A binding molecule targeting ROR1 and its applications

Abstract

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

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

[0005] ROR1 is a conserved embryonic protein, and its expression gradually decreases during embryonic development and is almost absent or low-expressed in most adult tissues. However, more and more literature has found that ROR1 is expressed in a variety of cancer cells, such as B-cell chronic lymphocytic leukemia (CLL) and other hematological malignancies, renal cell carcinoma, colon cancer, and certain 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 biomarker, ROR1 has become an ideal drug target for cancer treatment.

[0006] Although several antibody drugs targeting ROR1 have been disclosed in the prior art, as a tumor biomarker for pan-cancer, 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 ADCs in the field of tumor treatment, there is still an urgent need for ADCs containing ROR1 with effective therapeutic effects, and the present invention 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 for binding to ROR1 and binding to target cells expressing ROR1;

[0009] (2) Capable of entering cells through endocytosis;

[0010] (3) Suitable for constructing an antibody-drug conjugate for effective treatment.

[0011] In one embodiment, the present invention provides an anti-ROR1 antibody specifically binding to ROR1 and its antigen-binding fragment, which comprises:

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

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

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

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

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

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

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

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

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

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

[0028] 17) The three heavy-chain CDRs (HCDR1, HCDR2, HCDR3) contained in the heavy-chain variable region as shown in SEQ ID NO:78 and the 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 antigen-binding fragment thereof that specifically binds to ROR1, comprising:

[0031] 1) HCDR1, HCDR2, HCDR3 comprising a sequence as shown in SEQ ID NOs: 1, 2, and 3, or a sequence having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions or insertions relative to said sequence; and LCDR1, LCDR2, LCDR3 comprising a sequence as shown in SEQ ID NOs: 28, 29, and 30, or a sequence having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions or insertions relative to said sequence;

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

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

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

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

[0036] 6) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 15, 16, and 17, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences shown in SEQ ID NO: 38, 32, and 33, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0037] 7) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 18, 8, and 19, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences shown in SEQ ID NO: 31, 32, and 33, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0038] 8) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 20, 21, and 12, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences shown in SEQ ID NO: 31, 32, and 33, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0039] 9) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO: 22, 23, and 24, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences shown in SEQ ID NO: 39, 40, and 41, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0040] 10) HCDR1, HCDR2, and HCDR3 comprising the sequences set forth in SEQ ID NO: 4, 25, and 26, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences set forth in SEQ ID NO: 35, 32, and 33, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0041] 11) HCDR1, HCDR2, and HCDR3 comprising the sequences set forth in SEQ ID NO: 18, 25, and 27, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences set forth in SEQ ID NO: 35, 32, and 33, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0042] 12) HCDR1, HCDR2, and HCDR3 comprising the sequences set forth in SEQ ID NO: 4, 5, and 6, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences set forth in SEQ ID NO: 31, 45, and 48, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0043] 13) HCDR1, HCDR2, and HCDR3 comprising the sequences set forth in SEQ ID NO: 4, 5, and 6, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences set forth in SEQ ID NO: 43, 46, and 48, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0044] 14) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO:4, 5, and 6, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences shown in SEQ ID NO:44, 47, and 48, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0045] 15) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO:4, 42, and 6, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences shown in SEQ ID NO:31, 45, and 48, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0046] 16) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO:4, 42, and 6, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences shown in SEQ ID NO:43, 46, and 48, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences;

[0047] 17) HCDR1, HCDR2, and HCDR3 comprising the sequences shown in SEQ ID NO:4, 42, and 6, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; and LCDR1, LCDR2, and LCDR3 comprising the sequences shown in SEQ ID NO:44, 47, and 48, or sequences having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequences; or

[0048] 18) HCDR1, HCDR2, and HCDR3 comprising a sequence as shown in SEQ ID NO: 1, 2, and 3, or a sequence having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequence; and LCDR1, LCDR2, and LCDR3 comprising a sequence as shown in SEQ ID NO: 49, 50, and 30, or a sequence having one or more and no more than 3 amino acid substitutions (such as conservative substitutions), deletions, or insertions relative to said sequence.

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

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

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

[0052] 3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 59, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity 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 an amino acid sequence as shown in SEQ ID NO: 61, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 61, or consists of SEQ ID NO: 61;

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

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

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

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

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

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

[0060] 11) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 shown in SEQ ID NO: 83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 shown in SEQ ID NO: 84, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 shown in SEQ ID NO: 85, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 shown in SEQ ID NO: 75, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 shown in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 shown in SEQ ID NO: 77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77; or

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

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

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

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

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

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

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

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

[0075] 7) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 69, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 69, or consists of SEQ ID NO: 69;

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

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

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

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

[0081] 13) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 82, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 86, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 86, or consists of SEQ ID NO: 86;

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

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

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

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

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

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

[0090] 3) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 59, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 59, or consists of SEQ ID NO: 59, and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 58, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 as shown in SEQ ID NO: 61, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 as shown in SEQ ID NO: 60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 as shown in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 as shown in SEQ ID NO: 62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 shown in SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 65, or consists of SEQ ID NO: 65, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 64, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 64, or consists of SEQ ID NO: 64;

[0094] 7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 67, or consists of SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 68, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 68, or consists of SEQ ID NO: 68, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 as shown in SEQ ID NO: 70, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 70, or consists of SEQ ID NO: 70. The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 69, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 69, or consists of SEQ ID NO: 69;

[0097] 10) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 72, or consists of SEQ ID NO: 72. The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 71, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 71, or consists of SEQ ID NO: 71;

[0098] 11) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 74, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 74, or consists of SEQ ID NO: 74. The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 73, or consists of SEQ ID NO: 73;

[0099] 12) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 75, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 75, or consists of SEQ ID NO: 75. The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 79, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 79, or consists of SEQ ID NO: 79;

[0100] 13) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 76, or consists of SEQ ID NO: 76. The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 80, or consists of SEQ ID NO: 80;

[0101] 14) The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 76, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 76, or consists of SEQ ID NO: 76. The light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 81, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 80, or consists of SEQ ID NO: 80;

[0103] 16) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 77, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 77, or consists of SEQ ID NO: 77, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 78, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 78, or consists of SEQ ID NO: 78, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 80, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 80, or consists of SEQ ID NO: 80;

[0106] 19) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 78, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 78, or consists of SEQ ID NO: 78, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 81, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 78, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 78, or consists of SEQ ID NO: 78, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 83, or consists of SEQ ID NO: 83, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 86, or consists of SEQ ID NO: 86;

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

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

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

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

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

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

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

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

[0117] It should be understood that sequence variants of these constant region domains can also be used, for example, those containing one or more amino acid modifications, where the 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 its antigen-binding fragment that specifically binds to ROR1, which comprises a heavy chain and a light chain, wherein:

[0119] 1) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 93, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 as shown in SEQ ID NO: 92, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 as shown in SEQ ID NO: 91, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with 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 as shown in SEQ ID NO: 90, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 90, or consists of SEQ ID NO: 90;

[0121] 3) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 95, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 95, or consists of SEQ ID NO: 95, and the light chain comprises the amino acid sequence as shown in SEQ ID NO: 94, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 94, or consists of SEQ ID NO: 94;

[0122] 4) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 97, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 97, or consists of SEQ ID NO: 97. The light chain comprises the amino acid sequence as shown in SEQ ID NO: 96, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 96, or consists of SEQ ID NO: 96;

[0123] 5) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 99, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 99, or consists of SEQ ID NO: 99. The light chain comprises the amino acid sequence as shown in SEQ ID NO: 98, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 98, or consists of SEQ ID NO: 98;

[0124] 6) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 101, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 101, or consists of SEQ ID NO: 101. The light chain comprises the amino acid sequence as shown in SEQ ID NO: 100, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 100, or consists of SEQ ID NO: 100;

[0125] 7) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 103, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 103, or consists of SEQ ID NO: 103. The light chain comprises the amino acid sequence as shown in SEQ ID NO: 102, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 102, or consists of SEQ ID NO: 102;

[0126] 8) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 104, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 104, or consists of SEQ ID NO: 104. The light chain comprises the amino acid sequence as shown in SEQ ID NO: 102, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 102, or consists of SEQ ID NO: 102;

[0127] 9) The heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 106, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 106, or consists of SEQ ID NO: 106. The light chain comprises the amino acid sequence as shown in SEQ ID NO: 105, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 105, or consists of SEQ ID NO: 105;

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0147] In certain embodiments of the antibody of any of the foregoing, 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 covers 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, and the antibody-drug conjugate has the following advantages:

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

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

[0153] (3) Treating, preventing, or improving a disorder associated with abnormal function or expression of ROR1 in a subject (such as cancer, such as blood cancer, solid tumor), or treating, preventing, or improving 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 of tumors expressing ROR1 (such as long-term regression);

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

[0157] In one embodiment, the present application provides a conjugate comprising the anti-ROR1 antibody described in 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 its antigen-binding fragment described in the first aspect and at least one therapeutic active substance or pharmaceutically active ingredient, having the structure of Ab-(L-D)n, wherein: Ab is an antibody or its antigen-binding fragment that binds to ROR1 as described in the first aspect of the present invention; L is a linker; D is a therapeutic active substance or pharmaceutically active ingredient, and n represents an integer from 1 to 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 therapeutic active substances or pharmaceutically active ingredients, or a combination of the same therapeutic active substance or pharmaceutically active ingredient. In one embodiment, the antibody-drug conjugate has a drug / antibody ratio (DAR) of 1-20, such as a DAR with a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In one embodiment, the DAR is the average DAR. In one embodiment, the average DAR is 1-15, such as 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 therapeutic active substance or pharmaceutical active ingredient is a cytotoxin, phytotoxin, small molecule toxin, radioisotope, maytansine alkaloid, etc. In a specific embodiment, the cytotoxin is dolastatin and its auristatin derivatives, such as 0101 (2-methylpropanoyl-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-oxoheptan-4-yl]-N-methyl-L-valinamide), 8261 (2-methylpropanoyl-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-oxoheptan-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 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 its antigen-binding fragment by means of a linker in a non-site-specific manner or a site-specific manner.

[0162] In one embodiment, the linker is selected from maleimidocaproyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminopolyethylene glycol 6-propionyl, and maleimidocaproic acid group (mc), maleimidopropionyl group (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yl dithio)-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 sulfhydryl group of cysteine on the anti-ROR1 antibody through an MC-VC-PAB linker, having the structure of anti-ROR1 antibody-MC-VC-PAB-MMAE. IgG1 antibodies have 16 pairs of cysteine residues, which exist in the form of 12 intra-chain and 4 inter-chain disulfide bonds. The inter-chain disulfide bonds are solvent-accessible and can be reduced by a reducing agent to form eight sulfhydryl groups, which then become the conjugation targets (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 sulfhydryl group of cysteine on B62-H3L3 through an MC-VC-PAB linker.

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

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

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

[0168] In a fifth aspect, the present invention provides a vector comprising the nucleic acid molecule according to 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 according to the fifth aspect or the nucleic acid molecule according to the fourth aspect. 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 ROR1 expression in a subject in need thereof, comprising administering to the subject a prophylactically effective amount or a therapeutically effective amount of the antibody or an antigen-binding fragment thereof of the present invention, or a prophylactically effective amount or a therapeutically effective amount of the antibody-drug conjugate of the present invention, or a prophylactically effective amount or a therapeutically effective amount of the pharmaceutical composition of the present invention.

[0171] In one embodiment, the disease associated with abnormal ROR1 expression is a cancer with high ROR1 expression, 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 molecule or the pharmaceutical composition 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 related 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 the antibody or an antigen-binding fragment thereof of the present invention, or an effective amount of the antibody-drug conjugate of the present invention, or an effective amount of the pharmaceutical composition of the present invention.

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

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

[0176] Figure 1 shows the binding activity of each murine antibody clone Fab obtained in this application to huROR1-HEK293 cells measured by the FACS method; Figure 1A Shows 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; Figure 1B Shows the binding activity of C42 Fab and C77 Fab lysates to huROR1-HEK293 cells; NC represents the negative control, and MFI represents the median fluorescence intensity.

[0177] Figures 2A - 2L Shows the SEC-HPLC results of the antibodies in this application; Figure 2A Shows the SEC-HPLC results of B31; Figure 2B Shows the SEC-HPLC results of B32; Figure 2C Shows the SEC-HPLC results of B34; Figure 2D Shows the SEC-HPLC results of B39; Figure 2E Shows the SEC-HPLC results of B62; Figure 2F Shows the SEC-HPLC results of B74; Figure 2G Shows the SEC-HPLC results of C38; Figure 2H Shows the SEC-HPLC results of M71; Figure 2I Shows the SEC-HPLC results of M78; Figure 2J Shows the SEC-HPLC results of C42; Figure 2K Shows the SEC-HPLC results of C77; Figure 2L Shows the SEC-HPLC results of 99961.1.

[0178] Figures 3A - 3B Shows the binding activity of the antibodies in this application to the antigen protein huROR1-His measured by the ELISA method; Figure 3A Shows the binding activity of B31, B32, B34, B39, B62 and B74 to the antigen protein huROR1-His;Figure 3B showed the binding activities of C38, C42, C77, M71 and M78 with the antigen protein huROR1-His.

[0179] Figures 4A - 4B showed the binding activities of the antibodies of this application with A549 tumor cells determined by FACS method; Figure 4A showed the binding activities of B31, B32, B34, B39, B62 and B74 with A549 tumor cells; Figure 4B showed the binding activities of C38, C42, C77, M71 and M78 with A549 tumor cells.

[0180] Figures 5A - 5B showed the binding activities of the antibodies of this application with huROR1-HEK293 cells determined by FACS method; Figure 5A showed the binding activities of B31, B32, B34, B39, B62 and B74 with huROR1-HEK293 cells; Figure 5B showed the binding activities of C38, C42, C77, M71 and M78 with huROR1-HEK293 cells.

[0181] Figures 6A - 6B showed the binding activities of the antibodies of this application with the antigen protein MusROR1-His determined by ELISA method; Figure 6A showed the binding activities of B31, B32, B34, B39 and B62 with the antigen protein MusROR1-His; Figure 6B showed the binding activities of B74, C38, C42, C77, M71 and M78 with the antigen protein MusROR1-His.

[0182] Figures 7A - 7B showed the binding activities of the antibodies of this application with the antigen protein huROR2-His determined by ELISA method; Figure 7A showed the binding activities of B31, B32, B34, B39 and B62 with the antigen protein huROR2-His; Figure 7B showed the binding activities of B74, C38, C42, C77, M71 and M78 with the antigen protein huROR2-His.

[0183] Figures 8A - 8B showed the endocytosis efficiency of the antibodies of this application on huROR1-HEK293 cells determined by FACS method; Figure 8A showed the endocytosis efficiency of B62 and C42 on huROR1-HEK293 cells; Figure 8BShows the endocytosis efficiency of B31, B32, B34, B39, B74, C38, C77, M71 and M78 on huROR1-HEK293 cells.

[0184] Figures 9A - 9F Shows the endocytosis efficiency of the antibody of the present application on huROR1-HEK293 cells determined by the Fab-Zap method; Figure 9A Shows the endocytosis efficiency of B31 and B32 on huROR1-HEK293 cells; Figure 9B Shows the endocytosis efficiency of B34 and B39 on huROR1-HEK293 cells; Figure 9C Shows the endocytosis efficiency of B74 and C38 on huROR1-HEK293 cells; Figure 9D Shows the endocytosis efficiency of C77 and M71 on huROR1-HEK293 cells; Figure 9E Shows the endocytosis efficiency of M78 on huROR1-HEK293 cells; Figure 9F Shows the endocytosis efficiency of B62 and C42 on huROR1-HEK293 cells.

[0185] Figures 10A - 10E Shows the binding activity of the humanized antibody to the antigen protein huROR1-His determined by the ELISA method; Figure 10A Shows the binding activity of B31-H2L2, B31-H1L1 and B31-H2L3 to the antigen protein huROR1-His; Figure 10B Shows the binding activity of B31-H3L2, B31-H3L3 and B31-H3L4 to the antigen protein huROR1-His; Figure 10C Shows the binding activity of B31-H4L2, B31-H4L3 and B31-H4L4 to the antigen protein huROR1-His; Figure 10D Shows the binding activity of B62-H1L1, B62-H1L2, B62-H2L2 and B62-H2L3 to the antigen protein huROR1-His; Figure 10E Shows the binding activity of B62-H2L4, B62-H3L2, B62-H3L3 and B62-H3L4 to the antigen protein huROR1-His.

[0186] Figures 11A - 11B Shows the binding activity of the humanized antibody to A549 tumor cells determined by the FACS method; Figure 11AShowed the binding activities of B31-H1L1, B31-H2L2, B31-H2L3, B31-H3L2, B31-H3L3, B31-H3L4, B31-H4L2, B31-H4L3 and B31-H4L4 with A549 tumor cells; Figure 11B Showed the binding activities of B62-H1L1, B62-H1L2, B62-H2L2, B62-H2L3, B62-H2L4, B62-H3L2, B62-H3L3 and B62-H3L4 with A549 tumor cells.

[0187] Figure 12 Showed the endocytosis efficiencies of B31-H3L3 and B62-H3L3 on huROR1-HEK293 cells determined by the Fab-Zap method.

[0188] Figures 13A - 13B Showed the binding activities of the ADC of the present application with A549 and HT29 tumor cells determined by the FACS method; Figure 13A Showed the binding activities of B31-H3L3-MMAE and B62-H3L3-MMAE with A549 tumor cells; Figure 13B Showed the binding activities of B31-H3L3-MMAE and B62-H3L3-MMAE with HT29 tumor cells.

[0189] Figures 14A - 14D Showed the tumor cell killing effects of the ADC of the present application detected by the MTS method; Figure 14A Showed the killing effect of B31-H3L3-MMAE on A549 tumor cells; Figure 14B Showed the killing effect of B62-H3L3-MMAE on A549 tumor cells; Figure 14C Showed the killing effect of B31-H3L3-MMAE on HT29 tumor cells; Figure 14D Showed the killing effect of B62-H3L3-MMAE on HT29 tumor cells.

[0190] Figures 15A - 15C Showed the killing effects of the ADC of the present application on three tumor cells Jeko-1, MDA-MB-468 and NCI-H1944 determined by the CCK8 method; Figure 15A Showed the killing effects of B31-H3L3-MMAE and B62-H3L3-MMAE on Jeko-1 cells; Figure 15B Showed the killing effects of B31-H3L3-MMAE and B62-H3L3-MMAE on Jeko-1 cells; Figure 15CShows the killing effects of B31-H3L3-MMAE and B62-H3L3-MMAE on NCI-H1944 cells.

[0191] Figure 16 Shows the antigen-dependent killing effects of the ADC of the present application on huROR1-HEK293 cells determined by the CCK8 method.

[0192] Figures 17A - 17B Shows the endocytosis efficiency of the ADC of the present application on A549 and HT-29 tumor cells determined by the FACS method; Figure 17A Shows the endocytosis efficiency of B31-H3L3-MMAE and B62-H3L3-MMAE on A549 tumor cells; Figure 17B Shows the endocytosis efficiency of B31-H3L3-MMAE and B62-H3L3-MMAE on HT-29 tumor cells.

[0193] Figures 18A - 18C Shows the tumor suppression effect of the ADC of the present application in mice with HT-29 tumor models; Figure 18A Shows the changes in tumor volume in mice of different experimental groups during the experimental period, and the arrow indicates the time point of drug administration; Figure 18B Shows the changes in body weight of mice in different experimental groups during the experimental period; Figure 18C Shows the tumor weights in mice of different experimental groups after the experiment.

[0194] Figures 19A - 19C Shows the tumor suppression effect of the ADC of the present application in mice with A549 tumor models; Figure 19A Shows the changes in tumor volume in mice of different experimental groups during the experimental period; Figure 19B Shows the changes in body weight of mice in different experimental groups during the experimental period; Figure 19C Shows the tumor weights in mice of different experimental groups after the experiment.

[0195] Figures 20A - 20B Shows the tumor suppression effect of the ADC of the present application in mice with NCI-N87 tumor models; Figure 20A Shows the changes in tumor volume in mice of different experimental groups during the experimental period, and the arrow indicates the time point of drug administration; Figure 20B Shows the changes in body weight of mice in different experimental groups during the experimental period.

[0196] Figures 21A - 21B Shows the tumor suppression effect of the ADC of the present application in mice with MDA-MB-231 tumor models; Figure 21A Shows the changes in tumor volume in mice of different experimental groups during the experimental period, and the arrow indicates the time point of drug administration; Figure 21BShows the changes in the body weights of mice in different experimental groups during the experimental period.

[0197] Figures 22A - 22B Shows the antitumor effect of the ADC of the present application in mice of the MDA-MB-468 tumor model; Figure 22A Shows the changes in the tumor volumes in mice in different experimental groups during the experimental period, with the arrow indicating the time point of drug administration; Figure 22B Shows the changes in the body weights of mice in different experimental groups during the experimental period.

[0198] Figures 23A - 23B Shows the antitumor effect of the ADC of the present application in mice of the Jeko-1 tumor model; Figure 23A Shows the changes in the tumor volumes in mice in different experimental groups during the experimental period, with the arrow indicating the time point of drug administration; Figure 23B Shows the changes in the body weights of mice in different experimental groups during the experimental period. Detailed Description of the Invention

[0199] Before the present invention is described in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because such methods and conditions can be changed. Additionally, the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive.

[0200] I. Definitions

[0201] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0202] The term "about", when used in conjunction with a numerical value, means to cover a 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.

[0203] The term "and / or", when used to connect two or more alternatives, should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0204] As used herein, the term "comprising" or "including" means including the recited elements, integers or steps, but not excluding any other elements, integers or steps. In this text, when the term "comprising" or "including" is used, unless otherwise specified, the case consisting of the recited elements, integers or steps is also covered. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover an antibody variable region consisting of that specific sequence.

[0205] The term "ROR1" refers to any recombinant or naturally occurring form of receptor tyrosine kinase-like orphan receptor 1 (ROR1), its variants or homologs, said variants or homologs maintaining an activity of, for example, at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the ROR1 activity. The variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the naturally occurring ROR1 protein in the full sequence or a partial sequence (e.g., a partial sequence of 50, 100, 150 or 200 consecutive amino acids). In one embodiment, the ROR1 protein comprises the amino acid sequence of Uniprot ID: Q01973.

[0206] ROR1 is highly expressed in embryos, after which its expression level significantly decreases in the adult stage. However, it is found that the expression of ROR1 is significantly increased in a variety of hematological cancers and solid tumors. Hematological cancers with high expression of ROR1 include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL) and myeloid hematological cancers. Among solid tumors, cancer types expressing ROR1 include colorectal cancer, breast cancer, intestinal cancer, lung cancer, pancreatic cancer, ovarian cancer and other cancers.

[0207] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, provided that 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 in some cases may include fewer chains, for example, antibodies naturally occurring in camels may contain only heavy chains.

[0208] 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 an ROR1 antibody, an anti-ROR1 antibody can inhibit 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 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the ROR1 activity compared to a control.

[0209] The term "antibody fragment" refers to a molecule that is different from a full antibody, which comprises a part of a full antibody and is capable of binding to an antigen that the full antibody binds to. 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; bispecific or bivalent antibodies or fragments thereof; camelid antibodies (heavy-chain antibodies); and multispecific antibodies formed by antibody fragments (e.g., bispecific antibodies).

[0210] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody generally have similar structures, each domain comprising four conserved framework regions (FRs) and three complementarity-determining regions (CDRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain is sufficient to confer antigen-binding specificity.

[0211] "Complementary determining region" or "CDR region" or "CDR" is the region in the variable domain of an antibody that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen - contacting residues ("antigen - contact points"). CDRs are mainly responsible for binding to epitopes. The CDRs in the variable domain are commonly referred to as CDR1, CDR2, and CDR3, 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 many well - known antibody CDR assignment systems, including, for example: Chothia, based on the three - dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877 - 883, Al - Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927 - 948 (1997)), Kabat, based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0212] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above - mentioned ways.

[0213] CDRs can also be determined based on having the same AbM - numbered positions as a reference CDR sequence (e.g., any of the CDRs exemplified in the present invention). In one embodiment, the CDRs of the antibodies of the present invention are positioned according to the AbM numbering scheme.

[0214] Unless otherwise indicated, in the present invention, when referring to the residue positions in the antibody variable regions and CDRs (including the heavy chain variable region residues), it refers to the numbered positions according to the AbM numbering system.

[0215] A "humanized antibody" refers to a chimeric antibody that contains 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., CDR) in the humanized antibody correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the 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.

[0216] As used herein, the terms "bind" or "specifically bind" mean that the binding interaction 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 biomolecular layer interferometry or MSD assay or surface plasmon resonance (SPR).

[0217] The term "half maximal effective concentration (EC 50 )" refers to the concentration of a drug, antibody, or agent that induces a 50% response between baseline and maximum after a specific exposure time.

[0218] The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0219] The term "antibody-drug conjugate" or "ADC" refers to an antibody or antibody fragment that is covalently conjugated to a therapeutically active substance or active pharmaceutical ingredient such 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 can be a cytotoxin capable of killing the cells (preferably cancer cells) targeted by the ADC. The covalent linkage of the therapeutically active substance, active pharmaceutical ingredient, or cytotoxin can be carried out in a non-site-specific manner using a linker, or in a site-specific manner.

[0220] The term "site-specific conjugation" refers to the mode of linking a therapeutically active substance or active pharmaceutical ingredient specifically to a specific site of an antibody. In one embodiment, the conjugation is accomplished by means of a linker.

[0221] The term "cytotoxic agent" can be used interchangeably with "cytotoxin" and refers in the context of the present invention to a substance that inhibits or disrupts cellular function and / or causes cell death or destruction. In one embodiment, the cytotoxic agent can include, but is not limited to, bacterial toxins (such as diphtheria toxin), plant toxins (such as ricin), small molecule toxins, radioisotopes, maytansine alkaloids, and specifically, for example, anthracycline, camptothecin, combretastain, dolastatin and its auristatin derivatives, duocarmycin, enediyne, geldanamycin, indolino-benzodiazepine dimer, maytansine, puromycin, pyrrolobenzodiazepine dimer, taxane, vinca alkaloid, tubulysin, hemiasterlin, spliceostatin, pladienolide, and calicheamicin.

[0222] Any antibody-drug conjugate of the present invention can be prepared by conjugating dolastatin and its auristatin derivatives with an antibody. 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 anti-fungal activities. Modifications of their skeletons have been widely reported in the literature, mainly for the terminal subunits: P1 (N-terminus) and P5 (C-terminus). Modifications of the central peptide subunit also result in effective cytotoxic activity of such substances in vitro. In one aspect, dolastatin and its auristatin derivatives can be, for example, 0101 (2-methylpropanoyl-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-oxoheptan-4-yl]-N-methyl-L-valinamide), 8261 (2-methylpropanoyl-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-oxoheptan-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 (AF HPA), and other auristatins (such as auristatins described in U.S. Publication No. 20130129753), etc.

[0223] Monomethyl auristatin (MMAE), which is desmethyl-auristatin E, is a well-known member of the auristatin compound family, and its structural formula is as follows:

[0224]

[0225] MMAE inhibits microtubule polymerization effectively and plays a role in mitotic inhibition. Due to its cytotoxicity, it cannot be used as a drug, but it is widely used to prepare antibody-drug conjugates. MMAE is conjugated with a monoclonal antibody (MAB) through a linker to form MMAE-MAB. Generally, MMAE-MAB is targeted to tumor cells by the antibody. After MMAE-MAB enters the tumor cells, the linker is cleaved, releasing MMAE, which then exerts its cytotoxic effect to kill tumor cells.

[0226] The terms "linker" and "connector" are used interchangeably in this application and refer to a chemical module that covalently links an antibody to a therapeutically active substance or an active pharmaceutical ingredient in an ADC. In one embodiment, the linker may comprise amino acid residues that link the antigen to the payload. The amino acid residues can form dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide units. Amino acid residues include those that occur naturally as well as unnatural amino acid analogs, such as citrulline or β-amino acids, such as β-alanine, or ω-amino acids such as 4-aminobutyric acid.

[0227] Classified by nature, the linkers suitable for the present invention can be cathepsin-degradable linkers, such as valine-citrulline (val-cit) linker, cBu-Cit linker and CX linker; non-cleavable linkers such as SMCC linker or MD linker; acid-sensitive linkers, silicone grease structure linkers, disulfide-carbamate linkers, MC-GGFG linkers, TRX linkers, galactoside-containing linkers, pyrophosphate linkers, near-infrared sensitive linkers, ultraviolet sensitive linkers such as PC4AP.

[0228] The linker of the present invention can also be a combination of one or more linkers. For example, a cathepsin-degradable linker can be combined with other types of linkers to form a new linker. Therefore, the "linker" described in the present invention encompasses a single type of linker or a combination of different types of linkers, as long as it can conjugate the antibody of the present invention with the drug.

[0229] In specific embodiments, the linker includes but is not limited to maleimidocaproyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), aminopolyethylene glycol 6-propionyl, and maleimidocaproic acid group (mc), maleimidopropionyl group (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), N-succinimidyl 4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

[0230] The term "payload" or "drug payload" or "active payload" refers to the average number of active payloads per antibody within the ADC molecule (wherein "active payload" may be used interchangeably herein with "therapeutically active substance or active pharmaceutical ingredient"). The drug payload can range from 1 to 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) conjugated to the antibody. The ADCs described herein typically have a DAR of 1 to 20, and in certain specific embodiments have 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, and are typically represented as a combination of the letter D and a number, where the number represents the numerical value of the DAR, e.g., D2 represents a drug / antibody ratio with a DAR value of 2. In some embodiments, the DAR is an average DAR, i.e., determined by a detection method (e.g., by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and characterization with HPLC. Quantitative DAR values can also be measured. The DAR may be limited by the number of conjugation sites on the antibody. For example, in the case where the conjugation 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 thiol groups with sufficient reactivity (through which the linker unit can be conjugated). In some embodiments, the average DAR value of the conjugate of the present invention is from 1 to 20, such as 2-18, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0, and ranges with two of these values as endpoints.

[0231] The term "treatment" refers to slowing, interrupting, arresting, alleviating, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired treatment effects include, but are not limited to, preventing the emergence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. In some embodiments, the antibodies of the invention are used to retard disease development or to slow the progression of the disease.

[0232] The term "prevention" includes the inhibition of the occurrence or development of a disease or disorder or the symptoms of a particular disease or disorder. In some embodiments, a subject with a family history of cancer is a candidate for a preventive regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a medicament prior to the manifestation of the signs or symptoms of cancer, particularly in a subject at risk of cancer.

[0233] The term "effective amount" refers to the amount or dose of an antibody or conjugate or composition of the invention that, upon administration to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. The effective amount can be readily determined by an attending physician, who is a person skilled in the art, by considering a variety of factors such as the species of mammal; body weight, age, and general health; the specific disease involved; the degree 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 dosing regimen selected; and the use of any concomitant therapies.

[0234] The term "therapeutically effective amount" refers to the amount that, at the required dosage and for the required period of time, effectively achieves the desired therapeutic result. The therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition can vary depending on a variety of factors such as the disease state, the age, sex, and weight of the individual, and the ability of the antibody or antibody moiety to elicit the desired response in the individual. The therapeutically effective amount is also an amount in which any toxic or detrimental effects of the antibody or antibody fragment or its conjugate or composition are less than the therapeutic beneficial effects. Relative to an untreated subject, 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 predictive of efficacy in human tumors.

[0235] The term "preventively effective amount" refers to the amount that, at the required dosage and for the required period of time, effectively achieves the desired preventive result. Generally, since preventive doses are used in a subject prior to or at an earlier stage of the disease, the preventively effective amount will be less than the therapeutically effective amount.

[0236] The term "pharmaceutical composition" refers to a composition that is in a form that allows the biological activity of the active ingredient(s) contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0237] II. Compositions of the Invention

[0238] In some embodiments, the present invention provides a composition comprising any anti-ROR1 antibody described herein, or an ADC molecule thereof, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient, such as a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient known in the art, including buffers. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises a combination of an anti-ROR1 antibody of the present invention, or an ADC molecule thereof, and one or more other therapeutic agents.

[0239] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible.

[0240] For the use and application of pharmaceutically acceptable excipients, see also "Handbook of Pharmaceutical Excipients", 8th Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0241] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposome formulations and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0242] The route of administration of the compositions of the present invention is according to known methods, e.g., oral, by intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional routes; by sustained release systems or by implantable devices. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implantable devices.

[0243] The subject can be a mammal, such as, for example, a primate, preferably a higher primate, such as, for example, a human (e.g., an individual having a disease described herein or at risk of having a disease described herein). In one embodiment, the subject has a disease described herein (e.g., cancer) or is at risk of having a disease described herein. In certain embodiments, the subject has received or has previously received other treatments, such as chemotherapy treatment and / or radiotherapy. In some embodiments, the subject has previously received or is receiving immunotherapy.

[0244] A drug comprising an antibody described herein can be prepared by mixing an anti-ROR1 antibody of the present invention having the desired purity, or an ADC molecule thereof, with one or more optional pharmaceutically acceptable excipients, preferably in the form of a lyophilized preparation or an aqueous solution.

[0245] The pharmaceutical composition or formulation of the present invention may further comprise more than one active ingredient that is required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, including chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists), etc. The active ingredients are present in a suitable combination in an amount effective for the intended use.

[0246] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules.

[0247] III. Preparation of the Antibodies and Antibody-Drug Conjugates of the Present Invention

[0248] 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 the 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 certain embodiments, the method further comprises recovering the anti-ROR1 antibody from the host cell (or host cell culture medium).

[0249] To recombinantly produce the anti-ROR1 antibody of the present invention, first, the nucleic acid encoding the anti-ROR1 antibody of the present invention is isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes that can specifically bind to the nucleic acid encoding the anti-ROR1 antibody of the present invention.

[0250] The anti-ROR1 antibodies of the present invention prepared as described herein can be purified by known prior art methods 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 one of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0251] The prior art describes a variety of methods for conjugating cytotoxic agents or other therapeutic agents to antibodies. For example, conjugation can occur in the antibody via the amino group of the lysine side chain and the amino group at the N-terminus of the antibody, the carboxyl groups of aspartic acid, glutamic acid and the C-terminus, or the sulfhydryl group of activated cysteine, so that the conjugation reaction occurs. Examples

[0252] The following examples further illustrate the present invention. However, it should be understood that the examples are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art.

[0253] Unless expressly indicated to the contrary, the practice of the present invention will employ conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art.

[0254] Example 1 Preparation and Identification of Raw Materials

[0255] 1.1 Preparation and Identification of ROR1 Control Antibody

[0256] Preparation of ROR1 control antibody: In this application, the anti-ROR1 antibody Cirmtuzumab was used as a positive control antibody. The gene synthesis of the target fragment was carried out by General Biosciences 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. The constructed recombinant protein expression vector was transformed into Escherichia coli DH5α respectively and cultured overnight at 37°C. Then, the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain the desired expression vector expressing cetuximab, and the clone number was 99961.1. The cetuximab expressed below will be simply referred to as 99961.1. By ExpiFectamine TMThe CHO transfection kit (Thermo Fisher, A29129) was used to transfect 293 cells with the expression vector to express cetuximab. Seven days after transfection, the cell culture supernatant was collected and centrifuged at 15,000 g for 10 min. The obtained supernatant was filtered through a 0.22-μm filter membrane, and the antibody in the supernatant was affinity purified using a Protein A / G affinity chromatography column. The target antibody was eluted with 100 mM glycine salt (pH 3.0), and the eluted antibody was exchanged into PBS buffer using an ultrafiltration concentrator tube (Millipore, UFC901096).

[0257] Identification of ROR1 control antibody: The activity of the prepared positive control antibody 99961.1 (IgG1) was detected using the purchased huROR1-His antigen protein (Abnova, ROR-HM401). The specific method was as follows: huROR1-His (2 μg / mL, 30 μL / well) was coated on a 96-well ELISA plate and incubated overnight at 4°C; after washing the plate 3 times, it was blocked with 5% skim milk prepared with PBS at room temperature for 1 hour; after washing the plate 3 times, the control antibody 99961.1 diluted with PBS in gradient 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, then the plate was washed 6 times and developed with TMB for 5 - 20 min. After terminating the color development, the data was read at OD450 using an enzyme-linked immunosorbent assay reader, and the data was processed and graphed 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.

[0258] 1.2 Preparation and identification of antigen protein

[0259] 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 sequences of human ROR1 protein huROR1 ECD AA30 - 406 (Uniprot ID: Q01973), mouse ROR1 protein MusROR1 ECD AA30 - 406 (Uniprot ID: Q9Z139), and human ROR2 protein huROR2 ECD AA34 - 403 (Uniprot ID: Q01974), respectively. The obtained nucleic acid sequences were respectively constructed into the pcDNA3.4 vector, then transformed into Escherichia coli DH5α, cultured overnight at 37°C, and then the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950 - 01). The obtained plasmid was used with ExpiFectamine TM293 transfection kit (Gibco TM , A14524) was transiently transfected into HEK293 cells( CRL-1573 TM ). After 7 days of expression, the supernatant of the cell culture 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 100 mM glycine salt (pH = 3.0), concentrated, and the buffer was exchanged to finally obtain the antigen protein (huROR1-huFc). 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. Each eluted protein was exchanged into PBS buffer through an ultrafiltration concentrator tube (Millipore, UFC901096) to finally obtain the antigen proteins (huROR1-His, MusROR1-His, huROR2-His).

[0260] Antigen identification: The prepared antigens (huROR1-His, huROR1-huFc) were detected with the qualified antibody 99961.1 (IgG1) obtained in Example 1.1. The specific method is as follows: ELISA plates were coated with 2 μg / mL huROR1-His and huROR1-huFc respectively overnight at 4°C, and the purchased antigen proteins huROR1-His and huROR1-huFc (KaiKa Biotech ROR-HM201) were used as positive controls; after washing the plates 3 times, they were blocked with 5% skim milk prepared with PBS at room temperature for 1 hour; after washing the plates 3 times, antibody 99961.1 diluted with PBS gradient was added and incubated at room temperature for 1 hour; after washing the plates, 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, then the plates were washed 6 times and TMB was added for color development for 5 - 20 min, the color development reaction was terminated, and data was read at OD450 with an enzyme-linked immunosorbent assay reader, and the data was processed and graphed with 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.

[0261] Example 2 Construction and identification of a cell line overexpressing human ROR1

[0262] 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 onto the pLVX-puro plasmid (Clontech, Cat#632164). Then, the obtained plasmid was electrotransformed into HEK293 cells ( TM Transfection System, MP922947) by an electroporator (Invitrogen, Neon CRL-1573 TM ). After electroporation, the obtained cells were respectively transferred into DMEM medium (Gibco, 11995065) containing 10% (v / v) FBS (Gibco, 15140-141) and no antibiotics, and then the cells were transferred into 10×10 cm cell culture dishes and cultured for 48 hours. Then, the cells were dispensed into 96-well cell culture plates at a density of 10 4 cells / well, and puromycin with a final concentration of 2 μg / mL was added as a screening pressure. After about 2 weeks, the cell lines forming clones were picked for identification.

[0263] Flow cytometry identification of huROR1-HEK293 cells: The cells of the above cell line in the logarithmic growth phase were digested and plated into 96-well plates. After washing with FACS buffer (1×PBS buffer containing 2% (v / v) FBS), the primary antibody (99961.1) diluted with PBS gradient was added and incubated at 4°C for 30 min; after washing, the prepared fluorescent secondary antibody anti-human IgGFc (abcam, 98596) was added and incubated at 4°C for 30 min; finally, the detection was performed by a flow cytometer (Beckman, CytoFLEXAOO-1-1102). The detection results showed that the huROR1-HEK293 cell line with high surface expression of human ROR1 was obtained.

[0264] Example 3 Animal immunization and immune library construction

[0265] 3.1 Immunization protocol

[0266] Three Balb / C mice (Shanghai Lingchang Biotechnology Co., Ltd.) were cross-immunized by subcutaneous injection and intraperitoneal injection with huROR1-huFc and huROR1-His antigens, once every two weeks for a total of 4 times. One week after the 4th immunization, the mouse blood was taken for detection of immune titer. Finally, the mice were boosted with huROR1-huFc once again.

[0267] 3.2 Detection of serum antibody titer of immunized mice

[0268] Coat an ELISA plate with 2 μg / mL huROR1-His and huROR1-huFc overnight at 4°C (30 μL / well). After washing the plate 3 times, block it with 5% skim milk prepared with PBS at room temperature for 1 hour. After washing the plate 3 times, add mouse serum diluted with PBS in gradients, and at the same time add antibody 99961.1 as a positive control, and incubate at room temperature for 1 hour. After washing the plate, add 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 with PBS and incubate at room temperature for 1 hour. Then wash the plate 6 times, add TMB for color development for 5 - 20 min. After terminating the color development reaction, read the data at OD450 using an enzyme-linked immunosorbent assay (ELISA) reader, and process the data to plot a graph using Graphpad prism. The results show that the serum titers of all 3 mice meet the standards.

[0269] 3.3 Construction of a phage display antibody gene library

[0270] After immunization, take the spleens of mice, collect splenocytes after grinding and filtering, and add 1 mL of TRIzol TM Reagent (Thermo Fisher, 15596026) to lyse the splenocytes, extract total RNA by the phenol-chloroform method, and reverse transcribe the extracted RNA into cDNA using a reverse transcription kit (TaKaRa, 6210A). Then, using the cDNA as a PCR template, specifically amplify the variable region genes of the light and heavy chains of the antibody with primers specific to the murine antibody sequence. The PCR products are digested with NcoI and NotI to obtain antibody gene fragments, which are inserted into the phage display vector and ligated with T4 ligase. The ligation products are recovered using a DNA recovery kit (Omega, D6492-02), and finally transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) using an electroporator (Bio-Rad, MicroPulser). Spread the electroporated bacteria on a solid plate of 2-YT (C + / K + 2-YT) containing ampicillin and tetracycline to amplify the SS320 bacteria with correctly transformed antibody plasmids, and package them using VSCM13 helper phage (purchased from Stratagene) to obtain a phage display library containing Fab sequences.

[0271] Example 4 Screening of the phage display antibody gene library

[0272] 4.1 Screening of the phage display antibody gene library by the cell method

[0273] Culture hROR1-HEK293T cells in a T25 culture flask. When the cell growth density approaches 90%, remove the culture supernatant, wash once with PBS (Source Culture, B310KJ), then add 2 mL of 4% paraformaldehyde (Sangon Biotech, E672002-0500) and fix for 0.5 hour. Finally, wash twice with PBS and use it as the screening raw material. During screening, incubate the phage display library with the fixed hROR1-HEK293T cells at room temperature for 1 hour. After washing three times with 1×PBS, add 2 mL of glycine-HCl (pH = 2.0), gently mix for 10 minutes to elute the phages specifically binding to human ROR1. Then, infect the log-phase SS320 bacteria (Lucigen, 60512-1) with the eluted supernatant, let it stand for 30 minutes, then culture at 37°C and 220 rpm for 1 hour. Add VSCM13 helper phage, let it stand for 30 minutes, continue to culture at 37°C and 220 rpm for 1 hour, centrifuge and transfer to + / K + 2-YT medium. The finally obtained phages are continued to be used for the second round of screening. Repeat the screening multiple times. At the same time, randomly select 10 clones for sequence analysis in each round to evaluate the library. After 3 rounds of screening, the sequences in the library are significantly enriched.

[0274] 4.2 Screening of phage display antibody gene library by immunotube method and magnetic bead method

[0275] Enrich specific antibodies against the antigen by the immunotube method and the magnetic bead method, and the two methods complement and verify each other.

[0276] The screening by the immunotube method is a panning process of coating the antigen protein huROR1-His or huROR1-huFc on the surface of an immunotube with high adsorption capacity, adding the phage display antibody library into the immunotube and incubating, washing and eluting with the antigen protein adsorbed on the surface of the immunotube. After 2-4 rounds of panning, finally enrich the specific monoclonal antibody Fab against the antigen. In this example, after 3 rounds of panning, the monoclonal antibody Fab against human ROR1 is enriched. For the specific method, refer to Example 2.4.2 in Patent CN112250763B.

[0277] Magnetic bead screening is a panning process in which the antigen protein huROR1-His is biotinylated and then combined with magnetic beads conjugated with streptavidin. Through incubation, washing, and elution of the magnetic beads bound to the antigen with the antibody gene phage display library. Usually, 3-4 rounds of panning are experienced, and specific monoclonal antibodies against the antigen can be enriched in large quantities. In this example, biotinylated huROR1-His was used for screening the phage display library. After 3 rounds of panning, the primary screening of monoclonal antibodies Fab against human ROR1 was carried out. The specific method refers to Example 2.4.1 in Patent CN112250763B.

[0278] 4.3 Selection of Monoclonal

[0279] ELISA was used to detect each round of eluted phage pools to evaluate the enrichment effect. 10 clones were randomly selected from each round of screened phage pools for sequence analysis. By comprehensively analyzing the enrichment effect and the repeatability ratio of the measured sequences, a suitable round was selected for monoclonal selection.

[0280] For the primary screening of ELISA monoclonal antibodies, the antigen protein huROR1-His was used. The antibody Fab that bound to huROR1-His obtained from the primary screening was prepared into a Fab lysate, and then detected and verified by flow cytometry analysis (FACS) using the overexpressing cells huROR1-HEK293 prepared in Example 2.1. A total of 11 antibody Fab molecules that specifically bound to human ROR1 were screened, and the 11 murine antibody Fabs obtained were named with the corresponding clone numbers (B62, B31, B32, B74, B34, B39, C38, C77, C42, M71, and M78). The specific FACS results are shown in Figures 1A - 1B . The CDR amino acid sequences of the obtained murine antibody Fabs are shown in Table 1. The AbM method was used to define the CDR to determine the CDR sequences.

[0281] Table 1 Amino Acid Sequences of the CDR Regions of Murine Antibodies

[0282]

[0283]

[0284]

[0285] Example 5 Antibody Construction, Expression, and Purification

[0286] 5.1 Plasmid Construction

[0287] The VH-encoding sequence in the Fab sequences of the monoclonal B62, B31, B32, B74, B34, B39, C38, C77, C42, M71, and M78 obtained by screening was ligated to the encoding sequence of the heavy-chain constant region of human IgG1 (SEQ ID NO: 52) to obtain the encoding sequence of the heavy chain of the chimeric antibody. The VL-encoding sequence in the Fab sequence was ligated to the encoding sequence of the Kappa type of the human light-chain constant region (CL) (SEQ ID NO: 53) to obtain the encoding sequence of the light chain of the chimeric antibody. The encoding sequences of the heavy and light chains of the antibody were respectively inserted into the eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen), transformed into Escherichia coli DH5α, and cultured overnight at 37°C. The plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain an endotoxin-free antibody plasmid for eukaryotic expression.

[0288] 5.2 Expression and purification of antibodies

[0289] The full-length antibody sequence obtained above was expressed through the Expi CHO transient expression system (Thermo Fisher, A29133). The specific method is as follows: On the day of transfection, confirm that the density of CHO cells is about 7×10 6 to 1×10 7 viable cells / mL, and the cell viability > 98%. At this time, adjust the cells to a final concentration of 6×10 6 cells / mL with fresh ExpiCHO expression medium pre-warmed at 37°C. Dilute the target plasmid with OptiPRO TM SFM (add 1 μg of plasmid to 1 mL of the medium), and at the same time dilute ExpiFectamine TM CHO reagent with OptiPRO TM SFM. Then mix the two in equal volumes and gently pipette to mix evenly to prepare the ExpiFectamine TM CHO / plasmid DNA mixture. Incubate at room temperature for 1 - 5 min, slowly add it to the prepared cell suspension while gently shaking, and finally place it in a cell culture shaker and culture at 37°C and 8% CO2.

[0290] 18 - 22 h after transfection, add ExpiCHO TM Enhancer reagent and ExpiCHO TM Feed reagent to the culture medium, and place the shake flask in a shaker at 32°C and 5% CO2 and continue to culture. On the 5th day after transfection, add the same volume of ExpiCHO TMThe Feed reagent was slowly added while gently mixing the cell suspension. Seven days after transfection, the cell culture supernatant expressing the target protein was collected and centrifuged at 15,000 g for 10 min. The resulting supernatant was affinity purified using MabSelect SuRe LX (GE, 17547403), and then the target protein was eluted with 100 mM sodium acetate (pH 3.0), followed by neutralization with 1 M Tris-HCl. Finally, the obtained protein was exchanged into PBS buffer using an ultrafiltration concentrator tube (Millipore, UFC901096).

[0291] Detection of the Physicochemical Properties of the Antibody in Example 6

[0292] In this example, SDS-PAGE and SEC-HPLC were used to detect the relative molecular weight and purity of the obtained antibody above.

[0293] 6.1 Identification of the Antibody by SDS-PAGE

[0294] Preparation of non-reducing solution: 1 μg of each of the obtained antibodies and the control product IPI (Ipilimumab) were respectively added to 5×SDS loading buffer and 40 mM iodoacetamide, heated in a dry bath at 75 °C for 10 min, cooled to room temperature, and then centrifuged at 12,000 rpm for 5 min to take the supernatant.

[0295] Preparation of reducing solution: 2 μg of each of the obtained antibodies and the control product IPI were respectively added to 5×SDS loading buffer and 5 mM DTT, heated in a dry bath at 100 °C for 10 min, cooled to room temperature, and then centrifuged at 12,000 rpm for 5 min to take the supernatant. The supernatant was added to a Bis-tris 4-15% gradient gel (GenScript) for gel electrophoresis, and the protein bands were visualized by Coomassie Brilliant Blue staining.

[0296] The protein gel with visualized protein bands (decolorized with decolorizing solution until the gel background was transparent) was scanned using an EPSON V550 color scanner, and the purity of the reducing and non-reducing bands was calculated by the peak area normalization method using ImageJ.

[0297] The test results showed that the bands of the non-reducing gel of each antibody were around 150 kD, and the bands of the reducing gel were around 55 kD and 25 kD, which were in line with the expected sizes. The purity of all the antibodies obtained in this application detected by the reducing gel was greater than 95% (Table 2).

[0298] 6.2 Identification of the Monomer Purity of the Antibody by SEC-HPLC

[0299] Material preparation: 1. Mobile phase: 150 mmol / L phosphate buffer, pH 7.4; 2. Sample preparation: Dilute each antibody and the control product IPI to 0.5 mg / mL with the mobile phase solution. Agilent HPLC 1100 or Shimadzu LC2030C PLUS liquid chromatograph, the chromatographic column is XBridge BEH (SEC 3.5 μm, 7.8 mm I.D.×30 cm), the flow rate of Waters is set at 0.8 mL / min, the injection volume is 20 μL, and the wavelengths of the VWD detector are 280 nm and 214 nm. Inject the blank solution, the IPI control product solution and the antibody sample solution in sequence. Calculate the percentages of high molecular polymers, antibody monomers and low molecular substances in the sample according to the area normalization method.

[0300] The results are as Figures 2A - 2K shown in Table 2. Except for antibody B74, the SEC monomer purity of the other antibodies is greater than 96%.

[0301] Table 2 Antibody expression levels and physicochemical properties obtained in this application

[0302] 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

[0303] Example 7 Detection of antigen-binding activity of antibodies

[0304] In this example, the binding of the 11 expressed antibodies (B62, B31, B32, B34, B39, B74, C38, C42, C77, M71 and M78) to the 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 a human non-small cell lung cancer cell line that overexpresses human ROR1.

[0305] 7.1 Detection of the binding ability of antibodies to the antigen protein huROR1-His based on ELISA

[0306] Coat a 96-well ELISA plate (30 μL / well) with 2 μg / mL of huROR1-His and incubate overnight at 4°C. The next day, wash the wells 3 times with PBST and then block with 5% skim milk for 2 h. After washing the plate 3 times with PBST, add various antibodies at gradient dilutions (3.00000, 0.33333, 0.11111, 0.03704, 0.01235, 0.00412, 0.00046, 0.00005 μg / mL) and the positive control antibody 99961.1 and incubate for 1 h. Then wash 3 times with PBST, add the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) and incubate for 1 h. After incubation, wash the plate 6 times with PBST, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development result, add 2 M HCl to terminate the reaction, and read the data at OD450 using an ELISA reader (Molecular Devices, SpecterMax 190).

[0307] The results are as Figures 3A - 3B shown. All the antibodies and the antigen protein huROR1-His obtained in this application have good affinity activities, and the affinities of each antibody are comparable to that of 99961.1.

[0308] 7.2 Detection of the binding ability of antibodies to huROR1-HEK293 and A549 tumor cells based on FACS

[0309] In this example, the binding activities of antibodies were evaluated using two types of cells: human ROR1-overexpressing cells huROR1-HEK293 and A549 tumor cells.

[0310] The specific method is as follows: Prepare single-cell suspensions of huROR1-HEK293 cells or A549 cells in the logarithmic growth phase, adjust the density to 1×10 6 cells / mL, add 100 μL per well to a 96-well round-bottom plate, centrifuge at 4°C and 300 g, and remove the supernatant. Add the antibodies obtained in this application at gradient dilutions and the positive control antibody 99961.1 to the corresponding wells, mix well, and incubate at 4°C for 30 min. After washing the incubated cell mixture 3 times, add 100 μL of a 1:300 dilution of the secondary antibody Goat F(ab’)2 Anti-Human IgG-Fc (abcam, ab98596), incubate at 4°C in the dark for 30 min, and then detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102) after washing 3 times.

[0311] The results are shown in the figure. On A549 cells ( Figures 4A - 4B), except for C42, the affinity of other antibodies is better than that of the control antibody 99961.1; on huROR1-HEK293 cells ( Figures 5A - 5B ), the affinity of all antibodies is comparable to that of the control antibody 99961.1.

[0312] Example 8 Detection of Antibody Species and Cross-Reactivity within the Same Family

[0313] In this example, the cross-reactivity of each antibody obtained in this application was detected among species and within the same family. The mouse ROR1 antigen protein MusROR1-His and human ROR2 antigen protein huROR2-His prepared in Example 1.2 were used to identify the cross-reactivity of the antibodies obtained in this application among species and within the same family.

[0314] 8.1 Identification of Cross-Reactivity of Antibodies among Species

[0315] Coat a 96-well ELISA plate (30 μL / well) with 2 μg / mL of MusROR1-His at 4°C overnight. The next day, wash the plate 3 times with PBST and then block it with 5% skim milk for 2 h. After washing the plate 3 times with PBST, add various antibodies and the positive control antibody 99961.1 at gradient dilutions (1.00000, 0.11111, 0.03704, 0.01235, 0.00412, 0.00137, 0.00015, 0.00002 μg / mL) and incubate for 1 h. Then, after washing 3 times with PBST, add the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) and incubate for 1 h. After incubation, wash the plate 6 times with PBST, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, add 2 M HCl to terminate the reaction, and read the data at OD450 using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, SpecterMax 190).

[0316] The results are as Figures 6A - 6B and shown in Table 3. B62, B32, C38, and C42 bind to the antigen protein MusROR1-His, showing good cross-reactivity with mice. However, other antibodies and the positive control antibody 99961.1 do not bind to the mouse-derived antigen protein. Therefore, the antibodies B62, B32, C38, and C42 obtained in this application have wide applications in experiments and tests based on mouse models.

[0317] 8.2 Detection of Cross-Reactivity within the Same Family of the Antibodies in this Application

[0318] Coat a 96-well ELISA plate with 2 μg / mL of huROR2-His (30 μL / well) and incubate overnight at 4°C. The next day, wash the wells 3 times with PBST, then block with 5% skim milk for 2 h. After washing the plate 3 times with PBST, add serially diluted antibodies and the positive control antibody 99961.1 and incubate for 1 h. Then, after washing 3 times with PBST, add the secondary antibody Goat-anti-human Fc-HRP (abcam, ab97225) and incubate for 1 h. After incubation, wash the plate 6 times with PBST, add TMB (SurModics, TMBS-1000-01) for color development. According to the color development results, add 2 M HCl to terminate the reaction, and read the data at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0319] The results are as Figures 7A - 7B shown in Table 3, and none of the antibodies bind to the antigen protein huROR2-His, indicating that the antibodies prepared in this application can specifically bind to human ROR1.

[0320] Table 3 Species cross-reactivity and homologous cross-reactivity of the antibodies obtained in this application

[0321] Clone number Species MusROR1 - His huROR2 - His B31 Mouse Does not bind Does not bind B62 Mouse Binds Does not bind B32 Mouse Binds Does not bind B34 Mouse Does not bind Does not bind B39 Mouse Does not bind Does not bind B74 Mouse Does not bind Does not bind C38 Mouse Binds Does not bind C42 Mouse Binds Does not bind C77 Mouse Does not bind Does not bind M71 Mouse Does not bind Does not bind M78 Mouse Does not bind Does not bind

[0322] Example 9 Detection of antibody endocytosis efficiency

[0323] In this example, two detection methods, FACS and Fab-Zap, were used to detect the endocytosis efficiency of the antibodies obtained in this application. The FACS method for endocytosis detection binds cells with supersaturated antibodies to detect the endocytosis efficiency of antibodies in a short time; the Fab-Zap method binds cells with antibodies conjugated with Fab-Zap toxin at different concentrations, and the toxin is brought in through endocytosis to kill target cells, and this method reflects the long-term cumulative effect of endocytosis efficiency.

[0324] 9.1 Detection of the endocytosis efficiency of the antibodies in this application by the FACS method

[0325] Antibody dilution: Dilute the test antibody with DMEM complete medium to a final concentration of 10.0000 μg / mL.

[0326] Cell treatment: Digest huROR1-HEK293 cells and add DMEM complete medium. After thoroughly mixing the cells, count the cells and determine their viability. Take 10 6 % of the cells and add them to a 1.5 mL centrifuge tube, centrifuge at 300 g for 5 minutes, discard the supernatant, resuspend with 1 mL of pre-cooled DMEM medium, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0327] Primary antibody incubation: Take 1000 μL of the pre-cooled diluted antibody mentioned above, add it to the centrifuge tube containing cells to prepare an antibody-cell suspension, add the antibody-cell suspension to a 96-well plate, and incubate at 4°C.

[0328] Extracellular secondary antibody incubation: Quickly transfer the suspension in the 96-well plate to a second 96-well plate. Add 180 μL of pre-cooled FACS buffer to each well of the second 96-well plate and wash twice. Then add 100 μL of diluted secondary antibody FITC-labeled anit-huFc or RPE-labeled anit-huFc (the secondary antibody is diluted 1:150 with FACS buffer) to each well; incubate at 4°C for 30 min.

[0329] Cell fixation: After incubation, centrifuge and discard the supernatant, add 180 μL of pre-cooled FACS buffer to each well, and wash the cells twice. Centrifuge to remove the supernatant, and fix the cells with 100 μL of 4% paraformaldehyde at room temperature for 30 min.

[0330] Cell membrane disruption: After fixation, add 180 μL of FACS buffer and wash twice. Add 100 μL of pre-heated 0.5% Triton X-100 to each well and perforate at room temperature for 5 min.

[0331] Intracellular secondary antibody incubation: After washing the 96-well plate, add 180 μL of pre-heated PermeabilizationBuffer (Invitrogen TM eBioscience TM , 00-8333-56) to each well and wash twice. Add 100 μL of diluted secondary antibody RPE-labeled anit-huFc (diluted 1:150 with Perm buffer) to each well. Incubate at room temperature for 60 min.

[0332] Fluorescence detection: After washing the 96-well plate, resuspend with 100 μL of FACS buffer and detect by flow cytometry.

[0333] In this experiment, two different fluorescence settings were used to stain each sample. The first set was FITC+PE, that is, first stain the extracellular primary antibody with FITC secondary antibody, and after membrane disruption, stain the intracellular primary antibody with PE secondary antibody. In this group, PE is the intracellular signal and FITC is the extracellular signal; the second set was PE+PE, that is, first stain the extracellular primary antibody with PE secondary antibody, and after membrane disruption, stain the intracellular primary antibody with PE secondary antibody. In this group, PE is the sum of the intracellular and extracellular signals. At the same time, both groups of samples were detected with FITC and PE channels, and the calculated value of the endocytosis rate was calculated from the detection value of the PE channel. The specific formula is:

[0334] Endocytosis rate = (FITC + PE) PE channel of one group / (PE + PE) PE channel of the second group × 100%.

[0335] The results are as Figures 8A - 8B shown. The results of this example show that the endocytosis efficiency of all antibodies prepared in this application within a short period of time (within 3 h) is better than that of the control antibody 99961.1.

[0336] 9.2 Detection of antibody endocytosis efficiency by Fab-Zap method

[0337] In this experiment, the endocytic activity of antibodies was detected by the cytotoxicity of antibody-mediated Fab-ZAP endocytosis. Fab-ZAP is a Fab fragment conjugated with 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 with chimeric antibodies, Fab-ZAP makes the chimeric antibodies carry 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 was detected by MTS (Promega, G3580) to detect whether the antibodies were endocytosed.

[0338] The specific experimental method is as follows: First, dilute Fab-Zap with DMEM complete medium to 0.4 nM, and then prepare antibody dilutions of each antibody obtained in this application and the positive control antibody by gradient dilution of 0.4 nM Fab-Zap (0.020000, 0.006667, 0.002222, 0.000741, 0.000247, 0.000082, 0.000027, 0.000009 μg / mL). Prepare a single-cell suspension of huROR1-HEK293 cells in the logarithmic growth phase, adjust the density to 6×10 6 cells / mL, inoculate 50 μL per well into a 96-well plate, and then take the aforementioned antibody dilutions and add 50 μL per well to the cell culture plate, and mix well by pipetting. Place the cell culture plate in a 37 °C cell incubator and incubate for 48 hours. After incubation, add 7.5 μL of Triton X-100 solution to each well, gently tap and mix well, and place the cell culture plate in a 37 °C cell incubator and incubate for 0.5 hour. Then continue to add 20 μL of MTS to each well and incubate at 37 °C for 1 - 4 hours. Finally, centrifuge the cell culture plate at 1000 rpm for 5 minutes, read the data with an enzyme-linked immunosorbent assay reader, and detect the wavelength A492.

[0339] The results are as Figures 9A - 9FAs shown, it indicates that under the antibody concentration conditions defined in this embodiment, 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 above a certain threshold, it is difficult to show a significant difference when the accumulated toxin differences are small.

[0340] Example 10 Detection of Antibody Affinity Kinetics (Gator)

[0341] In this embodiment, the affinity of the antibody obtained in this application and the positive control antibody 99961.1 with the antigen protein huROR1-His was detected based on the Gator device.

[0342] First, PBS (10 mM pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from Yuanpei) were configured into Q buffer. The stock solution of the antibody to be tested was diluted to a working solution with a final concentration of 30 nM using the prepared Q buffer; the stock solution of the antigen protein huROR1-His was configured into working solutions with serial dilutions (480, 240, 120, 60, 30, 15, 7.5 nM) using Q buffer. Then, using the Gator instrument and its supporting software, the Advanced Kinetics experimental mode was selected for detection and analysis. The test results are shown in Table 4.

[0343] The results showed that: except that the C42 antibody had comparable affinity with the control antibody 99961.1, the affinities of the other antibodies were better than that of the control antibody 99961.1 by one order of magnitude or more. Among them, the affinity of the B62 antibody was better than that of the control antibody 99961.1 by about two orders of magnitude, and the affinity reached 9.84E-10.

[0344] Table 4 Detection Results of Antibody Affinity Kinetics Obtained in this Application

[0345]

[0346]

[0347] Example 11 Analysis of Antibody Affinity Kinetics (Biacore)

[0348] In this embodiment, the affinity of the antibody obtained in this application and the positive control antibody 99961.1 with the antigen protein huROR1-His was detected based on the Biacore device.

[0349] Coupling of the protein: Dilute the huROR1-His protein produced in Example 1.2 with NaAc buffer at pH 5.0 to 5.6 μg / mL, set the flow rate to 10 μL / min, the activation time of the chip with the mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is the default value of 420 s, and fix the antigen protein huROR1-His to about 75 RU level by the coupling mode with the preset coupling amount. Block the activated groups that have not bound the test sample with ethanolamine.

[0350] Sample test conditions: Use PBS buffer (pH 7.4) containing 0.05% Tween-20 as the running buffer, use the running buffer as the control test sample, set a series of antibody concentrations (4 nM, 20 nM), set the flow rate to 30 μL / min during sample analysis, the binding time is 120 s, and the dissociation time is 360 s. After the dissociation is completed, regenerate with 10 mM Gly-HCl (pH 2.0) for 20 s to completely remove the antibody bound to the ligand.

[0351] Parameter fitting: The experiment adopts multi-cycle operation, with the response signal taking the analysis time as the abscissa and the response value as the ordinate. After the obtained data is subjected to double-reference subtraction, it is fitted by the BIAcore T200 analysis software. The fitting model used is the 1:1 Langmuir binding model to determine the affinity indexes such as its binding and dissociation constants.

[0352] The results are shown in Table 5. The affinity of antibody B62 is about two orders of magnitude better than that of the control antibody 99961.1, and its KD reaches 6.39E-11, while the KD of the control antibody 99961.1 is only 1.98E-9; in addition, the affinity of antibody B31 is comparable to that of the control antibody 99961.1.

[0353] Table 5 Kinetic detection results of the affinity of the antibodies obtained in this application

[0354] 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

[0355] Epitope grouping by affinity kinetics in Example 12

[0356] In this example, the epitopes of the antibodies obtained in this application and the positive control antibody 99961.1 are grouped by the method of affinity kinetics.

[0357] The specific test method is as follows: First, PBS (10 mM, pH 7.4) (IgG-free, purchased from Jackson ImmunoResearch Lab) + 0.02% Tween 20 (purchased from thermo) + 0.2% BSA (purchased from Yuanpei) are configured into Q buffer. The stock solution of the antibody to be tested is diluted with the prepared Q buffer to a working solution with a final concentration of 100 nM. The stock solution of the antigen huROR1-His is configured into a working solution of 50 nM with Q buffer, and then detected and analyzed using the Gator instrument and its supporting software based on the Tandem setting of the Epitope Binning experimental mode.

[0358] The specific detection and analysis steps are as follows:

[0359] Equilibration stage 1: The probe is equilibrated in Q buffer at a rotation speed of 1000 rpm for 60 s.

[0360] Antigen immobilization stage: After equilibration stage 1, the probe is immobilized in Q buffer containing 50 nM antigen at a rotation speed of 400 rpm for 90 s.

[0361] Equilibration stage 2: After the antigen immobilization stage, the probe is equilibrated in Q buffer at a rotation speed of 1000 rpm for 60 s.

[0362] Antibody 1 binding stage: After equilibration stage 2, the probe is bound in Q buffer containing 100 nM antibody 1 at a rotation speed of 1000 rpm for 180 s.

[0363] Equilibration stage 3: After the antibody 1 binding stage, the probe is equilibrated in Q buffer at a rotation speed of 1000 rpm for 30 s.

[0364] Antibody 2 binding stage: After equilibration stage 3, the probe is bound in Q buffer containing 100 nM antibody 2 at a rotation speed of 1000 rpm for 180 s.

[0365] Regeneration stage: From equilibration stage 1 to the antibody 2 binding stage is one Assay. Before the probe for one Assay is used for the next Assay, it needs to be regenerated in Q buffer at a rotation speed of 1000 rpm for 50 s.

[0366] The results of the epitope grouping of each antibody obtained are shown in Table 6. The results show that the antibody of the present application and the control antibody 99961.1 are divided into 2 epitope groups in total. Only C42 has the same binding epitope as the control antibody, and the binding epitopes of the remaining antibodies are different from those of the control antibody.

[0367] Table 6 Summary of epitope grouping results

[0368] Epitope grouping Clone number 1 99961.1、C42 2 B62, B32, C38, B74, M71, B34, M78, C77, B39, B31

[0369] Example 13 Humanization of Murine Antibodies

[0370] The VH and VL sequences of murine antibodies B31 and B62 screened in Example 4 were respectively aligned with a known human antibody database to find the germline gene VH and VL sequences of the human species with the highest homology to the murine VH and VL sequences. The framework regions of the corresponding germline gene VH and VL sequences were selected (using AbM to define CDRs and framework regions), and the complementary determining region (CDR) sequences of this germline gene were replaced with the corresponding CDR sequences in murine antibodies B31 and B62 of the present application. Then, with the aid of computer prediction and simulation, murine amino acids in the framework regions of murine antibodies B31 and B62 that have an important impact on antigen binding were retained by back mutation. The amino acid sequences of the CDR regions of the humanized antibodies obtained after humanizing murine antibodies B31 and B62 are shown in Table 7. Multiple antibodies after humanizing B31 and B62 were constructed, expressed, and purified by the method of Example 5, and the multiple antibody proteins obtained after humanizing B31 and B62 were identified by SDS-PAGE and SEC-HPLC. The results are shown in Table 8. The results show that the bands of each antibody in the non-reducing gel are around 150 kD, and the bands in the reducing gel are around 55 kD and 25 kD, which are in line with the expected sizes. The purity of all antibodies obtained in the present application detected by the reducing gel is greater than 95%; the SEC monomer purity of all antibodies is greater than 96%.

[0371] Table 7 Amino Acid Sequences of CDR Regions of Humanized Antibodies

[0372]

[0373]

[0374] Table 8 Physicochemical Properties of Humanized Antibodies

[0375] 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

[0376] Example 14 Detection of Antigen Affinity of Humanized Antibodies

[0377] In this example, the affinity effect of the humanized antibody with the human ROR1 antigen protein huROR1-His was detected based on the ELISA method, and the affinity ability of the humanized antibody with A549 tumor cells was detected based on the FACS method. The specific test method refers to Example 7.

[0378] The ELISA detection results are as Figures 10A - 10E shown. The affinity of the humanized antibody with the antigen protein is comparable to that of the corresponding parental molecule and the control antibody 99961.1.

[0379] The results of the FACS test are as Figures 11A - 11B shown. Except for B31-H3L4, B31-H4L3, B31-H4L2, and B31-H4L4, the affinity of the humanized antibody for the antigen protein on A549 tumor cells is comparable to that of the corresponding parental molecule and the control antibody 9996.1.

[0380] Example 15 Detection of the Endocytosis Efficiency of Humanized Antibodies

[0381] In this example, the Fab-Zap method was used to detect the endocytosis of the humanized antibodies B31-H3L3 and B62-H3L3 in Example 13. The specific method was referred to Example 9. The results are as Figure 12 shown. The endocytosis effects of B31-H3L3 and B62-H3L3 are comparable to those of the control antibody 99961.1.

[0382] Example 16 Preparation of ADC

[0383] In this example, the antibodies B62-H3L3, B31-H3L3, and the control antibody 99961.1 were conjugated with the toxin MMAE (a tubulin inhibitor with anti-cancer activity) to construct antibody-drug conjugates. MMAE was connected to the linker MC-VC-PAB to form MC-VC-PAB-MMAE, and was covalently linked to the sulfhydryl group on the cysteine of the antibody through the linker, so that the antibody was conjugated with MMAE to obtain the antibody-drug conjugate. IgG1-type antibodies have 16 pairs of cysteine residues, which exist in the form of 12 intra-chain and 4 inter-chain disulfide bonds. The inter-chain disulfide bonds are solvent-accessible and can be reduced by reducing agents to form eight sulfhydryl groups, which then become the conjugation targets (McCombs J, Owen S. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015; 17: 339-51).

[0384] The specific preparation method is as follows:

[0385] Antibodies B62-H3L3, B31-H3L3 and control antibody 99961.1 were taken out from the -80 °C refrigerator. After melting, they were respectively transferred into 15 mL 30KD ultrafiltration centrifugal tubes, and coupling buffer (per 1 L content: 6.86 g of Na2HPO4·2H2O, 1.58 g of NaH2PO4·H2O, made up to 1000 g with purified water, pH 7.4) was added to make up to 15 mL. Centrifuged at 4500 rpm for about 30 min, concentrated to 2 - 3 mL, and then dialysis buffer (per 1 L content: 0.73 g of histidine, 1.12 g of histidine monohydrochloride monohydrate, made up to 1000 g with purified water, pH 6.0) was added again to make up to 15 mL. Dialyzed repeatedly 8 - 10 times to obtain the antibody stock solution, and the antibody concentration after dialysis was detected.

[0386] The antibody stock solution, 10 mM disulfide bond reducing agent TCEP stock solution (tris(2-carboxyethyl)phosphine hydrochloride stock solution, per 1 L content: 6.86 g of Na2HPO4·2H2O, 1.58 g of NaH2PO4·H2O, made up to 1000 g with purified water), 10 mM DTPA stock solution (diethylenetriaminepentaacetic acid stock solution, per 1 L content: 3.90 g of DTPA, 1.20 g of NaOH, made up to 1000 g with purified water) and coupling buffer were added in sequence to form a reduction reaction system to reduce the sulfhydryl groups on the cysteine of the antibody. The addition amounts of each component in the reduction reaction system are shown in Table 9, so that the antibody concentration in the reduction reaction system is 5 mg / mL, the final concentration of DTPA is 1 mM, 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 mixing well, the reduction reaction system was placed in a 25 °C constant temperature mixer with a rotation speed of 400 rpm, and the reduction reaction was carried out for 2 h.

[0387] MC-VC-PAB-MMAE was weighed and dissolved with DMSO to prepare a 5 mM MC-VC-PAB-MMAE stock solution. After the reduction reaction was completed, the MC-VC-PAB-MMAE stock solution was added to the reduction reaction system in an ice-water bath in sequence, and the addition amount is shown in Table 10 to form a coupling reaction system. After mixing well, the coupling reaction system was placed in a 25 °C constant temperature mixer at 400 rpm, and the coupling reaction was carried out for 1 h to obtain a solution containing ADC.

[0388] After the conjugation was completed, the solution containing the ADC was centrifuged and filtered to obtain an ADC sample, which was transferred to a 15 mL 30 KD ultrafiltration centrifugal tube. The dialysis buffer was added to 15 mL, and centrifuged at 4500 rpm for 20 min to concentrate it to 2 - 3 mL. Then the dialysis buffer was added again to 15 mL, and repeated dialysis was performed 8 - 10 times. The dialyzed ADC sample was 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 showed that an ADC with a purity of over 99% was obtained.

[0389] Table 9 Composition of the reduction reaction system

[0390]

[0391] Table 10 Addition amount of the MC - VC - PAB - MMAE mother liquor

[0392] Clone number B62 - H3L3 B31 - H3L3 99961.1 Molar ratio of MC - VC - PAB - MMAE to antibody 6.0 6.0 6.0 Volume of 5 mM MC - VC - PAB - MMAE stock solution added (μL) 620 384 642

[0393] Table 11 Test results of the ADC sample

[0394]

[0395] Detection of the antigen - binding activity of the ADC in Example 17

[0396] 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.

[0397] The specific method is as follows: Single - cell suspensions of A549 cells or HT - 29 cells in the logarithmic growth phase were prepared, and the density was adjusted to 1×10 6Cells / mL, add 100 μL per well to a 96-well round bottom plate, centrifuge at 4°C and 300 g, and remove the supernatant. Add gradient dilutions (1.0000, 0.3333, 0.1111, 0.0370, 0.0123, 0.0041, 0.0014, 0.0001 μg / mL) of the antibodies of the present application (B62-H3L3 and B31-H3L3), the ADCs of the present application (B62-H3L3-MMAE, B31-H3L3-MMAE), 99961.1, 99961.1-MMAE, and negative control to the corresponding wells, mix well, and incubate at 4°C for 30 min. Wash the incubated cell mixture 3 times, then add 100 μL of a 1:300 diluted secondary antibody, Goat F(ab’)2 Anti-Human IgG-Fc (abcam, ab98596), incubate at 4°C in the dark for 30 min, wash 3 times, and detect by flow cytometry (Beckman, CytoFLEX AOO-1-1102).

[0398] The results are as Figures 13A - 13B shown: The results of binding to A549 cells are shown in Figure 13A . After conjugation, the binding activities of B62-H3L3-MMAE and 99961.1-MMAE to A549 cells were slightly worse than those of the corresponding naked antibodies, and the binding activities of B62-H3L3 and B62-H3L3-MMAE to A549 cells were better than those of the corresponding positive controls; the results of binding to HT-29 cells are shown in Figure 13B : The binding activities of the naked antibodies and the corresponding ADCs to HT-29 cells were comparable, and the binding activities of B62-H3L3 and B62-H3L3-MMAE to HT-29 cells were slightly better than those of the corresponding positive controls.

[0399] Example 18 Detection of the Tumor Cell Killing Effect of ADC Based on the MTS Method

[0400] In this example, the A549 and HT-29 cells were used to detect the killing of tumor cells by the ADCs of the present application and control ADCs, respectively.

[0401] The specific method is as follows: Prepare a single-cell suspension of A549 cells or HT-29 cells in the logarithmic growth phase, adjust the density of A549 to 1×10 4 cells / mL, and adjust the density of HT-29 to 1.5×10 4Cells / mL, add 100 μL per well to a 96-well cell culture plate, and incubate at 37 °C with 5% CO2 for 12 h. Then add ADC samples with gradient dilution (2000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 1.953 nM), and incubate at 37 °C with 5% CO2 for 72 h (A549 cells) or 96 h (HT-29 cells). Then add 40 μL of MTS (Promega, G3580) to each well, incubate at 37 °C for 1 h, and read the data at OD492 using a microplate reader.

[0402] The results are as Figures 14A - 14D shown: The killing effect of B31-H3L3-MMAE on the two tumor cells A549 ( Figure 14A ) and HT-29 ( Figure 14C ) is slightly better than that of the control ADC 99961.1-MMAE, while the killing effect of B62-H3L3-MMAE on A549 ( Figure 14B ) and HT-29 ( Figure 14D ) is slightly worse than that of the control ADC 99961.1-MMAE.

[0403] Example 19 Detection of the killing effect of ADC on tumor cells based on the CCK8 method

[0404] 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 the control ADC.

[0405] The specific method is as follows: Prepare single-cell suspensions of Jeko-1 cells, MDA-MB-468 cells, or NCI-H1944 cells in the logarithmic growth phase. Adjust the density of Jeko-1 to 1×10 5 cells / mL, the density of MDA-MB-468 to 2×10 5 cells / mL, and the density of NCI-H1944 to 1.2×10 5 cells / mL. Add 90 μL per well to a 96-well cell culture plate, and incubate at 37 °C with 5% CO2 for 12 h. Then add ADC samples, and dilute the drug concentration gradient to 500, 158, 50, 15.8, 5, 1.58, 0.5, 0.158, 0.05 nM, and incubate at 37 °C with 5% CO2 for 72 h. Then add 10 μL of CCK8 (Bimake, B34304) to each well, incubate at 37 °C for 1 h, and read the data at OD450 using a microplate reader.

[0406] The test results are as follows Figures 15A - 15C shown. The killing effects of B31-H3L3-MMAE and B62-H3L3-MMAE on three tumor cells Jeko-1 ( Figure 15A ), MDA-MB-468 ( Figure 15B ), and NCI-H1944 ( Figure 15C ) are comparable to those of the control ADC99961.1-MMAE. Among them, the EC 50 values of B31-H3L3-MMAE on Jeko-1, MDA-MB-468, and NCI-H1944 are 38.24 nM, 15.24 nM, and 117.3 nM respectively, and the EC 50 values of B62-H3L3-MMAE on Jeko-1, MDA-MB-468, and NCI-H1944 are 50.14 nM, 28.64 nM, and 128.6 nM respectively.

[0407] Example 20 Detection of the antigen-dependent killing effect of ADC based on the CCK8 method

[0408] In this example, huROR1-HEK293 cells were used to detect the antigen-dependent killing effects of B31-H3L3-MMAE and the control ADC.

[0409] The specific method is as follows: Prepare a single-cell suspension of huROR1-HEK293 cells in the logarithmic growth phase, adjust the cell density to 6×10 4 cells / mL, add 50 μL per well to a 96-well cell culture plate, culture at 37 °C and 5% CO2 for 24 h, then add 50 μL of antibody B31-H3L3 and positive control antibody 99961.1 with a concentration of 100 μg / mL to each well. After incubating at 37 °C for 2 h, add the corresponding ADC B31-H3L3-MMAE and control ADC 99961.1-MMAE with gradient dilutions (42.6667, 21.3333, 10.6667, 5.3333). Culture at 37 °C and 5% CO2 for 96 h, then add 30 μL of CCK8 (absin / Aibixin, abs50003) to each well. After incubating at 37 °C for 1 - 4 h, read the plate at OD450 using an enzyme-linked immunosorbent assay reader. A system with only ADC added without antibody was used as the control group.

[0410] The results are as follows Figure 16 shown. The competition between antibodies B31-H3L3 and 99961.1 can significantly inhibit the killing effects of their corresponding ADCs on huROR1-HEK293 cells, proving that the ADC prepared in this application kills cells depending on the binding of the antibody conjugated to the ADC to the antigen on the cell surface. Therefore, it is antigen-dependent killing rather than non-specific killing by the conjugated toxin.

[0411] Example 21 Detection of the Endocytosis Efficiency of ADC

[0412] In this example, the endocytosis efficiency of the ADC obtained in this application was detected using A549 and HT-29 tumor cells based on the FACS method. For the specific experimental method, refer to Example 9.

[0413] The experimental results showed that on A549 cells ( Figure 17A ), the endocytosis efficiencies of B31-H3L3-MMAE and the control ADC 99961.1-MMA were comparable, the endocytosis efficiency of B62-H3L3-MMAE was slightly lower than that of the positive control ADC, and the endocytosis efficiencies of the ADCs were all higher than those of their corresponding antibodies; on HT-29 cells ( Figure 17B ), the endocytosis efficiency of B31-H3L3-MMAE was slightly higher than that of the positive control ADC, the endocytosis efficiency of B62-H3L3-MMAE was slightly lower than that of the positive control ADC, and the endocytosis efficiencies of the antibodies were generally lower than those of their corresponding ADCs.

[0414] Example 22 Detection of the Efficacy of ADC in Inhibiting HT-29 Mouse Tumor-Bearing Models

[0415] In this example, the antitumor effects of two candidate ADCs (B31-H3L3-MMAE and B62-H3L3-MMAE) in animals were verified, with 99961.1-MMAE as the positive control, and the tumor cells used were colon cancer cells HT-29 (BNCC337732).

[0416] The specific method is as follows: Female Balb / C nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) at 6-8 weeks of age and weighing about 20 g were used. Each nude mouse was subcutaneously injected with 1×10 6 HT-29 cells. When the tumor-bearing volume reached about 100 mm 3 , they were randomly grouped and caged. There were 6 tumor-bearing nude mice in each group, and a total of 9 groups, including a PBS negative control group, 5 candidate ADC groups (B31-H3L3-MMAE 0.4 mg / kg (mpk), B31-H3L3-MMAE 2 mg / kg, B31-H3L3-MMAE 10 mg / kg, B62-H3L3-MMAE 2 mg / kg, and B62-H3L3-MMAE 10 mg / kg), and 3 positive control ADC groups (99961.1-MMAE 0.4 mg / kg, 99961.1-MMAE 2 mg / kg, and 99961.1-MMAE 10 mg / kg). The administration method was intravenous injection via the tail vein. The tumor volume was measured twice a week and the drug was administered twice a week for a total of 8 times / 4 weeks (BIW*4). The calculation method of the tumor volume (V) was: V = L×W 2 / 2 (where L is the longest among the tumor diameters and W is the shortest among the tumor diameters). The mice were euthanized 1 week after the end of drug administration, and the tumors were taken and weighed. The data of tumor volume, tumor weight and mouse body weight changes were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 18A - 18C and Table 12.

[0417] The experimental results showed that there were no significant differences in the body weights of the mice in each group, and there were no significant changes in the body weights of the mice in each group during the treatment period, indicating that the mice had good tolerance to the ADC ( Figure 18B ); B31-H3L3-MMAE, B62-H3L3-MMAE and the control ADC 99961.1-MMAE all showed significant tumor inhibition effects under the condition of high dose (10 mpk). Under the condition of high dose, the tumor inhibition effects of B31-H3L3-MMAE and 99961.1-MMAE were comparable, and the tumor inhibition rate of B62-H3L3-MMAE was slightly weaker than that of the control ADC 99961.1-MMAE. While under the condition of lower dose (2 mpk), the tumor inhibition effect of B62-H3L3-MMAE was significantly better than that of the control ADC 99961.1-MMAE and B62-H3L3-MMAE ( Figure 18A 、 Figure 18C and Table 12).

[0418] Table 12 Tumor inhibition rate of ADC in mice

[0419]

[0420]

[0421] Example 23 Pharmacodynamic detection of ADC inhibiting A549 mouse tumor-bearing model

[0422] In this example, the tumor inhibition effects of 2 candidate ADCs (B31-H3L3-MMAE and B62-H3L3-MMAE) in animals were detected. The tumor cells used were non-small cell lung cancer cells A549 (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, C2107019), and 99961.1-MMAE was used as a positive control.

[0423] The specific method is as follows: Female nude mice (Balb / C, Beijing Vital River Laboratory Animal Technology Co., Ltd.) at 6-8 weeks old and weighing 18-20 g were subcutaneously injected with 1×10 6 A549 cells on the right dorsal side of each nude mouse. When the tumor-bearing volume reached 100 mm 3When the tumor volume reached about [X] mm³, random grouping and cage separation were carried out. Six tumor-bearing nude mice were in each group, with a total of 4 groups, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 10 mpk (mg / kg) and B62-H3L3-MMAE 10 mpk), and 1 positive control ADC group (99961.1-MMAE 10 mpk). The administration method was intravenous injection via the tail vein. The tumor volume was measured twice and the drug was administered twice a week for a total of 6 times / 3 weeks (BIW * 3). The calculation formula for tumor volume (V) was: V = L × W 2 / 2 (where L is the longest of the tumor diameters and W is the shortest of the tumor diameters). After the administration ended, the mice were euthanized after observing for a certain period of time, and the tumors were taken and the tumor weights were measured. The data of tumor volume, tumor weight, and mouse body weight changes were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 19A - 19C and Table 13.

[0424] The experimental results showed that there were no significant changes in the body weights of the mice in each group during the treatment period, indicating that the mice had good tolerance to the ADC ( Figure 19B ); B62-H3L3-MMAE showed an antitumor effect equivalent to that of the positive control ADC at the same dose, and B31-H3L3-MMAE showed an antitumor effect superior to that of the positive control ADC ( Figure 19A , 19C and Table 13).

[0425] Table 13 Tumor inhibition rate of ADC in mice

[0426]

[0427]

[0428] Example 24 Pharmacodynamic detection of ADC inhibiting the NCI-N87 mouse tumor-bearing model

[0429] In this example, the antitumor effect of 1 candidate ADC (B31-H3L3-MMAE) in animals was detected. The tumor cells used were gastric cancer cells NCI-N87 (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, C2009021, P3), and 99961.1-MMAE was used as the positive control.

[0430] The specific method was as follows: Female nude mice (Balb / C, Beijing Vital River Laboratory Animal Technology Co., Ltd.) at 6 - 8 weeks old and weighing 18 - 20 g were used. Each nude mouse was subcutaneously injected with 1×10 6 NCI-N87 cells on the back. When the tumor-bearing volume reached 100 mm 3When the tumor volume reached about 100 mm³, random grouping and cage separation were carried out. There were 10 tumor-bearing nude mice in each group, with a total of 5 groups, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 5 mpk (mg / kg) and B31-H3L3-MMAE 10 mpk), and 2 positive control ADC groups (99961.1-MMAE 5 mpk and 99961.1-MMAE 10 mpk). The administration method was intravenous injection via the tail vein. The tumor volume was measured twice a week and the drug was administered once a week for a total of 3 times / 3 weeks (QW*3). The calculation method of tumor volume (V) was: V = L×W 2 / 2 (where L is the longest of the tumor diameters and W is the shortest of the tumor diameters). Analyze the data of tumor volume and mouse body weight changes, and calculate the tumor inhibition rate. The results are shown in Figures 20A - 20B and Table 14

[0431] The experimental results showed that there were no significant changes in the body weights of the mice in each group during the treatment period, indicating that the mice had good tolerance to the ADC( Figure 20B ); compared with the PBS group, each dose group had a significant tumor inhibition effect, and the tumor inhibition effects of the B31-H3L3-MMAE molecule and the 99961.1-MMAE molecule were equivalent at 10 mpk( Figure 20A and Table 14).

[0432] Table 14 Tumor inhibition rate of ADC in mice

[0433]

[0434]

[0435] Example 25 Pharmacodynamic detection of ADC inhibiting the MDA-MB-231 mouse tumor-bearing model

[0436] In this example, the tumor inhibition effect of 1 candidate ADC (B31-H3L3-MMAE) in animals was detected. The tumor cells used were triple-negative breast cancer cells MDA-MB-231 (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, C2006040), and 99961.1-MMAE was used as a positive control

[0437] The specific method was as follows: Female nude mice (NSG, Beijing Vital River Laboratory Animal Technology Co., Ltd.) at 6-8 weeks of age and weighing 20-22 g were used. Each nude mouse was subcutaneously injected with 1×10 6 MDA-MB-231 cells. When the tumor-bearing volume reached 100 mm 3Around [time], random grouping and cage separation were carried out. There were 10 tumor-bearing nude mice in each group, with a total of 5 groups, including a PBS negative control group, 2 ADC groups (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 administration method was intravenous injection via the tail vein. The drug was administered once a week and the tumor volume was measured twice. A total of 3 doses were administered over 3 weeks (QW*3). The calculation method for tumor volume (V) was: V = L × W 2 / 2 (where L is the longest of the tumor diameters and W is the shortest of the tumor diameters). Analyze the data on tumor volume and mouse body weight changes, and calculate the tumor inhibition rate. The results are shown in Figures 21A - 21B and Table 15.

[0438] The experimental results showed that there were no significant differences among the mice in each group in the early stage of drug administration. The body weight of the PBS group began to decrease on the 35th day, presumably due to excessive tumor burden. There were no significant changes in the body weight of the mice in the ADC groups and the positive control groups, indicating that the mice had good tolerance to ADC ( Figure 21B ); compared with the PBS group, the high-dose groups of the positive control and the test ADC had a comparable tumor inhibitory effect, and the effect was significant; under the low-dose condition, the test ADC (B31-H3L3-MMAE) showed a better tumor inhibitory effect than the positive control ADC. There was a certain rebound of the tumor tissue in the low-dose group after drug withdrawal ( Figure 21A and Table 15).

[0439] Table 15 Tumor inhibition rate of ADC in mice

[0440]

[0441]

[0442] Example 26 Pharmacodynamic detection of ADC in inhibiting the MDA-MB-468 mouse tumor-bearing model

[0443] In this example, the tumor inhibitory effect of 1 candidate ADC (B31-H3L3-MMAE) in animals was detected. The tumor cells used were triple-negative breast cancer cells MDA-MB-468 (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, TCHu136), and 99961.1-MMAE was used as the positive control.

[0444] The specific method was as follows: Female nude mice (NOD SCID, Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) at 6-8 weeks of age and weighing 21-25 g were used. Each nude mouse was subcutaneously injected with 1×10 7MDA-MB-468 cells were used. When the tumor volume reached about 200 mm3, the mice were randomly grouped and caged. There were 6 tumor-bearing nude mice in each group, and a total of 5 groups, including a PBS negative control group, 2 ADC groups (B31-H3L3-MMAE 5 mpk (mg / kg) (QW*3), B31-H3L3-MMAE 10 mpk (QW*3)), and 2 positive control ADC groups (99961.1-MMAE 5 mpk (QW*3), 99961.1-MMAE 10 mpk (QW*3)); There were 3 tumor-bearing nude mice in each group, and a total of 2 groups, including B31-H3L3-MMAE 10 mpk (single dose) and 99961.1-MMAE 10 mpk (single dose). The administration method was intravenous injection via the tail vein, and the drug was administered 3 times / 3 weeks (QW*3) or as a single dose (single dose). The calculation method of tumor volume (V) was: V = L × W 2 / 2 (where L is the longest of the tumor diameters and W is the shortest of the tumor diameters). The data of tumor volume and mouse body weight changes were analyzed, and the tumor inhibition rate was calculated. The results are shown in Figures 22A - 22B and Tables 16 - 17.

[0445] The experimental results showed that there were no significant changes in the body weights of the mice in each group during the treatment period, indicating that the mice had good tolerance to the ADC ( Figure 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 complete remission (CR) was achieved in all mice approximately 18 days after drug administration; The tumor inhibition effect of the group administered with 5 mg / kg B31-H3L3-MMAE by QW*3 was better than that of the group administered with 5 mg / kg 99961.1-MMAE by QW*3, and CR was achieved in 4 / 6 mice 25 days after drug 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. In the B31-H3L3-MMAE group, all mice achieved CR 18 days after drug administration, while tumor recurrence occurred in the 99961.1-MMAE group 20 days after drug administration. In this experiment, no tumor recurrence was observed in all B31-H3L3-MMAE groups ( Figure 22A and Tables 16 - 17).

[0446] Table 16 Tumor Inhibition Rate of ADC in Mice

[0447]

[0448]

[0449] Table 17 Complete Remission Rate of ADC in Mice

[0450]

[0451] Example 27: Efficacy Detection of ADC in Jeko-1 Mouse Tumor-bearing Model

[0452] In this example, the antitumor effect of one candidate ADC (B31-H3L3-MMAE) in animals was detected. The tumor cells used were mantle cell lymphoma cells Jeko-1 (ATCC, CRL-3006), and 99961.1-MMAE was used as a positive control.

[0453] The specific method is as follows: Female nude mice (BALB / c, Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) at 6-8 weeks of age and weighing 21-25 g were subcutaneously injected with 1×10 7 Jeko-1 cells on the back of each nude mouse. When the tumor-bearing volume reached about 100 mm 3 , they were randomly grouped and caged. There were 7 tumor-bearing nude mice in each group, and a total of 9 groups, 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-MMAE 10 mpk (single dose), and B31-H3L3-MMAE 2.5 + 3.5 mpk (Q2W*2, that is, 2.5 mpk was administered on the 15th day and 3.5 mpk was administered on the 29th day, 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 10 mpk (single dose), and 99961.1-MMAE 2.5 + 3.5 mpk (Q2W*2, that is, 2.5 mpk was administered on the 15th day and 3.5 mpk was administered on the 29th day, abbreviated as D15 2.5 mpk + D29 3.5 mpk)). The administration method was intravenous injection through the tail vein, and the drug was administered 3 times / 3 weeks (QW*3) or single dose or administered 2 times / 3 weeks (Q2W*2). The calculation method of tumor volume (V): V = L×W 2 / 2 (where L is the longest of the tumor diameters and W is the shortest of the tumor diameters). Analyze the data of tumor volume and mouse body weight changes, and calculate the tumor inhibition rate. The results are shown in Figures 23A - 23B and Tables 18-19.

[0454] The test results showed that there were no significant changes in the body weights of the mice in each group during the treatment period, indicating that the mice had good tolerance to ADC( Figure 23B);In the QW*3 dosing group, the tumor growth inhibition rate (TGI) of mice in the B31-H3L3-MMAE-10mpk and 99961.1-MMAE-10mpk dose groups was approximately 96%, and the tumor suppression rates were comparable. The tumors did not recur 23 days after dosing; the tumor suppression effects of the B31-H3L3-MMAE-2.5mpk and 99961.1-MMAE-2.5mpk dose groups were comparable, with TGIs of 60.47% and 59.22% respectively. In the single-dose group, the TGIs of the B31-H3L3-MMAE (10mpk) and 99961.1-MMAE (10mpk) dose groups were 79.24% and 91.74% respectively, and the tumors did not recur 23 days after dosing. In the Q2W*2 dosing group, the tumor suppression effect of the B31-H3L3-MMAE (2.5 + 3.5mpk) dose group was comparable to that of the 99961.1-MMAE (2.5 + 3.5mpk) group, with TGIs of 34.96% and 34.60% respectively ( Figure 23A and Tables 18 - 19).

[0455] Table 18 Tumor Suppression Rate of ADC in Mice

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[0457] Table 19 Tumor Suppression Rate of ADC in Mice

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[0459]

[0460] Sequence Listing

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Claims

1. An anti-ROR1 antibody and antigen-binding fragment thereof that specifically binds to ROR1, comprising: HCDR1 of the sequence shown in SEQ ID NO: 4; HCDR2 of the sequence shown in SEQ ID NO: 5; HCDR3 of the sequence shown in SEQ ID NO: 6; LCDR1 of the sequence shown in SEQ ID NO: 43; LCDR2 of the sequence shown in SEQ ID NO: 46; and LCDR3 of the sequence shown in SEQ ID NO:

48.

2. The anti-ROR1 antibody and antigen-binding fragment thereof according to claim 1, which 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, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 77 and comprising HCDR1, HCDR2, and HCDR3 of the sequences 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 having at least 90% identity with the amino acid sequence of SEQ ID NO: 81 and comprising LCDR1, LCDR2, and LCDR3 of the sequences shown in SEQ ID NOs: 43, 46, and 48, or consists of SEQ ID NO:

81.

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

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

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

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

53.

7. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which comprises 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 having at least 90% identity with the amino acid sequence of SEQ ID NO: 113, or consists of SEQ ID NO:

113. The light chain comprises the amino acid sequence shown in SEQ ID NO: 117, or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 117, or consists of SEQ ID NO:

117.

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

9. The antibody-drug conjugate according to claim 8, wherein the dolastatin and its auristatin derivatives are 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).

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

11. The antibody-drug conjugate according to any one of claims 8-10, wherein the linker is selected from 1) cathepsin-degradable linkers valine-citrulline (val-cit) linker, cBu-Cit linker or CX linker; 2) non-cleavable linkers SMCC linker or MD linker; 3) acid-sensitive linkers; 4) linkers with a silicone grease structure; 5) disulfide-carbamate linkers; 6) MC-GGFG linkers; 7) TRX linkers; 8) linkers containing galactoside; 9) pyrophosphate linkers; 10) near-infrared sensitive linkers; 11) ultraviolet sensitive linker PC4AP.

12. The antibody-drug conjugate according to claim 11, wherein the linker is selected from maleimidocaproyl-valine-citrulline-p-aminobenzyloxy (mc-vc-PAB), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-VC-PABC), amino-PEG6-propionyl, and maleimidocaproic acid group (mc), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)-propionate (SPDP).

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

14. The antibody-drug conjugate according to any one of claims 8-10, wherein the Ab comprises: HCDR1 of the sequence shown in SEQ ID NO:4; HCDR2 of the sequence shown in SEQ ID NO:5; HCDR3 of the sequence shown in SEQ ID NO:6; LCDR1 of the sequence shown in SEQ ID NO:43; LCDR2 of the sequence shown in SEQ ID NO:46; and LCDR3 of the sequence shown in SEQ ID NO:

48.

15. The antibody-drug conjugate according to claim 14, 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, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

81.

16. The antibody-drug conjugate according to claim 15, wherein the Ab comprises: a heavy chain of the amino acid sequence shown in SEQ ID NO:113, and a light chain of the amino acid sequence shown in SEQ ID NO:

117.

17. The antibody-drug conjugate according to any one of claims 14-16, wherein the linker is MC-VC-PAB and the cytotoxin is MMAE.

18. A pharmaceutical composition, comprising: (1) The anti-ROR1 antibody or an antigen-binding fragment thereof according to any one of claims 1-7, or the antibody-drug conjugate according to any one of claims 8-17, and; (2) a pharmaceutically acceptable carrier.

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

20. A vector comprising the nucleic acid molecule according to claim 19.

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

22. A host cell comprising the vector according to claim 20 or 21, or the nucleic acid molecule according to claim 19.

23. Use of the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1-7 in the preparation of an antibody-drug conjugate for preventing or treating cancers with high ROR1 expression.

24. Use of the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1-7 in the preparation of a drug for preventing or treating cancers with high ROR1 expression.

25. Use of the antibody-drug conjugate according to any one of claims 8-17 in the preparation of a drug for preventing or treating cancers with high ROR1 expression.

26. The use according to any one of claims 23-25, wherein the cancers with high ROR1 expression are chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma, renal cell carcinoma, colon cancer, breast cancer, neuroblastoma, lung cancer, gastric cancer, head and neck cancer, and melanoma.

27. A non-therapeutic method of 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 1-7, or an effective amount of the antibody-drug conjugate according to any one of claims 8-17, or an effective amount of the pharmaceutical composition according to claim 18.

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