material preparation: 1. Mobile phase: 150mmol / L phosphate buffer, pH7.4; 2. Sample preparation: dilute each antibody and quality control product IPI to 0.5mg / mL with mobile phase solution. Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph, chromatographic column is XBridge BEH (SEC 3.5μm, 7.8mm ID×30cm), Waters flow rate is set to 0.8mL / min, injection volume is 20μL, VWD detector wavelength is 280nm and 214nm. Inject blank solution, IPI quality control solution and antibody sample solution in turn. Calculate the percentage of high molecular weight polymers, antibody monomers and low molecular weight substances in the sample according to the area normalization method.

[0301] The results are as follows Figures 2A-2K As shown in Table 2, except antibody B74, the SEC monomer purity of the remaining antibodies was greater than 96%.

[0302] Table 2 Antibody expression and physicochemical properties obtained in this application

[0303] Clone number SDS-PAGE (%) SEC-HPLC(%) 99961.1 >95.0 98.68 B31 >95.0 96.45 B62 >95.0 99.1 B32 >95.0 98.79 B34 >95.0 99.29 B39 >95.0 98.6 B74 >95.0 Abnormal peak shape C38 >95.0 98.9 C42 >95.0 99.39 C77 >95.0 100 M71 >95.0 100 M78 >95.0 98.93

[0304] Example 7 Antibody Antigen Binding Activity Detection

[0305] In this example, the binding of 11 expressed antibodies (B62, B31, B32, B34, B39, B74, C38, C42, C77, M71 and M78) to human ROR1 antigen protein huROR1-His was detected based on the ELISA method, and the binding ability of the antibodies (B62, B31, B32, B34, B39, B74, C38, C42, C77, M71 and M78) to human ROR1 overexpressing cells huROR1-HEK293 and A549 tumor cells was also detected based on the FACS method. A549 tumor cells are human non-small cell lung cancer cell lines that overexpress human ROR1.

[0306] 7.1 ELISA-based detection of antibody binding ability to antigen protein huROR1-His

[0307] 96-well ELISA plates (30 μL / well) were coated with 2 μg / mL huROR1-His at 4°C overnight. The next day, the plates were washed 3 times with PBST and blocked with 5% skim milk for 2 hours. After washing the plates 3 times with PBST, the gradient dilutions (3.00000, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00046, 0.00005 μg / mL) of each antibody and the positive control antibody 99961.1 were added and incubated for 1 hour. After washing 3 times with PBST, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After incubation, the plates were washed 6 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0308] The results are as follows Figure 3A-3B As shown, all antibodies and antigen protein huROR1-His obtained in the present application have good affinity activity, and the affinity of each antibody is equivalent to 99961.1.

[0309] 7.2 FACS-based detection of antibody binding ability to huROR1-HEK293 and A549 tumor cells

[0310] In this example, the binding activity of the antibody was evaluated using two types of cells, human ROR1 overexpressing cells huROR1-HEK293 and A549 tumor cells.

[0311] The specific method is as follows: huROR1-HEK293 cells or A549 cells in the logarithmic growth phase were prepared into a single cell suspension, and the density was adjusted to 1×10 6 cells / mL, 100 μL per well was added to a 96-well round-bottom plate, centrifuged at 4°C, 300g and the supernatant was removed. The gradient dilution of the antibody obtained in this application and the positive control antibody 99961.1 were added to the corresponding wells, mixed and incubated at 4°C for 30 minutes. After washing the incubated cell mixture 3 times, 100 μL of a 1:300 diluted secondary antibody Goat F(ab')2Anti-Human IgG-Fc (abcam, ab98596) was added, incubated at 4°C in the dark for 30 minutes, washed 3 times and detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0312] The results are shown in the figure. Figures 4A-4B), except C42, the affinity of other antibodies was better than that of the control antibody 99961.1; on huROR1-HEK293 cells ( Figure 5A-5B ), the affinities of all antibodies were comparable to the control antibody 99961.1.

[0313] Example 8 Antibody species and homologous cross-reactivity detection

[0314] In this example, the cross-reactivity of each antibody obtained in this application in species and homology was detected. The mouse ROR1 antigen protein MusROR1-His and the human ROR2 antigen protein huROR2-His prepared in Example 1.2 were used to identify the species and homology cross-reactivity of the antibodies obtained in this application.

[0315] 8.1 Identification of Antibody Species Cross-Reactivity

[0316] 96-well ELISA plates (30 μL / well) were coated with 2 μg / mL MusROR1-His at 4°C overnight. The next day, the plates were washed 3 times with PBST and blocked with 5% skim milk for 2 hours. After washing the plates 3 times with PBST, the gradient dilutions (1.00000, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, 0.00015, 0.00002 μg / mL) of each antibody and positive control antibody 99961.1 were added and incubated for 1 hour. After washing 3 times with PBST, the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) was added and incubated for 1 hour. After incubation, the plates were washed 6 times with PBST and TMB (SurModics, TMBS-1000-01) was added for color development. According to the color development results, 2M HCl was added to terminate the reaction, and the data were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0317] The results are as follows Figure 6A-6B As shown in Table 3, B62, B32, C38 and C42 bind to the antigen protein MusROR1-His, showing good mouse cross-reactivity. Other antibodies and the positive control antibody 99961.1 do not bind to the mouse antigen protein. Therefore, the antibodies B62, B32, C38 and C42 obtained in this application have a wide range of uses in experiments and tests based on mouse models.

[0318] 8.2 Detection of cross-reactivity of antibodies of the present application

[0319] Coat a 96-well ELISA plate (30 μL / well) with 2 μg / mL huROR2-His at 4°C overnight. The next day, wash the plate three times with PBST and block with 5% skim milk for 2 hours. After washing the plate three times with PBST, add gradient dilutions of antibodies and positive control antibody 99961.1 and incubate for 1 hour. After washing three times with PBST, add the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) and incubate for 1 hour. After incubation, wash the plate 6 times with PBST and add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, add 2M HCl to terminate the reaction, and read the data at OD450 using an enzyme reader (Molecular Devices, SpecterMax 190).

[0320] The results are as follows Figure 7A-7B As shown in Table 3, all antibodies did not bind to the antigen protein huROR2-His, indicating that the antibodies prepared in the present application can specifically bind to human ROR1.

[0321] Table 3 Antibody cross-species and homologous cross-family activities obtained in this application

[0322] Clone number Species MusROR1-His huROR2-His B31 mouse No combination No combination B62 mouse Combination No combination B32 mouse Combination No combination B34 mouse No combination No combination B39 mouse No combination No combination B74 mouse No combination No combination C38 mouse Combination No combination C42 mouse Combination No combination C77 mouse No combination No combination M71 mouse No combination No combination M78 mouse No combination No combination

[0323] Example 9 Antibody endocytosis efficiency detection

[0324] In this embodiment, the endocytosis efficiency of the antibody obtained in this application was detected by using two detection methods, FACS and Fab-Zap. The FACS method endocytosis detection uses supersaturated antibodies to bind cells and detects the antibody endocytosis efficiency in a short period of time; the Fab-Zap method uses antibodies bound to Fab-Zap toxins at different concentrations to bind cells, and the toxins are brought in to kill target cells through endocytosis. This method reflects the long-term cumulative effect of endocytosis efficiency.

[0325] 9.1 FACS method to detect the endocytic efficiency of the antibody of the present application

[0326] Antibody dilution: Dilute the test antibody to a final concentration of 10.0000 μg / mL in DMEM complete medium.

[0327] Treat cells: Digest huROR1-HEK293 cells and add DMEM complete medium. After thoroughly mixing the cells, count the cells and determine their viability. Take 10 6 Add the cells to a 1.5 mL centrifuge tube, centrifuge at 300 g for 5 minutes, discard the supernatant, resuspend in 1 mL of pre-cooled DMEM medium, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0328] Primary antibody incubation: Take 1000 μL of the pre-cooled diluted antibody and add it to the centrifuge tube containing cells to prepare the antibody-cell suspension. Add the antibody-cell suspension to a 96-well plate and incubate at 4°C.

[0329] Incubation with extracellular secondary antibody: Quickly transfer the suspension in the 96-well plate to a second 96-well plate. Add 180 μL of pre-cooled FACS buffer to each well of the second 96-well plate and wash twice. Then add 100 μL of diluted secondary antibody FITC-labeled anit-huFc or RPE-labeled anit-huFc (diluted 1:150 with FACS buffer) to each well; incubate at 4°C for 30 min.

[0330] Cell fixation: After incubation, centrifuge and discard the supernatant, add 180 μL of pre-cooled FACS buffer to each well, wash the cells twice, centrifuge and discard the supernatant, and fix the cells with 100 μL of 4% paraformaldehyde at room temperature for 30 minutes in each well.

[0331] Disrupt cell membrane: After fixation, add 180 μL FACS buffer to wash twice. Add 100 μL preheated 0.5% Triton X-100 to each well and perforate for 5 minutes at room temperature.

[0332] Intracellular secondary antibody incubation: After washing the 96-well plate, add 180 μL of preheated Permeabilization Buffer (Invitrogen TM eBioscience TM , 00-8333-56) were washed twice. 100 μL of diluted secondary antibody RPE-labeled anit-huFc (diluted 1:150 with Perm buffer) was added to each well. Incubate at room temperature for 60 min.

[0333] Detect fluorescence: After washing the 96-well plate, resuspend it in 100 μL FACS buffer and detect it by flow cytometry.

[0334] This experiment used two different fluorescence settings to stain each sample. The first set was FITC+PE, that is, FITC secondary antibody was used to stain the extracellular primary antibody first, and PE secondary antibody was used to stain the intracellular primary antibody after the membrane was broken. In this set, PE is the intracellular signal and FITC is the extracellular signal; the second set was PE+PE, that is, PE secondary antibody was used to stain the extracellular primary antibody first, and PE secondary antibody was used to stain the intracellular primary antibody after the membrane was broken. In this set, PE is the sum of intracellular and extracellular signals. At the same time, both sets of samples were detected using FITC and PE channels, and the calculation value of the internalization rate was calculated by calculating the PE channel detection value. The specific formula is:

[0335] Internalization rate = one group (FITC + PE) PE channel / two groups (PE + PE) PE channel × 100%.

[0336] The results are as follows Figures 8A-8B As shown, the results of this example show that the endocytosis efficiency of all antibodies prepared in this application is better than that of the control antibody 99961.1 in a shorter period of time (within 3 hours).

[0337] 9.2 Fab-Zap method to detect antibody internalization efficiency

[0338] This experiment detects the endocytic activity of antibodies through the cytotoxicity of antibody-mediated Fab-ZAP endocytosis. Fab-ZAP is a Fab fragment connected to saporin, which is a ribosome inhibitor that can inhibit protein synthesis and cause cell death. The Fab-ZAP used in this experiment is a Fab fragment that can bind to the human Fc of chimeric antibodies. After incubation of Fab-ZAP and chimeric antibodies, the chimeric antibodies are charged with toxins. When the chimeric antibodies are endocytosed, the toxins enter the cells along with the chimeric antibodies, causing cell death. Then, the activity of the cells is detected by MTS (Promega, G3580) to detect whether the antibodies are endocytosed.

[0339] The specific experimental method is as follows: First, Fab-Zap was diluted to 0.4 nM with DMEM complete medium, and then 0.4 nM Fab-Zap was gradiently diluted (0.020000, 0.006667, 0.002222, 0.000741, 0.000247, 0.000082, 0.000027, 0.000009 μg / mL) to prepare antibody dilution solution. HuROR1-HEK293 cells in logarithmic growth phase were prepared into single cell suspension and the density was adjusted to 6×10 6 cells / mL, inoculated into a 96-well plate at 50 μL per well, then took the aforementioned antibody dilution and added 50 μL per well to the cell culture plate, and mixed thoroughly by blowing. The cell culture plate was placed in a 37°C cell culture incubator for 48 hours. After the incubation, 7.5 μL TritonX-100 solution was added to each well, gently tapped to mix, and the cell culture plate was placed in a 37°C cell culture incubator for 0.5 hours. Then, 20 μL 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 an ELISA reader, and the detection wavelength was A492.

[0340] The results are as follows Figures 9A-9FAs shown, it is shown that under the antibody concentration conditions specified in this example, the endocytosis effect of the antibody prepared in this application is comparable to that of the control antibody. Considering that cells are sensitive to toxins only when they react above a certain threshold, it is difficult to increase the difference when the accumulated toxins are not much different.

[0341] Example 10 Antibody Affinity Kinetics (Gator) Detection

[0342] In this example, the affinity of the antibody obtained in this application and the positive control antibody 99961.1 to the antigen protein huROR1-His was detected based on the Gator device.

[0343] First, PBS (10mM pH7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from source culture) was configured into Q buffer, and the storage solution of the antibody to be tested was diluted to a working solution with a final concentration of 30nM with the configured Q buffer; the storage solution of the antigen protein huROR1-His was configured into a working solution with multiple dilutions (480, 240, 120, 60, 30, 15, 7.5nM) with Q buffer, and then the Gator instrument and its supporting software were used to select the Advanced Kinetics experimental mode for detection and analysis. The test results are shown in Table 4.

[0344] The results showed that except for C42 antibody, which had comparable affinity to the control antibody 99961.1, the affinities of the other antibodies were superior to that of the control antibody 99961.1 by one order of magnitude or more. Among them, the affinity of B62 antibody was superior to that of the control antibody 99961.1 by about two orders of magnitude, reaching 9.84E-10.

[0345] Table 4 Affinity kinetic test results of antibodies obtained in this application

[0346]

[0347]

[0348] Example 11 Antibody Affinity Kinetics (Biacore) Analysis

[0349] In this example, the affinity of the antibodies obtained in this application and the positive control antibody 99961.1 to the antigen protein huROR1-His was detected based on the Biacore device.

[0350] Protein coupling: The huROR1-His protein produced in Example 1.2 was diluted to 5.6 μg / mL with NaAc buffer at pH 5.0, the flow rate was set to 10 μL / min, the chip activation time was set to the default value of 420 s with a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the antigen protein huROR1-His was fixed to a level of about 75 RU using a coupling mode with a preset coupling amount, and the activated groups that were not bound to the test sample were blocked with ethanolamine.

[0351] Sample test conditions: PBS buffer (pH 7.4) containing 0.05% Tween-20 was used as the running buffer, and the running buffer was used as the control test sample. A series of antibody concentrations (4 nM, 20 nM) were set. During sample analysis, the flow rate was set to 30 μL / min, the binding time was 120 s, and the dissociation time was 360 s. After the dissociation was completed, 10 mM Gly-HCl (pH 2.0) was used for regeneration for 20 s to completely remove the antibody bound to the ligand.

[0352] Parameter fitting: The experiment was run in multiple cycles, with the response signal taking the analysis time as the horizontal axis and the response value as the vertical axis. After double reference subtraction, the obtained data were fitted by BIAcore T200 analysis software, and the fitting model used was the 1:1 Langmuir binding model to determine the affinity indexes such as the binding dissociation constant.

[0353] The results are shown in Table 5. The affinity of antibody B62 is about 2 orders of magnitude better than that of control antibody 99961.1, with a KD of 6.39E-11, while the KD of control antibody 99961.1 is only 1.98E-9; in addition, the affinity of antibody B31 is comparable to that of control antibody 99961.1.

[0354] Table 5 Affinity kinetic test results of antibodies obtained in this application

[0355] Clone number ka(1 / Ms) kd(1 / s) t 1 / 2(s) KD(M) Rmax(RU) <![CDATA[Chi 2 (RU 2 )]]> 99961.1 6.94E+05 1.37E-03 504.4 1.98E-09 Local Fit 3.13 B31 6.53E+05 8.17E-04 848.8 1.25E-09 Local Fit 1.61 B62 1.21E+06 7.74E-05 8954.3 6.39E-11 Local Fit 0.92

[0356] Example 12 Affinity Kinetics Epitope Grouping

[0357] This example uses the affinity kinetics method to group the epitopes of the antibodies obtained in this application and the positive control antibody 99961.1.

[0358] The specific experimental method is as follows: first, PBS (10mM PH7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from source culture) is configured into Q buffer, and the storage solution of the antibody to be tested is diluted with the configured Q buffer to a working solution with a final concentration of 100nM; the antigen huROR1-His storage solution is configured with Q buffer to a 50nM working solution, and then the Gator instrument and its supporting software are used for detection and analysis based on the Tandem setting of the Epitope Binning experimental mode.

[0359] The specific detection and analysis steps are as follows:

[0360] Equilibrium stage 1: The probe was equilibrated in Q buffer at 1000 rpm for 60 s;

[0361] Immobilization antigen stage: After the completion of equilibrium 1 stage, the probe was immobilized in Q buffer containing 50 nM antigen at a speed of 400 rpm for 90 s;

[0362] Equilibrium 2 stage: After the antigen immobilization stage, the probe was equilibrated in Q buffer at a speed of 1000 rpm for 60 s;

[0363] Antibody 1 binding stage: After the completion of equilibrium 2, the probe was subjected to binding reaction at 1000 rpm in Q buffer containing 100 nM antibody 1 for 180 s;

[0364] Equilibrium 3 stage: After the antibody 1 binding stage was completed, the probe was equilibrated in Q buffer at a speed of 1000 rpm for 30 s;

[0365] Antibody 2 binding stage: After the completion of the equilibrium 3 stage, the probe was subjected to binding reaction in Q buffer containing 100 nM antibody 2 at a speed of 1000 rpm for 180 s;

[0366] Regeneration phase: From equilibrium 1 phase to antibody 2 binding phase is one assay. After completing one assay, the probe needs to be regenerated in Q buffer at a speed of 1000 rpm for 50 seconds before the next assay.

[0367] The epitope grouping results of each antibody thus obtained are shown in Table 6. The results show that the antibodies of the present application and the control antibody 99961.1 are divided into 2 epitope groups, only C42 and the control antibody have the same binding epitope, and the binding epitopes of the remaining antibodies are different from those of the control antibody.

[0368] Table 6 Summary of epitope grouping results

[0369] Epitope Grouping Clone number 1 99961.1、C42 2 B62, B32, C38, B74, M71, B34, M78, C77, B39, B31

[0370] Example 13 Humanization of murine antibodies

[0371] The VH and VL sequences of the murine antibodies B31 and B62 obtained by screening in Example 4 were compared with the known human antibody database, and the human germline gene VH and VL sequences with the highest homology to the murine VH and VL sequences were found. The framework regions of the corresponding human germline gene VH and VL sequences (using AbM to define CDR and framework regions) were selected, and the complementary determining region (CDR) sequences of the germline genes were replaced with the corresponding CDR sequences in the murine antibodies B31 and B62 of the present application, and then the murine amino acids in the framework regions of the murine antibodies B31 and B62 that have an important influence on antigen binding were retained by means of computer prediction simulations. The amino acid sequences of the humanized antibody CDR regions obtained after humanization of murine antibodies B31 and B62 are shown in Table 7. The multiple antibodies obtained after humanization of B31 and B62 were constructed, expressed and purified by the method of Example 5, and the multiple antibody proteins obtained after humanization of B31 and B62 were identified by SDS-PAGE and SEC-HPLC, and the results are shown in Table 8. The results show that the bands of non-reducing gel of each antibody are around 150kD, and the bands of reducing gel are around 55kD and 25kD, which are in line with the expected size. The purity of all antibodies obtained in this application detected by reducing gel is greater than 95%; the SEC monomer purity of all antibodies is greater than 96%.

[0372] Table 7 Amino acid sequences of CDR regions of humanized antibodies

[0373]

[0374]

[0375] Table 8 Physicochemical properties of humanized antibodies

[0376] Clone number SDS-PAGE (%) SEC(%) B31 >95.0 100 B31-H1L1 >95.0 98.34 B31-H2L2 >95.0 100 B31-H2L3 >95.0 100 B31-H3L2 >95.0 100 B31-H3L3 >95.0 100 B31-H3L4 >95.0 100 B31-H4L2 >95.0 100 B31-H4L3 >95.0 99.1 B31-H4L4 >95.0 100 B62 >95.0 98.72 B62-H1L1 >95.0 100 B62-H1L2 >95.0 100 B62-H2L2 >95.0 100 B62-H2L3 >95.0 100 B62-H2L4 >95.0 96.73 B62-H3L2 >95.0 100 B62-H3L3 >95.0 100 B62-H3L4 >95.0 97.07

[0377] Example 14 Antigen affinity detection of humanized antibodies

[0378] In this example, the affinity of the humanized antibody to the human ROR1 antigen protein huROR1-His was tested based on the ELISA method, and the affinity of the humanized antibody to A549 tumor cells was tested based on the FACS method. The specific test methods are described in Example 7.

[0379] ELISA test results Figures 10A-10E As shown, the affinity of the humanized antibody to the antigen protein is comparable to that of the corresponding parental molecule and the control antibody 99961.1.

[0380] FACS test results Figures 11A-11B As shown, except for B31-H3L4, B31-H4L3, B31-H4L2 and B31-H4L4, the affinity of the humanized antibodies to the antigenic proteins on A549 tumor cells was comparable to that of the corresponding parental molecules and the control antibody 9996.1.

[0381] Example 15 Detection of humanized antibody endocytosis efficiency

[0382] In this example, the humanized antibodies B31-H3L3 and B62-H3L3 in Example 13 were subjected to Fab-Zap method endocytosis detection. The specific method is referred to Example 9. The results are as follows: Fig.12 As shown, the internalization effects of B31-H3L3 and B62-H3L3 were comparable to that of the control antibody 99961.1.

[0383] Example 16 ADC Preparation

[0384] In this embodiment, antibodies B62-H3L3, B31-H3L3 and control antibody 99961.1 were coupled with toxin MMAE (a tubulin inhibitor with anticancer activity) to construct an antibody-drug conjugate, MMAE was connected with a linker MC-VC-PAB to form MC-VC-PAB-MMAE, and the linker was covalently linked to the sulfhydryl group on the cysteine ​​of the antibody, so that the antibody was conjugated with MMAE to obtain an antibody-drug conjugate. IgG1 type antibodies have 16 pairs of cysteine ​​residues, which exist in the form of 12 intrachain and 4 interchain disulfide bonds. The interchain disulfide bond has solvent accessibility and can be reduced by a reducing agent to form eight sulfhydryl groups, which then become the coupling target (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015; 17: 339-51).

[0385] The specific preparation method is as follows:

[0386] Antibodies B62-H3L3, B31-H3L3 and control antibody 99961.1 were taken out of the -80°C refrigerator, thawed and transferred to 15 mL 30KD ultrafiltration centrifuge tubes, respectively, and coupling buffer (content per 1L: Na2HPO4·2H2O 6.86 g, NaH2PO4·H2O 1.58 g, purified water to 1000 g, pH 7.4) was added to 15 mL, centrifuged at 4500 rpm for about 30 min, concentrated to 2-3 mL, and dialysate (content per 1L: histidine 0.73 g, histidine hydrochloride monohydrate 1.12 g, purified water to 1000 g, pH 6.0) was added to 15 mL, dialyzed repeatedly 8-10 times to obtain antibody stock solution, and the antibody concentration after dialysis was detected.

[0387] The antibody stock solution, 10mM disulfide bond reducing agent TCEP mother solution (tris (2-carboxyethyl) phosphine hydrochloride mother solution, content per 1L: Na2HPO4 · 2H2O 6.86g, NaH2PO4 · H2O 1.58g, purified water is diluted to 1000g), 10mM DTPA mother solution (diethylenetriamine pentaacetic acid mother solution, content per 1L: DTPA 3.90g, NaOH 1.20g, purified water is diluted to 1000g) and coupling buffer are added in sequence to form a reduction reaction system to reduce the sulfhydryl group on the cysteine ​​of the antibody. The amount of each component added in the reduction reaction system is shown in Table 9, so that the antibody concentration in the reduction reaction system is 5mg / mL, the final concentration of DTPA is 1mM, the molar ratio of TCEP to B62-H3L3 or B31-H3L3 is 2, and the molar ratio of TCEP to 9996.1 is 2.2. After being thoroughly mixed, the reduction reaction system was placed in a constant temperature mixer at 25°C, with a rotation speed of 400 rpm and the reduction reaction was carried out for 2 hours.

[0388] Weigh MC-VC-PAB-MMAE, dissolve it in DMSO, and prepare a 5mM MC-VC-PAB-MMAE mother solution. After the reduction reaction is completed, add the MC-VC-PAB-MMAE mother solution to the reduction reaction system in an ice-water bath in sequence, and the addition amount is shown in Table 10 to prepare a coupling reaction system. After sufficient mixing, place the coupling reaction system in a 25°C constant temperature mixer, 400 rpm, and perform the coupling reaction for 1 hour to obtain a solution containing ADC.

[0389] After the coupling is completed, the solution containing ADC is centrifuged and filtered to obtain an ADC sample, which is transferred to a 15mL 30KD ultrafiltration centrifuge tube. The dialysate is added to 15mL, centrifuged at 4500rpm for 20min, concentrated to 2-3mL, and the dialysate is added again to 15mL, and the dialysate is repeated 8-10 times. The dialyzed ADC sample is subjected to SEC-HPLC detection, HIC-HPLC detection, concentration detection, free drug detection, etc. The test results are shown in Table 11. The test results show that an ADC with a purity of more than 99% is obtained.

[0390] Table 9 Reduction reaction system composition

[0391]

[0392] Table 10 MC-VC-PAB-MMAE mother liquor addition amount

[0393] Clone number B62-H3L3 B31-H3L3 99961.1 MC-VC-PAB-MMAE to antibody molar ratio 6.0 6.0 6.0 5mM MC-VC-PAB-MMAE stock solution addition volume (μL) 620 384 642

[0394] Table 11 ADC sample test results

[0395]

[0396] Example 17 Antigen Binding Activity Detection of ADC

[0397] In this example, the binding ability of the prepared ADCs (B62-H3L3-MMAE, B31-H3L3-MMAE) to human ROR1 on tumor cells A549 and HT-29 (human colon cancer cells) was detected based on the FACS method.

[0398] The specific method is 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 6Cells / mL, 100 μL per well was added to a 96-well round bottom plate, centrifuged at 4°C, 300 g 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 application antibodies (B62-H3L3 and B31-H3L3), the present application ADC (B62-H3L3-MMAE, B31-H3L3-MMAE), 99961.1, 99961.1-MMAE and negative control were added to the corresponding wells, mixed and incubated at 4°C for 30 min. The incubated cell mixture was washed three times and then 100 μL of 1:300 diluted secondary antibody Goat F(ab')2 Anti-Human IgG-Fc (abcam, ab98596) was added. The mixture was incubated at 4°C in the dark for 30 min. After washing three times, the cells were detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0399] The results are as follows Figures 13A-13B Results of binding to A549 cells are shown in Fig.13A After coupling, the binding activity of B62-H3L3-MMAE and 99961.1-MMAE to A549 cells was slightly worse than that of the corresponding naked antibodies, and the binding activity of B62-H3L3 and B62-H3L3-MMAE to A549 cells was better than that of the corresponding positive control; the results of binding to HT-29 cells are shown in Fig. 13B : The binding activities of naked antibody and corresponding ADC to HT-29 cells are comparable, and the binding activities of B62-H3L3 and B62-H3L3-MMAE to HT-29 cells are slightly better than those of the corresponding positive controls.

[0400] Example 18 Detection of ADC tumor cell killing effect based on MTS method

[0401] In this example, A549 and HT-29 cells were used to detect the killing effects of the ADC of the present application and the control ADC on tumor cells.

[0402] The specific method is as follows: A549 cells or HT-29 cells in the logarithmic growth phase are prepared into a single cell suspension, and the density of A549 is adjusted to 1×10 4 cells / mL, and the HT-29 density was adjusted to 1.5×10 4Cells / mL, 100 μL per well was added to a 96-well cell culture plate, 37°C, 5% CO2, and cultured for 12 hours. Then, a gradient dilution (2000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 1.953 nM) of ADC samples was added, 37°C, 5% CO2, and cultured for 72 hours (A549 cells) or 96 hours (HT-29 cells). Then, 40 μL of MTS (Promega, G3580) was added to each well, and after incubation at 37°C for 1 hour, the data was read at OD492 by a microplate reader.

[0403] The results are as follows Figures 14A-14D As shown: B31-H3L3-MMAE in two tumor cells A549 ( Fig.14A ) and HT-29( Fig. 14C ) was slightly better than the control ADC 99961.1-MMAE, while B62-H3L3-MMAE had a slightly better killing effect on A549 ( Fig. 14B ) and HT-29( Fig.14D ) is slightly worse than the control ADC 99961.1-MMAE.

[0404] Example 19 Detection of ADC tumor cell killing effect based on CCK8 method

[0405] 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 of tumor cells by B31-H3L3-MMAE, B62-H3L3-MMAE and control ADC.

[0406] The specific method is 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 cells / mL, and the density of MDA-MB-468 was adjusted to 2×10 5 cells / mL, and the density of NCI-H1944 was adjusted to 1.2×10 5 Cells / mL, 90 μL per well was added to a 96-well cell culture plate, 37°C, 5% CO2, and cultured for 12 hours. Then, ADC samples were added, and the drug concentration was gradiently diluted to 500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, 0.05 nM, 37°C, 5% CO2, and cultured for 72 hours. Then, 10 μL CCK8 (Bimake, B34304) was added to each well, incubated at 37°C for 1 hour, and the data was read at OD450 by a microplate reader.

[0407] The test results are as follows Figures 15A-15C As shown, B31-H3L3-MMAE and B62-H3L3-MMAE were expressed in three tumor cells, Jeko-1 ( Fig.15A ) and MDA-MB-468( Fig. 15B ) and NCI-H1944( Fig. 15C ) was comparable to the control ADC99961.1-MMAE, among which B31-H3L3-MMAE had an EC of 0. 50 The EC values ​​of B62-H3L3-MMAE on Jeko-1, MDA-MB-468 and NCI-H1944 were 38.24 nM, 15.24 nM and 117.3 nM, respectively. 50 They are 50.14nM, 28.64nM and 128.6nM respectively.

[0408] Example 20 Detection of ADC antigen-dependent killing effect based on CCK8 method

[0409] In this example, huROR1-HEK293 cells were used to detect the antigen-dependent killing effects of B31-H3L3-MMAE and control ADC.

[0410] The specific method is as follows: huROR1-HEK293 cells in the logarithmic growth phase were prepared into a single cell suspension, and the cell density was adjusted to 6×10 4 Cells / mL, 50 μL per well was added to a 96-well cell culture plate, 37°C, 5% CO2, cultured for 24 hours, 50 μL of antibody B31-H3L3 at a concentration of 100 μg / mL and positive control antibody 99961.1 were added to each well, incubated at 37°C for 2 hours, and the corresponding ADC B31-H3L3-MMAE and control ADC 99961.1-MMAE with gradient dilutions (42.6667, 21.3333, 10.6667, 5.3333) were added, 37°C, 5% CO2, cultured for 96 hours, 30 μL CCK8 (absin / Aibixin, abs50003) per well, incubated at 37°C for 1-4 hours, and the plate was read at OD450 by a microplate reader. The system without antibody and only ADC was used as the control group.

[0411] The results are as follows Fig.16 As shown, the competition between antibodies B31-H3L3 and 99961.1 can significantly inhibit the killing effect of their corresponding ADCs on huROR1-HEK293 cells, proving that the cell killing of the ADC prepared in the present application depends on the binding of the antibody connected to the ADC with the antigen on the cell surface, and is therefore antigen-dependent killing rather than non-specific killing by the connected toxin.

[0412] Example 21 ADC endocytosis efficiency detection

[0413] In this example, A549 and HT-29 tumor cells were used to detect the endocytosis efficiency of the ADC obtained in this application based on the FACS method. For specific experimental methods, see Example 9.

[0414] The experimental results showed that on A549 cells ( Fig.17A ), the endocytosis efficiency of B31-H3L3-MMAE and the control ADC 99961.1-MMA was similar, the endocytosis efficiency of B62-H3L3-MMAE was slightly lower than that of the positive control ADC, and the endocytosis efficiency of the ADCs was higher than that of their corresponding antibodies; on HT-29 cells ( Fig. 17B ), the endocytosis efficiency of B31-H3L3-MMAE was slightly higher than that of the positive control ADC, and the endocytosis efficiency of B62-H3L3-MMAE was slightly lower than that of the positive control ADC, and the endocytosis efficiency of antibodies was generally lower than that of their corresponding ADCs.

[0415] Example 22 ADC efficacy test in inhibiting HT-29 mouse tumor model

[0416] In this example, the tumor inhibition effects of two candidate ADCs (B31-H3L3-MMAE and B62-H3L3-MMAE) in animals were verified, 99961.1-MMAE was used as a positive control, and the tumor cells used were colon cancer cells HT-29 (BNCC337732).

[0417] The specific method is as follows: 6-8 week old female Balb / C nude mice weighing about 20 g (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used, and 1×10 6 HT-29 cells, when the tumor volume reaches 100mm 3 Around 14:00, the mice were randomly divided into groups and cages. There were 6 tumor-bearing nude mice in each group, for a total of 9 groups, including a PBS negative control group, 5 candidate ADC groups (B31-H3L3-MMAE 0.4mg / kg (mpk), B31-H3L3-MMAE 2mg / kg, B31-H3L3-MMAE 10mg / kg, B62-H3L3-MMAE 2mg / kg and B62-H3L3-MMAE 10mg / kg) and 3 positive control ADC groups (99961.1-MMAE0.4mg / kg, 99961.1-MMAE 2mg / kg and 99961.1-MMAE 10mg / kg). The administration method was tail vein injection, 2 times a week and the tumor volume was measured 2 times, for a total of 8 times / 4 weeks (BIW*4). Tumor volume (V) calculation method: 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 the administration, the mice were euthanized, and the tumors were removed and the tumor weight was measured. The tumor volume, tumor weight and mouse body weight change data were analyzed and the tumor inhibition rate was calculated. The results are shown in Figures 18A-18C and Table 12.

[0418] The test results showed that there was no significant difference in the body weight of mice in each group, and there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well ( Fig.18B ); B31-H3L3-MMAE, B62-H3L3-MMAE and control ADC 99961.1-MMAE all showed significant anti-tumor effects under high-dose conditions (10 mpk). Under high-dose conditions, B31-H3L3-MMAE and 99961.1-MMAE had comparable anti-tumor effects, and the anti-tumor rate of B62-H3L3-MMAE was slightly weaker than that of the control ADC 99961.1-MMAE. However, under low-dose conditions (2 mpk), the anti-tumor effect of B62-H3L3-MMAE was significantly better than that of the control ADC 99961.1-MMAE and B62-H3L3-MMAE ( Fig.18A , Fig.18C and Table 12).

[0419] Table 12 Tumor inhibition rate of ADC in mice

[0420]

[0421]

[0422] Example 23 ADC inhibition of A549 mouse tumor model efficacy test

[0423] This example detects the tumor inhibition effects of two candidate ADCs (B31-H3L3-MMAE and B62-H3L3-MMAE) in animals. The tumor cells used are non-small cell lung cancer cells A549 (Shanghai Cell Bank, Chinese Academy of Sciences, C2107019), and 99961.1-MMAE is used as a positive control.

[0424] The specific method is as follows: 6-8 week old female nude mice (Balb / C Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) weighing 18-20 g were used, and 1×10 6 A549 cells, when the tumor volume reaches 100mm 3Around 24 hours after the injection, the mice were randomly divided into groups and cages. There were 6 tumor-bearing nude mice in each group, and there were 4 groups in total, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 10mpk (mg / kg) and B62-H3L3-MMAE 10mpk) and 1 positive control ADC group (99961.1-MMAE 10mpk). The administration method was tail vein injection, 2 times a week and the tumor volume was measured 2 times, for a total of 6 times / 3 weeks (BIW*3). Tumor volume (V) calculation method: V = L × W 2 / 2 (where L is the longest tumor diameter and W is the shortest tumor diameter). After a certain period of observation after the end of the administration, the mice were euthanized, the tumors were removed and the tumor weight was measured. The tumor volume, tumor weight and mouse body weight change data were analyzed and the tumor inhibition rate was calculated. The results are shown in Figures 19A-19C and Table 13.

[0425] The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well ( Fig.19B ); B62-H3L3-MMAE showed an anti-tumor effect comparable to that of the positive control ADC at the same dose, and B31-H3L3-MMAE showed an anti-tumor effect superior to that of the positive control ADC ( Fig.19A , 19C and Table 13).

[0426] Table 13 Tumor inhibition rate of ADC in mice

[0427]

[0428]

[0429] Example 24 ADC inhibition of NCI-N87 mouse tumor model efficacy test

[0430] This example detects the tumor inhibition effect of a candidate ADC (B31-H3L3-MMAE) in animals. The tumor cells used are gastric cancer cells NCI-N87 (Shanghai Cell Bank of the Chinese Academy of Sciences, C2009021, P3), and 99961.1-MMAE is used as a positive control.

[0431] The specific method is as follows: 6-8 weeks old female nude mice (Balb / C Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) weighing 18-20 g were used, and 1×10 6 NCI-N87 cells, when the tumor volume reaches 100mm 3Around 1:10 pm, the mice were randomly divided into groups and cages. There were 5 groups with 10 tumor-bearing nude mice in each group, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 5mpk (mg / kg) and B31-H3L3-MMAE 10mpk) and 2 positive control ADC groups (99961.1-MMAE 5mpk and 99961.1-MMAE 10mpk). The drugs were administered by tail vein injection, once a week and the tumor volume was measured twice, for a total of 3 times / 3 weeks (QW*3). Tumor volume (V) calculation method: V = L × W 2 / 2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed and the tumor inhibition rate was calculated. The results are shown in Figures 20A-20B and Table 14.

[0432] The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well ( Fig. 20B ); Compared with the PBS group, each dose group had a significant tumor inhibition effect, and the tumor inhibition effects of B31-H3L3-MMAE and 99961.1-MMAE molecules were comparable at 10 mpk ( Fig. 20A and Table 14).

[0433] Table 14 Tumor inhibition rate of ADC in mice

[0434]

[0435]

[0436] Example 25 ADC inhibition of MDA-MB-231 mouse tumor model efficacy test

[0437] This example detects the tumor inhibition effect of a candidate ADC (B31-H3L3-MMAE) in animals. The tumor cells used are triple-negative breast cancer cells MDA-MB-231 (Shanghai Cell Bank, Chinese Academy of Sciences, C2006040), and 99961.1-MMAE is used as a positive control.

[0438] The specific method is as follows: 6-8 week old female nude mice weighing 20-22 g (NSG Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were used, and 1×10 6 MDA-MB-231 cells, when the tumor volume reaches 100mm 3Around 1:10 pm, the mice were randomly divided into groups and cages. There were 5 groups with 10 tumor-bearing nude mice in each group, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 5mpk (mg / kg) and B31-H3L3-MMAE 10mpk) and 2 positive control ADC groups (99961.1-MMAE 5mpk and 99961.1-MMAE 10mpk). The drugs were administered by tail vein injection, once a week and the tumor volume was measured twice, for a total of 3 times / 3 weeks (QW*3). Tumor volume (V) calculation method: V = L × W 2 / 2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed and the tumor inhibition rate was calculated. The results are shown in Figures 21A-21B and Table 15.

[0439] The test results showed that there was no significant difference in the early stage of drug administration among the mice in each group. The PBS group began to lose weight on the 35th day, which was expected to be caused by excessive tumor load. The weight of the mice in the ADC group and the positive control group did not change significantly, indicating that the mice tolerated ADC well. Fig. 21B ); Compared with the PBS group, the positive control and the high-dose group of the test ADC had a considerable tumor inhibition effect, and the effect was significant; the test ADC (B31-H3L3-MMAE) under low-dose conditions showed a tumor inhibition effect that was superior to the positive control ADC. The low-dose group had a certain tumor tissue rebound after drug withdrawal ( Fig.21A and Table 15).

[0440] Table 15 Tumor inhibition rate of ADC in mice

[0441]

[0442]

[0443] Example 26 ADC inhibition of MDA-MB-468 mouse tumor model efficacy test

[0444] This example detects the tumor inhibition effect of a candidate ADC (B31-H3L3-MMAE) in animals. The tumor cells used are triple-negative breast cancer cells MDA-MB-468 (Shanghai Cell Bank, Chinese Academy of Sciences, TCHu136), and 99961.1-MMAE is used as a positive control.

[0445] The specific method is as follows: 6-8 weeks old female nude mice (NOD SCID Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) weighing 21-25 g were used, and 1×10 7When the tumor volume reached about 200 mm3, the cells were randomly divided into groups and cages. There were 6 tumor-bearing nude mice in each group, for a total of 5 groups, including a PBS negative control group, 2 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); 3 tumor-bearing nude mice in each group, for a total of 2 groups, including B31-H3L3-MMAE 10mpk (single administration) and 99961.1-MMAE 10mpk (single administration), and the administration method was tail vein injection, for a total of 3 times / 3 weeks (QW*3) or single administration (singledose). 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 tumor volume and mouse body weight change data were analyzed and the tumor inhibition rate was calculated. The results are shown in Figures 22A-22B and Tables 16-17.

[0446] The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well ( Fig. 22B ); The tumor inhibition rate of the group administered with 10 mpk B31-H3L3-MMAE by QW*3 was slightly higher than that of the group administered with 10 mpk 99961.1-MMAE by QW*3 (Table 17), but all mice achieved complete remission (CR) about 18 days after administration; The group administered with 5 mg / kg B31-H3L3-MMAE by QW*3 had a better tumor inhibition effect than the group administered with 5 mg / kg 99961.1-MMAE by QW*3, and 4 / 6 mice achieved CR 25 days after administration. The group administered with a single dose of 10 mg / kg B31-H3L3-MMAE showed a better tumor inhibition effect than the group administered with a single dose of 10 mg / kg 99961.1-MMAE, among which the B31-H3L3-MMAE group achieved CR in all mice 18 days after administration, while the 99961.1-MMAE group had tumor recurrence 20 days after administration. In this experiment, no tumor recurrence was observed in all B31-H3L3-MMAE groups ( Fig.22A and Tables 16-17).

[0447] Table 16 Tumor inhibition rate of ADC in mice

[0448]

[0449]

[0450] Table 17 Complete remission rate of ADC in mice

[0451]

[0452] Example 27 ADC inhibition of Jeko-1 mouse tumor model efficacy test

[0453] This example detects the tumor-suppressing effect of a candidate ADC (B31-H3L3-MMAE) in animals. The tumor cells used are mantle cell lymphoma cells Jeko-1 (ATCC, CRL-3006), and 99961.1-MMAE is used as a positive control.

[0454] The specific method is as follows: 6-8 weeks old female nude mice (BALB / c Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) weighing 21-25 g were used, and 1×10 7 Jeko-1 cells, when the tumor volume reaches 100mm 3 Around 14:00 p.m., the mice were randomly divided into groups and cages. There were 9 groups with 7 tumor-bearing nude mice in each group, including a PBS negative control group, 4 ADC groups (B31-H3L3-MMAE 2.5 mpk (mg / kg) (QW*3), B31-H3L3-MMAE 10 mpk (QW*3), B31-H3L3-MMAE10 mpk (single dose) and B31-H3L3-MMAE 2.5+3.5 mpk (Q2W*2, i.e., 2.5 mpk on day 15 and 3.5 mpk on day 29, abbreviated as D15 2.5 mpk+D29 3.5 mpk)) and 4 positive control ADC groups (99961.1-MMAE 2.5 mpk (QW*3), 99961.1-MMAE 10 mpk (QW*3), 99961.1-MMAE 10mpk (single dose) and 99961.1-MMAE2.5+3.5mpk (Q2W*2, i.e. 2.5mpk on the 15th day and 3.5mpk on the 29th day, abbreviated as D15 2.5mpk+D293.5mpk)), the administration method is tail vein injection, a total of 3 times / 3 weeks (QW*3) or single dose (singledose) or 2 times / 3 weeks (Q2W*2). Tumor volume (V) calculation method: V = L × W 2 / 2 (where L is the longest tumor diameter and W is the shortest tumor diameter). The tumor volume and mouse body weight change data were analyzed and the tumor inhibition rate was calculated. The results are shown in Figures 23A-23B and Tables 18-19.

[0455] The test results showed that there was no significant change in the body weight of mice in each group during the treatment period, indicating that the mice tolerated ADC well ( Fig. 23B); QW*3 administration group, B31-H3L3-MMAE-10mpk and 99961.1-MMAE-10mpk dose groups had a tumor growth inhibition rate of about 96% TGI, and the tumor inhibition rate was comparable. No tumor recurred 23 days after administration; B31-H3L3-MMAE-2.5mpk and 99961.1-MMAE-2.5mpk dose groups had comparable tumor inhibition effects, with TGIs of 60.47% and 59.22%, respectively. Single administration group, B31-H3L3-MMAE (10mpk) and 99961.1-MMAE (10mpk) dose groups had TGIs of 79.24% and 91.74%, respectively. No tumor recurred 23 days after administration. In the Q2W*2 dosing group, the tumor inhibition effect of the B31-H3L3-MMAE (2.5+3.5mpk) dosage group was comparable to that of the 99961.1-MMAE (2.5+3.5mpk) group, with TGIs of 34.96% and 34.60%, respectively. Fig.23A and Tables 18-19).

[0456] Table 18 Tumor inhibition rate of ADC in mice

[0457]

[0458] Table 19 Tumor inhibition rate of ADC in mice

[0459]

[0460]

[0461] Sequence Listing

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

Claims

1. An anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising three heavy chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy chain variable region as shown in SEQ ID NO: 77 and three light chain CDRs (LCDR1, LCDR2, LCDR3) contained in the light chain variable region as shown in SEQ ID NO:

81.

2. An anti-ROR1 antibody and an antigen-binding fragment thereof that specifically binds to ROR1, comprising HCDR1, HCDR2, HCDR3 of the sequences shown in SEQ ID NOs: 4, 5 and 6; and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOs: 43, 46 and 48.

3. The anti-ROR1 antibody and antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 77 and comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 4, 5, and 6, or consists of SEQ ID NO: 77; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 81 and comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 43, 46, and 48, or consists of SEQ ID NO:

81. 4 . The isolated anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 , wherein the antigen-binding fragment is selected from Fab, Fab′-SH, Fv (eg, scFv), or (Fab′) 2 fragment. 5 . The isolated anti-ROR1 monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 4 , 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 any one of claims 1 to 5, 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. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising a heavy chain and a light chain, wherein: The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 113, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 113, or consists of SEQ ID NO: 113, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 117, or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 117, or consists of SEQ ID NO:

117.

8. An antibody-drug conjugate having an Ab-(LD)n structure, wherein Ab is an anti-ROR1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7, L is a linker, D is a therapeutically active substance or a pharmaceutically active ingredient, and n represents an integer of 1 to 20.

9. The antibody-drug conjugate according to claim 8, wherein the therapeutically active substance or pharmaceutically active ingredient is a cytotoxin, a plant toxin, a small molecule toxin, or a radioactive isotope.

10. The antibody-drug conjugate according to claim 8 or 9, wherein the therapeutically active substance or pharmaceutically active ingredient is Dolastatin and its auristatin derivatives, such as Dolastatin 10, Dolastatin 15, auristatin E, auristatin PE, monomethyl auristatin D (MMAD), monomethylauristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), auristatin F hydroxypropylamide (AF HPA).

11. The antibody-drug conjugate as described in any one of claims 8 to 10, wherein the dolastatin toxin (Dolastatin) and auristatin derivatives thereof are MMAE, MMAF, auristatin F hydroxypropylamide or auristatin F phenylenediamine.

12. The antibody-drug conjugate of any one of claims 8 to 11, wherein the linker is a cathepsin-degradable linker (e.g., a valine-citrulline (val-cit) linker, a cBu-Cit linker, and a CX linker), a non-cleavable linker (e.g., an SMCC linker or an MD linker), an acid-sensitive linker, a silicone-structured linker, a disulfide-carbamate linker, an MC-GGFG linker, a TRX linker, a galactoside-containing linker, a pyrophosphate linker, a near-infrared-sensitive linker, or a UV-sensitive linker (e.g., PC4AP).

13. The antibody-drug conjugate of any one of claims 8 to 12, wherein the linker is selected from maleimido-caproyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminoPEG6-propionyl, and maleimidocaproyl (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (al a-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

14. The antibody-drug conjugate of any one of claims 8 to 13, wherein the linker is MC-VC-PAB, SMCC or MC-GGFG.

15. The antibody-drug conjugate of any one of claims 8 to 14, wherein the Ab comprises HCDR1, HCDR2, HCDR3 of the sequences shown in SEQ ID NOs: 4, 5 and 6 and LCDR1, LCDR2, LCDR3 of the sequences shown in SEQ ID NOs: 43, 46 and 48.

16. The antibody-drug conjugate according to any one of claims 8 to 15, wherein the Ab comprises a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

81.

17. The antibody-drug conjugate according to any one of claims 8 to 16, wherein the Ab comprises: The heavy chain has the amino acid sequence shown in SEQ ID NO:113 and the light chain has the amino acid sequence shown in SEQ ID NO:

117.

18. The antibody-drug conjugate according to any one of claims 15 to 17, wherein the linker is MC-VC-PAB and the cytotoxin is MMAE.

19. A pharmaceutical composition comprising: (1) the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the antibody-drug conjugate according to any one of claims 8 to 18, and; (2) Pharmaceutically acceptable carrier.

20. An isolated polynucleotide molecule encoding the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

21. A vector comprising the nucleic acid molecule according to claim 20, preferably, the vector is an expression vector.

22. A host cell comprising the vector of claim 21 or the nucleic acid molecule of claim 20.

23. Use of the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the preparation of an antibody-drug conjugate for preventing or treating cancer, or in the preparation of a medicament for preventing or treating a disease associated with abnormal expression of ROR1.

24. Use of the antibody-drug conjugate according to any one of claims 8 to 18 in the preparation of a medicament for preventing or treating a disease associated with abnormal expression of ROR1.

25. The use according to claim 24, wherein the disease associated with abnormal expression of ROR1 is cancer with high expression of ROR1.

26. The use according to claim 25, wherein the cancer that highly expresses 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 method for killing cells expressing ROR1 or inhibiting the growth of cells expressing ROR1, comprising contacting the cells with an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or an effective amount of the antibody-drug conjugate according to any one of claims 8 to 18, or an effective amount of the pharmaceutical composition according to claim 19.

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