Ligand-cytotoxic drug conjugates and pharmaceutical uses thereof

Abstract

The invention discloses an anti-receptor tyrosine kinase-like orphan receptor 1 antibody and a conjugate of the anti-receptor tyrosine kinase-like orphan receptor 1 antibody and a small molecule drug. Also disclosed is the use of the ligand-cytotoxic drug conjugate and a pharmaceutical composition comprising the same in the preparation of a medicament for the treatment of cancer.

Description

Technical Field

[0001] The present invention relates to a novel ROR1 antibody or a functional fragment thereof, which comprises an engineered heavy chain and a light chain. The present invention further relates to a conjugate of an improved ROR1 antibody and a small molecule drug. The present invention further relates to the use of the antibody and the conjugate in the manufacture of a drug for treating cancer. Background Art

[0002] Receptor tyrosine kinase-like orphan receptors (RORs) are transmembrane proteins of the receptor tyrosine kinase (RTK) family, and there are ROR1 and ROR2, which are type I transmembrane receptor tyrosine kinases. Since overexpression of ROR1 is observed in various cancer cells, it is classified as an oncofetal gene. In particular, ROR1 has attracted attention as an anticancer antibody target because it was found to be overexpressed in chronic lymphocytic leukemia (CLL). It is reported that in addition to chronic lymphocytic leukemia (CLL), it is not only overexpressed in hematological malignancies (such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), etc.), but also overexpressed in solid cancers (including breast cancer, ovarian cancer, gastric cancer, lung cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colon cancer, etc.). This cancer cell-specific expression of ROR1 suggests that ROR1 can be an effective cancer target, so it is necessary to develop antibodies that specifically recognize it. This oncofetal protein is an attractive target for cancer therapy.

[0003] ROR1 antibodies have been described in documents WO2014031174 (UC961), WO2017127664 (XBR1-402), etc. And a humanized mouse anti-ROR1 antibody UC961 has entered clinical trials for relapsed or refractory chronic lymphocytic leukemia. Monoclonal antibodies have received increasing attention due to their high target specificity, low side effects and other characteristics. However, when used alone, their therapeutic effect is limited. Therefore, most monoclonal antibody drugs are used in combination with chemotherapy. So far, the main way to improve the therapeutic effect of monoclonal antibodies is antibody-drug conjugates (ADCs).

[0004] Antibody-drug conjugates (ADCs) are a new type of anticancer drug that consists of three parts: antibodies, cytotoxins, and linkers that connect the two parts. Monoclonal antibodies are coupled to cytotoxins through chemical coupling. Antibody-drug conjugation technology fuses small molecule drugs with biological proteins, which can have the advantages of both and significantly improve the efficacy of drugs and reduce side effects.

[0005] Since ROR1 is not expressed at high levels in most tumor cells, ADCs targeting ROR1 may require antibodies that can effectively trigger internalization, small molecule compounds with higher toxicity, coupling methods with higher DARs, linkers that release poisons more effectively, etc. Examples include NBE-002 and LCB71 / ABL202, both of which use highly toxic small molecule compounds. At the same time, in order to avoid off-target toxicity caused by the release of small molecule drugs, both use very stable linkers to ensure that the drug is released only in tumor cells. Given that antibody selection, coupling sites, linkers, and small molecule compounds all play a key role in ADC design, there is an urgent need to use more diverse components and combinations to develop more advanced anti-ROR1 ADCs. The present invention provides a technical solution that meets this need. Summary of the invention

[0006] The anticancer efficacy of antibody-drug conjugates is believed to rely on the uptake of surface-expressed antigens by cancer cells, and thus the insufficient internalization of monoclonal antibodies targeting ROR1 is an urgent issue to be addressed.

[0007] The technical problem to be solved by the present invention is to develop an advanced anti-ROR1 ADC that exhibits very strong anti-tumor effects in cancer. The anti-tumor effect may be due to the high DAR value and strong endocytosis as well as the potential effect of the antibody itself on the inhibition of Wnt5a signaling.

[0008] Specifically, the present invention covers the following aspects:

[0009] The present disclosure provides an antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof

[0010] Ab-(L2-L1-D) y

[0011] (A)

[0012] in:

[0013] D is a cytotoxic drug;

[0014] L1 and L2 are connection units;

[0015] y is a number from 1 to 20;

[0016] Ab is an anti-ROR1 antibody or an antigen-binding fragment thereof, which comprises: an antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions, and an antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: a) HCDR1 is as shown in SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05 or SEQ ID NO: 06; b) HCDR2 is as shown in SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13; c) HCDR3 is as shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20; d) LCDR1 is as shown in SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 NO:24, SEQ ID NO:25 or SEQ ID NO:26; e) LCDR2 is shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32; f) LCDR3 is shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39.

[0017] In some embodiments, the heavy chain variable region of Ab in general formula (A) comprises: HCDR1 as shown in SEQ ID NO: 01, HCDR2 as shown in SEQ ID NO: 07, and HCDR3 as shown in SEQ ID NO: 14; or HCDR1 as shown in SEQ ID NO: 02, HCDR2 as shown in SEQ ID NO: 08, and HCDR3 as shown in SEQ ID NO: 15; or HCDR1 as shown in SEQ ID NO: 03, HCDR2 as shown in SEQ ID NO: 09, and HCDR3 as shown in SEQ ID NO: 16; or HCDR1 as shown in SEQ ID NO: 04, HCDR2 as shown in SEQ ID NO: 10, and HCDR3 as shown in SEQ ID NO: 17; or HCDR1 as shown in SEQ ID NO: 05, HCDR2 as shown in SEQ ID NO: 11, and HCDR3 as shown in SEQ ID NO: 18; or HCDR1 as shown in SEQ ID NO: 05, HCDR2 as shown in SEQ ID NO: 12, and HCDR3 as shown in SEQ ID NO: 19; or HCDR1 as shown in SEQ ID NO: 05, HCDR2 as shown in SEQ ID NO: 13, and HCDR3 as shown in SEQ ID NO: 14; HCDR1 as shown in SEQ ID NO:06, HCDR2 as shown in SEQ ID NO:13 and HCDR3 as shown in SEQ ID NO:20.

[0018] In some embodiments, the light chain variable region of Ab in Formula (A) comprises: LCDR1 as shown in SEQ ID NO:21, LCDR2 as shown in SEQ ID NO:27, and LCDR3 as shown in SEQ ID NO:33; or LCDR1 as shown in SEQ ID NO:22, LCDR2 as shown in SEQ ID NO:28, and LCDR3 as shown in SEQ ID NO:34; or LCDR1 as shown in SEQ ID NO:23, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:35; or LCDR1 as shown in SEQ ID NO:24, LCDR2 as shown in SEQ ID NO:30, and LCDR3 as shown in SEQ ID NO:36; or LCDR1 as shown in SEQ ID NO:24, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:37; or LCDR1 as shown in SEQ ID NO:25, LCDR2 as shown in SEQ ID NO:32, and LCDR3 as shown in SEQ ID NO:38; or LCDR1 as shown in SEQ ID NO:2 LCDR1 as shown in SEQ ID NO:26, LCDR2 as shown in SEQ ID NO:32 and LCDR3 as shown in SEQ ID NO:39.

[0019] In a preferred embodiment, Ab in general formula (A) comprises: a) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 07 and SEQ ID NO: 14, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 33, respectively; or b) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 02, SEQ ID NO: 08 and SEQ ID NO: 15, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 34, respectively; or c) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 03, SEQ ID NO: 09 and SEQ ID NO: 10, respectively. NO: 16; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 29 and SEQ ID NO: 35, respectively; or d) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 10 and SEQ ID NO: 17, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 30 and SEQ ID NO: 36, respectively; or e) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 11 and SEQ ID NO: 18, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 31 and SEQ ID NO: NO: 37; or f) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 12 and SEQ ID NO: 19, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 25, SEQ ID NO: 32 and SEQ ID NO: 38, respectively; or g) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 13 and SEQ ID NO: 20, respectively;and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 32 and SEQ ID NO: 39, respectively. ;

[0020] In a preferred embodiment, Ab in the general formula (A) is selected from a murine antibody, a chimeric antibody, a humanized antibody, a human antibody or an antigen-binding fragment thereof.

[0021] In some embodiments, Ab in general formula (A) comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 41, 43, 45, 47, 49, 51 and 53, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0022] In some embodiments, Ab in general formula (A) comprises: a) a heavy chain variable region as shown in SEQ ID NO: 40, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO: 41, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or b) a heavy chain variable region as shown in SEQ ID NO: 42, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO: 43, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or c) a heavy chain variable region as shown in SEQ ID NO: 44, or having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO: NO:45, or a light chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; or d) a heavy chain variable region as set forth in SEQ ID NO:46, or a sequence identity thereto; and / or a light chain variable region as set forth in SEQ ID NO:47, or a sequence identity thereto; or e) a heavy chain variable region as set forth in SEQ ID NO:48, or a sequence identity thereto; and / or a light chain variable region as set forth in SEQ ID NO:49, or a sequence identity thereto; or f) a heavy chain variable region as set forth in SEQ ID NO:41, or a sequence identity thereto; NO:50, or a heavy chain variable region having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:51, or a sequence identity thereto; or g) a heavy chain variable region as shown in SEQ ID NO:52, or a sequence identity thereto; and / or a light chain variable region as shown in SEQ ID NO:53, or a sequence identity thereto.

[0023] In a preferred embodiment, Ab in general formula (A) comprises: a) a heavy chain variable region as shown in SEQ ID NO: 40 and a light chain variable region as shown in SEQ ID NO: 41; b) a heavy chain variable region as shown in SEQ ID NO: 42 and a light chain variable region as shown in SEQ ID NO: 43; c) a heavy chain variable region as shown in SEQ ID NO: 44 and a light chain variable region as shown in SEQ ID NO: 45; d) a heavy chain variable region as shown in SEQ ID NO: 46 and a light chain variable region as shown in SEQ ID NO: 47; e) a heavy chain variable region as shown in SEQ ID NO: 48 and a light chain variable region as shown in SEQ ID NO: 49; f) a heavy chain variable region as shown in SEQ ID NO: 50 and a light chain variable region as shown in SEQ ID NO: 51; g) a heavy chain variable region as shown in SEQ ID NO: 52 and a light chain variable region as shown in SEQ ID NO: 53.

[0024] In some embodiments, Ab in general formula (A) is a full-length antibody, which further comprises a human antibody constant region; preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and conservative variants thereof, and the light chain constant region of the human antibody constant region is selected from the κ chain and λ chain constant regions of human antibodies and conservative variants thereof; more preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 68 and the human light chain constant region of SEQ ID NO: 69.

[0025] In a preferred embodiment, Ab in general formula (A) comprises: a) a heavy chain as shown in SEQ ID NO: 54 and a light chain as shown in SEQ ID NO: 55; b) a heavy chain as shown in SEQ ID NO: 56 and a light chain as shown in SEQ ID NO: 57; c) a heavy chain as shown in SEQ ID NO: 58 and a light chain as shown in SEQ ID NO: 59; d) a heavy chain as shown in SEQ ID NO: 60 and a light chain as shown in SEQ ID NO: 61; e) a heavy chain as shown in SEQ ID NO: 62 and a light chain as shown in SEQ ID NO: 63; f) a heavy chain as shown in SEQ ID NO: 64 and a light chain as shown in SEQ ID NO: 65; g) a heavy chain as shown in SEQ ID NO: 66 and a light chain as shown in SEQ ID NO: 67.

[0026] In some embodiments, Ab in the general formula (A) is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (diabody), disulfide stabilized Fv (dsFv) and CDR-containing peptide.

[0027] In some embodiments, the cytotoxic drug is selected from the group consisting of a toxin, a chemotherapeutic agent, an antibiotic, a radioisotope, and a nucleolytic enzyme.

[0028] In a further embodiment, the cytotoxic drug is selected from a tubulin inhibitor or a topoisomerase inhibitor; preferably an auristatin analog or a camptothecin derivative; more preferably MMAE, MMAF or Exatecan.

[0029] In some embodiments, the antibody-drug conjugate is represented by the general formula (I):

[0030]

[0031] in:

[0032] L1 and L2 are connection units;

[0033] y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8 or 2 to 6, further preferably a number from 6 to 8, most preferably 4, 6 or 8;

[0034] Ab is the anti-ROR1 antibody or antigen-binding fragment mentioned above.

[0035] In another embodiment, the antibody-drug conjugate is represented by the general formula (II):

[0036]

[0037] in:

[0038] L1 and L2 are connection units;

[0039] y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6, 2 to 4, 3 to 6, 3 to 5, 4 to 6, 4 to 8, 6 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0;

[0040] Ab is the anti-ROR1 antibody or antigen-binding fragment mentioned above.

[0041] In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (IA):

[0042]

[0043] in:

[0044] L2 is the connection unit;

[0045] R1 is selected from hydrogen, C 1-6 Haloalkyl or C 3-8 Cycloalkyl;

[0046] R2 is selected from hydrogen, C 1-6 Haloalkyl or C 3-8 Cycloalkyl;

[0047] Or, R1 and R2 together with the carbon atom to which they are attached form C 3-8 Cycloalkyl;

[0048] y1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6, 2 to 4, 4 to 6, 4 to 8, 6 to 8, most preferably 2, 4, 6, 8;

[0049] Ab is the anti-ROR1 antibody or antigen-binding fragment mentioned above.

[0050] In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (IA):

[0051]

[0052] in:

[0053] R1 is selected from hydrogen, C 1-3 Haloalkyl or C 3-6 Cycloalkyl;

[0054] R2 is selected from hydrogen, C 1-3 Haloalkyl or C 3-6 Cycloalkyl;

[0055] Or, R1 and R2 together with the carbon atom to which they are attached form C 3-6 Cycloalkyl;

[0056] y1 and Ab are mentioned above.

[0057] In a preferred embodiment, the antibody-drug conjugate is represented by the general formula (II-A):

[0058]

[0059] in:

[0060] L2 is the connection unit;

[0061] y2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 6, 2 to 4, 3 to 6, 3 to 5, 4 to 6, 4 to 8, 6 to 8, and most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0;

[0062] Ab is the anti-ROR1 antibody or antigen-binding fragment mentioned above.

[0063] In some embodiments, the linking unit L2 is represented by the general formula (III):

[0064]

[0065] in:

[0066] s 1 and 2 Each independently represents an integer selected from 0 to 8. Preferably, s 1 and 2 Independently selected from 1, 2, 3, 4, 5 or 6;

[0067] or 1 is an integer from 1 to 8, s 2 is 0, preferably, s 1 is selected from 4, 5, 6, 7 or 8, and s 2 is 0;

[0068] or 2 An integer selected from 2 to 8, s 1 is 2, preferably, s 2 is selected from 2, 3, 4, 5 or 6, and s 1 Serves 2.

[0069] In one aspect, the antibody-drug conjugate has the following structure:

[0070]

[0071]

[0072]

[0073] in:

[0074] y1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8 or 2 to 6, further preferably a number from 6 to 8, and most preferably 4, 6 or 8;

[0075] y2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0;

[0076] Ab is the anti-ROR1 antibody or antigen-binding fragment mentioned above.

[0077] In one aspect, the antibody-drug conjugate is represented by formula (IB) or formula (II-B):

[0078]

[0079] in:

[0080] y 1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8 or 2 to 6, further preferably a number from 6 to 8, and most preferably 4, 6 or 8;

[0081] y 2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0;

[0082] Ab is the anti-ROR1 antibody or antigen-binding fragment mentioned above.

[0083] In one aspect, the antibody-drug conjugate is selected from the group consisting of:

[0084]

[0085]

[0086]

[0087]

[0088] in:

[0089] y 1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8 or 2 to 6, further preferably a number from 6 to 8, and most preferably 4, 6 or 8;

[0090] y 2 It is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, and most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0.

[0091] In some embodiments, the present disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate according to the present disclosure or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0092] In some embodiments, the present disclosure also provides a method for treating or preventing a disease associated with overexpression of ROR1, comprising the step of administering a therapeutically effective amount of an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate, or a pharmaceutical composition according to the present disclosure to a subject in need of treatment or prevention of the disease.

[0093] In some embodiments, the disease is a cancer with ROR1 expression, preferably the cancer is chronic lymphocytic leukemia (CLL), B cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) and marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

[0094] In some embodiments, the present disclosure also provides the use of the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate and the pharmaceutical composition in the manufacture of a medicament for treating or preventing a disease associated with human ROR1.

[0095] In some embodiments, the present disclosure also provides the use of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate and the pharmaceutical composition in the manufacture of a medicament for treating or preventing a cancer expressing ROR1; preferably, the cancer is chronic lymphocytic leukemia (CLL), B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

[0096] The antibody or antigen-binding fragment thereof can (1) specifically recognize or bind to ROR1 expressed on the surface of human-derived cells, or (2) specifically recognize or bind to the extracellular domain of ROR1 on a non-cell surface, or (3) have sufficient internalization in human cells.

[0097] The antibody-drug conjugate has high cytotoxic activity in tumor cells, and the tumor inhibitory effect of the antibody-drug conjugate is significantly strong at low doses in the Jeko-1 CDX model. The half-life of the antibody-drug conjugate is superior to that of VLS-101, thus showing significant long-term efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1-A ~C. In vitro binding characterization of ROR1 hybridoma clones to HT29 (A), HCC1187 (B), and T47D (C) cell lines using flow cytometric analysis.

[0099] Figure 2-A ~B. In vitro binding characterization of recombinant ROR1 antibodies to ROR1+Jeko-1 cells (A) and ROR1-T47D cells (B) was determined by flow cytometry analysis.

[0100] Figure 3 .Internalization kinetics of anti-ROR1 recombinant antibody in ROR1+Jeko1 cells.

[0101] Figure 4-A ~H. In vitro cytotoxic activity of ADCs against different tumor cell lines, as determined by the CellTiter-Glo luminescent viability assay.

[0102] Figure 5 .In vivo efficacy of ADC (5 mg / kg, Q4D) in the Jeko-1 tumor xenograft mouse model.

[0103] Figure 6 .In vivo efficacy of ADC (1 mg / kg or 2 mg / kg, Q1W) in the Jeko-1 tumor xenograft mouse model.

[0104] Figure 7 .In vivo efficacy of ADC (3 mg / kg, Q1W) in the NCI-H1975 (EGFR-L858R-T790M-C797S) tumor xenograft mouse model. DETAILED DESCRIPTION

[0105] The present invention is based on the development of an antibody that can specifically bind to ROR1. The antibody of the present invention may optionally be coupled to a growth inhibitory agent or a cytotoxic agent (such as a toxin, including, for example, auristatins or maytansinoids).

[0106] The titles used in this section are for convenience of description only and do not limit the present invention. Unless otherwise defined herein, the scientific terms and technical terms used herein have the same meanings as those generally understood by those skilled in the art. In addition, unless the context specifically requires, the singular includes the plural, and the plural includes the singular. The abbreviations of amino acid residues are standard three-letter and / or one-letter codes used in the art, which represent one of 20 common L-amino acids.

[0107] definition

[0108] The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH region and the VL region can be further subdivided into hypervariable regions called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0109] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., ROR1). It has been shown that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, which is composed of VL, VH, CL, and CH I (ii) F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fd fragment, which consists of VH and CH Idomains; (iv) Fv fragments, which consist of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of the VH domain; (vi) isolated complementarity determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by synthetic linkers using recombinant methods, enabling them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.

[0110] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0111] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated or isolated by recombinant means, such as (a) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) of human immunoglobulin genes or hybridomas prepared therefrom (further described in Section 1 below), (b) antibodies isolated from host cells transformed to express antibodies, such as from transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire in vivo.

[0112] The term "CDR" refers to one of the six hypervariable regions within an antibody variable domain that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (LCDR1, LCDR2, LCDR3 or L1, L2, L3) of a light chain variable domain, and CDR1, CDR2, and CDR3 (HCDR1, HCDR2, HCDR3 or H1, H2, H3) of a heavy chain variable domain.

[0113] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within a given HCVR and / or LCVR amino acid sequence disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, Chothia (Chothia et al. (1989) Nature 342:877-883) based on the three-dimensional structure of antibodies and the topology of CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics Database (IMGT) (imgt.cines.fr / on the World Wide Web), and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. One skilled in the art can readily identify the CDRs defined by each numbering system.

[0114] A useful comparison of CDR numbers is as follows:

[0115]

[0116] Note 1: Some of these definitions (particularly for the Chothia loop) vary depending on the individual publications examined; Note 2: Any numbering scheme can be used for these CDR definitions, except for the contact definition, which uses the Chothia or Martin (enhanced Chothia) definition; Note 3: When numbering using the Kabat numbering convention, the ends of the Chothia HCDR1 loop vary between H32 and H34, depending on the length of the loop. This is because the Kabat numbering scheme places the insertion at H35A and H35B.

[0117] As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, it is "operably linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to a coding sequence.

[0118] The method for preparing nucleic acid is a conventional method in the art. Preferably, it comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full-length sequence synthesis.

[0119] Those skilled in the art know that the base sequence encoding the protein amino acid sequence can be appropriately substituted, deleted, changed, inserted or added to provide a polynucleotide homologue. The homologue of the polynucleotide of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the antibody activity.

[0120] The term "ligand" is a macromolecular compound that can recognize and bind to a target cell-associated antigen or receptor. The role of the ligand is to deliver the drug to a target cell population that binds to the ligand. The ligand includes, but is not limited to, protein hormones, lectins, growth factors, antibodies, and other molecules that can bind to cells. In an embodiment of the present invention, the ligand is represented by Ab. A connecting bond can be formed between the heteroatom and the linking unit in the ligand.

[0121] As used herein, the term "connecting unit" refers to the part that connects the antibody to the drug in the antibody-drug conjugate (i.e., ADC), which can be cleavable or non-cleavable. A cleavable linker (i.e., a cleavable linker or a biodegradable linker) can be destroyed in or on a target cell, and the drug is released thereby. In some embodiments, the connecting unit or linker of the present invention has very good stability, and greatly reduces the release of the drug during delivery to the target (e.g., in the blood), thereby reducing side effects and toxicity. In some specific embodiments, the connecting unit or linker of the present invention is selected from a cleavable linker, such as a disulfide-based linker (which is selectively destroyed in tumor cells at higher thiol concentrations), a peptide linker (which is enzymatically cleaved in tumor cells) and a hydrazone linker.

[0122] The term "cytotoxic drug" refers to a chemical molecule that can strongly destroy the normal growth of tumor cells. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations, but due to lack of specificity, when killing tumor cells, it also causes apoptosis of normal cells, resulting in serious side effects. In an embodiment of the present invention, the cytotoxic drug is represented by D, and non-limiting examples include tubulin inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, topoisomerase inhibitors and DNA synthesis inhibitors, preferably tubulin inhibitors and topoisomerase inhibitors.

[0123] The term "ligand-cytotoxic drug conjugate" refers to a ligand connected to a biologically active drug via a linker. In some specific embodiments, the "ligand-cytotoxic drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment connected to a biologically active cytotoxic drug via a linker.

[0124] The term "drug-antibody ratio (DAR)" refers to the average number of cytotoxic drugs loaded on each ligand, and can also be expressed as the ratio of the amount of drug and the amount of antibody. It can be an integer or a non-integer. The drug loading range of each ligand (Ab) can be 1-20 cytotoxic drugs (D). In an embodiment of the present invention, the drug-antibody ratio is expressed as y, and y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 4 to 6, 4 to 8, 6 to 8, further preferably 4 to 8 or 6 to 8, and most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0. The average number of drugs in each ADC molecule after the coupling reaction can be identified by conventional methods, such as UV / visible spectroscopy, mass spectrometry, ELISA test and HPLC feature identification.

[0125] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group having 1 to 12 carbon atoms, more preferably an alkyl group having 1 to 10 carbon atoms, and most preferably an alkyl group having 1 to 6 carbon atoms (having 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched chain isomers thereof. More preferably, alkyl is a low alkyl group having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available point of attachment. The substituents are preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocyclothio and oxo.

[0126] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0127] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent having 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (having 3, 4, 5, 6, 7 or 8 carbon atoms). Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc. Polycyclic cycloalkyls include cycloalkyls having spirocyclic, condensed or bridged rings.

[0128] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293 cells, plant cells, or fungi (including yeast cells), that express the antibody.

[0129] The sequence of the DNA molecule of the antibody according to the present invention or its fragment can be obtained by conventional techniques (for example, as the method of PCR amplification or genomic library screening).In addition, the sequence encoding light chain and heavy chain can be fused together to form a single-chain antibody.

[0130] Once the related sequence is obtained, it can be obtained in bulk using recombinant methods. This is usually done by cloning the sequence into a vector, transforming cells with the vector, and then isolating the related sequence from the propagated host cells by conventional methods.

[0131] In addition, related sequences can be synthesized artificially, especially when the fragment length is short. Usually, several small fragments are synthesized first and then connected together to obtain a long sequence fragment.

[0132] At present, the DNA sequence encoding the antibody of the present invention (or its fragment or derivative thereof) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or, for example, vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0133] Typically, the host cells obtained are cultured under conditions suitable for expressing the antibodies according to the invention. The antibodies of the invention are then purified using conventional immunoglobulin purification steps, such as conventional separation and purification means well known to those skilled in the art, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

[0134] The obtained monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined by, for example, Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)).

[0135] The antibodies according to the present invention can be expressed in cells or on cell membranes, or can be secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods according to the physical, chemical and other characteristics of the recombinant protein. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel chromatography), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and any other liquid chromatography, and combinations thereof.

[0136] The term "variant" of a polypeptide (e.g., an antigen binding fragment, a protein, or an antibody) is a polypeptide in which one or more amino acid residues are inserted, deleted, added, and / or substituted compared to another polypeptide sequence, and includes fusion polypeptides. In addition, protein variants include protein variants that are modified by proteinase cleavage, phosphorylation, or other post-translational modifications, but retain the biological activity of the antibodies disclosed herein, such as binding and specificity to ROR1. The variant may be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% identical to the sequence of the antibody or antigen binding fragment thereof disclosed herein. Identity or homology percentage (%) can be calculated with reference to the following description.

[0137] In one embodiment, the homology or identity percentage can be calculated as 100×[(same positions) / min(TGA, TGB)], and in this formula, TGA, TGB are the sum of the residue numbers and internal gap positions of the compared sequences A and B (Russell et al., J. Mol Biol., 244:332-350 (1994)).

[0138] In the present invention, the antibodies of the present invention also include conservative variants thereof, which means that compared with the amino acid sequence of the antibodies of the present invention, up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are substituted by amino acids with similar or similar properties to form polypeptides. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0139] Table A

[0140] Original residue Representative replacement Preferred substitution Ala Val; Leu; Ile Val Arg Lys; Gln; Asn Lys Asn Gln; His; Lys; Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn Asn Glu Asp Asp Gly Pro; Ala Ala His Asn; Gln; Lys; Arg Arg Ile Leu; Val; Met; Ala; Phe Leu Leu Ile; Val; Met; Ala; Phe Ile Lys Arg; Gln; Asn Arg Met Leu; Phe; Ile Leu Phe Leu; Val; Ile; Ala; Tyr Leu Pro Ala Ala Ser Thr Thr Thr Ser Ser Trp Tyr; Phe Tyr Tyr Trp; Phe; Thr; Ser Phe Val Ile; Leu; Met; Phe; Ala Leu

[0141] As used herein, the term "K D "(M) is intended to refer to the dissociation equilibrium constant for a particular antibody-antigen interaction. "K D ” refers to the dissociation constant, which is composed of K d With K a The ratio of K d / K a ) and expressed as molar concentration (M). In view of the present disclosure, the K of an antibody can be determined using methods in the art. D For example, the K D It can be determined by using surface plasmon resonance, such as by using a biosensor system (eg, the RAPID® system) or using bio-layer interferometry (eg, the Octet RED96 system).

[0142] The term "affinity" is the strength of the interaction between an antibody or its antigen-binding fragment and an antigen, which is determined by the characteristics of the antigen, such as the size, shape and / or charge of the antigen, and the CDR sequence of the antibody or antigen-binding fragment. Methods for determining affinity are known in the art, and reference may be made to the following.

[0143] When the dissociation constant (K D ) <l0 6 M, an antibody or antigen-binding fragment thereof is said to "specifically bind" to its target (e.g., antigen). D <l0 9 When M, the antibody binds specifically to its target with "high affinity".

[0144] As used herein, the term "pharmaceutical composition" is intended to refer to a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs thereof and other chemical components (such as physiologically / pharmaceutically acceptable carriers and excipients). The purpose of the pharmaceutical composition is to facilitate administration to an organism, which is beneficial to the absorption of the active ingredient and the exertion of biological activity.

[0145] "Administering" and "treating" when applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid means contacting the animal, human, subject, cell, tissue, organ, or biological fluid with an exogenous pharmaceutical agent, therapeutic agent, diagnostic agent, or composition. "Administering" and "treating" may refer, for example, to therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treatment of cells encompasses contacting cells with agents, as well as contacting fluids with agents, wherein the fluids are in contact with cells. "Administering" and "treating" also refer to in vitro and ex vivo treatments, such as in vitro and ex vivo treatments of cells by reagents, diagnostic agents, binding compositions, or by another cell. When applied to humans, veterinary medicine, or research subjects, "treatment" refers to therapeutic treatments, preventive or prophylactic measures, research and diagnostic applications.

[0146] In addition, the present disclosure includes a drug for treating a disease associated with ROR1, which comprises the antibody, antigen-binding fragment thereof, or antibody-drug conjugate of the present disclosure as an active ingredient.

[0147] There is no limitation on the disease associated with ROR1, as long as it is a disease associated with ROR1, for example, the therapeutic response induced by the molecules disclosed in the present disclosure can be reduced by binding to human ROR1. Therefore, when in a formulation suitable for therapeutic application, the molecules disclosed in the present disclosure are very useful for those suffering from tumors, cancer or infectious diseases.

[0148] In addition, the present disclosure relates to a method for immunodetection or measurement of ROR1, a reagent for immunodetection or measurement of ROR1, a method for immunodetection or measurement of cells expressing ROR1, and a diagnostic reagent for diagnosing a disease associated with ROR1-positive cells, which comprises the antibody or antigen-binding fragment of the present disclosure that specifically recognizes human ROR1 as an active ingredient.

[0149] In the present disclosure, the method for detecting or determining the amount of ROR1 may be any known method. For example, it includes immunoassay or determination.

[0150] Immunoassay or assay is a method for detecting or determining the amount of an antibody or antigen by using a labeled antigen or antibody. Examples of immunoassay or assay include radioactive labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, Western blotting, physicochemical methods, etc.

[0151] The above-mentioned diseases associated with ROR1-positive cells can be diagnosed by detecting or measuring cells expressing ROR1 using the antibodies or antibody fragments thereof of the present invention.

[0152] In order to detect cells expressing the polypeptide, known immunoassays can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistochemical staining, etc. can be used. In addition, a fluorescent antibody staining method or the like can be used, which uses the FMAT8100HTS system (Applied Bio system).

[0153] Example

[0154] The present invention is further illustrated by the following specific examples. It will be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods without detailed conditions in the following examples generally comply with the conditions described in the conventional conditions, such as Sambrook.J et al. "Molecular Cloning Experiment Guide" (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer (e.g., product instructions). Unless otherwise stated, percentages and parts are all by weight. Unless otherwise stated, the experimental materials and reagents used in the following examples are all commercially available.

[0155] The room temperature described in the examples is a conventional room temperature in the art, and is usually 10-30°C.

[0156] Example 1: Immunization and antibody screening

[0157] Example 1-1: Preparation of immunogen

[0158] A combination of the recombinant protein antigen huROR1-Fc (R&D systems, catalog number 9490-RO), engineered huROR1-HEK293 cells and HT-29 cells (ATCC, catalog number HTB38) was used to immunize mice. For the engineered huROR1-HEK293 cell line, HEK293 cells were transduced with a lentivirus containing a gene sequence encoding the extracellular domain of human ROR1 and a puromycin resistance gene. A single clone with the highest huROR1 expression was selected from the transduced HEK293 cells.

[0159] Example 1-2: Immunization

[0160] Immunization regimen:

[0161] Anti-ROR1 antibodies were obtained by immunizing genetically modified mice encoding human immunoglobulin heavy chain and kappa light chain variable regions with a combination of recombinant protein antigen huROR1-Fc (R&D systems, catalog number 9490-RO), engineered huROR1-HEK293 cells and HT-29 cells (ATCC, catalog number HTB38). Antibody immune responses were monitored by ROR1-specific immunoassays. When the desired immune response was achieved, splenocytes were harvested from each mouse and fused with mouse myeloma cells to maintain their viability and form hybridoma cells, and screened for ROR1 specificity.

[0162] Example 1-3: Spleen cell fusion

[0163] By electrofusion or PEG fusion, spleen lymphocytes and myeloma cells Sp2 / 0 ( CRL-158) to obtain hybridoma cells. TM PEG fusion was performed by HY technology (STEMCELL technologies). The ratio of primary cells: mouse myeloma cell line for electrofusion was 1:1, and the ratio for PEG fusion was 10:1.

[0164] Example 1-4: Screening of hybridoma clones specifically binding to human ROR1 protein by ELISA

[0165] ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated with 1 μg / ml human ROR1-Fc (9490-R0-050), human ROR2-Fc (8609-RO-050) or BSA overnight. Excess unbound protein was washed away by washing the plate three times with wash buffer and then blocked at room temperature for 1 hour. 50 μl of ROR1 hybridoma supernatant was added to two duplicate wells and incubated for 1 hour. Excess unbound antibody was washed away, and 50 μl of a 1:30000 diluted secondary goat anti-mouse IgG Fc-HRP (ab5870) was added to each well for 1 hour. According to the manufacturer's protocol, the plate was washed and 50 μL of chemiluminescent agent (color A and color B) was added. The reaction was stopped using 25 μL of 2N sulfuric acid. The optical density of the sample at 450 nm was measured by an ELISA reader (PerkinElmer). All clones tested selectively bound to human ROR1 but not to human ROR2 or BSA, demonstrating ROR1 specificity.

[0166] Table 1. ELISA determination (OD 450 ) Characterization of the binding of hybridoma clones to human ROR1 and human ROR2

[0167]

[0168] Example 1-5: Screening of hybridoma clones that specifically bind to cancer cells expressing ROR1 by flow cytometry

[0169] Hybridoma supernatants were tested for binding on ROR1+ cell lines HT29 (ATCC, HTB-38), HCC1187 (ATCC, CRL-2322) and ROR1-ROR2+ ​​cell lines T47D (ATCC, HTB-133) using flow cytometry analysis. Briefly, 50 μL of cells (2×10 6 Cells / mL) were mixed with 50 μL of undiluted hybridoma supernatant. The mixture was incubated on ice for 20 min and then washed twice with ice-cold staining buffer. The cells were then stained with 50 μL of PE-labeled secondary antibody (1:400 dilution, BioLegend, Cat#405307) for 20 min. After washing with staining buffer and fixing with 4% PFA, the cells were analyzed by flow cytometry. Purified anti-human ROR1 antibody was used as a positive control (BioLegend, Cat#357802). Purified mouse IgG1 antibody was used as an isotype control (Biolegend, Cat#400102). Figure 1-A Figure 4 shows an example of selected cell binding signals measured by flow cytometry. It was identified that clones 1E9, 8B10, 15G4, 29D4, 31G8, 37F9, 38H10 had enhanced binding to HT29 cells and HCC1187 cells compared to T47D cells.

[0170] Example 2 Sequencing of positive hybridoma clones

[0171] The process of cloning sequences from positive hybridomas is as follows. Hybridoma cells in the logarithmic growth phase are collected, RNA is extracted, reverse transcribed, and then the VDJ region is amplified. Next-generation sequencing is performed on the cDNA library amplified from each clone. The amino acid sequences of the heavy chain and light chain variable region DNA sequences corresponding to antibodies 1E9, 8B10, 15G4, 29D4, 31G8, 37F9 and 38H10 were obtained. After manufacturability evaluation, several mutations were made in the FR region, and the amino acid sequences and CDR sequences of the heavy chain variable region and light chain variable region of each antibody are shown in the following table. The amino acid residues of the CDR in VH / VL are numbered and annotated according to the Kabat and Wu numbering systems.

[0172] Table 2. CDR sequences of heavy chain variable domains of ROR1 hybridoma clones

[0173]

[0174] Table 3. CDR sequences of light chain variable domains of ROR1 hybridoma clones

[0175]

[0176] Table 4. Sequences of the heavy and light chain variable domains of ROR1 hybridoma clones

[0177]

[0178]

[0179] Example 3 Construction and expression of anti-ROR1 recombinant antibodies

[0180] Example 3-1: Molecular cloning of recombinant antibodies

[0181] The cDNA sequence of VH and VL district encoding the selected clone is directly synthesized as a DNA fragment with a 5' end in-frame leader sequence (MGWSCIILFLVATATGVHS). These DNA fragments are cloned into the selected vector using NEBuilder DNA assembly cloning kit (New England Biolabs). The VH district is cloned into the pFUSE-CHIg_hG1 vector (InvivoGen # pfuse-hchg1), and the hIgG1 heavy chain constant region in the VH district and the vector is in frame. The VL district is cloned into the pFUSE2-CLIg_hk vector (InvivoGen, # pfuse2-hclk), and the hIgκ light chain constant region in the VL district and the vector is in frame.

[0182] The IgG form of the antibody is disclosed as the following full-length heavy and light chains, in the order of 1E9, 8B10, 15G4 (N65S), 29D4, 31G8, 37F9 and 38H10 (N81K, T83S), respectively: SEQ ID NO: 54 (heavy chain) and 55 (light chain); SEQ ID NO: 56 (heavy chain) and 57 (light chain); SEQ ID NO: 58 (heavy chain) and 59 (light chain); SEQ ID NO: 60 (heavy chain) and 61 (light chain); SEQ ID NO: 62 (heavy chain) and 63 (light chain); SEQ ID NO: 64 (heavy chain) and 65 (light chain); SEQ ID NO: 66 (heavy chain) and 67 (light chain).

[0183] Table 5. Sequences of the constant regions of recombinant antibody IgG

[0184]

[0185] Table 6. Full-length sequences of heavy and light chains of anti-ROR1 recombinant antibodies

[0186]

[0187]

[0188]

[0189]

[0190] Example 3-2: Expression and purification of recombinant antibodies

[0191] Heavy chain expression plasmid and light chain plasmid were co-transfected into Expi293F cells (ThermoFisher, #A14527) using ExpiFectamine 293 transfection kit (ThermoFisher, A14524) or co-transfected into ExpiCHO-S cells (ThermoFisher, #A29127) using ExpiFectamine CHO transfection kit (ThermoFisher, A29129). Based on the manufacturer's instructions, the plasmid DNA concentration reached 1.0 ug per ml of suspended cells, and the LC:HC vector ratio was 1:1. Transfected cells were cultured on an orbital shaker at 37°C, 8% CO2 for 5 to 7 days. Conditioned media were collected and antibodies were purified using HiTrap MabSelect SuRe chromatography columns (Cytiva, #17549112) on an AKTA Pure 25 machine (Cytiva). The eluted antibody was neutralized with Tris buffer (pH 9.0) and subjected to PBS buffer exchange. The product concentration was measured by UV absorption, and the quality was determined by SDS-PAGE and HPLC.

[0192] Example 4: Characterization of binding of anti-ROR1 recombinant antibodies to ROR1+ and ROR1- cell lines by flow cytometry

[0193] Binding of hIgG1 mAb to cell surface ROR1 was determined by FACS analysis using different cancer cell lines positive for ROR1 or ROR2 expression, including Jeko-1 and T-47D cells.

[0194] Jeko-1 cells (ROR1 positive) were maintained in RPMI-1640 medium supplemented with 20% FBS and 1% penicillin and streptomycin. T-47D cells (ROR2 positive) were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin. Both cell lines were cultured at 37°C in a humidified atmosphere of 5% CO2.

[0195] To determine the binding of hIgG1 mAb to the cell surface ROR1 receptor, cells were first harvested and cultured at 2 × 10 6 Cells were resuspended in cell staining buffer (BioLegend) at 100 cells / mL. The cells were then treated with human Fc receptor blocking reagent (BioLegend) on ice for 10 min. The resulting cell suspension was divided into 50 μL aliquots. 50 μL of different concentrations of hIgG1 mAb were mixed with the cell aliquots, and the final concentration of hIgG1 mAb in the mixture ranged from 1.1 pM to 200 nM. The cells were incubated on ice for 1 hour and then washed twice with cell staining buffer. 50 μL of secondary antibody (PE-conjugated goat anti-human Fc, eBioscience TM , 1:200 dilution) to resuspend the cells. The cells were incubated on ice for another 30 min. The cells were then washed twice with cell staining buffer and resuspended in 4% PFA to fix the cells. The samples were analyzed using iQue3 to measure the median fluorescence intensity using the corresponding channels. The results are published in Figure 2-A , Figure 2-B and Table 7. The results confirmed that the anti-ROR1 antibody of the present invention can specifically bind to the ROR1 protein expressed in situ in human cells in a concentration-dependent manner, and confirmed that the antibody does not bind to the ROR1 family member protein, human ROR2 protein.

[0196] Table 7. K of anti-ROR1 recombinant antibodies binding to ROR1+Jeko-1 cells D value

[0197] Antibody <![CDATA[K D (nM)]]> 1E9 0.26 8B10 1.93 15G4(N65S) 2.49 29D4 1.08 31G8 0.47 37F9 1.17 38H10(N81K / T83S) 0.20

[0198] Example 5: Characterization of cellular internalization of anti-ROR1 recombinant antibodies in cells expressing ROR1

[0199] To explore the possibility of developing an ADC strategy using the ROR1 antibodies of the present invention, the inventors evaluated the endocytosis of antibodies induced by ROR1 antibody binding. In the assay, Jeko-1 cells were collected and cultured at 2×10 6Cells / mL were suspended in ice-cold staining buffer (PBS with 0.5% FBS) and then treated with human Fc receptor blocking reagent (BioLegend, 422302). The cells were then pre-incubated on ice with antibodies labeled with Alexa Fluor 488 for 1 hour. After incubation, the cells were transferred to a 37°C incubator. At predetermined time points (0min, 15min, 30min, 45min, 1 hour, 2 hours, 4 hours, 6 hours), 50 μL of cell suspension was aliquoted and transferred to ice for incubation until the end of incubation for internalization. Then, all aliquots were washed twice with ice-cold staining buffer. The Alexa Fluor 488 signal bound to the cell surface was quenched by anti-Alexa Fluor antibody (Invitrogen, A-11094). The remaining intracellular signal caused by internalization was then analyzed by flow cytometry. Another 50 μL aliquot of cells was collected at 0min, stored on ice, and analyzed by flow cytometry without quenching. This sample served as a control for 100% cell surface binding signal. The amount of internalized antibody was calculated and normalized to 100% cell surface binding signal for each antibody as calculated by the following formula:

[0200] Normalized internalization (%) = (MFI t –MFI t 0) / (MFI 总计 –MFI t0 )×100%

[0201] Internalization results are based on median fluorescence intensity (MFI). t is the MFI of the sample collected at time t; MFI t0 is the MFI of the sample collected at time 0 minutes. MFI 总计 is the MFI of an additional sample collected and served as a control for 100% cell surface-bound signal.

[0202] Table 8. Internalization kinetics of anti-ROR1 recombinant antibodies in ROR1+Jeko1 cells.

[0203]

[0204]

[0205] like Figure 3 As shown in Table 8, strong internalization signals were observed for the anti-ROR1 antibodies 1E9, 15G4 (N65S), 29D4, 31G8, 37F9, and 38H10 (N81K, T83S) in Jeko-1 cells.

[0206] Example 6: Epitope Characterization of Anti-ROR1 Recombinant Antibodies

[0207] Example 6-1: ELISA binding to human ROR1 protein subdomain

[0208] ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated overnight with 1 μg / ml human ROR1 protein (RO1-H522y) or human ROR1 subdomain Ig-like (RO1-H5221), Frizzled (RO1-H5222) and Kringle (RO1-H5223). Excess unbound protein was washed away by washing the plate three times with wash buffer and then blocked at room temperature for 1 hour. 50 μl of 100 μg / mL ROR1 recombinant antibody was added to duplicate wells and incubated for 1 hour. Excess unbound antibody was washed away, and 50 μl of a 1:2000 diluted secondary antibody goat anti-human IgG Fc-HRP (ab6858) was added to each well for another 1 hour. According to the manufacturer's protocol, the plate was washed and 50 μL of chemiluminescent agent (color A and color B) was added. The reaction was stopped using 25 μL of 2N sulfuric acid. The optical density of the samples was measured at 450 nm by a microplate reader (PerkinElmer). All recombinant antibodies tested bound to the full-length human ROR1 protein. 1E9, 8B10, 15G4 (N65S), 29D4, 31G8, 37F9, and UC-961 bound to the Ig-like subdomain of the human ROR1 protein, while 38H10 (N81K / T83S) bound to the Kringle subdomain of the human ROR1 protein.

[0209] Table 9. Results of ELISA (OD 450 ) Characterization of binding of ROR1 recombinant antibodies to full-length or subdomain human ROR1 protein

[0210]

[0211] Example 6-2: Octet typing (binning)

[0212] A total of five clones (15G4 (N65S), 29D4, 31G8, 37F9 and 38H10 (N81K / T83S)) were epitope typed and compared with the anti-ROR1 antibody UC961. Antibody epitope typing was performed using the Octet Red384 system equipped with anti-species and Ni-NTA sensors from Pall Life Sciences (Menlo Park, CA). The experiment was performed as a tandem typing assay. The assay consisted of a five-step binding cycle: 1) establish a buffer baseline for 30 seconds, 2) couple 50 nM ROR1 antigen (RO1-H522y) to the Ni-NTA octet sensor for 5 minutes using standard 1× assay buffer (PBS + 0.02% Tween 20, 0.1% BSA, 0.05% sodium azide) diluted from a 10× kinetic buffer stock (ForteBio), 3) load 250 nM of each antibody (saturated mAb) to saturate the immobilized antigen for 10 minutes, 4) allow 250 nM of each antibody (competing mAb) to bind for 5 minutes, 5) regenerate the capture sensor for 30 seconds. As shown in Tables 10 and 11, the five anti-ROR1 recombinant antibodies can be divided into 2 different epitope bins. 15G4 (N65S), 29D4, 31G8, and 37F9 are in the same bin 1, while 38H10 (N81K / T83S) is in a different bin 2. Furthermore, UC-961 binding did not compete with any of the five anti-ROR1 recombinant antibodies (15G4 (N65S), 29D4, 31G8, 37F9, and 38H10 (N81K / T83S)), indicating that the UC-961 binding epitope is located in a separate epitope subtype 3.

[0213] Table 10. Epitope typing of recombinant antibodies binding to human ROR1 protein by BLI using the Octet Red384 system.

[0214]

[0215] Signal displacement unit: nm

[0216] Table 11. Epitope typing of anti-ROR1 recombinant antibodies against human ROR1

[0217] Epitope typing Anti-ROR1 Antibody 1 15G4(N65S), 29D4, 31G8, 37F9 2 38H10(N81K / T83S) 3 UC961

[0218] Example 7: Cross-reactivity of anti-ROR1 recombinant antibodies with mouse ROR1 protein

[0219] ELISA was performed using the DuoSet ELISA Auxiliary Kit (R&D System, DY008). ELISA plates were coated with 1 μg / ml mouse ROR1 protein (RO1-M5221), rat ROR1 protein (RO1-R5221) or control BSA overnight. Excess unbound protein was washed away by washing the plate three times with wash buffer and then blocked at room temperature for 1 hour. 50 μl of 25 μg / ml ROR1 recombinant antibody was added to the double wells and incubated for 1 hour. Excess unbound antibody was washed away, and 50 μl of a 1:2000 diluted secondary goat anti-human IgG Fc-HRP (ab6858) was added to each well for another 1 hour. According to the manufacturer's protocol, the plate was washed and 50 μL of chemiluminescent agent (color A and color B) was added. The reaction was stopped using 25 μL of 2N sulfuric acid. The optical density of the sample at 450 nm was measured by an enzyme reader (PerkinElmer). Recombinant antibodies 15G4 (N65S), 29D4, 31G8, 37F9 and 38H10 (N81K / T83S) were able to bind to both mouse and rat ROR1 proteins. In contrast, UC961 showed no cross-reactivity to either mouse or rat ROR1 proteins.

[0220] Table 12. Results of ELISA (OD 450 ) Characterization of binding of ROR1 recombinant antibody clones to mouse ROR1 or rat ROR1 protein

[0221]

[0222] Example 8: Preparation of anti-ROR1 antibody drug conjugate (ADC)

[0223] The antibody of the present invention has cell affinity activity and endocytosis activity, making it suitable for being coupled with a drug to form an antibody-drug conjugate for treating ROR1-mediated diseases.

[0224] Purification of monoclonal antibodies

[0225] 15G4 (N65S), 29D4, 31G8, 37F9 and 38H10 (N81K / T83S) were purified for drug conjugation with reference to the preparation process of anti-ROR1 antibodies described in Example 3. Other antibodies used for conjugation can include any of the antibodies described herein (see Example 3).

[0226] Preparation of intermediates for pharmaceuticals

[0227] The following intermediate compounds are used to prepare antibody drug conjugates (ADCs) of anti-ROR1 antibodies, and compound D is prepared by the method disclosed in the PCT patent application. (See application PCT / CN2022 / 073943 filed on January 26, 2022).

[0228]

[0229] The antibody drug conjugates (ADCs) against ROR1 antibodies described herein can use MC-val-cit(vc)-PAB-MMAE drug linker, and the preparation method thereof is similar to the method described in US Pat. No. US2005 / 0238649.

[0230]

[0231] Conjugation of monoclonal antibodies to drug molecules

[0232] Anti-ROR1 antibodies (5 mg / mL in PBS, pH 7.4) were treated with sufficient molar equivalents of tris(2-carboxyethyl)phosphine (TCEP, 10 mM) at 37°C for 1 hour. Sufficient molar equivalents of a drug linker (e.g., MC-val-cit(vc)-PAB-MMAE or a DMSO solution of compound D) were added to the reduced antibody in PBS. After one hour of incubation, the reaction mixture was purified using size exclusion chromatography (SEC) to separate ADC and free, unconjugated drug linkers. The drug-antibody ratio (DAR) of the ADC was determined using a TOF LC / MS system (Agilent), and the average DAR values ​​are summarized in Table 13. The average DAR values ​​of anti-ROR1 antibodies conjugated to MC-val-cit(vc)-PAB-MMAE ranged from 3.5 to 4.5. The average DAR values ​​of anti-ROR1 antibodies conjugated to compound D were approximately 8.0 or 6.0.

[0233]

[0234] Table 13: Conjugation of monoclonal antibodies to linker / payload moieties

[0235]

[0236]

[0237] Example 9: Inhibitory effect of ADC on tumor cell growth

[0238] This example analyzes ADC-01 to ADC-11 immunoconjugates with different cytotoxic moieties and linker chemistries. VLS-101 is a positive control drug. The ROR1-mediated cytotoxicity of these conjugates was tested in cell culture to define the efficacy of various linker-cytotoxic agent combinations. Six different types of cancer cell lines were tested: colon cancer cell line HT-29, gastric cancer cell line AGS, NSCLC cell line NCI-H1975, TNBC cell line HS578T, MCL cell line Jeko-1, and ALL cell line Kasumi-2.

[0239] This example analyzes ADC-03, ADC-05, ADC-12, ADC-13 immunoconjugates with different antibodies and DAR values, wherein ADC-12 has a different DAR value from ADC-03, and ADC-13 has a different DAR value from ADC-05. VLS-101 is a positive control drug. The ROR1-mediated cytotoxicity of these conjugates was tested in cell culture. The cancer cell line tested was the human testicular embryonic carcinoma cell line NTERA-2.

[0240] Cells were collected in logarithmic phase growth and distributed into 96-well plates at 1000-2000 cells / well. After overnight incubation, immunoconjugates were added to each well. The final concentration of immunoconjugates in the wells ranged from 0.01 nM to 666.7 nM. After incubation for 4 to 7 days, cell viability in each well was determined by Cell Titer Glo 2.0 assay (Promega).

[0241] Sigmoidal dose-response nonlinear regression fitting was used to generate curves and IC in GraphPad Prism 50 The data from these experiments are summarized in Tables 14-16. Figure 4-A Figure 4 shows the representative IC values ​​of all immunoconjugates tested against the indicated cell lines. 50 Fig. B12-ADC is a negative control. All ADC-07 to ADC-13 showed good potential toxicity to tumor cells, and the immunoconjugates with different DAR values ​​showed considerable potential toxicity to tumor cells. ADC-03, ADC-05, ADC-12, and ADC-13 showed better targeted killing than VLS-101.

[0242] Table 14: Evaluation of cytotoxicity of antibody-drug conjugates against tumor cells

[0243]

[0244]

[0245] Table 15: Evaluation of cytotoxicity of antibody-drug conjugates against tumor cells

[0246]

[0247] Table 16: Cytotoxicity of Antibody-Drug Conjugates with Different DAR Values ​​on Tumor Cells

[0248]

[0249] Example 10: Tumor inhibition experiment of ADC on ROR1 positive cancer cell subcutaneous transplanted tumor model in nude mice

[0250] 1. Efficacy of anti-ROR1 ADC in mantle cell lymphoma (MCL) xenograft mouse model

[0251] Jeko-1 cells (MCL) were subcutaneously transplanted into mice (CB17 / SCID). When the tumor volume reached approximately 120-180 mm 3 The transplanted mice were randomly divided into six groups (5 mice per group). The groups were vehicle control, VLS-101, ADC-07, ADC-08, ADC-09 and ADC-10. The mice were treated with ADC (5 mg / kg) intravenously Q4D. The average tumor growth inhibition (TGI) was calculated using the following formula:

[0252] TGI = ((mean value (C) - mean value (C0)) - (mean value (T) - mean value (T0))) / (mean value (C) - mean value (C0)) * 100%; T is the current group value, C is the control group value, T0 and C0 represent the tumor volume at the beginning of the test.

[0253] The results of the study are shown in Table 17 and Figure 5 . Mice treated with the six ADCs showed statistically significant differences in tumor growth compared to control mice, resulting in partial or even complete regression of tumors. Subsequent tumor inhibition studies with VLS-101 and ADC-10 were performed in the same Jeko-1 xenograft mouse model. Jeko-1 xenograft mice were randomly divided into five groups (5 mice per group), namely vehicle control group, 2mg / kg VLS-101 group, 1mg / kg VLS-101 group, 2mg / kg ADC-10 group and 1mg / kg ADC-10 group. Mice were treated with ADCs intravenously Q1W. The results of the subsequent studies are shown in Tables 18 and Figure 6 Among them, ADC-10 treatment at 1 mg / kg showed stronger tumor growth inhibition with a TGI value of 36.9%, while the tumor growth inhibition rate of mice treated with 1 mg / kg of VLS-101 was 3.8%.

[0254] Table 17. Efficacy of high-dose ADC on Jeko-1 xenograft tumors in tumor-bearing mice

[0255]

[0256] Table 18. Efficacy of low-dose ADC on Jeko-1 xenograft tumors in tumor-bearing mice

[0257]

[0258] 2. Xenograft (CDX) model derived from NCI-H1975 (EGFR-L858R-T790M-C797S) cell line

[0259] NCI-H1975 (EGFR-L858R-T790M-C797S) cells were subcutaneously transplanted into mice (NOD / SCID). When the tumor volume reached approximately 10-160 mm 3 The transplanted mice were randomly divided into seven groups (5 mice per group). The groups were vehicle control, VLS-101, ADC-07, ADC-08, ADC-09, ADC-10, and ADC-11. The mice were treated with ADC (3 mg / kg) intravenously Q1W. The results of the study are shown in Tables 18 and Figure 7 Tumor growth in mice treated with ADC-07, ADC-08, and ADC-10 showed statistically significant differences compared to mice treated with VLS-101, with TGI values ​​of 95.4%, 94.5%, and 96.8%, respectively, while tumors in mice treated with VLS-101 showed a TGI of 78.4%.

[0260] Table 18. Efficacy of medium-dose ADC against NCI-H1975 (EGFR-TM) xenograft tumors in tumor-bearing mice

[0261]

[0262] Example 11: Pharmacokinetic (PK) Determination of Humanized Anti-ROR1 Antibodies and Antibody-Drug Conjugates in Mice

[0263] Female Balb / C mice (6-8 weeks old) were used to evaluate the pharmacokinetics of antibody-drug conjugates. Blank serum was collected before administration. ADC was administered intravenously at 3 mg / kg. After injection, serum was collected from designated mice at predetermined time points (1, 4, 24, 48, 72, 120, 168, 336 and 504 hours) and immediately stored at -80°C until further analysis.

[0264] Serum concentration of ADC is measured by ELISA. Briefly, in order to measure the total antibody concentration in serum, the assay plate is coated with 1 μg / mL of ROR1-his protein (Acro Biosystems, Cat#RO1-H522y), and then blocked with blocking buffer. After washing the plate 5 times, 30 μL of sample is added to each well and incubated at room temperature for 1 hour. The plate is washed 5 times and then incubated with HRP-coupled goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Cat#109-035-008). Finally, TMB substrate is added to each well. The reaction is stopped after 10 minutes. The absorbance at 450nm is measured to calculate the sample concentration. In order to measure the concentration of payload-coupled antibodies, anti-payload antibodies are coated on the assay plate to capture payload-coupled antibodies. HRP-coupled goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, Cat#109-035-097) is used to detect captured analytes.

[0265] Pharmacokinetic parameters were calculated by PK Solver 2.0. The main pharmacokinetic parameters are shown in Tables 19 and 20.

[0266] Table 19. Pharmacokinetic parameters of total antibody in mice

[0267]

[0268]

[0269] Table 20. Pharmacokinetic parameters of ADC in mice

[0270]

[0271] ROR1 antibodies UC961, 29D4, 31G8, and 37F9 had similar AUCs in mice. The half-lives of ADC-08, ADC-09, and ADC-10 were superior to that of VLS-101, which may contribute to their superior in vivo therapeutic efficacy.

Claims

1. An antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, <h2 style=";text-align:left;direction:ltr">Ab-(L2-L1-D)<h2 style=";text-align:left;direction:ltr"> y (A) in: D is a cytotoxic drug; L1 and L2 are connection units; y is a number from 1 to 20, preferably from 2 to 10, more preferably from 2 to 8, further preferably from 2 to 6 or about 4 to 8, most preferably about 2, 4, 6, 8; Ab is an anti-ROR1 antibody or an antigen-binding fragment thereof, comprising: an antibody heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 regions and an antibody light chain variable region comprising LCDR1, LCDR2 and LCDR3 regions, wherein: HCDR1 is shown in SEQ ID NO: 04, HCDR2 is shown in SEQ ID NO: 10, and HCDR3 is shown in SEQ ID NO: 17; or HCDR1 is shown in SEQ ID NO: 05, HCDR2 is shown in SEQ ID NO: 11, and HCDR3 is shown in SEQ ID NO: 18; or HCDR1 is shown in SEQ ID NO: 05, HCDR2 is shown in SEQ ID NO: 12, and HCDR3 is shown in SEQ ID NO: 19; or HCDR1 is shown in SEQ ID NO: 03, HCDR2 is shown in SEQ ID NO: 09, and HCDR3 is shown in SEQ ID NO: 16; or HCDR1 is shown in SEQ ID NO: 01, HCDR2 is shown in SEQ ID NO: 07, and HCDR3 is shown in SEQ ID NO: 14; or HCDR1 is shown in SEQ ID NO: 02, HCDR2 is shown in SEQ ID NO: 08, and HCDR3 is shown in SEQ ID NO: 15; or HCDR1 is shown in SEQ ID NO: 06, HCDR2 is shown in SEQ ID NO: 13, and HCDR3 is shown in SEQ ID NO: 20; and / or LCDR1 is shown in SEQ ID NO: 24, LCDR2 is shown in SEQ ID NO: 30, and LCDR3 is shown in SEQ ID NO: 36; or LCDR1 is shown in SEQ ID NO: 24, LCDR2 is shown in SEQ ID NO: 31, and LCDR3 is shown in SEQ ID NO: 37; or LCDR1 is shown in SEQ ID NO: 25, LCDR2 is shown in SEQ ID NO: 32, and LCDR3 is shown in SEQ ID NO: 38; or LCDR1 is shown in SEQ ID NO: 23, LCDR2 is shown in SEQ ID NO: 29, and LCDR3 is shown in SEQ ID NO:35; or LCDR1 is shown in SEQ ID NO: 21, LCDR2 is shown in SEQ ID NO: 27, and LCDR3 is shown in SEQ ID NO: 33; or LCDR1 is shown in SEQ ID NO: 22, LCDR2 is shown in SEQ ID NO: 28, and LCDR3 is shown in SEQ ID NO: 34; or LCDR1 is shown in SEQ ID NO: 26, LCDR2 is shown in SEQ ID NO: 32, and LCDR3 is shown in SEQ ID NO:

39.

2. The antibody-drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab comprises: a) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 04, SEQ ID NO: 10 and SEQ ID NO: 17, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 30 and SEQ ID NO: 36, respectively; or b) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 11 and SEQ ID NO: 18, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 31 and SEQ ID NO: 37, respectively; or c) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 05, SEQ ID NO: 12 and SEQ ID NO: 19, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 25, SEQ ID NO: 32 and SEQ ID NO: 38, respectively; d) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 03, SEQ ID NO: 09 and SEQ ID NO: 16, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 29 and SEQ ID NO: 35, respectively; or e) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 01, SEQ ID NO: 07 and SEQ ID NO: 14, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 27 and SEQ ID NO: 33, respectively; or f) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 02, SEQ ID NO: 08 and SEQ ID NO: 15, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 34, respectively; or g) a heavy chain variable region sequence comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 06, SEQ ID NO: 13 and SEQ ID NO: 20, respectively; and a light chain variable region sequence comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 32 and SEQ ID NO: 39, respectively.

3. The antibody-drug conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the Ab is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antigen-binding fragment thereof.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, wherein the Ab comprises a heavy chain variable region of the following sequence: SEQ ID NO: 46, 48, 50, 44, 40, 42 and 52, or has at least 80%, 85%, 90%, 95% or 99% sequence identity thereto; and / or The Ab comprises a light chain variable region of SEQ ID NO: 47, 49, 51, 45, 41, 43 and 53, or has at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 4, wherein the Ab comprises: a) a heavy chain variable region as set forth in SEQ ID NO:46, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as set forth in SEQ ID NO:47, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; or b) a heavy chain variable region as set forth in SEQ ID NO:48, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as set forth in SEQ ID NO:49, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; or c) a heavy chain variable region as set forth in SEQ ID NO:50, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as set forth in SEQ ID NO:51, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; or d) a heavy chain variable region as set forth in SEQ ID NO:44, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as set forth in SEQ ID NO:45, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; or e) a heavy chain variable region as set forth in SEQ ID NO:40, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as set forth in SEQ ID NO:41, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; or f) a heavy chain variable region as set forth in SEQ ID NO:42, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as set forth in SEQ ID NO:43, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; or g) a heavy chain variable region as shown in SEQ ID NO:52, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%; and / or a light chain variable region as shown in SEQ ID NO:53, or a sequence identity thereof of at least 80%, 85%, 90%, 95% or 99%.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 5, wherein the Ab comprises: a) a heavy chain variable region as shown in SEQ ID NO: 46 and a light chain variable region as shown in SEQ ID NO: 47; b) a heavy chain variable region as set forth in SEQ ID NO:48 and a light chain variable region as set forth in SEQ ID NO:49; c) a heavy chain variable region as shown in SEQ ID NO: 50 and a light chain variable region as shown in SEQ ID NO: 51; d) a heavy chain variable region as shown in SEQ ID NO: 44 and a light chain variable region as shown in SEQ ID NO: 45; e) a heavy chain variable region as shown in SEQ ID NO:40 and a light chain variable region as shown in SEQ ID NO:41; f) a heavy chain variable region as shown in SEQ ID NO:42 and a light chain variable region as shown in SEQ ID NO:43; g) a heavy chain variable region as shown in SEQ ID NO: 52 and a light chain variable region as shown in SEQ ID NO:

53.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 6, wherein the Ab is a full-length antibody further comprising a human antibody constant region; Preferably, the heavy chain constant region of the human antibody constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and conservative variants thereof, and the light chain constant region of the human antibody constant region is selected from the constant regions of κ and λ chains of human antibodies and conservative variants thereof; More preferably, the full-length antibody comprises the human antibody heavy chain constant region of SEQ ID NO: 68 and the human light chain constant region of SEQ ID NO:

69.

8. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 7, wherein the Ab comprises: a) a heavy chain as shown in SEQ ID NO: 60 and a light chain as shown in SEQ ID NO: 61; b) a heavy chain as shown in SEQ ID NO: 62 and a light chain as shown in SEQ ID NO: 63; c) a heavy chain as shown in SEQ ID NO: 64 and a light chain as shown in SEQ ID NO: 65; d) a heavy chain as shown in SEQ ID NO: 58 and a light chain as shown in SEQ ID NO: 59; e) a heavy chain as shown in SEQ ID NO:54 and a light chain as shown in SEQ ID NO:55; f) a heavy chain as shown in SEQ ID NO: 56 and a light chain as shown in SEQ ID NO: 57; g) the heavy chain shown in SEQ ID NO: 66 and the light chain shown in SEQ ID NO:

67.

9. The antibody-drug conjugate according to any one of claims 1 to 8, or a pharmaceutically acceptable salt or solvate thereof, wherein the antigen binding fragment is selected from Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), dimerization domain V (diabody), disulfide stabilized Fv (dsFv) and CDR-containing peptides.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 9, wherein the cytotoxic drug is selected from a toxin, a chemotherapeutic agent, an antibiotic, a radioisotope and a nucleolytic enzyme.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 10, wherein the cytotoxic drug is selected from a tubulin inhibitor or a topoisomerase inhibitor; preferably an auristatin analog or a camptothecin derivative; more preferably SN-38, MMAE, MMAF or exitecan.

12. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 11, wherein the antibody-drug conjugate is represented by general formula (I) or (II): in: L1 and L2 are connection units; y1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8 or 2 to 6, further preferably a number from 6 to 8, and most preferably 4, 6 or 8; y2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0; Ab is the anti-ROR1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 12, wherein the antibody-drug conjugate is represented by general formula (IA) or (II-A): in: L2 is the connection unit; R1 is selected from hydrogen, C 1-6 Haloalkyl or C 3-8 Cycloalkyl; R2 is selected from hydrogen, C 1-6 Haloalkyl or C 3-8 Cycloalkyl; Or, R1 and R2 together with the carbon atom to which they are attached form C 3-8 Cycloalkyl; y1, y2 and Ab are as defined in claim 12.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 13, wherein: R1 is selected from hydrogen, C 1-3 Haloalkyl or C 3-6 Cycloalkyl; R2 is selected from hydrogen, C 1-3 Haloalkyl or C 3-6 Cycloalkyl; Or, R1 and R2 together with the carbon atom to which they are attached form C 3-6 Cycloalkyl.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 12 to 14, wherein L2 is represented by general formula (III): in: s 1 and 2 Each independently represents an integer selected from 0 to 8. Preferably, s 1 and 2 Independently selected from 1, 2, 3, 4, 5 or 6; or 1 is an integer from 1 to 8, s 2 is 0, preferably, s 1 is selected from 4, 5, 6, 7 or 8, and s 2 is 0; or 2 An integer selected from 2 to 8, s 1 is 2, preferably, s 2 is selected from 2, 3, 4, 5 or 6, and s 1 Serves 2.

16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 12 to 15, wherein the antibody-drug conjugate has the following structure: in: y1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8 or 2 to 6, further preferably a number from 6 to 8, and most preferably 4, 6 or 8; y2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.0; Ab is the anti-ROR1 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 16, wherein the antibody-drug conjugate is selected from the following compounds: in: y1 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8 or 2 to 6, further preferably a number from 6 to 8, most preferably 4, 6 or 8; y2 is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably 2 to 4, 2 to 6, 3 to 6, 3 to 5, 4 to 6, 4 to 8, most preferably 2.0, 3.8, 3.9, 4.0, 4.1, 4.3, 6.0 or 8.

0.

18. A pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 17, and one or more pharmaceutically acceptable carriers, diluents or excipients.

19. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 17, or the pharmaceutical composition according to claim 18, in the manufacture of a medicament for treating or preventing a disease associated with human ROR1.

20. The use according to claim 19, characterized in that Use in the manufacture of a medicament for treating or preventing a cancer expressing ROR1; Preferably, the cancer is chronic lymphocytic leukemia (CLL), B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and marginal zone lymphoma (MZL), breast cancer, kidney cancer, ovarian cancer, stomach cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

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