Treatment of drug-resistant cancer based on Anti-ROR1 antibody-drug conjugate

Abstract

An anti-human ROR1 antibody, an antibody-drug conjugate comprising the anti-human ROR1 antibody, and use for treating drug-resistant cancer.

Description

Treatment of drug-resistant cancers based on anti-ROR1 antibody-drug conjugates Technical Field

[0001] This disclosure pertains to the biomedical field and relates to the treatment of drug-resistant cancers, particularly acquired drug-resistant cancers, based on antibody-drug conjugates formed by linking anti-ROR1 antibodies to cytotoxic drugs via a linker unit. Background Technology

[0002] The statements herein are provided only to provide background information in connection with this disclosure and to aid in understanding this disclosure, and do not necessarily constitute prior art.

[0003] ROR1 is a transmembrane receptor tyrosine kinase protein belonging to the type I receptor tyrosine kinase (RTK) family. Human ROR1 consists of an extracellular immunoglobulin-like domain (Ig), two cysteine-rich domains (FZD), a juxtamembrane kringle domain, a single transmembrane structure, an intracellular tyrosine kinase domain (TKD), two serine / threonine-rich domains (S / TRD), and a proline-rich domain (PRD). ROR1 is highly expressed during embryonic and infant development and plays a crucial role in various physiological processes, including regulating cell division, proliferation, migration, and chemotaxis, and is essential for the formation of neural, skeletal, and vascular organs. In subsequent development, ROR1 expression significantly decreases, and it is absent or poorly expressed in normal adult tissues. However, ROR1 is highly expressed in various hematologic malignancies and solid tumors. Hematologic malignancies with high ROR1 expression include B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid hematologic cancers. In solid tumors, ROR1-expressing tumors include triple-negative breast cancer, colon cancer, lung cancer, pancreatic cancer, and ovarian cancer, and ROR1 expression is closely related to disease progression and treatment efficacy. Studies have found that ROR1 can participate in the Wnt5a-mediated non-canonical Wnt signaling pathway. Wnt5a activates receptors ROR1 or FZD5, leading to Dvl2 / 3 activation and Akt phosphorylation. Akt then promotes IKKα phosphorylation, activating the IKK complex. The IKK complex degrades IκBα and promotes phosphorylation of the NF-κB subunit p65. Phosphorylated p65 translocates to the nucleus, promoting the transcriptional expression of target genes, including Wnt5a. Wnt5a secretion, in turn, promotes a new round of autonomous feedback loops. Activation of the autonomous feedback loop ROR1 / Akt / p65 pathway further promotes the secretion of pro-inflammatory factors (such as IL-6) and chemokines (such as CCL2). Furthermore, some studies suggest that ROR1 is associated with epithelial-mesenchymal transition (EMT) in tumor cells or with the activation of YAP / TAZ transcription, thereby enhancing tumorigenesis and chemotactic resistance.

[0004] Cancer drug resistance is a major obstacle to cancer treatment. It has been reported that approximately 15% of HER2-overexpressing breast cancer patients who have received a wide range of existing anticancer therapies respond to trastuzumab, while about 85% of this group do not respond or respond only weakly to trastuzumab treatment. Mechanisms of such resistance include drug efflux, acquisition of drug-binding defective mutants of the target, occupation of alternative survival pathways, and epigenetic alterations. For example, RAF inhibitors are used to target malignant melanomas with the B-raf V600E mutation; however, their clinical success is hampered by acquired resistance. Therefore, new treatment approaches are needed to address the heterogeneity and resistance to drug therapies in cancer cell populations. Summary of the Invention

[0005] Through extensive and in-depth research, the inventors have discovered that anti-ROR1 antibodies or anti-ROR1 antibody-drug conjugates (also referred to as "anti-ROR1-ADCs" in this disclosure) can be used to treat cancers resistant to anticancer therapeutics, particularly to cancers resistant to topoisomerase I inhibitors or their derivatives, or antibody-drug conjugates containing topoisomerase I inhibitors or their derivatives.

[0006] On one hand, this disclosure provides a method for treating cancer, comprising administering an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate to a subject in need, wherein the subject has cancer resistant to anticancer therapeutic agents. In some embodiments, the anticancer therapeutic agent is:

[0007] (i) an inhibitor of topoisomerase I or a derivative thereof; or

[0008] (ii) Antibody-drug conjugates containing a topoisomerase I inhibitor or a derivative thereof (i.e., the drug portion of the antibody-drug conjugate is a topoisomerase I inhibitor or a derivative thereof).

[0009] In some embodiments, the anticancer therapeutic agent is DXd or a derivative thereof. In some embodiments, the anticancer therapeutic agent is an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof, wherein the antibody-drug conjugate targets HER2.

[0010] In some embodiments, the anticancer therapeutic agent is an anti-HER2 antagonist. In some embodiments, the anticancer therapeutic agent is an anti-HER2 antibody. In some embodiments, the anticancer therapeutic agent is an anti-HER2 antibody-drug conjugate. In some embodiments, the anticancer therapeutic agent is trastuzumab. In some embodiments, the anticancer therapeutic agent is a small molecule HER2 inhibitor.

[0011] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises a first antigen-binding domain and a second antigen-binding domain; wherein the first antigen-binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding domain comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein:

[0012] The heavy chain variable region VH1 includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:38, and the light chain variable region VL1 includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23; or

[0013] The heavy chain variable region VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:22, and the light chain variable region VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23; and the second antigen-binding domain comprises:

[0014] The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:72, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:71; or

[0015] The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:73, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:71; or

[0016] The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:74, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:75; or

[0017] The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:74, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:76; or

[0018] The heavy chain variable region VH2 includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:74, and the light chain variable region VL2 includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:71.

[0019] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate includes a first antigen-binding domain and a second antigen-binding domain; wherein the first antigen-binding domain includes a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding domain includes a heavy chain variable region VH2 and a light chain variable region VL2, wherein:

[0020] The heavy chain variable region VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:38, and the light chain variable region VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23; and

[0021] The heavy chain variable region VH2 includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:72, and the light chain variable region VL2 includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:71.

[0022] The amino acid sequences of the aforementioned HCDR and LCDR are determined according to the Kabat, Chothia, AbM, or IMGT numbering systems. Various numbering systems for HCDR and LCDR are well known to those skilled in the art; in one embodiment, the amino acid sequences of the HCDR and LCDR are determined according to the Kabat numbering system.

[0023] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises a first antigen-binding domain and a second antigen-binding domain; wherein the first antigen-binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding domain comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein:

[0024] The heavy chain variable region VH1 includes HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:10; or

[0025] The heavy chain variable region VH1 includes HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:18, and HCDR3 as shown in SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:10; and

[0026] The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:61, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:50; or

[0027] The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:66, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:50; or

[0028] The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:67, LCDR2 as shown in SEQ ID NO:68, and LCDR3 as shown in SEQ ID NO:50; or

[0029] The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:69, and LCDR3 as shown in SEQ ID NO:50; or

[0030] The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:50.

[0031] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate includes a first antigen-binding domain and a second antigen-binding domain; wherein the first antigen-binding domain includes a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding domain includes a heavy chain variable region VH2 and a light chain variable region VL2, wherein:

[0032] The heavy chain variable region VH1 includes HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:10; and

[0033] The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:61, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:50.

[0034] In some embodiments, the anti-ROR1 antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0035] In some embodiments, the anti-ROR1 antibody is a humanized antibody and includes the FR region of a human antibody.

[0036] In some embodiments, the first antigen-binding domain of the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate includes:

[0037] The heavy chain variable region VH1 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:38, and the light chain variable region VL1 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:23; or

[0038] The heavy chain variable region VH1 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:22, and the light chain variable region VL1 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:23; and the second antigen-binding domain of the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises:

[0039] The heavy chain variable region VH2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:72, and the light chain variable region VL2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:71; or

[0040] The heavy chain variable region VH2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:73, and the light chain variable region VL2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:71; or

[0041] The heavy chain variable region VH2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:74, and the light chain variable region VL2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:75; or

[0042] The heavy chain variable region VH2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:74, and the light chain variable region VL2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:76; or

[0043] The heavy chain variable region VH2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:74, and the light chain variable region VL2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:71.

[0044] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises:

[0045] The heavy chain variable region VH1 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:38, and the light chain variable region VL1 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:23; and

[0046] The heavy chain variable region VH2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:72, and the light chain variable region VL2 comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:71.

[0047] In some embodiments, the first antigen-binding domain of the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises:

[0048] The heavy chain variable region VH1 contains the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region VL1 contains the amino acid sequence shown in SEQ ID NO:23; or

[0049] The heavy chain variable region VH1 comprises the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region VL1 comprises the amino acid sequence shown in SEQ ID NO:23; and the second antigen-binding domain of the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises:

[0050] The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:72, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:71; or

[0051] The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:73, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:71; or

[0052] The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:74, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:75; or

[0053] The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:74, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:76; or

[0054] The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:74, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:71.

[0055] In some embodiments, the first antigen-binding domain of the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises:

[0056] The heavy chain variable region VH1 comprises the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region VL1 comprises the amino acid sequence shown in SEQ ID NO:23; and the second antigen-binding domain comprises:

[0057] The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:72, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:71.

[0058] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate includes a heavy chain constant region; in some embodiments, the heavy chain constant region is an IgG1 or IgG4 subtype.

[0059] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate includes a light chain constant region; in some embodiments, the light chain constant region is κ-type.

[0060] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:11 or a variant thereof; and / or the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:12 or a variant thereof. In some embodiments, both the heavy chain constant region variant and the light chain constant region variant are conventional variants.

[0061] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises:

[0062] The heavy chain with an amino acid sequence as shown in SEQ ID NO:77 and the light chain with an amino acid sequence as shown in SEQ ID NO:78; or

[0063] The heavy chain with an amino acid sequence as shown in SEQ ID NO:30 and the light chain with an amino acid sequence as shown in SEQ ID NO:31; or

[0064] The heavy chain with an amino acid sequence as shown in SEQ ID NO:44 and the light chain with an amino acid sequence as shown in SEQ ID NO:31.

[0065] In some embodiments, the anti-ROR1 antibody is an antibody fragment; in some embodiments, the antibody fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv and dAb.

[0066] In some embodiments, the first antigen-binding domain and the second antigen-binding domain of the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate bind to different epitopes on human ROR1; in some embodiments, the first antigen-binding domain does not block the second antigen-binding domain from binding to human ROR1; in some embodiments, the first antigen-binding domain binds to amino acids 130-165 of human ROR1, and the second antigen-binding domain binds to amino acids 70-130 of human ROR1, wherein the amino acid sequence of human ROR1 is shown in SEQ ID NO:87.

[0067] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises an Fc region containing a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein each of Fc1 and Fc2 independently has one or more amino acid substitutions that reduce homodimerization of the Fc region. In some embodiments, wherein Fc1 has a knotted structure according to the knife-in-hole technique, and Fc2 has a holed structure according to the knife-in-hole technique.

[0068] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 has a protruding structure according to the mortar and pestle technique, and Fc2 has a porous structure according to the mortar and pestle technique; in some embodiments, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index.

[0069] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate includes an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 comprises the amino acid sequence of SEQ ID NO: 79 and Fc2 comprises the amino acid sequence of SEQ ID NO: 80.

[0070] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises four chains as shown in (a)-(d) below:

[0071] (a) [heavy chain variable region VH1]-[CH1]-[Fc1],

[0072] (b) [Light chain variable region VL1]-[CL1],

[0073] (c)[heavy chain variable region VH2]-[CH1]-[Fc2], and

[0074] (d) [Light chain variable region VL2]-[CL2]; or

[0075] The anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate comprises the four chains shown in (e), (b), (f), and (d) below:

[0076] (e)[heavy chain variable region VH1]-[CH1]-[Fc2],

[0077] (b) [Light chain variable region VL1]-[CL1],

[0078] (f)[heavy chain variable region VH2]-[CH1]-[Fc1], and

[0079] (d)[Light chain variable region VL2]-[CL2];

[0080] In this context, the structures shown in formulas (a), (b), (c), (d), (e), and (f) are arranged from the N-terminus to the C-terminus; CL1 and CL2 are each independently the light chain constant region of the antibody, and their amino acid sequences may be the same or different; CH1 is the first part of the heavy chain constant region of the antibody.

[0081] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate, wherein CH1 is the CH1 sequence of IgG. In some embodiments, CH1 is the CH1 of IgG1. In some embodiments, CH1 comprises the amino acid sequence shown in SEQ ID NO: 88.

[0082] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate, wherein CL1 and CL2 are light chain constant regions of the antibody. In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate, wherein CL1 or CL2 is a light chain constant region of kappa or lambda. In some embodiments, wherein CL1 and / or CL2 comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0083] In some embodiments, the anti-ROR1 antibody or anti-ROR1 antibody drug conjugate comprises the four chains described below:

[0084] Chain 1, which contains the amino acid sequence shown in SEQ ID NO:81;

[0085] Chain 2, which contains the amino acid sequence shown in SEQ ID NO:82;

[0086] Chain 3, comprising the amino acid sequence shown in SEQ ID NO:83; and

[0087] Chain 4, which contains the amino acid sequence shown in SEQ ID NO:84.

[0088] In some embodiments, the anti-ROR1 antibody-drug conjugate has the structure shown in the following formula:

[0089] in:

[0090] Ab represents anti-ROR1 antibody; L represents linker; D represents drug; n represents an integer or decimal from 1 to 10.

[0091] In some embodiments, the L has -L a -L b -L c -L d - structure, where L a Linked to antibodies, L d Related to drugs, including:

[0092] L a Selected from Among them, the wavy line * indicates the connection point with Ab, and * indicates the connection point with L. b The connection point;

[0093] L b Selected from -(CH2)mC(O)-, -NH-(CH2-CH2-O)p-(CH2)sC(O)-, -C(O)-NH-(CH2)qC(O)-, -NH-(CH2)rC(O)- and bonds, wherein:

[0094] m is an integer from 0 to 10 (including 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10), preferably m is 0, 2, 3, or 5; p is an integer from 1 to 8 (including 1, 2, 3, 4, 5, 6, 7, and 8), and s is an integer from 0 to 6 (including 0, 1, 2, 3, 4, 5, and 6); preferably p is 4 and s is 2.

[0095] q is an integer from 1 to 5 (inclusive), preferably q is 2;

[0096] r is an integer from 1 to 5 (inclusive of 1, 2, 3, 4 and 5), preferably r is 2;

[0097] L c Oligopeptides, or L-amino acids, are composed of 1 to 7 amino acids (including 1, 2, 3, 4, 5, 6, and 7). c The amino acid is valine, citrulline, glycine, phenylalanine, alanine, proline, isoleucine, lysine, serine, glutamic acid, and aspartic acid, wherein the amino acid is unsubstituted or independently substituted by one or more substituents, wherein each substituent is independently selected from halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, Halogenated C 1- 6-alkyl, C 1-6 Alkoxy and C 3-7 cycloalkyl;

[0098] L d Selected from -NH-CH2-O-CH2-C(O)-, -NH-R a -CH2-OC(O)- and bonds, where R a It is a phenyl or a 5-6 membered heterocyclic group, wherein the phenyl and the 5-6 membered heterocyclic group are unsubstituted or independently substituted by one or more substituents, wherein each substituent is independently selected from... Halogen, oxo group, hydroxyl group, cyano group, amino group, C 1-6 Alkyl, Halogenated C 1-6 Alkyl and C 1-6 Alkoxy, where # represents the linking site with a phenyl or 5-6 membered heterocyclic group.

[0099] In some embodiments, the L has a structure selected from the group consisting of:

[0100] i)L a for L b For -C(O)- or -C(O)-NH-(CH2)2-C(O)-, L c It is one of the following oligopeptides: -glycine-glycine-phenylalanine-glycine-, -valine-citrulline-, or -glycine-, or L c For key, and L d for -NH-CH2-O-CH2-C(O)- or bond; or

[0101] ii)L a for L b The expression is -(CH2)mC(O)-, where m is 2 or 5, and L c For -valine-citrulline- or -glycine-, and L d for or

[0102] iii)L a for L b For -NH-(CH2-CH2-O)4-(CH2)2-C(O)-, -NH-(CH2)2-C(O)- or bonds, L c It is one of the following oligopeptides: -valine-citrulline-, -glycine-, or -glycine-glycine-phenylalanine-glycine-, or L c For key, and L d for Or -NH-CH2-O-CH2-C(O)-; and

[0103] iiii)L a for L b For -(CH2)3-C(O)-, L c For -valine-citrulline-, and L d for

[0104] Among them, wavy lines * indicates the connection point with Ab, and * indicates the connection point with L. b The connection point, a * Indicates with L c The connection point, b * Indicates the connection point with the drug.

[0105] In some embodiments, the L has the following structure:

[0106] L a for L b For -C(O)-, L c For -valine-citrulline-, and L d for

[0107] In some embodiments, the L has the following structure:

[0108] Where 1 represents the binding point with the anti-ROR1 antibody Ab, and 2 represents the binding point with D (drug).

[0109] In some embodiments, the drug in the anti-ROR1 antibody-drug conjugate is selected from cytotoxic compounds, immunomodulators, enzyme and hormone inhibitors. In some embodiments, the drug in the anti-ROR1 antibody-drug conjugate is a cytotoxic compound.

[0110] In some embodiments, the drug in the anti-ROR1 antibody-drug conjugate is selected from eryribulin, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), exatecan, maytansine and its analogues, SN-38 or combinations thereof.

[0111] In some embodiments, the drug in the anti-ROR1 antibody-drug conjugate is selected from eryribulin, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), exatecan, maytansine, DM1, DM4, SN-38, or combinations thereof.

[0112] In some embodiments, the drug in the anti-ROR1 antibody-drug conjugate is eryribulin.

[0113] In some embodiments, the anti-ROR1 antibody-drug conjugate has the following structure:

[0114] In some embodiments, the Ab comprises a heavy chain as shown in SEQ ID NO:77 and a light chain as shown in SEQ ID NO:78. In some embodiments, the Ab comprises four polypeptide chains as described below:

[0115] Chain 1, comprising the amino acid sequence shown in SEQ ID NO:81; chain 2, comprising the amino acid sequence shown in SEQ ID NO:82; chain 3, comprising the amino acid sequence shown in SEQ ID NO:83; and chain 4, comprising the amino acid sequence shown in SEQ ID NO:84; n is 3.5-4.5.

[0116] In some implementation schemes, the anticancer therapeutic agent is:

[0117] (i) an inhibitor of topoisomerase I or a derivative thereof; or

[0118] (ii) Antibody-drug conjugates containing a topoisomerase I inhibitor or a derivative thereof (i.e., the drug portion of the antibody-drug conjugate is a topoisomerase I inhibitor or a derivative thereof).

[0119] In some embodiments, the topoisomerase I inhibitor includes, but is not limited to, DXd (Exatecan derivative) or a derivative thereof, SN-38 or a derivative thereof, Exatecan or a derivative thereof, Rubitecan or a derivative thereof, Topotecan or a derivative thereof, Irinotecan or a derivative thereof, and Camptothecin or a derivative thereof. In some embodiments, the topoisomerase I inhibitor is DXd or a derivative thereof.

[0120] In some embodiments, the antibody comprising a topoisomerase I inhibitor or a derivative thereof is an anti-tumor-associated antigen antibody. In some embodiments, the antibody is an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0121] The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 128, HCDR2 shown in SEQ ID NO: 129, and HCDR3 shown in SEQ ID NO: 130, and the light chain variable region includes LCDR1 shown in SEQ ID NO: 131, LCDR2 shown in SEQ ID NO: 132, and LCDR3 shown in SEQ ID NO: 133.

[0122] In some embodiments, the anti-HER2 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 134 and a light chain variable region as shown in SEQ ID NO: 135.

[0123] In some embodiments, the anti-HER2 antibody comprises a heavy chain as shown in SEQ ID NO: 136 and a chain as shown in SEQ ID NO: 136.

[0124] The light chain shown in ID NO: 137.

[0125] In some embodiments, the antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof is selected from trastuzumab deruxtecan, trastuzumab rezetecan, JSKN-003 (Anbenitamab repodatecan), IBI-354, TQB-2101, BL-M07D1, BNT-323 (Trastuzumab Pamirtecan), FDA022, GQ1005, DAN-311, T-PL1, PRO1102, and MTX-1000.

[0126] In some embodiments, the antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof is trastuzumab deruxtecan.

[0127] In some implementations, the anticancer therapeutic agent is a HER2 antagonist.

[0128] In some implementations, the HER2 antagonist is an anti-HER2 antibody, an anti-HER2 antibody-drug conjugate, or a small molecule HER2 inhibitor.

[0129] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein:

[0130] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as specified in SEQ ID NO: 134, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as specified in SEQ ID NO: 135. The amino acid sequences of the HCDR and LCDR are determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0131] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein:

[0132] The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 128, HCDR2 shown in SEQ ID NO: 129, and HCDR3 shown in SEQ ID NO: 130, and the light chain variable region includes LCDR1 shown in SEQ ID NO: 131, LCDR2 shown in SEQ ID NO: 132, and LCDR3 shown in SEQ ID NO: 133.

[0133] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region as shown in SEQ ID NO:134 and a light chain variable region as shown in SEQ ID NO:135.

[0134] In some embodiments, the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain as shown in SEQ ID NO:136 and a light chain as shown in SEQ ID NO:137.

[0135] In some implementation schemes, the anticancer therapeutic agent is trastuzumab.

[0136] In some implementations, the anticancer therapeutic agent is an anti-HER2 antibody-drug conjugate.

[0137] In some embodiments, the anticancer therapeutic agent is an anti-HER2 antibody-drug conjugate, wherein the drug is a microtubule inhibitor or a topoisomerase I inhibitor. In some embodiments, the microtubule inhibitor includes, but is not limited to, maytansine compounds (such as DM1 and DM4) and aurestatin derivatives (such as MMAE and MMAF); the topoisomerase I inhibitor includes, but is not limited to, camptothecin compounds (such as esaxatecan, irinotecan, and SN-38), chachiomycin compounds (such as chachiomycin γ1I and N-acetyl-γ1I chachiomycin), and aprotinin derivatives (such as PBD derivatives).

[0138] In some embodiments, the anticancer therapeutic agent is an anti-HER2 antibody-drug conjugate, wherein the drug is selected from exatecan, MMAE, MMAF, maytansine compounds, SN-38, irinotecan, and topotecan. In some embodiments, the anticancer therapeutic agent is an anti-HER2 antibody-drug conjugate, wherein the drug is exatecan.

[0139] In some embodiments, the anti-HER2 antibody-drug conjugate has the following structure:

[0140] Where n is a decimal or integer between 2 and 8, preferably a decimal or integer between 3 and 8, more preferably a decimal or integer between 7 and 8, further preferably a decimal or integer between 7.5 and 8, and most preferably, n is about 8.

[0141] In some embodiments, the anticancer therapeutic agent is an anti-HER2 antibody. In some embodiments, the anti-HER2 antibody is selected from trastuzumab, pertuzumab, zanidatamab, zenocutuzumab, inetetamab, margetuximab, HLX22, IAH0968, BAT1006, B002T, HK001, TrasGEX (timigutuzumab), and FS102. The aforementioned anti-HER2 antibodies include their respective biosimilar products.

[0142] In some implementations, the anticancer therapeutic agent is an anti-HER2 antibody-drug conjugate. In some embodiments, the anti-HER2 antibody-drug conjugate is selected from trastuzumab (DS-8201), vedicitumab, trastuzumab rezetecan, SYD985 (trastuzumab duocarmazine), trastuzumab botidotin, BAT8001, TAA013, MRG002 (trastuzumab vedotin), LCB14-0110, SYA1501, DB-1303, JSKN-003 (Anbenitamab repodatecan), BL-M07D1, TQB2102, GQ1005, IBI354, NCB001 (anvatabart opadotin), MM-302, DX126-262, or combinations thereof. The aforementioned anti-HER2 antibody-drug conjugates include their respective biosimilar products.

[0143] In some embodiments, the anticancer therapeutic agent is a small molecule HER2 inhibitor. In some embodiments, the small molecule HER2 inhibitor is selected from neratinib and lapatinib. Canertinib, zongertinib, and irbinitinib.

[0144] In some embodiments, the anticancer therapeutic agent is trastuzumab deruxtecan (DS-8201), marketed as Urogene. It is an antibody-drug conjugate targeting HER2; the trastuzumab includes its biosimilar products.

[0145] In some implementations, the subject has cancer resistant to anticancer agents, wherein the cancer is selected from: breast cancer, gastric cancer, lung cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, endometrial cancer, pancreatic cancer, cervical cancer, squamous cell carcinoma, small cell lung cancer, gastric / esophageal junction (GEJ) adenocarcinoma, esophageal squamous cell carcinoma, esophageal cancer, bladder cancer, salivary gland cancer, biliary tract cancer, Paget's disease, hepatocellular carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, colon cancer, rectal cancer, glioma, mesothelioma, head and neck cancer, skin cancer, uterine cancer, peritoneal cancer, liver cancer, vulvar cancer, melanoma, laryngeal tumors, pharyngeal tumors, oral tumors, urothelial carcinoma, neuroblastoma, lymphoma, leukemia, sarcoma, plasmacytoma, triple-negative breast cancer, triple-positive breast cancer, HER2-positive breast cancer, hormone receptor-positive breast cancer, and multiple myeloma. In some implementations, the drug resistance is acquired due to treatment with anticancer therapeutic agents.

[0146] In some implementations, the drug resistance is not acquired due to treatment with anticancer agents.

[0147] In some implementations, the drug resistance is acquired due to treatment with an anti-HER2 antagonist.

[0148] In some implementations, the drug resistance is not acquired due to treatment with an anti-HER2 antagonist.

[0149] In some implementations, the drug resistance is acquired due to treatment with anti-HER2 antibodies.

[0150] In some implementations, the drug resistance is acquired due to treatment with the anti-HER2 antibody-drug conjugate.

[0151] In some implementations, the drug resistance is acquired due to treatment with trastuzumab (DS8201).

[0152] In some implementations, the drug resistance is acquired due to treatment with trastuzumab.

[0153] In some embodiments, the drug resistance is acquired due to treatment with an antibody-drug conjugate containing a topoisomerase I inhibitor or a derivative thereof.

[0154] In some embodiments, the drug resistance is acquired due to treatment with a topoisomerase I inhibitor or a derivative thereof.

[0155] In some embodiments, the topoisomerase I inhibitors include, but are not limited to, DXd (Exatecan derivative), SN-38, Exatecan, Rubitecan, Topotecan, Irinotecan, and Camptothecin.

[0156] In some implementations, the drug resistance is not acquired due to treatment with an antibody-drug conjugate containing a topoisomerase I inhibitor or a derivative thereof.

[0157] In some implementations, the resistance is acquired due to treatment with DXd, SN-38, irinotecan, or topotecan.

[0158] In some implementations, the subject has cancer resistant to anticancer treatments, wherein the cancer is a hematologic malignancy.

[0159] In some implementations, the tumor or cancer is a hematologic malignancy, including lymphoma and leukemia.

[0160] In some implementations, the subject has cancer resistant to anticancer agents, wherein the cancer is selected from non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloid hematologic, pre-B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL).

[0161] In some implementations, the subject has cancer that is resistant to anticancer treatments, wherein the cancer is a solid tumor.

[0162] In some implementations, the subject has cancer resistant to anticancer agents, wherein the cancer is selected from: breast cancer, gastric cancer, lung cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, endometrial cancer, pancreatic cancer, cervical cancer, squamous cell carcinoma, small cell lung cancer, gastric / esophageal junction (GEJ) adenocarcinoma, esophageal squamous cell carcinoma, esophageal cancer, bladder cancer, salivary gland cancer, biliary tract cancer, Paget's disease, hepatocellular carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, colon cancer, rectal cancer, glioma, mesothelioma, head and neck cancer, skin cancer, uterine cancer, peritoneal cancer, liver cancer, vulvar cancer, melanoma, laryngeal tumors, pharyngeal tumors, oral tumors, urothelial carcinoma, neuroblastoma, malignant lymphoma, sarcoma, plasmacytoma, triple-negative breast cancer, triple-positive breast cancer, HER2-positive breast cancer, hormone receptor-positive breast cancer, and multiple myeloma.

[0163] In some implementations, the subject has cancer resistant to anticancer treatments, wherein the cancer is selected from lung cancer, breast cancer, stomach cancer, ovarian cancer, endometrial cancer, and prostate cancer.

[0164] In some implementations, the subject has cancer that is resistant to anticancer treatments, wherein the cancer is breast cancer.

[0165] In some implementations, the subject has cancer resistant to anticancer treatments, wherein said cancer is triple-negative breast cancer.

[0166] In some implementations, the subject has cancer resistant to anticancer treatment agents, wherein the cancer is unresectable locally advanced or metastatic (LA / M) triple-negative breast cancer (TNBC).

[0167] In some embodiments, the cancer is HER2-expressing cancer. In some embodiments, the cancer is HER2-overexpressing cancer. In some embodiments, the HER2-overexpressing cancer is cancer in which HER2 expression is determined to be 3+ by immunohistochemistry.

[0168] In some embodiments, the cancer is a cancer with low HER2 expression. In some embodiments, the cancer with low HER2 expression is a cancer in which HER2 expression is 2+ as determined by immunohistochemistry and HER2 expression is negative as determined by in situ hybridization. In some embodiments, the cancer with low HER2 expression is a cancer in which HER2 expression is 1+ as determined by immunohistochemistry.

[0169] In some implementations, the cancer cells of the cancer express ROR1.

[0170] In some embodiments, the subject has cancer containing one or more cells expressing ROR1. In some embodiments, at least about 0.01%, at least about 0.1%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cancer cells express ROR1.

[0171] In some implementations, the aforementioned methods for treating cancer also include administering additional therapeutic agents to the subject.

[0172] In some embodiments, the additional therapeutic agent is selected from one or more of the following: immune checkpoint inhibitors, epidermal growth factor receptor (EGFR) inhibitors, B-cell antigen inhibitors, BTK inhibitors, BCL-2 inhibitors, CDK4 / 6 inhibitors, and chemotherapeutic agents.

[0173] In some embodiments, the additional therapeutic agent is selected from one or more of SIRPα binding antagonists, PD-1 binding antagonists, PD-L1 binding antagonists, CD20 binding antagonists, CD47 binding antagonists, and chemotherapeutic agents.

[0174] In some embodiments, the additional therapeutic agent is selected from anti-SIRPα antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, and anti-CD47 antibody.

[0175] In some embodiments, the additional therapeutic agent is a SIRPα binding antagonist. In some embodiments, the SIRPα binding antagonist is an anti-SIRPα antibody. Anti-SIRPα antibodies known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein.

[0176] In some embodiments, the SIRPα binding antagonist is an anti-SIRPα antibody, including but not limited to BR105, CC-95251, HCB-101, BI765063, GS-0189, IBI397, BI-770371, APX-700, ES-004, ADU1805, ELA-026, and BYON-4228. The aforementioned anti-SIRPα antibodies include their respective biosimilar products.

[0177] In some embodiments, the SIRPα binding antagonist is the anti-SIRPα antibody BR105, which can be prepared with reference to WO2022121980A1.

[0178] Additional anti-SIRPα antibodies that can be used in this method include those described in the following patents: WO200140307, ​​WO2002092784, WO2007133811, WO2009046541, WO2010083253, WO2011076781, WO2013056352, WO2015138600, WO2016179399, WO2016205042, W O2017178653, WO2018026600, WO2018057669, WO2018107058, WO2018190719, WO2018210793, WO2019023347, WO2019042470, WO2019175218, WO2019183266, WO2020013170, WO2020068752 and WO2020088580.

[0179] In some embodiments, the SIRPα binding antagonist is a SIRPα-Fc fusion protein. SIRPα-Fc fusion proteins known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein. In some embodiments, the SIRPα-Fc fusion protein is selected from ALX-148 (evorpacept), timdarpacept, TTI-621, TTI-622, JMT601 (CPO107), SL-172154, SIRPα-F8, JMT601 (CPO107), SS002M91, SIRPα-lgG4-Fc-Fc, and hCD172a (SIRPα)-Fc-LIGHT. Exemplary SIRPα-Fc fusion proteins include ALX-148 (also known as evorpacept, described in WO2013109752), tidapacept, TTI-621, or TTI-622 (described in WO2014094122). The aforementioned SIRPα-Fc fusion proteins comprise their respective biosimilar products.

[0180] In some embodiments, the SIRPα binding antagonist is selected from: BR105, CC-95251, HCB-101, BI765063, GS-0189, IBI397, BI-770371, APX-700, ES-004, ADU1805, ELA-026, BYON-4228, ALX-148 (evorpacept), timdarpacept, TTI-621, TTI-622, JMT601 (CPO107), SL-172154, SIRPα-F8, JMT601 (CPO107), SS002M91, SIRPα-lgG4-Fc-Fc, and hCD172a (SIRPα)-Fc-LIGHT; the above SIRPα binding antagonists include their respective biosimilar products.

[0181] In some embodiments, the PD-1 binding antagonist is an anti-PD-L1 antibody that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a PD-1 binding moiety fused to a constant region (such as the Fc region of an immunoglobulin).

[0182] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. Anti-PD-1 antibodies known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein.

[0183] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 bispecific antibody. Any anti-PD-1 bispecific antibody known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein.

[0184] In some embodiments, the PD-1 binding antagonist is selected from pembrolizumab. MK-3475, SCH900475), zimberelimab (AB122, GLS-010, WBP-3055), nivolumab ( BMS-936558, MDX-1106), cimipril (cemiplimab) Cimiprizumab (rwlc, REGN-2810), pidilizumab (CT-011), AMG-404, MEDI0680 (AMP-514), spartalizumab (PDR001), tislelizumab (BGB-A317), toripalimab (JS-001), genolimzumab (CBT-501, APL-501, GB 226), camrelizumab (SHR-1210), sintilimab (… IBI-308, dostarlimab (TSR-042, WBP-285), lambrolizumab (MK-3475); sasanlimab (PF-06801591), cetrelimab (JNJ-63723283), serplulimab (HLX-10), retifanlimab (MGA-012), balstilimab (AGEN2034), prolgolimab (BCD) 100), budigalimab (ABBV-181), vopratelimab (JTX-4014), retifanlimab, catonilimab, BMS-986213 (Relatlimab + Nivolumab), ivonescimab, geptanolimab, QL-1604 (iparomlimab), pucotetenlimab, AK-105, CS-1003, BI-754091, LZM-009, Sym-021, BA T-1306, PD-1-PIK, tebotelimab (MGD013; PD-1 / LAG-3), RO-7247669 (PD-1 / LAG-3), FS-118 (LAG-3 / PD-L1), RO-7121661 (PD-1 / TIM-3), RG7769 (PD-1 / TIM-3), PF-06936308 (PD-1 / CTLA4), MGD-019 (PD-1 / CTLA4), KN-046 (PD-1 / CTLA4), XmAb-20717 (PD-1 / CTLA4), AK-104 (CTLA4 / PD-1), and MEDI-5752 (CTLA4 / PD-1). The above PD-1 binding antagonists include their respective biosimilar products.

[0185] In some implementations, the PD-1 binding antagonist is pembrolizumab.

[0186] In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody that inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a PD-L1 binding moiety fused to a constant region (e.g., the Fc region of an immunoglobulin)).

[0187] In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. Anti-PD-L1 antibodies known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein.

[0188] In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 bispecific antibody. Any anti-PD-L1 bispecific antibody known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein.

[0189] In some embodiments, the PD-L1 binding antagonist is selected from: avelumab ( MSB0010718C, atezolizumab Envafolimab (ASC22) and Durvalumab (ASC22) MEDI-4736, adebrelimab, BMS-936559 (MDX1105), cosibelimab (CK-301), lodapolimab (LY 3300054), garivulimab (BGB A333), envafolimab (KN035), opucolimab (HLX 20), manelimab (BCD 135), CX-072, CBT-502 (TQB2450), MSB-2311, sugemalimab (CS-1001; WBP3155), A167 (KL-A167, HBM) The list includes PD-L1 binding antagonists such as 9167, STI-A1015 (IMC-001), FAZ-053, BMS-936559 (MDX1105), INCB086550, GEN-1046 (PD-L1 / 4-1BB), FPT-155 (CTLA4 / PD-L1 / CD28), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM-3 / PDL1), INBRX-105 (4-1BB / PDL1), and GNS-1480 (PD-L1 / EGFR). These PD-L1 binding antagonists include their respective biosimilar products.

[0190] In some implementations, the PD-L1 binding antagonist is atezolizumab.

[0191] In some implementations, the PD-L1 binding antagonist is avelumab.

[0192] In some embodiments, the CD47 binding antagonist is an anti-CD47 antibody that inhibits the binding of SIRPα to CD47. In some embodiments, the CD47 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a CD47 binding portion fused to a constant region (e.g., the Fc region of an immunoglobulin).

[0193] In some embodiments, the CD47 binding antagonist is an anti-CD47 antibody. Anti-CD47 antibodies known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein. In some embodiments, the anti-CD47 antibody includes, but is not limited to, IBI-188 (letaplimab), TJC-4 (lemzoparlimab), SHR-1603, HLX-24, LQ-001, IMC-002, ZL-1201, B6H12, GenSci-059 (gentulizumab), TAY-018, PT-240, 1F8-GMCSF, SY-102, and KD-015.

[0194] Other anti-CD47 antibodies used in this method include antibodies described in the following patents: WO199727873, WO199940940, WO2002092784, WO2005044857, WO2009046541, WO2010070047, WO2011143624, WO2012170250, WO2013109752, WO2013119714, WO2014087248, WO201 5191861, WO2016022971, WO2016023040, WO2016024021, WO2016081423, WO2016109415, WO2016141328, WO 2016188449, WO2017027422, WO2017049251, WO2017053423, WO2017121771, WO2017194634, WO2017196793 , WO2017215585, WO2018075857, WO2018075960, WO2018089508, WO2018095428, WO2018137705, WO2018233 575. WO2019027903, WO2019034895, WO2019042119, WO2019042285, WO2019042470, WO2019086573, WO2019 108733, WO2019138367, WO2019144895, WO2019157843, WO2019179366, WO2019184912, WO2019185717, WO2019201236, WO2019238012, WO2019241732, WO2020019135, WO2020036977, WO2020043188 and WO2020009725.

[0195] In some embodiments, the CD47 binding antagonist is an anti-CD47 bispecific antibody. Any anti-CD47 bispecific antibody known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein. Examples of bispecific antibodies targeting CD47 include, but are not limited to, seralimumab (IBI-322) (CD47 / PD-L1), IMM-0306 (CD47 / CD20), TJ-L1C4 (CD47 / PD-L1), HX-009 (CD47 / PD-1), PMC-122 (CD47 / PD-L1), PT-217 (CD47 / DLL3), IMM-26011 (CD47 / FLT3), IMM-0207 (CD47 / VEGF), IMM-2902 (CD47 / HER2), BH29xx (CD47 / PD-L1), IMM-03 (CD47 / CD20), IMM-2502 (CD47 / PD-L1), HMBD-004B (CD47 / BCMA), and HMBD-004A (CD47 / CD33).

[0196] In some embodiments, the CD47 binding antagonist is selected from: magrolimab, lemzoparlimab, letaplimab, ligufalimab, AO-176, simridarlimab (IBI-322), gentulizumab, ZL-1201, IMC-002, SRF-231, CC-90002 (also known as INBRX-103), NI-1701 (also known as TG-1801), STI-6643, SHR-1603, HLX-24, LQ-001, B6H12, TAY-018, PT-240, 1F8-GMCSF, SY-102, and KD-015; the above CD47 binding antagonists include their respective biosimilar products.

[0197] In some embodiments, the CD20 binding antagonist is an anti-CD20 antibody. In some embodiments, the CD20 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a CD20 binding portion fused to a constant region (e.g., the Fc region of an immunoglobulin).

[0198] In some embodiments, the CD20 binding antagonist is an anti-CD20 antibody. Anti-CD20 antibodies known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein.

[0199] In some embodiments, the CD20 binding antagonist is an anti-CD20 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 118, HCDR2 shown in SEQ ID NO: 119, and HCDR3 shown in SEQ ID NO: 120, and the light chain variable region comprises LCDR1 shown in SEQ ID NO: 121, LCDR2 shown in SEQ ID NO: 122, and LCDR3 shown in SEQ ID NO: 123.

[0200] In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region as shown in SEQ ID NO:124 and a light chain variable region as shown in SEQ ID NO:125.

[0201] In some embodiments, the anti-CD20 antibody comprises a heavy chain as shown in SEQ ID NO:126 and a light chain as shown in SEQ ID NO:127.

[0202] In some embodiments, the CD20 binding antagonist is an anti-CD20 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 139, HCDR2 shown in SEQ ID NO: 140, and HCDR3 shown in SEQ ID NO: 141, and the light chain variable region comprises LCDR1 shown in SEQ ID NO: 142, LCDR2 shown in SEQ ID NO: 143, and LCDR3 shown in SEQ ID NO: 144.

[0203] In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region as shown in SEQ ID NO:145 and a light chain variable region as shown in SEQ ID NO:146.

[0204] In some embodiments, the anti-CD20 antibody comprises a heavy chain as shown in SEQ ID NO:147 and a light chain as shown in SEQ ID NO:148.

[0205] In some embodiments, the CD20 binding antagonist is selected from zuberitamab, obinutuzumab, ibritumomab, ofatumumab, tositumomab, ocrelizumab, ubliximab, and rituximab. The aforementioned anti-CD20 antibodies include their respective biosimilar products.

[0206] In some implementations, the anti-CD20 antibody is zabetulimab.

[0207] In some implementations, the anti-CD20 antibody is rituximab.

[0208] In some embodiments, the additional therapeutic agent is a chemotherapy drug. Chemotherapy drugs known in the art can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein.

[0209] In some implementation schemes, the chemotherapeutic drugs are selected from platinum-based antitumor drugs, camptothecin-based antitumor drugs, taxane-based antitumor drugs, nitrogen mustard-based antitumor drugs, antimetabolites, anthracyclines, vinblastine-based antitumor drugs, podophyllotoxin-based antitumor drugs, hormone-based antitumor drugs, and combinations thereof.

[0210] In some embodiments, the hormonal antitumor drug is a glucocorticoid antitumor drug. In some embodiments, the chemotherapeutic drug is selected from platinum-based antitumor drugs (including but not limited to oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycloplatin, miplatin, lobaplatin, and picoplatin), camptothecin-based antitumor drugs (including but not limited to camptothecin, hydroxycamptothecin, aminocamptothecin, irinotecan, topotecan, ethanotecan, rubitecan, lurtotecan, gemmatinecan, and karenitecin), taxane-based antitumor drugs (including but not limited to paclitaxel, paclitaxel liposomes, albumin-bound paclitaxel, and docetaxel), nitrogen mustard-based antitumor drugs (including but not limited to cyclophosphamide, ifosfamide, chlorambucil, melphalan, and bendamustine), and antimetabolites. Oncology drugs (including but not limited to fluorouracil (including but not limited to 5-fluorouracil, tegafur, capecitabine, tegafur), methotrexate, cytarabine, gemcitabine, fludarabine, azacitidine), anthracycline antitumor drugs (including but not limited to doxorubicin, epirubicin, pirarubicin, amorubicin, arubicin, idarubicin, daunorubicin, mitoxantrone), vinblastine alkaloids (including but not limited to vinblastine, vincristine, vindesine, and vinorelbine), podophyllodoalkaloids (including but not limited to etoposide and teniposide), hormonal antitumor drugs (including but not limited to glucocorticoids such as prednisone, prednisolone, dexamethasone, methylprednisolone sodium succinate), procarbazine, hexamethylpyrimethamine, and dacarbazine, one or more of these.

[0211] In some embodiments, the chemotherapeutic agent is a platinum-based antitumor drug, including but not limited to oxaliplatin, cisplatin, carboplatin, nedaplatin, bicycloplatin, miplatin, lobaplatin, and picoplatin. In some embodiments, the chemotherapeutic agent is carboplatin or cisplatin.

[0212] In some embodiments, the chemotherapeutic agent is a podophyllodes alkaloid antitumor drug, including but not limited to etoposide and teniposide. In some embodiments, the chemotherapeutic agent is etoposide.

[0213] In some embodiments, the chemotherapeutic agent is a nitrogen mustard antitumor drug. In some embodiments, the chemotherapeutic agent is cyclophosphamide.

[0214] In some embodiments, the chemotherapeutic agent is an anthracycline antitumor drug. In some embodiments, the chemotherapeutic agent is doxorubicin.

[0215] In some embodiments, the chemotherapy drug is a hormonal antitumor drug. In some embodiments, the chemotherapy drug is a glucocorticoid antitumor drug. In some embodiments, the chemotherapy drug is prednisone or prednisolone.

[0216] In some embodiments, the chemotherapy drug is a taxane-based antitumor drug. In some embodiments, the chemotherapy drug is paclitaxel.

[0217] In some embodiments, the chemotherapy drug is an antimetabolite antitumor drug. In some embodiments, the chemotherapy drug is gemcitabine.

[0218] In some embodiments, the chemotherapy drug is a vincristine antitumor drug. In some embodiments, the chemotherapy drug is vincristine, vinorelbine, vindesine, or vinorelbine. In some embodiments, the chemotherapy drug is vincristine.

[0219] In some embodiments, the chemotherapeutic agent is selected from nitrogen mustard antitumor drugs, anthracycline antitumor drugs, and hormonal antitumor drugs. In some embodiments, the chemotherapeutic agent is selected from nitrogen mustard antitumor drugs, anthracycline antitumor drugs, and glucocorticoid antitumor drugs. In some embodiments, the chemotherapeutic agent is selected from cyclophosphamide, doxorubicin, and prednisone / prednisolone.

[0220] In some embodiments, the chemotherapeutic agent is selected from nitrogen mustard antitumor drugs, anthracycline antitumor drugs, vincristine antitumor drugs, and hormonal antitumor drugs. In some embodiments, the chemotherapeutic agent is selected from nitrogen mustard antitumor drugs, anthracycline antitumor drugs, vincristine antitumor drugs, and glucocorticoid antitumor drugs. In some embodiments, the chemotherapeutic agent is selected from cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0221] In some implementations, the chemotherapeutic agent is selected from paclitaxel, albumin-bound paclitaxel, vincristine, vinorelbine tartrate, gemcitabine, etoposide, azacitidine, palbociclib, cyclophosphamide, doxorubicin, doxorubicin, cisplatin, carboplatin, prednisone, prednisolone, irinotecan, topotecan, esaxatecan, rubitecan, and combinations thereof.

[0222] In some implementations, the additional therapeutic agent is a BTK inhibitor.

[0223] In some embodiments, the BTK inhibitor is ibrutinib (1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one). In another embodiment, the BTK inhibitor is Roche BTKi RN486. In yet another embodiment, the BTK inhibitor is acalatinib (4-[8-amino-3-[(2S)-1-but-2-ynylpyrrolidine-2-yl]imidazo[1,5-a]pyrazin-1-yl]-N-pyridin-2-ylbenzamide). In yet another embodiment, the BTK inhibitor is zanubrutinib (S)-7-(1-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidin-3-carboxamide). Other BTK inhibitors that can be used in combination with the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate disclosed herein are CT-1530, DTRMWXHS-12, and spebrutinib benzyl sulfonate. besylate), vecabrutinib, evobrutinib, tirabrutinib, fenebrutinib, poseltinib, BMS-986142, ARQ531, LOU-064, PRN-1008, ABBV-599, AC-058, BIIB-068, BMS-986l95, HWH-486, PRN-2246, TAK-020, GDC-0834, BMX-IN-l, RN486, SNS-062, LFM-A13, and PCI-32765.

[0224] In some embodiments, the BTK inhibitor is selected from ibrutinib, BTKi RN486, netabrutinib, acalabrutinib, zanubrutinib, and orelabrutinib.

[0225] In some implementations, the additional therapeutic agent is a BCL-2 inhibitor.

[0226] In some implementations, the BCL-2 inhibitor is veneclax (ABT-199), ABT-737 (Oltersdorf, T. et al. (2005) Nature 435:677-681), navitoclax / ABT-263 (Tse, C. et al. (2008) Cancer Res. 68:3421-3428), BM-1197 (Bai, L. et al. (2014) PLoS ONE 9:e99404), S44563 (Nemati, F. et al. (2014) PLoS ONE 9:e80836), BCL2-32 (Adam, A. et al. (2014) Blood 124:5304), AZD4320 (Hennessy, EJ et al. (2015) ACS Medicinal Chemistry annual meeting). https: / / www.acsmedchem.org / ama / orig / abstracts / mediabstractf 2015.pdf_abstr.24) and S55746 (International Standard Randomized Controlled Trial Number Registry.ISRCTN http: / / www.isrctn.com / ISRCTN04804337(2016)).

[0227] In some embodiments, the BCL-2 inhibitor is selected from Veneclare and Navecilla.

[0228] In some implementations, the BCL-2 inhibitor is Veneclare.

[0229] In some implementations, the additional therapeutic agent is a CDK4 / 6 inhibitor.

[0230] In some embodiments, the CDK4 / 6 inhibitor is selected from: palbociclib, abecilib, ribociclib, trelaciclib, lerocilib, avozidil, ronicillin, pyrosinib, trelaciclib, riviciclib, milkiclib, RGB-286638, NSN3106729, PHA-793887, R547, indirubin, NU6102, bohemine, CDK9-IN-7, CGP60474, purvalanol A, PF-06873600, neem lactone, FN-1501, AG-024322, ON123300, G1T28, G1T38, AMG925, SHR-6390, BPI-1178, BPI-16350, FCN437, BEBT-209, Ty-302, TQB-3616, HS-10342, PF-06842874, CS-2002, MM-D37K, CDK4 / 6-IN-2, SU9516 and AT7519.

[0231] In some implementations, the CDK4 / 6 inhibitor is selected from palbociclib, abeciclib, ribociclib, trelaciclib, lerocilib, avozidil, ronizil, piroxicillin, and trelaciclib.

[0232] In some implementations, the additional therapeutic agent is selected from a combination of anti-CD20 antibodies and chemotherapeutic agents.

[0233] In some embodiments, the additional therapeutic agents are selected from anti-CD20 antibodies, nitrogen mustard antitumor drugs, anthracycline antitumor drugs, and hormonal antitumor drugs. In some embodiments, the additional therapeutic agents are selected from anti-CD20 antibodies, nitrogen mustard antitumor drugs, anthracycline antitumor drugs, and glucocorticoid antitumor drugs. In some embodiments, the chemotherapeutic agents are selected from zabetuzumab or rituximab, cyclophosphamide, doxorubicin, and prednisone / prednisolone.

[0234] In some embodiments, the additional therapeutic agent is selected from anti-CD20 antibodies, nitrogen mustard antitumor drugs, anthracycline antitumor drugs, vinblastine alkaloid antitumor drugs, and corticosteroid antitumor drugs. In some embodiments, the additional therapeutic agent is selected from anti-CD20 antibodies, nitrogen mustard antitumor drugs, anthracycline antitumor drugs, vinblastine alkaloid antitumor drugs, and glucocorticoid antitumor drugs. In some embodiments, the chemotherapy drug is selected from zabetumumab or rituximab, cyclophosphamide, doxorubicin, cyclophosphamide, and prednisone / prednisolone. In some embodiments, the additional therapeutic agent is selected from a combination of zabetumumab or rituximab and a chemotherapy drug.

[0235] In some embodiments, the additional therapeutic agent is a combination of zabetuzumab and a glucocorticoid antitumor drug. In some embodiments, the additional therapeutic agent is a combination of rituximab and a glucocorticoid antitumor drug.

[0236] In some embodiments, the additional therapeutic agent is a combination of zabetuzumab and cyclophosphamide; in some embodiments, the additional therapeutic agent is a combination of zabetuzumab and doxorubicin; in some embodiments, the additional therapeutic agent is a combination of zabetuzumab and prednisone / prednisolone.

[0237] In some embodiments, the additional therapeutic agent is a combination of anti-CD20 antibody, cyclophosphamide, doxorubicin, and prednisone or their equivalents; in some embodiments, the additional therapeutic agent is a combination of zabetutumab, cyclophosphamide, doxorubicin, and prednisone / prednisolone; in some embodiments, the additional therapeutic agent is a combination of rituximab, cyclophosphamide, doxorubicin, and prednisone / prednisolone.

[0238] In some embodiments, the additional therapeutic agent is a combination of anti-CD20 antibody, cyclophosphamide, doxorubicin, vincristine, and prednisone or their equivalents; in some embodiments, the additional therapeutic agent is a combination of zabetutumab, cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; in some embodiments, the additional therapeutic agent is a combination of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone.

[0239] In some implementations, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate and an additional therapeutic agent are administered sequentially.

[0240] In some implementations, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate is administered simultaneously with an additional therapeutic agent.

[0241] In some implementations, the anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate is administered separately from the additional therapeutic agent.

[0242] In another aspect, this disclosure provides anti-ROR1 antibodies or anti-ROR1 antibody-drug conjugates as agents for treating cancers resistant to anticancer therapies.

[0243] In a third aspect, this disclosure provides the use of anti-ROR1 antibodies or anti-ROR1 antibody-drug conjugates in the preparation of pharmaceutical agents for treating cancers resistant to anticancer therapeutics. In some embodiments, the anticancer therapeutic agent is a HER2 antagonist. In some embodiments, the anticancer therapeutic agent is an anti-HER2 antibody, an anti-HER2 antibody-drug conjugate, or a small molecule HER2 inhibitor. In some embodiments, the anticancer therapeutic agent is a topoisomerase I inhibitor. In some embodiments, the anticancer therapeutic agent is an antibody-drug conjugate containing a topoisomerase I inhibitor. In some embodiments, the topoisomerase I inhibitor is DXd. In some embodiments, the antibody containing the topoisomerase I inhibitor targets a tumor-associated antigen. In some embodiments, the antibody containing the topoisomerase I inhibitor is an anti-HER2 antibody.

[0244] This disclosure provides a kit for treating drug-resistant cancers. The kit may comprise (a) a container containing an antibody or antibody-drug conjugate and optionally one or more containers containing additional therapeutic agents (such as anti-SIRPα antibodies) and optional additional chemotherapeutic agents. If desired, the kit may further comprise one or more of a variety of conventional pharmaceutical kit components, such as containers having one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to a person skilled in the art. The kit may also include printed instructions as inserts or labels indicating the amount of components to be administered, administration guidelines, and / or guidelines for mixing components.

[0245] It should be understood that, within the scope of this disclosure, the various technical features described above and those specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0246] Figure 1 shows the tumor-suppressive activity of ADC in the HCC1187 breast cancer model of female SCID mice.

[0247] Figure 2 shows the tumor suppressor activity of ADC in the Jeko-1 mantle cell lymphoma model in female SCID mice.

[0248] Figure 3 shows the tumor-suppressive activity of ADC in a mouse model transplanted with the PA-1 ovarian cancer cell line.

[0249] Figure 4A shows the phagocytic effect of combined administration of ADC-3 and anti-SIRPα antibody 14# on gastric cancer cells SNU-601; Figure 4B shows the phagocytic effect of combined administration of ADC-3 and anti-SIRPα antibody 14# on endometrial cancer cells RL95-2; Figure 4C shows the phagocytic effect of combined administration of ADC-3 and anti-SIRPα antibody 14# on non-small cell lung cancer HCC827; where * indicates a statistically significant difference between the combined administration group and the ADC-3 monotherapy group, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0250] Figure 5A shows the killing effect of combined administration of ADC-3 and pembrolizumab on human breast cancer cells MDA-MB-468; Figure 5B shows the killing effect of combined administration of ADC-3 and avelumab on human breast cancer cells MDA-MB-468.

[0251] Figure 6 shows the killing effect of combined administration of ADC-3 and avelumab on human gastric cancer cells SNU-601.

[0252] Figure 7 shows the killing effect of combined administration of ADC-3 and vincristine on breast cancer cells MDA-MB-468.

[0253] Figure 8A shows the killing effect of ADC-3 and vincristine combined on diffuse large B-cell lymphoma cells Ri-1; Figure 8B shows the killing effect of ADC-3 and cisplatin combined on mantle cell lymphoma cells Jeko-1; Figure 8C shows the killing effect of ADC-3 and etoposide combined on mantle cell lymphoma cells Jeko-1.

[0254] Figure 9 shows the killing effect of combined administration of the ADC-3 and BCL-2 inhibitor Veneclare on Ri-1 cells of diffuse large B-cell lymphoma in breast cancer.

[0255] Figure 10 shows the in vitro killing effect of ADC-3, zabetoin, and pegphosphamide combined on NU-DUL-1 diffuse large B-cell lymphoma cells.

[0256] Figure 11 shows the in vitro killing effect of ADC-3, zabetoumab, and doxorubicin combined administration on NU-DUL-1 diffuse large B-cell lymphoma cells.

[0257] Figure 12 shows the in vitro killing effect of ADC-3, zabetoumab, and prednisolone combined on NU-DUL-1 diffuse large B-cell lymphoma cells.

[0258] Figure 13 shows the in vitro killing effect of ADC-3 and gemcitabine combined administration on human breast cancer cells MDA-MB-468.

[0259] Figure 14 shows the in vitro killing effect of ADC-3 and paclitaxel combined administration on human breast cancer cells MDA-MB-468.

[0260] Figure 15 shows the in vitro killing effect of ADC-3 and Hi-CHP / R-CHP combined administration on NU-DUL-1 diffuse large B-cell lymphoma cells.

[0261] Figure 16 shows the in vitro killing effect of ADC-3 and Hi-CHOP / R-CHOP combined administration on NU-DUL-1 diffuse large B-cell lymphoma cells.

[0262] Figure 17 shows the in vitro proliferation inhibition of trastuzumab on NCI-H2170 and NCI-H2170 / Enhertu-R resistant cells at a resistance concentration of 5.23 nM.

[0263] Figure 18 shows the in vitro proliferation inhibition of trastuzumab on NCI-H2170 and NCI-H2170 / Enhertu-R resistant cells at a resistance concentration of 5.23 nM.

[0264] Figure 19 shows the in vitro proliferation inhibition effect of Dxd on NCI-H2170 and NCI-H2170 / Enhertu-R resistant cells at a drug resistance concentration of 5.23 nM.

[0265] Figure 20 shows the in vitro proliferation inhibition of trastuzumab on NCI-H2170 and NCI-H2170 / Enhertu-R resistant cells at a resistance concentration of 13.1 nM.

[0266] Figure 21 shows the in vitro proliferation inhibition effect of Dxd on NCI-H2170 and NCI-H2170 / Enhertu-R resistant cells at a drug resistance concentration of 13.1 nM.

[0267] Figure 22 shows the in vitro proliferation inhibition of trastuzumab on NCI-H2170 and NCI-H2170 / Enhertu-R resistant cells at a resistance concentration of 6.54 nM.

[0268] Figure 23 shows the in vitro proliferation inhibition effect of Dxd on NCI-H2170 and NCI-H2170 / Enhertu-R resistant cells at a drug resistance concentration of 6.54 nM.

[0269] Figure 24 shows the in vitro proliferation inhibition of ADC-3 and detrastuzumab on NCI-H2170 / Enhertu-R resistant cells at a resistance concentration of 6.54 nM.

[0270] Figure 25 shows the in vitro proliferation inhibition of ADC-3 and detrastuzumab on NCI-H2170 / Enhertu-R resistant cells at a concentration of 13.1 nM.

[0271] Figure 26A shows the in vitro inhibitory effect of trastuzumab on the proliferation of BT474 and BT474 / Enhertu-R resistant cells at a resistance concentration of 0.52 nM. Figure 26B shows the in vitro inhibitory effect of Dxd on the proliferation of BT474 and BT474 / Enhertu-R resistant cells at a resistance concentration of 0.52 nM.

[0272] Figure 27 shows the in vitro proliferation inhibition of ADC-3 and detrastuzumab on BT474 / Enhertu-R resistant cells at a concentration of 0.2 nM.

[0273] Figure 28A shows the inhibitory effect of trastuzumab on the in vitro proliferation of HCC1954 and HCC1954 / Enhertu-R resistant cells. Figure 28B shows the inhibitory effect of DXd on the in vitro proliferation of HCC1954 and HCC1954 / Enhertu-R cells.

[0274] Figure 29 shows the in vitro proliferation inhibition of ADC-3 and trastuzumab on HCC1954 / Enhertu-R resistant cells. Detailed Implementation

[0275] Through extensive and in-depth research, this disclosure discloses the design and construction of a series of anti-ROR1 antibodies with novel CDR sequences that specifically bind to two epitopes of human ROR1 with high affinity. These anti-ROR1 antibodies specifically bind to epitopes located at amino acid positions 130-165 and 70-130 of the human ROR1 protein. Furthermore, this disclosure also discloses the construction of anti-ROR1 antibody-drug conjugates. In vitro and in vivo experiments show that the antibody-drug conjugates disclosed herein exhibit significant tumor-suppressive effects against cell lines derived from various cancers or tumors (e.g., breast cancer, ovarian cancer, and lymphocytic carcinoma) and xenograft mouse models, outperforming positive controls. This indicates that the antibody-drug conjugates disclosed herein can serve as drugs for the treatment of various solid tumors and hematological malignancies, and can be used for tumor or cancer therapy. Furthermore, this disclosure reveals that the anti-ROR1 antibody or its drug conjugate disclosed herein, when used in combination with another therapeutic agent, particularly anti-SIRPα antibody, anti-PD-1 antibody, anti-PD-L1 antibody, and chemotherapy drugs, exhibits an additive or synergistic effect, resulting in more efficient killing of tumor cells. Moreover, the inventors of this disclosure have discovered that, even in cancers resistant to HER2 antagonists or topoisomerase I inhibitors, the anti-ROR1 antibody or its drug conjugate disclosed herein can still inhibit the growth of drug-resistant cancer cells, demonstrating promising application prospects.

[0276] Based on this, this disclosure has been completed.

[0277] the term

[0278] To better understand this disclosure, the following terms are defined.

[0279] Unless otherwise stated, all singular terms also include the plural, active, and past tense forms of the terms.

[0280] Unless the context clearly indicates otherwise, the term “about” includes values ​​within the standard deviation range of the stated values.

[0281] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.” For example, for HCDR1 containing the amino acid sequence of SEQ ID NO: 45, it explicitly covers the amino acid sequence of HCDR1 as shown in SEQ ID NO: 45.

[0282] According to this disclosure, “subject” or “patient” refers to animals, including human patients in need of cancer treatment or therapy. In some respects, this disclosure may also be applied in veterinary practice to any mammal or other animal in need of such ROR1-targeted cancer therapy. This may include, for example, non-human primates, dogs, felines, pigs, horses, and any other animals receiving ROR1-targeted cancer therapy.

[0283] The term "drug resistance" is used to indicate a lack of response to a therapeutic agent. It can also be expressed as "non-responsive" or "unresponsive." The term "drug resistance" can mean "a tumor or cancer that has acquired resistance due to treatment with a therapeutic agent (such as an anticancer agent)" or it can mean "resistance inherent in a tumor or cancer independent of treatment with a therapeutic agent (such as an anticancer agent)." Tumors resistant to therapeutic agents (such as anticancer agents) include tumors or cancers that do not respond to anticancer agent treatment and / or have a reduced ability to produce a significant response to anticancer agent treatment (e.g., partial response and / or complete response).

[0284] The determination or assessment of "drug resistance" is known in the art and is described in the examples. The acquisition of drug resistance can be assessed by measuring the growth of drug-tolerant persisters as described in Example 19. In some embodiments, it can be assessed via IC50. 50 EC 50 Changes in resistance can indicate drug resistance. In some embodiments, the change is greater than any one of about 50%, 100%, and / or 200%. Furthermore, changes in the acquisition of resistance and / or maintenance of sensitivity can be assessed in vivo, for example, by assessing the response to anticancer drug treatment, duration of response, and / or time to progression, such as partial and complete responses. Changes in the acquisition of resistance and / or maintenance of sensitivity can be based on changes in the response to anticancer drug treatment, duration of response, and / or time to progression in a population of individuals, such as the number of partial and complete responses.

[0285] The term "HER2 antagonist resistance" refers to a lack of response to treatment with HER2 antagonists.

[0286] The terms “HER2 receptor antagonist” and “HER2 antagonist” refer to compounds that inhibit the expression or function of the HER2 protein or gene. For the purposes of this disclosure, HER2 antagonists refer to receptor tyrosine kinase inhibitors, particularly HER2 receptor protein inhibitors, examples of which include anti-HER2 antibodies, anti-HER2 antibody-drug conjugates, and small molecule inhibitors that inhibit HER2 activity.

[0287] "Anti-HER2 antibody" or "HER2 antibody" is an antibody that binds to the HER2 receptor. Optionally, the HER2 antibody further interferes with the activation or function of HER2. Various anti-HER2 antibodies are known in the art, and preferably, such antibodies are monoclonal antibodies; they can be so-called chimeric antibodies, humanized antibodies, or fully human antibodies; they can be full-length anti-HER2 antibodies, anti-HER2 antibody fragments with the same biological activity, including amino acid sequence variants and / or glycosylated variants of such antibodies or fragments. Known examples of humanized anti-HER2 antibodies include trastuzumab and pertuzumab.

[0288] Suitable anti-HER2 antibody-drug conjugates (ADCs) include trastuzumab (DS-8201), an ADC composed of huMAb4D5-8 (HERCEPIN™, i.e., trastuzumab) and ezetidine, which is approved for the treatment of metastatic breast cancer. Other suitable anti-HER2 ADCs include T-DM1 (trastuzumab emtansine), A166 (trastuzumab botidotin), RC48-ADC, and SHR-A1811.

[0289] The terms “trastuzumab,” “pertuzumab,” “T-DM1,” and “DS8201” encompass all corresponding anti-HER2 antibodies that meet the requirements necessary to obtain marketing authorization as identical or biosimilar products in countries or territories selected from the United States, Europe, and Japan. Trastuzumab has the CDR region defined in U.S. Patent 5,821,337. Pertuzumab has the CDR region defined in WO 01 / 00245.

[0290] The term "antibody" is used in the broadest sense and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. For example, a natural IgG antibody is a heterotetraglycosyl protein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL). The light chains are either κ or λ. The heavy chain is divided into γ, μ, α, δ, or ε, and antibody isotypes are defined as IgG, IgM, IgA, IgD, and IgE, respectively. In both the light and heavy chains, the variable and constant regions are linked by a “J” region of approximately 12 or more amino acids, and the heavy chain also includes a “D” region of approximately 10 or more amino acids. (Generally, see *Basic Immunology*, Paul W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7; all are incorporated herein by reference for various purposes.)

[0291] The term "antibody fragment" refers to a molecule that is distinct from the complete antibody but contains a portion of the complete antibody that retains the antigen-binding ability of the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), biantibodies, linear antibodies, and single-chain antibody molecules (e.g., scFv). The fragments are: (i) Fab fragments having VL, CL, VH, and CH1 domains and a disulfide bond between the heavy and light chains; (ii) Fab' fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments having VL and VH domains of one arm of the antibody; (vi) dAb fragments consisting of a VH domain; (vii) hingeless antibodies containing at least VL, VH, CL, and CH1 domains and lacking a hinge region; (vii) i) F(ab)2 fragment, which is a bivalent fragment containing two Fab' fragments connected by a disulfide bridge in the hinge region; (ix) single-chain antibody molecule (e.g., single-chain Fv; scFv); (x) "diabodies" having two antigen-binding sites, including a heavy chain variable domain (VH) and a light chain variable domain (VL) connected to each other in the same polypeptide chain; (xi) "linear antibody" containing a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions; (xii) dsFv refers to a fragment formed by replacing one amino acid residue in each of VH and VL with a cysteine ​​residue via an SS bond between the cysteine ​​residues.

[0292] Monoclonal antibodies typically require isolation and purification. This means that the purity of interfering proteins and other contaminants generated during antibody production or purification is usually at least 50%, but the possibility of the monoclonal antibody binding to excess drug-acceptable carriers or other carriers intended to facilitate its use cannot be ruled out. Sometimes, the monoclonal antibody is at least 60%, 70%, 80%, 90%, 95%, or 99% w / w purity of the interfering proteins and contaminants produced or purified.

[0293] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies; based on the number of antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or more bispecific antibodies; and based on whether the structure is symmetrical, they can be classified into symmetrical and asymmetrical bispecific antibodies. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. These antibodies have low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively large molecular weight. The Fc fragment helps in antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, and 1Fab-IgG. TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb , IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).

[0294] The specific binding of an antibody to its target antigen means that it has at least 10 6 10 7 10 8 10 9 ,or 10 10 M -1The affinity of a monoclonal antibody for a target is higher. Specific binding is detectable on a higher order of magnitude, unlike nonspecific binding, which binds to at least one unrelated target. Specific binding may result from the formation of bonds between specific functional groups or specific spatial matches (e.g., lock and key types), while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that a monoclonal antibody binds to one and only one target.

[0295] The term "variable region" or "variable domain" refers to the domain in an antigen-binding molecule that binds to the antigen. Both VH and VL contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. The sequence from N-terminus to C-terminus for each VH and VL is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0296] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering system, the "ABM" numbering system, the "contact" numbering system (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering system (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77(2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art. Exemplary numbering rules disclosed herein are shown in Table 1 below.

[0297] Table 1. Relationship between CDR numbering systems

[0298] Those skilled in the art will understand that even under a specific numbering rule, the position number of the CDR for a particular antibody may differ by a few amino acids. For example, under the Kabat rule, some antibodies have LCDR1 at positions 24-33 and HCDR1 at positions 98-106. Unless otherwise stated, the variable regions and CDR sequences in the embodiments disclosed herein are governed by the "Kabat" numbering rule. Although a numbering system (such as Kabat) is used to define amino acid residues in specific embodiments, the corresponding technical solutions using other numbering systems are considered equivalent.

[0299] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise stated, the Fc region is numbered using the EU index. The C-terminus of the Fc region can be a full C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. Thus, in some embodiments, the composition of a complete antibody may comprise an antibody population with all K447 residues and / or G446+K447 residues removed. In some embodiments, the composition of intact antibodies may include an antibody population without the removal of K447 residues and / or G446+K447 residues. In some embodiments, the composition of intact antibodies has an antibody population consisting of a mixture of antibodies with and without K447 residues and / or G446+K447 residues.

[0300] In some embodiments, the Fc region disclosed herein includes a modification according to the knock-in-hole (KIH) technique, which involves introducing a knot structure at the interface of the first subunit and a hole structure at the interface of the second subunit. This allows the knot structure to be positioned within the hole structure, promoting the formation of heterodimers and inhibiting the formation of homodimers. The knot structure is constructed by replacing a small amino acid side chain from the interface of the first subunit with a larger side chain (e.g., tyrosine or tryptophan). The hole structure is created at the interface of the second subunit by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The knot structure and hole structure are prepared by altering the nucleic acid encoding the polypeptide; optional amino acid substitutions are shown in Table 2 below.

[0301] Table 2. KIH mutation combinations

[0302] Besides the mortar and pestle technique, other techniques for modifying the CH3 domain of heavy chains to achieve heterodimerization are also known in the art, such as those described in WO1996027011A1, WO1998050431, EP1870459, WO2007110205, WO2009089004, WO2010129304, WO201190754, WO2011143545, WO2012058768, WO2013157954 and WO2013096291.

[0303] The term "epitope" refers to a site on an antigen where an antibody binds. Epitopes can be formed from adjacent or discontinuous amino acids through the ternary folding of one or more proteins. Epitopes formed from adjacent amino acids are generally preserved in denaturing solvents, while epitopes formed from ternary folding are generally lost in denaturing solvents. An epitope typically consists of at least three, and more commonly, at least five or eight to ten amino acids forming a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0304] Antibodies that recognize the same or overlapping epitopes can be detected using simple immunoassays, which test the competitive ability of one antibody to bind to the target antigen. Epitopes of antibody-antigen binding can also be determined by X-ray crystallography to identify interacting residues. Alternatively, if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then the two antibodies have the same epitope. If some amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then the two antibodies have overlapping epitopes.

[0305] Competition between antibodies can be experimentally detected by assessing whether a test antibody inhibits the specific binding of a reference antibody to a common antigen (e.g., Junghans et al., Cancer Res. 50:1495, 1990). In competitive binding assays, if the test antibody is in excess of the reference antibody (e.g., at least 2, 5, 10, 20, or 100 times), the test antibody inhibits the binding of the reference antibody by at least 50% when competing with the reference antibody, but preferably 75%, 90%, or 99% in competitive binding assays. Antibodies that can be identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, as well as antibodies that bind to adjacent epitopes of the reference antibody.

[0306] To distinguish between conserved and non-conserved substitutions in amino acids, they are classified as follows: Category I (hydrophobic side chains): Met, Ala, Val, Leu, Ile; Category II (neutral hydrophilic side chains): Cys, Ser, Thr; Category III (acidic side chains): Asp, Glu; Category IV (basic side chains): Asn, Gln, His, Lys, Arg; Category V (residues affecting chain orientation): Gly, Pro; Category VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions include substitutions between amino acids of the same class. Non-conservative substitutions are substitutions between amino acids of different classes.

[0307] A “biosimilar” or “biosimilar product” refers to an antibody having the same primary amino acid sequence as a reference antibody (e.g., trastuzumab) and optionally having detectable differences in post-translational modifications (e.g., glycosylation and / or phosphorylation) compared to the reference antibody (e.g., a different glycoform). In some embodiments, a biosimilar is an antibody or antigen-binding fragment thereof having a light chain having the same primary amino acid sequence as a reference antibody (e.g., trastuzumab) and a heavy chain having the same primary amino acid sequence as the reference antibody. In some instances, a biosimilar is an antibody or antigen-binding fragment thereof whose light chain contains the same light chain variable domain sequence as the reference antibody (e.g., trastuzumab) and whose heavy chain contains the same heavy chain variable domain sequence as the reference antibody. In some embodiments, a biosimilar may have a similar glycosylation pattern compared to a reference antibody (e.g., trastuzumab). In other embodiments, a biosimilar may have a different glycosylation pattern compared to a reference antibody (e.g., trastuzumab).

[0308] "Chemotherapy agents" or "chemotherapeutic agents" refer to chemical compounds that can be used to treat cancer, regardless of their mechanism of action. Categories of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, and kinase inhibitors. Chemotherapy agents include compounds used in "targeted therapy" and conventional chemotherapy. Examples of chemotherapeutic agents include: erlotinib (…). Genentech / OSIPharm), docetaxel Sanofi-Aventis, 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (gemcitabine, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatin(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel, Bristol-Myers Squibb Oncology, Princeton, NJ), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentaazabicyclo[4.3.0]non-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1), Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethylamine), ), and doxorubicin, Akti-1 / 2 and rapamycin.

[0309] More examples of chemotherapy agents include: oxaliplatin, Sanofi), bortezomib Millennium Pharm.), sutent( SU11248 (Pfizer), letrozole (letrozole) Novartis), imatinib mesylate, Novartis), XL-518 (MEK inhibitor, Exelixis, WO 2007 / 044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787 / ZK222584 (Novartis), fulvestrant AstraZeneca, leucovorin (folinic acid), rapamycin (sirolimus) Wyeth), lapatinib GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR TM ,SCH 66336, Schering Plow), sorafenib( BAY43-9006, Bayer Labs), gefitinib( AstraZeneca), irinotecan CPT-11, Pfizer), tipifarnib (ZARNESTRA TM (Johnson & Johnson), ABRAXANE TM Free of cremophor, albumin-modified nanoparticle formulation pallitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474), AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus ( Wyeth), pazopanib (GlaxoSmithKline), canfosfamide ( Telik, thiotepa, and cyclophosphamide Alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; anechoic acid lactones (ac etogenins, especially bulbatacin and bulbatacinone; camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil. Mustard; nitrosoureas, such as carmustine, chlorozotocin, formustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediynes (e.g., calicheamicin, calicheamicin γ1I, calicheamicin ωI1 (Angew Chem. Intl. Ed. Engl., (1994) 33: 183-186); anthracyclines, dynemicin A; bisphosphonates, such as clodronate; esperamicin;And neocarzinostatin chromophores and related chromogenic chromophores of alkenyne antibiotics, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycin, Actinomycin D, daunorubicin, detorubicin, 6-diaza-5-oxo-L-leucine, morpholino-daunorubicin, cyanomorpholino-daunorubicin, 2-pyrrole-daunorubicin and deoxydaunorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid Nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorozolidine Zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine.Pyrimidine analogues, such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenergics, such as aminoglutethimide, mitotane, and trilostane; and folic acid supplements, such as folinic acid. acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elfornithine; elliptinium acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (T-2 toxin, verrucarin A, roridin A, and anguidin); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; pipobroman; gacy tosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; thioguanine; mecaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone ); vincristine; vinorelbine Teniposide; Edatraxate; Daunomycin; Aminopterin; Capecitabine Roche; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above substances.

[0310] The definition of “chemotherapeutic agents” or “chemotherapeutic drugs” also includes: (i) antihormonal agents, which regulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen. Tamoxifen citrate, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and (ii) aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, (megestrol acetate) (Exemestane; Pfizer), Formestane, Fadrozole, (vorozole) (letrozole; Novartis), and (i) Anastrozole; AstraZeneca; (ii) Antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxane cytosine analog); (iii) Protein kinase inhibitors, such as MEK inhibitors (WO2007 / 044515); (iv) Lipid kinase inhibitors; (vi) Antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in aberrant cell proliferation, such as PKC-α, Raf, and H-Ras, such as oblimersen ( Genta Inc.); (vii) ribozymes, such as VEGF expression inhibitors (e.g., ... (viii) HER2 expression inhibitors; and vaccines, such as gene therapy vaccines, for example and rIL-2; topoisomerase 1 inhibitors, such as rmRH; (ix) anti-angiogenic agents, such as bevacizumab, Genentech); and any pharmaceutically acceptable salts, acids and derivatives of the above substances. (viiii) Glucocorticoids, including but not limited to prednisolone, prednisone, methylprednisolone, dexamethasone, allopregnane, beclomethasone, and betamethasone.

[0311] The term "glucocorticoid" generally refers to naturally occurring or synthetic steroid hormones that interact with glucocorticoid receptors. Exemplary examples include prednisolone, prednisone, methylprednisolone, dexamethasone, allopregnanolone, beclomethasone, betamethasone, pevidone, prednisolone sodium, nandrolone hemisuccinate, 19-nortestosterone hemisuccinate, deoxycorticosterone, dexamethasone:semine, corticosterone, and 11-deoxycorticosterone hemisuccinate. As used herein, "taxane" is a diterpene that can bind to tubulin, promote microtubule assembly and stabilization, and / or prevent microtubule depolymerization.

[0312] The term "CDK4 / 6 inhibitor" refers to a compound that can negatively regulate or inhibit all or part of the enzymatic activity of CDK4 and / or CDK6. Throughout this specification, CDK4 / 6 is used to refer to both CDK4 and CDK6. Any compound that blocks the formation of the CDK4 / 6-cyclin D complex and terminates cell division can be used as a CDK4 / 6 inhibitor without limitation; exemplary examples include abemaciclib, ribociclib, palbociclib, trilaciclib, PF-06873600, or pharmaceutically acceptable salts thereof.

[0313] The term "BTK inhibitor" includes, but is not limited to, compounds that have inhibitory activity against Bruton's tyrosine kinase (BTK), such as AVL-292 and ibrutinib.

[0314] The term "BCL-2 inhibitor" refers to drugs that inhibit the activity of the Bcl-2 protein. Typically, the IC50 of BCL-2 inhibitors is... 50 It ranges from approximately 0.001 μM to approximately 2 μM.

[0315] IC 50 "This refers to the concentration of a specific compound required to inhibit 50% of the measured specific activity."

[0316] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned. During alignment, gaps may be introduced where necessary to achieve the maximum percentage of sequence identity, but any conserved substitutions are not considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0317] Antibody effector functions refer to those generated by the Fc domain of Ig. These functions can include antibody-dependent cytotoxicity, antibody-dependent phagocytosis, or complement-dependent cytotoxicity. For example, the binding of the Fc effector domain to Fc receptors on immune cells with phagocytic or lytic activity, or the binding of the Fc effector domain to components of the complement system, can produce effector functions. Typically, actions mediated by Fc binding to cells or complement components lead to growth inhibition and / or apoptosis in ROR1-expressing target cells. The Fc region of an antibody can recruit Fc receptor (FcR)-expressing cells and pull them to the vicinity of antibody-bound target cells. Cells expressing FcRs on their membranes include FcγRIII (CD16), FcγRII (CD32), and FcγRI (CD64), which can act as effector cells that kill IgG-binding cells. These effector cells include monocytes, macrophages, natural killer cells, neutrophils, and eosinophils. IgG contact with FcγR can activate antibody-dependent cytotoxicity (ADCC) or antibody-dependent phagocytosis (ADCP). ADCC is mediated by CD16.sup.+ effector cells through secretory membrane porogens and proteases, while phagocytosis is mediated by CD32.sup.+ and CD64.sup.+ effector cells (see *Basic Immunology*, 4th ed., Pauled., Lippincott-Raven, New York, 1997, Chapters 3, 17, and 30; Uchida et al., 2004, J. Exp. Med. 199:1659-69; Akewanlop et al., 2001, Cancer Res. 61:4061-65; Watanabe et al., 1999, Breast Cancer Res. Treat. 53:199-207). In addition to ADCC and ADCP, the Fc region of the cell-bound antibody can also activate the classical complement pathway, triggering complement-dependent cytotoxicity (CDC). When an antibody forms a complex with an antigen, the C1q of the complement system binds to the Fc region of the antibody. The binding of C1q to the antibody on the bound cell initiates a cascade reaction, including the proteolytic activation of C4 and C2 to produce the C3 convertase. The C3 convertase cleaves C3 into C3b, which activates the terminal complement components, including C5b, C6, C7, C8, and C9. In general, these proteins form membrane-attack pores on the antibody-coated cell. These pores disrupt the cell membrane's integrity, killing the target cell (see *Immunobiology*, 6th ed., Janeway et al., Garland Science, New York, 2005, Chapter 2).

[0318] The term "antibody-dependent cytotoxicity," or ADCC, is a mechanism that induces cell death through the interaction of antibody-coated target cells with lysogenic immune cells (also known as effector cells). These effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells attach to the Fc effector domain of Ig, while IgG binds to target cells through antigen-binding sites. The effector cells exert their activity, leading to the death of the antibody-coated target cells.

[0319] The term "antibody-dependent phagocytosis," or ADCP, refers to the process by which antibody-encapsulated cells are wholly or partially internalized by phagocytic immune cells (such as macrophages, neutrophils, and dendritic cells) that bind to the Fc effector domain of Ig.

[0320] The term "complement-dependent cytotoxicity," or CDC, refers to a mechanism that induces cell death in which the Fc effector domain of an antibody binding to a target cell activates a series of enzymatic reactions that ultimately create pores in the target cell membrane. Typically, antigen-antibody complexes, such as those formed by antibodies encapsulating target cells, bind to and activate complement component C1q, thereby activating the complement cascade and leading to target cell death. Activation of complement can also result in the deposition of complement components on the target cell surface, promoting ADCC by binding to complement receptors (such as CR3) on leukocytes.

[0321] "Cytotoxicity" refers to the depletion, elimination, and / or killing of target cells. "Cytotoxic agents" are preparations that have cytotoxic effects on cells. Cytotoxic agents can be conjugated with antibodies or administered in combination with antibodies.

[0322] "Cellular inhibition" refers to the inhibition of cell proliferation. "Cellular inhibitors" are drugs that inhibit cell proliferation, thereby suppressing the growth and / or expansion of specific cell subpopulations. Cellular inhibitors can be conjugated with antibodies or administered in combination with antibodies.

[0323] The term "pharmaceutical acceptable" means something that is approved or permitted by a regulatory agency, or listed in a pharmacopoeia or other generally recognized pharmacopoeia, for use in animals, and particularly in humans. The term "pharmaceutical compatible ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier that binds to an anti-ROR1 antibody or an anti-ROR1 antibody-drug conjugate.

[0324] The term "pharmaceutically acceptable salt" refers to an organic or inorganic salt of a pharmaceutically acceptable anti-ROR1 antibody or its conjugate, or a formulation used in combination with an anti-ROR1 antibody or its conjugate. Exemplary salts include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinate, lactates, salicylates, citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates, ethylamine sulfonates, benzenesulfonates, p-toluenesulfonates, and pyrantel salts (i.e., 1,1'-dimethyldi(2-hydroxy-3-naphthyl)ate). Pharmaceutically acceptable salts may contain another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic portion that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. Instances where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0325] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to parental cells in their nucleic acid contents and may contain mutations. In this text, the term includes mutant progeny that have the same function or biological activity as cells screened or selected in primary transformed cells. Host cells include prokaryotic and eukaryotic host cells, with eukaryotic host cells including, but not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia membranaefaciens*, *Pichia minuta* (Ogataea minuta, *Pichia lindneri*), *Pichia xiaopuntiae*, *Pichia thermotolerans*, *Pichia salictaria*, *Pichia guercuum*, *Pichia pijperi*, *Pichia stiptis*, *Pichia methanolica*, *Pichia* genus, *Saccharomyces cerevisiae*, *Saccharomyces* genus, and *Hansenula*. The fungi include *Candida polymorpha*, *Kluyveromyces lactis*, *Candida albicans*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, *Chrysosporium lucknowense*, *Fusarium sp.*, *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, and *Neurospora crassa*. The following species are included: *Pichia*, any *Saccharomyces*, *Hansenula polymorpha*, any *Kluyveromyces*, *Candida albicans*, any *Aspergillus*, *Trichoderma reesei*, *Chrysosporium lucknowense*, any *Fusarium*, *Yarrowia lipolytica*, and *Neurospora crassa*. The host cell of this patent does not include subject matter not authorized under patent law.

[0326] The anti-ROR1 antibody disclosed herein is a heterodimer formed by combining a first Fc variant (Fc1) and a second Fc variant (Fc2) in a "knob-in-Hole" configuration, obtained through Fc mutation design. Fc variant mutation design technology has been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "knob-in-Hole" configuration proposed by Cater et al. (Protein Engineering vol. 9 no. 7 pp 617-621, 1996); the Fc-containing heterodimer configuration formed by electrostatic redirection by Amgen engineers (US2010286374A1); and the heterodimer configuration formed through IgG / IgA chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp. 1–8, 2010) (SEED). The bispecific molecules formed using the DuoBody platform technology of Genmab (Science, 2007, 317(5844)); the heterodimeric protein form formed by Xencor's engineers through comprehensive structural calculations and Fc amino acid mutations, combining different modes of action (mAbs3:6, 546-557; November / December 2011); the Fc modification method based on charge networks of Suzhou Corning Jerry Co., Ltd. (CN201110459100.7) to obtain the heterodimeric protein form; and other genetic engineering methods based on Fc amino acid changes or functional modifications to achieve the formation of heterodimeric functional proteins. The selection of specific immunoglobulin Fc regions from specific immunoglobulin classes and subclasses is within the scope of knowledge of those skilled in the art. The Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4 are preferred, with the Fc regions of human antibodies IgG1 and IgG4 being more preferred. A knob mutation is randomly selected from either the first or second Fc variant, and a hole mutation is performed on the other. In this embodiment, the first Fc variant undergoes a knot mutation; the second Fc variant undergoes a hole mutation.

[0327] In one specific embodiment of this disclosure, the anti-ROR1 antibody comprises four polypeptide chains: chain 1, which contains the amino acid sequence shown in SEQ ID NO:81; chain 2, which contains the amino acid sequence shown in SEQ ID NO:82; chain 3, which contains the amino acid sequence shown in SEQ ID NO:83; and chain 4, which contains the amino acid sequence shown in SEQ ID NO:84; wherein chain 1 contains an Fc knob, chain 3 contains an Fc hole, and chains 1 and 3 are combined together in a "Knob-in-Hole" manner to form a heterodimer.

[0328] This disclosure also provides antibody-drug conjugates (ADCs) based on the antibodies disclosed herein.

[0329] The term antibody-drug conjugate (ADC) refers to an antibody or antibody fragment linked to a biologically active toxic drug via a linker unit. The antibodies or antibody fragments described in this disclosure can be conjugated to effector molecules in any manner. For example, antibodies or antibody fragments can be attached to toxic drugs chemically or recombinantly. Chemical methods for preparing conjugates are known in the art. Methods for conjugating antibodies or antibody fragments to drugs must be able to link the antibody to the toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0330] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive agent. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have amino, carboxyl, thiol, hydroxyl, or ketone groups that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody. Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, etc.

[0331] Cytotoxic drugs are substances that inhibit or prevent cellular function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also induce apoptosis in normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re186 Re 188 、Sm 153 Bi 212 P 32 (and radioactive isotopes of Lu), chemotherapy drugs, antibiotics, and nucleolysins.

[0332] This disclosure allows for the conjugation of antibodies and cytotoxic drugs via conjugating agents. Examples of such conjugating agents include any one or more of non-selective conjugating agents, carboxyl-based conjugating agents, peptide chains, and disulfide-based conjugating agents. Non-selective conjugating agents are compounds that covalently link the effector molecule and the antibody, such as glutaraldehyde. Carboxyl-based conjugating agents can be any one or more of maleic aconitine-based conjugating agents (e.g., maleic aconitine) and acylhydrazone-based conjugating agents (with an acylhydrazone as the conjugation site).

[0333] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0334] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker (or linker) between the drug and the antibody. The terms "linker unit," "linking fragment," or "linking cell" refer to a chemical structural fragment or bond that is linked at one end to an antibody or its antigen-binding fragment and at the other end to a drug. Other linkers may also be attached before the drug is linked. Linkers can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine; or tripeptides such as glycine-phenylalanine-glycine; or tetrapeptides such as glycine-glycine-phenylalanine-glycine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release drugs under conditions where antibodies are hydrolyzed by proteases.

[0335] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0336] In this disclosure, the drug-connector compound can be used to form an ADC in a simple step. In other embodiments, the bifunctional linker compound can be used to form the ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form the ADC.

[0337] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0338] This disclosure also provides a method for preparing an ADC, which may further include binding an antibody to a drug-linker compound (or a drug-linker compound (LD), such as LD-1 to LD-17 shown in this disclosure) under conditions sufficient to form an antibody-drug conjugate (ADC).

[0339] In some embodiments, the method disclosed herein includes binding an antibody to a adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method disclosed herein further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0340] Drug loading, also known as drug-to-antibody ratio (DAR), is the average number of drugs conjugated to each antibody in an ADC. It can range from about 1 to about 10 drugs per antibody, and in some embodiments, from about 1 to about 8 drugs per antibody, preferably from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Exemplarily, the drug loading can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The general formula of the ADC disclosed herein includes a set of antibody-drug conjugates within the aforementioned range. In embodiments of the disclosure, the drug loading can be expressed as n, which can be a decimal or an integer. The drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC.

[0341] In one embodiment of this disclosure, the cytotoxic drug is coupled to the antibody via a linker unit.

[0342] The loading of ligand-drug conjugates can be controlled using the following non-limiting methods, including:

[0343] (1) Control the molar ratio of drug linker fragments to monoclonal antibodies,

[0344] (2) Control the reaction time and temperature.

[0345] (3) Choose different reaction reagents.

[0346] This disclosure also provides a composition. Preferably, the composition is a pharmaceutical composition containing the antibody or antibody-drug conjugate of this disclosure, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. In this disclosure, the term "pharmaceutical composition" refers to an antibody or antibody-drug conjugate of this disclosure that, together with a pharmaceutically acceptable carrier, can form a pharmaceutical formulation composition to exert its therapeutic effect more stably. These formulations can ensure the conformational integrity of the amino acid core sequence of the antibody or antibody-drug conjugate disclosed in this disclosure, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). The pharmaceutical compositions disclosed herein contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned anti-ROR1 antibody, antibody-drug conjugate, and pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions disclosed herein can be formulated as injections, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions should preferably be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, approximately 10 micrograms / kg body weight to approximately 50 milligrams / kg body weight per day. Furthermore, the pharmaceutical compositions disclosed herein can be used in conjunction with other therapeutic agents.

[0347] When using a pharmaceutical composition, a safe and effective amount of the antibody or its drug conjugate is administered to a mammal. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0348] The term "bond" refers to a group that does not exist, but whose two sides are directly connected to form a bond.

[0349] The term "alkyl" refers to a saturated, straight-chain or branched aliphatic hydrocarbon group having 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms (i.e., C2). 1- 20 Alkyl group). The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms (i.e., C12). 1-12 Alkyl groups, more preferably alkyl groups having 1 to 6 carbon atoms (i.e., C14-C6 ... 1-6 Alkyl groups). 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 their various branched isomers, etc. Alkyl groups can be substituted or unsubstituted. When substituted, they can be substituted at any usable connection point. The substituents are preferably selected from one or more of the following: D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0350] The term "alkoxy" refers to -O-(alkyl), where alkyl is defined as described above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy, etc. Alkoxy groups can be substituted or unsubstituted, and when substituted, they can be substituted at any usable linker. The substituent is preferably selected from one or more of the following: D atom, halogen, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0351] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic carbocyclic ring (i.e., monocyclic cycloalkyl) or polycyclic system (i.e., polycyclic cycloalkyl) having 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) ring atoms (i.e., 3 to 20-membered cycloalkyl). The cycloalkyl is preferably a cycloalkyl having 3 to 12 ring atoms (i.e., 3 to 12-membered cycloalkyl), more preferably a cycloalkyl having 3 to 8 ring atoms (i.e., 3 to 8-membered cycloalkyl, e.g., C12). 3-7 Cycloalkyl groups, most preferably cycloalkyl groups having 3 to 6 ring atoms (i.e., 3 to 6 membered cycloalkyl groups, such as C14-12 ... 3-6 cycloalkyl).

[0352] The term "aryl" refers to a monocyclic all-carbon aromatic ring (i.e., monocyclic aryl) or a polycyclic aromatic ring system (i.e., polycyclic aryl) having a conjugated π-electron system, having 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., 6 to 14-membered aryl). The aryl is preferably an aryl having 6 to 10 ring atoms (i.e., 6 to 10-membered aryl). The monocyclic aryl is, for example, phenyl. Non-limiting examples of the polycyclic aryl include naphthyl, anthraceneyl, phenanthrene, etc. The polycyclic aryl further includes fusion of the phenyl with one or more heterocyclic groups or cycloalkyl groups, or fusion of the naphthyl with one or more heterocyclic groups or cycloalkyl groups, wherein the bonding point is on the phenyl or naphthyl group, and in this case, the number of ring atoms continues to represent the number of ring atoms in the polycyclic aromatic ring system, non-limiting examples including:

[0353] wait.

[0354] The aryl group can be substituted or unsubstituted. When substituted, it can be substituted at any usable connection point. The substituent is preferably selected from one or more of the following: D atom, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxyl, hydroxyalkyl, oxo, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0355] The term "heteroaryl" refers to a monocyclic heteroaryl ring (i.e., monocyclic heteroaryl) or a polycyclic heteroaryl ring system (i.e., polycyclic heteroaryl) having a conjugated π-electron system, wherein the ring contains at least one (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur (the nitrogen may optionally be oxidized, i.e., to form nitrogen oxides; the sulfur may optionally be oxidized, i.e., to form sulfoxides or sulfones, but excluding -OO-, -OS-, or -SS-), having 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., 5 to 14-membered heteroaryl). The heteroaryl is preferably a heteroaryl having 5 to 10 ring atoms (i.e., 5 to 10-membered heteroaryl), more preferably a heteroaryl having 5 or 6 ring atoms (i.e., 5 or 6-membered heteroaryl).

[0356] The term "cycloalkyloxy" refers to cycloalkyl-O-, where the cycloalkyl group is as defined above.

[0357] The term “heterocyclic oxy group” refers to the heterocyclic group -O-, where the heterocyclic group is as defined above.

[0358] The term "aryloxy group" refers to aryl-O-, where the aryl group is as defined above.

[0359] The term “heteroaryloxy” refers to heteroaryl-O-, where the heteroaryl group is as defined above.

[0360] The term "alkylthio" refers to alkyl-S-, where the alkyl group is as defined above.

[0361] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0362] The term “deuterated alkyl” refers to an alkyl group that is replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

[0363] The term "haloalkoxy" refers to an alkoxy group that is substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0364] The term "hydroxyalkyl" refers to an alkyl group that is replaced by one or more hydroxyl groups, wherein the alkyl group is as defined above.

[0365] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0366] The term "hydroxyl group" refers to -OH.

[0367] The term "amino" refers to -NH2.

[0368] The term "cyano" refers to -CN.

[0369] The term "oxo" refers to =O.

[0370] N-Ethyldiisopropylamine is abbreviated as DIEA.

[0371] N,N-Dimethylformamide is abbreviated as DMF.

[0372] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphorus, abbreviated as HATU.

[0373] 1-Hydroxybenzotriazole is abbreviated as HOBt.

[0374] "Substituted" refers to one or more hydrogen atoms in a group, preferably 1 to 6, more preferably 1 to 3 hydrogen atoms, which are independently substituted by the corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions without much effort (through experimentation or theory). For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0375] The term "combination" refers to the use of more than one therapy or treatment agent; "combination" of the administration of one or more other treatment agents includes simultaneous (co-) administration and sequential administration in any order.

[0376] The present disclosure is illustrated below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the disclosure in any way.

[0377] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available products.

[0378] Example 1: Humanization and expression of D10 antibody

[0379] The mouse anti-ROR1 monoclonal antibody D10 was humanized using the CDR transplantation method. The heavy chain CDR and the light chain CDR were transplanted onto the matching heavy chain variable region and light chain variable region frame sequences, respectively, to obtain the heavy chain variable region sequence (as shown in SEQ ID NO:1) and the light chain variable region sequence (as shown in SEQ ID NO:2) of the humanized antibody BRHu-3.

[0380] BRHu-3-VH (SEQ ID NO:1):

[0381] BRHu-3-VL (SEQ ID NO:2):

[0382] D10-VH (SEQ ID NO:3):

[0383] D10-VL (SEQ ID NO:4):

[0384] Table 3. CDR region of antibody D10 / BRHu-3

[0385] The VH and VL regions of antibody BRHu-3 and the VH and VL regions of monoclonal antibody D10 were combined with the constant regions of the human heavy chain and κ light chain of IgG1, respectively, to obtain humanized antibody BRHu-3 and chimeric antibody ch-D10.

[0386] IgG1 heavy chain constant region (SEQ ID NO:11):

[0387] κ light chain constant region (SEQ ID NO:12): EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0388] The chimeric antibody ch-D10 (heavy chain amino acid sequence as shown in SEQ ID NO:15, light chain amino acid sequence as shown in SEQ ID NO:16) and the humanized antibody BRHu-3 (heavy chain amino acid sequence as shown in SEQ ID NO:13, light chain amino acid sequence as shown in SEQ ID NO:14) were expressed and purified in CHO-K1 cells. First, the vector containing the antibody heavy and light chain coding sequences was electroporated into CHO-K1 cells. After culturing at 37°C and 5% CO2 for 4 days, the cell culture supernatant was centrifuged at 3000 rpm for 10 min. The supernatant was collected and purified using protein A to achieve an antibody purity >95%.

[0389] BRHu-3 heavy chain sequence (SEQ ID NO:13):

[0390] BRHu-3 light chain sequence (SEQ ID NO:14):

[0391] Heavy chain sequence of chimeric antibody ch-D10 (SEQ ID NO:15):

[0392] The light chain sequence of the chimeric antibody ch-D10 (SEQ ID NO:16):

[0393] Example 2: Affinity maturation of humanized antibody BRHu-3

[0394] Affinity maturation of the humanized antibody BRHu-3 was performed using the following method:

[0395] Using the humanized antibody BRHu-3 as a mutation template, primers were designed, and random mutations were introduced into the six CDR regions of BRHu-3 by PCR. The mutant DNA was electroporated into SU320+ Helper phage supercompetent cells and cultured overnight at 37°C. The mutant library was then isolated and purified using the PEG8000 / NaCl precipitation method to obtain the phage display mutant library.

[0396] Phage display mutant libraries were biopanilated to enrich positive single clones. ROR2 antigen was coated at 500 ng / well in Maxi-Sorp plates and incubated overnight at 4°C. Maxi-Sorp plates were blocked with 1% PVA at room temperature for 2 h. Subsequently, the plates were incubated with the mutant phage library at 4°C for 2 h. The supernatant was collected and incubated with negative CHO-K1 cells at 4°C for 2 h. The supernatant was then centrifuged and incubated with CHO-K1-hROR1 cells (a stable cell line overexpressing human ROR1; the sequence of human ROR1 is shown in SEQ ID NO: 87) at 4°C for 2 h. Add PBS buffer (PBST) containing 0.05% Tween 20 pre-chilled at 4°C, wash 6-8 times, then infect 1 mL of NEBalpha5F' cells (purchased from NEB) with OD600 = 0.8, and incubate at 37°C for 1 h. Then add M13K07 helper phage (purchased from NEB), incubate at 37°C for 1 h, and transfer to 40 mL of 2YT / Carb / Kan medium for overnight expansion at 37°C. Plate the culture, and assess the enrichment level on day 3. Purify the phage using PEG 8000 / NaCl precipitation method for the next round of screening.

[0397] Perform five rounds of biological screening following the steps above to obtain successfully enriched bacteriophage clones.

[0398] From the enriched phage clones, single clones were randomly selected for phage ELISA experiments. Recombinant proteins ROR1 (purchased from Kaika Biotechnology) and ROR2 (purchased from Kaika Biotechnology) were coated onto ELISA plates at 100 ng / well, incubated overnight at 4°C, and then blocked with 1% PVA at room temperature for 2 hours. On the same day of coating, single phage clones were picked and cultured in deep-well plates at 37°C overnight. The deep-well plates were centrifuged, and 50 μL of the supernatant was transferred to ELISA plates at room temperature for 2 hours. The ELISA plates were washed with PBST, and 100 μL of Anti-M13 HRP antibody (purchased from Yiqiao Shenzhou) was added, incubated at room temperature for 1 hour. The ELISA plates were then washed with PBST, followed by PBS, and 100 μL of TMB was added. The plates were incubated at 37°C for 5–10 minutes. The reaction was terminated by adding 50 μL of 1M phosphate. The absorbance at 450 nm was measured using a microplate reader. Five clones with high affinity were selected for sequencing: YR-4, YR-8, YR-10, YR-11, and YR-21. The variable region and CDR region of the antibody are shown below:

[0399] Table 4. CDR region of antibody YR-8 Note: The underlined part indicates that the sequence is different from that of the parent antibody BRHu-3.

[0400] The variable region sequence of the antibody obtained from affinity maturation is as follows:

[0401] YR-8-VH (SEQ ID NO:22):

[0402] YR-8-VL (SEQ ID NO:23):

[0403] YR-4-VH (SEQ ID NO:24):

[0404] YR-4-VL (SEQ ID NO:25):

[0405] YR-10-VH (SEQ ID NO:26):

[0406] YR-10-VL (SEQ ID NO:27):

[0407] YR-11-VH (SEQ ID NO:28):

[0408] YR-11-VL (SEQ ID NO:23):

[0409] YR-21-VH (SEQ ID NO:29):

[0410] YR-21-VL(SEQ ID NO:23):

[0411] The VH and VL sequences described above were combined with the constant regions of the human heavy chain and κ light chain of IgG1 (as shown in Example 1) to obtain the full-length sequence of the antibody. The antibody was expressed and purified according to the method described in Example 1. The heavy and light chains of the exemplary antibody YR-8 are shown in SEQ ID NO:30 and SEQ ID NO:31, respectively.

[0412] Heavy chain sequence of YR-8 (SEQ ID NO:30):

[0413] The light chain sequence of YR-8 (SEQ ID NO:31):

[0414] Example 3: Modification of the affinity maturation antibody YR-8 molecule

[0415] Amino acid mutations were performed on the N at position 60 and the S at position 61 of the heavy chain of the YR-8 antibody (the amino acid positions were obtained according to the Kabat number). The mutation schemes included: mutating the N at position 60 to E, or mutating the S at position 61 to A, V, T, L, and I, respectively. The resulting antibodies were named YR-8-ES, YR-8-NA, YR-8-NV, YR-8-NT, YR-8-NL, and YR-8-NI, respectively. The HCDR2 and VH sequences of the mutated antibodies are as follows:

[0416] Table 5. HCDR2 sequence of YR-8 modified antibody Note: The underlined part indicates that the sequence is different from that of the parent antibody YR-8.

[0417] The VH sequence of the mutated antibody is as follows:

[0418] YR-8-ES-VH (SEQ ID NO:38):

[0419] YR-8-NA-VH (SEQ ID NO:39):

[0420] YR-8-NV-VH (SEQ ID NO:40):

[0421] YR-8-NT-VH (SEQ ID NO:41):

[0422] YR-8-NL-VH (SEQ ID NO:42):

[0423] YR-8-NI-VH (SEQ ID NO:43):

[0424] The mutated VH was combined with YR-8-VL (SEQ ID NO:23) and the constant regions of the human IgG1 heavy chain and κ light chain (as shown in Example 1) to obtain a complete antibody. The mutant was expressed and purified according to the method described in Example 1. The heavy and light chains of the exemplary YR-8-ES antibody obtained in this disclosure are shown in SEQ ID NO:44 and SEQ ID NO:31, respectively.

[0425] Heavy chain sequence of YR-8-ES (SEQ ID NO:44):

[0426] The light chain sequence of YR-8-ES (SEQ ID NO:31):

[0427] Example 4: Humanization of H10 antibody

[0428] The CDR transplantation method was used to humanize the mouse monoclonal antibody H10 against ROR1. Heavy chain CDRs and light chain CDRs were transplanted into matching heavy chain and light chain variable regions, respectively, resulting in 16 humanized molecules with different sequences. The variable region and CDR region sequences of the humanized antibodies are shown below:

[0429] Table 6. CDR regions of H10 / H10 humanized antibodies

[0430] H10 VH (SEQ ID NO:51):

[0431] H10 VL (SEQ ID NO:52):

[0432] H10-hVH1 (SEQ ID NO:53):

[0433] H10-hVH2 (SEQ ID NO:54):

[0434] H10-hVH3 (SEQ ID NO:55):

[0435] H10-hVH4 (SEQ ID NO:56):

[0436] H10-hVL1 (SEQ ID NO:57):

[0437] H10-hVL2 (SEQ ID NO:58):

[0438] H10-hVL4 (SEQ ID NO:59):

[0439] H10-hVL5 (SEQ ID NO:60):

[0440] The variable region of the humanized H10 antibody was combined with the constant regions of the human IgG1 heavy chain and κ light chain (as shown in Example 1) to obtain a complete antibody. The specific antibody is as follows:

[0441] Table 7. H10 Humanized Antibodies

[0442] Example 5: Affinity maturation of H10 humanized antibody

[0443] Six CDR regions of H10-hVH4-hVL4 were subjected to saturation mutagenesis. The mutated vector containing the antibody heavy and light chain coding sequences was electroporated into CHO-K1 cells. Cells were cultured at 37°C and 5% CO2 for 4 days. The supernatant was added to microplates coated with ROR1 (manufacturer: Kaika Biotechnology, catalog number: ROR-HM401) and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG (manufacturer: Jackson, 109-005-008) was added, and the reaction was carried out at 37°C for 1 hour. After washing, TMB solution was added, and the reaction was carried out at room temperature in the dark for 15 minutes. Then, ELISA Stopping Solution was added to terminate the reaction. The absorbance was measured at 450 nm using a microplate reader. Six clones with high affinity were sequenced, yielding six clones: AM3-ZH1, AM3-ZH3, AM3-ZH4, AM3-ZH5, AM3-ZH6, and AM3-ZH7. The relevant antibody sequences are as follows:

[0444] Table 8. CDR sequences of H10 affinity maturation antibodies Note: The underlined part indicates that the sequence is different from that of the parent antibody H10-hVH4-hVL4.

[0445] AM3-ZH1-VH (SEQ ID NO:70):

[0446] AM3-ZH1-VL (SEQ ID NO:71):

[0447] AM3-ZH3-VH (SEQ ID NO:72):

[0448] AM3-ZH3-VL (SEQ ID NO:71):

[0449] AM3-ZH4-VH (SEQ ID NO:73):

[0450] AM3-ZH4-VL (SEQ ID NO:71):

[0451] AM3-ZH5-VH (SEQ ID NO:74):

[0452] AM3-ZH5-VL (SEQ ID NO:75):

[0453] AM3-ZH6-VH (SEQ ID NO:74):

[0454] AM3-ZH6-VL (SEQ ID NO:76):

[0455] AM3-ZH7-VH (SEQ ID NO:74):

[0456] AM3-ZH7-VL (SEQ ID NO:71):

[0457] The variable region described above is combined with the constant regions of the human IgG1 heavy chain and κ light chain (as shown in Example 1) to obtain a complete antibody. The heavy chain and light chain sequences of the exemplary antibody AM3-ZH-3 obtained in this disclosure are shown in SEQ ID NO:77 and SEQ ID NO:78, respectively.

[0458] Heavy chain sequence of AM3-ZH3 (SEQ ID NO:77):

[0459] The light chain sequence of AM3-ZH3 (SEQ ID NO:78):

[0460] Example 6: Construction of a dual epitope antibody targeting ROR1

[0461] In this embodiment, a bispecific antibody targeting ROR1 was constructed using ROR1 antibodies derived from D10 and ROR1 antibodies derived from H10, wherein the ROR1 antibodies derived from D10 and ROR1 antibodies derived from H10 bind to different epitopes of ROR1, respectively.

[0462] This embodiment constructs a dual epitope antibody with a KIH structure, and an exemplary structure is shown below:

[0463] Structure 1 contains the following four chains:

[0464] Chain 1: YR8VH-CH1-Fcknob;

[0465] Chain 2: YR8VL-CL

[0466] Chain 3: ZH3VH-CH1-FcHole

[0467] Chain 4: ZH3VL-CL

[0468] Structure 2 contains the following four chains:

[0469] Chain 1: YR8VH-CH1-Fchole;

[0470] Chain 2: YR8VL-CL

[0471] Chain 3: ZH3VH-CH1-Fcknob

[0472] Chain 4: ZH3VL-CL

[0473] CH1 (SEQ ID NO:88)

[0474] The sequence of Fcknob (T366W) (SEQ ID NO:79):

[0475] The sequence of FcHole (T366S, L368A, Y407V) (SEQ ID NO:80):

[0476] To further prepare antibodies binding to different epitopes of ROR1, this disclosure also discloses the preparation of YR-8-ES variant 1, with its Fc region containing only the Knob (T366W) mutation, based on the YR-8-ES antibody, and the preparation of AM3-ZH3 variant 1, with its Fc region containing only the Hole (T366S, L368A, Y407V) mutation, based on the AM3-ZH3 antibody. The sequences are as follows:

[0477] YR-8-ES variant 1 heavy chain (SEQ ID NO:81):

[0478] YR-8-ES variant 1 light chain (SEQ ID NO:31):

[0479] AM3-ZH3 variant 1 heavy chain (SEQ ID NO:83):

[0480] AM3-ZH3 variant 1 light chain (SEQ ID NO:78):

[0481] For example, YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were dissolved separately in PBS at pH 7.2, mixed in a 1:1 molar ratio, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 10:1). After incubation at 25°C for approximately 16 hours, purification and desalting were performed by elution with G25 resin, and the mixture was filtered through a 0.2 μm filter under aseptic conditions to obtain the ROR1-targeting biepisode antibody KIH YR8 / ZH3, the sequence of which is as follows:

[0482] Chain 1 of KIH YR8 / ZH3 (SEQ ID NO:81):

[0483] Chain 2 of KIH YR8 / ZH3 (SEQ ID NO:82):

[0484] Chain 3 of KIH YR8 / ZH3 (SEQ ID NO:83):

[0485] Chain 4 of KIHYR8 / ZH3 (SEQ ID NO:84):

[0486] In addition, this disclosure uses UC961 as a positive control, the sequence of which is shown below:

[0487] UC961 heavy chain (SEQ ID NO:85):

[0488] UC961 light chain (SEQ ID NO:86):

[0489] The amino acid sequence of human ROR1 (SEQ ID NO:87):

[0490] This disclosure uses sequences from other antibodies or ADCs as follows:

[0491] (1) Anti-SIRPα antibody BR105 was prepared according to antibody 14# in WO2022121980A1, and its specific sequence is as follows:

[0492] HCDR1 of anti-SIRPα antibody 14# (SEQ ID NO: 138):

[0493] HCDR2 of anti-SIRPα antibody 14# (SEQ ID NO: 89):

[0494] HCDR3 of anti-SIRPα antibody 14# (SEQ ID NO: 90):

[0495] LCDR1 of anti-SIRPα antibody 14# (SEQ ID NO: 91):

[0496] LCDR2 of anti-SIRPα antibody 14# (SEQ ID NO: 92):

[0497] LCDR3 of anti-SIRPα antibody 14# (SEQ ID NO: 93):

[0498] VH of anti-SIRPα antibody 14# (SEQ ID NO: 94):

[0499] VL of anti-SIRPα antibody 14# (SEQ ID NO: 95):

[0500] Heavy chain of anti-SIRPα antibody 14# (SEQ ID NO: 96):

[0501] Light chain of anti-SIRPα antibody 14# (SEQ ID NO: 97):

[0502] (2) The anti-PD-1 antibody used in this disclosure is pembrolizumab (Keytruda), prepared according to the h409A11 antibody in WO2008156712A1, and its sequence is as follows:

[0503] HCDR1 of anti-PD-1 antibody (SEQ ID NO: 98):

[0504] HCDR2 of anti-PD-1 antibody (SEQ ID NO: 99):

[0505] HCDR3 of anti-PD-1 antibody (SEQ ID NO: 100):

[0506] LCDR1 of anti-PD-1 antibody (SEQ ID NO: 101):

[0507] LCDR2 (SEQ ID NO: 102), an anti-PD-1 antibody:

[0508] LCDR3 (SEQ ID NO: 103), an anti-PD-1 antibody:

[0509] VH (SEQ ID NO: 104) of anti-PD-1 antibody:

[0510] VL of anti-PD-1 antibody (SEQ ID NO: 105):

[0511] Heavy chain of anti-PD-1 antibody (SEQ ID NO: 106):

[0512] The light chain of the anti-PD-1 antibody (SEQ ID NO: 107):

[0513] (3) The anti-PD-L1 antibody used in this disclosure is avelumab, prepared according to antibody A09-246-2 in WO2013 / 079174A1, and its sequence is as follows:

[0514] Avirumab's HCDR1 (SEQ ID NO: 108):

[0515] Avirumab's HCDR2 (SEQ ID NO: 109):

[0516] Averumab's HCDR3 (SEQ ID NO: 110):

[0517] Averumab's LCDR1 (SEQ ID NO: 111):

[0518] Averumab's LCDR2 (SEQ ID NO: 112):

[0519] Avelumab's LCDR3 (SEQ ID NO: 113):

[0520] Averumab heavy chain variable region (SEQ ID NO: 114):

[0521] Averumab light chain variable region (SEQ ID NO: 115):

[0522] Averumab heavy chain (SEQ ID NO:116):

[0523] Avelumab light chain (SEQ ID NO:117):

[0524] (4) The anti-CD20 antibody used in this disclosure is zabetoin (HS006), which was prepared according to CN117402885 and its sequence is as follows;

[0525] Zabetoumab HCDR1 (SEQ ID NO: 118):

[0526] Zabetoumab HCDR2 (SEQ ID NO: 119):

[0527] Zabetoumab HCDR3 (SEQ ID NO: 120):

[0528] Zabetoumab LCDR1 (SEQ ID NO: 121):

[0529] Zabetoumab LCDR2 (SEQ ID NO: 122):

[0530] Zabetoumab LCDR3 (SEQ ID NO: 123):

[0531] Zabetoumab heavy chain variable region (SEQ ID NO:124):

[0532] Zabetoumab light chain variable region (SEQ ID NO:125):

[0533] Zabetoumab heavy chain (SEQ ID NO:126):

[0534] Zabetoumab light chain (SEQ ID NO:127):

[0535] Another anti-CD20 antibody used in this disclosure is rituximab, prepared according to US7381560B2, and its sequence is as follows:

[0536] Rituximab HCDR1 (SEQ ID NO:139):

[0537] Rituximab HCDR2 (SEQ ID NO:140):

[0538] Rituximab HCDR3 (SEQ ID NO:141):

[0539] Rituximab LCDR1 (SEQ ID NO:142):

[0540] Rituximab LCDR2 (SEQ ID NO:143):

[0541] Rituximab LCDR3 (SEQ ID NO:144):

[0542] Rituximab heavy chain variable region (SEQ ID NO:145):

[0543] Rituximab light chain variable region (SEQ ID NO:146):

[0544] Rituximab heavy chain (SEQ ID NO:147):

[0545] Rituximab light chain (SEQ ID NO:148):

[0546] (5) The sequence of trastuzumab used in this disclosure is as follows: HCDR1 of trastuzumab (SEQ ID NO:128):

[0547] HCDR2 of trastuzumab (SEQ ID NO:129):

[0548] HCDR3 of trastuzumab (SEQ ID NO:130):

[0549] The LCDR1 of trastuzumab (SEQ ID NO:131):

[0550] The LCDR2 of trastuzumab (SEQ ID NO:132):

[0551] The LCDR3 of trastuzumab (SEQ ID NO:133):

[0552] The heavy chain variable region of trastuzumab (SEQ ID NO:134):

[0553] The light chain variable region of trastuzumab (SEQ ID NO:135):

[0554] Heavy chain of trastuzumab (SEQ ID NO:136):

[0555] The light chain of trastuzumab (SEQ ID NO:137):

[0556] Example 7: Preparation of Linker-Drug

[0557] The toxins (drugs) used to prepare ADCs are pre-coupled with linkers to prepare linker-drug (LD) compounds LD-1 to LD-17; exemplary toxins are MMAE, Eribulin, and Exatecan.

[0558] In this embodiment, experimental methods without specific conditions are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents without a specific source are commercially available, standard reagents.

[0559] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6 The unit (ppm) is given. 1 The H NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide as the solvent and tetramethylsilane (TMS) as the internal standard.

[0560] MS measurements were performed using a Shimadzu LCMS-2020 Single Quadrupole LC-MS / MS system (manufacturer: Shimadzu, MS model: 2020 Single Quadrupole MS); column: Phenomenex Gemini NX 5μ, C18. 50 x 4.6 mm, mobile phase: 0.1% aqueous solution of formic acid / 0.1% acetonitrile (ACN) solution of formic acid, flow rate: 1 mL / min.

[0561] The RP-HPLC system used was the Shimadzu Nexera preparative liquid chromatography system, with a Phenomenex Gemini NX 5μ, C18 column. 150 x 50 mm, mobile phase: 0.1% aqueous solution of trifluoroacetic acid / 0.1% acetonitrile (ACN) solution of trifluoroacetic acid, flow rate: 50 mL / min.

[0562] 7.1 Preparation of LD-1

[0563] To 3 mL of anhydrous tetrahydrofuran containing a solution of 2-tert-butylhydrazine-1,2-dicarboxylic acid ester (76.8 mg, 0.33 mmol) and tert-butyl 3-bromo-2-(bromomethyl)propionate (200 mg, 0.66 mmol), NaH (60%, 80 mg, 2.0 mmol) was added. The mixture was stirred at room temperature for 15 minutes, and then the reaction was quenched with 1 mL of water containing 60 μL AcOH. The mixture was then purified by RP-HPLC. The purified fraction was lyophilized to give 204 mg of a white solid, Al.

[0564] MS m / z: 373.6 [M+H] + .

[0565] To 8.0 mL of acetic acid (AcOH) solution containing tert-butyl 2-[[tert-butyloxycarbonyl-(tert-butyloxycarbonylamino)amino]methyl]prop-2-enoate (A1) (204 mg, 0.55 mmol), 3,4-dibromofuran-2,5-dione (140 mg, 0.55 mmol) was added. The mixture was refluxed and stirred under argon atmosphere for 11 days, then concentrated to 3 mL and purified by RP-HPLC to give 43 mg of white solid A2 (yield: 22%).

[0566] MS m / z: 354.8 [M+H] + .

[0567] N-hydroxysuccinimide (230 mg) was added to a stirred solution of 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (A2, 350 mg) in anhydrous dichloromethane (DCM, 10 mL), followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI, 400 mg). The mixture was stirred at room temperature for 30 minutes, and the reaction was concentrated to dryness under reduced pressure. The residue was purified directly by RP-HPLC and lyophilized to give compound 4 as a white solid (337 mg).

[0568] MS m / z: 452.0 [M+H] + .

[0569] Compound 4 (12 mg) was added to a 1 mL DMF solution containing compound 6 (31 mg, purchased from MedChemExpress, catalog number: HY-100374), followed by 0.01 mL of DIEA. The reaction mixture was stirred at room temperature (22 °C). After 3 hours, the crude reaction mixture was purified directly by RP-HPLC, and after lyophilization, compound 8 (17 mg), i.e., LD-1, was obtained as a white solid.

[0570] MS m / z: 1243.6 [M+H] + .

[0571] 7.2 Preparation of LD-2

[0572] Saturated NaHCO3 (0.2 mL) and bromoacetic anhydride (9 mg) were added to an acetonitrile / water (6 / 4, v / v, 2 mL) solution containing compound 6 (20 mg). The reaction mixture was stirred at room temperature (22 °C) for 10 minutes, and then purified directly by RP-HPLC. After lyophilization, compound 7 (17 mg), i.e., LD-2, was obtained as a white solid.

[0573] MS m / z: 1243.6 [M+H] + .

[0574] 7.3 Preparation of LD-3

[0575] DIEA (0.025 mL) was added to an anhydrous DMF (1 mL) solution containing compound 6 (62 mg) and Fmoc-NH-PEG4-COOH (compound 9, 25 mg, purchased from PurePEG, catalog number 433704-1H), followed by HATU (20 mg). The reaction mixture was stirred at room temperature (22 °C). After 15 minutes, piperidine (0.1 mL) was added, and the reaction was continued for another 30 minutes. The crude reaction mixture was purified directly by RP-HPLC and lyophilized to give compound 10 (61 mg, TFA salt) as a white solid.

[0576] Saturated NaHCO3 (0.03 mL) and bromoacetic anhydride (7 mg) were added to an acetonitrile / water (6 / 4, v / v, 2 mL) solution containing compound 10 (37 mg). The reaction mixture was stirred at room temperature for 10 minutes, and the crude mixture was purified by RP-HPLC and lyophilized to give compound 11 (32 mg), i.e., LD-3, as a white solid.

[0577] MS m / z: 491.0 [M+H] + .

[0578] 7.4 Preparation of LD-4

[0579] Maleimide hexanoic acid (compound 18, 12 mg) was added to a solution of anhydrous DMF (2 mL) containing compound 6 (62 mg, TFA salt), followed by DIEA (0.02 mL) and HATU (20 mg). The reaction mixture was stirred at room temperature (22 °C). After 15 minutes, the crude reaction mixture was purified directly by RP-HPLC and lyophilized to give compound 19 (62 mg, TFA salt), i.e., LD-4, as a white solid.

[0580] MS m / z: 1316.6 [M+H] + .

[0581] 7.5 Preparation of LD-5

[0582] DIEA (0.002 mL) was added to an anhydrous DMF (2 mL) solution containing compound 12 (65 mg, prepared according to WO2022026915) and MMAE (72 mg), followed by HOBt (3 mg). The reaction mixture was stirred at room temperature (22 °C) for 18 hours and then diluted with water (20 mL). The reaction mixture was extracted with diethyl ether (40 mL), the organic phase was dried over Na2SO4, and concentrated to dryness under reduced pressure to give crude compound 13, which was finally dissolved in methanol (2 mL). Zinc powder (200 mg) was added to the methanol solution containing compound 13, followed by formic acid (0.2 mL). The reaction was stirred at room temperature for 30 minutes. The solid was removed by filtration, and the filtrate was directly purified by RP-HPLC and lyophilized to give a white solid compound 14 (72 mg). DIEA (0.025 mL) was added to an anhydrous DMF (2 mL) solution of compound 14 (TFA salt, 66 mg) and Fmoc-NH-PEG4-COOH (purchased from PurePEG, catalog number 433704, 25 mg), followed by HATU (20 mg). The mixture was stirred at room temperature. After 16 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 15 (72 mg) as a white powder.

[0583] Compound 15 (70 mg) was dissolved in acetonitrile / water (6 / 4, v / v, 3 mL), and NaOH (aq. 1 M, 0.3 mL) was added. The reaction mixture was stirred at room temperature (22 °C) to obtain compound 16. After 8 hours, hydrochloric acid (1 M, 0.12 mL) was added to the crude compound 16, followed by bromoacetic anhydride (14 mg), and the reaction was allowed to proceed for 0.5 hours. The crude reaction mixture was purified directly by RP-HPLC, and after lyophilization, compound 17 (46 mg), i.e., LD-5, was obtained as a white solid.

[0584] MS m / z: 1426.7 [M+H] + .

[0585] 7.6 Preparation of LD-6

[0586] To a solution of compound 20 (60 mg, purchased from InnoPep) and Exatecan Mesylate (compound 21, 53 mg, purchased from Advance ChemBlocks, catalog number 10484) in anhydrous N,N-dimethylformamide (2 mL), N-ethyldiisopropylamine (0.05 mL) was added, followed by O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphide (38 mg). The mixture was stirred at room temperature for 15 minutes. Piperidine (0.1 mL) was added. After 30 minutes, the crude mixture was purified by RP-HPLC and lyophilized to give compound 22 (67 mg, trifluoroacetate) as a yellow powder.

[0587] Compound 4 (25 mg, synthesized according to the method in PCT / US2022 / 078563) was added to a solution of compound 22 (44 mg) in N,N-dimethylformamide (2 mL), followed by N-ethyldiisopropylamine (0.02 mL). The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP-HPLC and lyophilized to give compound 23, LD-6, as a yellow solid (42 mg).

[0588] MS m / z: 1090.2 [M+H] + .

[0589] 7.7 Preparation of LD-14

[0590] To an anhydrous N,N-dimethylformamide (2 mL) solution containing compound 1 (77 mg, MedChemExpress, HY-41189), iribulin (compound 2, methanesulfonate, 82 mg, MedChemExpress, HY-13442) was added, followed by N-ethyldiisopropylamine (0.035 mL). The reaction mixture was stirred at room temperature (22 °C). After 6 hours, piperidine (0.1 mL) was added, and the reaction was stirred at room temperature for 15 minutes. The reaction mixture was purified directly by RP-HPLC, and after lyophilization, gave compound 3 (110 mg, trifluoroacetate) as a white solid.

[0591] Compound 4 (20 mg, synthesized according to the method reported in PCT / US2022 / 078563) was added to a solution of N,N-dimethylformamide (2 mL) containing compound 3 (50 mg), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 3 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 5 (43 mg), i.e., LD-14, as a white solid.

[0592] MS m / z: 1471.6 [M+H] + .

[0593] 1 HNMR (400MHz, DMSO) δ: 10.00 (s, 1H), 8.36 (d, J=8.8Hz, 1H), 8.25 (d, J=7.6Hz, 1H), 7.57 (d, J=8.4H z, 2H), 7.27 (d, J=8.4Hz, 2H), 7.08 (t, J=6Hz, 1H), 5.97 (t, J=5.6Hz, 1H), 5.41 (s, 2H), 5.05 (s, 1H), 5.00 (s, 1H), 4.97-4.89 (m, 2H), 4.83 (s, 1H), 4.75 (s, 1H), 4.63 (s, 1H), 4.55 (dd, J=7.2, 4.4Hz, 2H ), 4.38 (dd, J=13.2Hz, J=7.6Hz, 1H), 4.32–4.21 (m, 4H), 4.21–4.12 (m, 3H), 4.22-4.06 (m, 3H), 4.02 (t, J=7.6Hz, 1H), 3.86-3.73 (m, 2H), 3.73-3.62 (m, 2H), 3.58–3.45 (m, 3H), 3.30 (s, 1H), 3.28-3.1 8 (m, 4H), 3.06–2.90 (m, 4H), 2.84 (d, J=9.6Hz, 1H), 2.80-2.63 (m, 2H), 2.61-2.52 (m, 1H), 2.37–2.1 7(m, 5H), 2.17–2.08(m, 1H), 2.06–1.85(m, 7H), 1.78-1.54(m, 7H), 1.54-1.39(m, 4H), 1.39–1.26(m , 4H), 1.24-1.11 (m, 1H), 1.03 (d, J=6.4Hz, 3H), 1.01-0.92 (m, 1H), 0.86 (dd, J=12.4, J=6.8Hz, 6H).

[0594] 7.8 Preparation of LD-7

[0595] Compound 4 (12 mg) was added to a solution of compound 24 (21 mg, synthesized according to the procedure reported in US10155821B2) in N,N'-dimethylformamide (1 mL), followed by diisopropylethylamine (10 μL). The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP-HPLC and lyophilized to give compound 25 (i.e., LD-7, 23 mg) as a yellow solid.

[0596] MS m / z: 1177.4 [M+H] + .

[0597] 7.9 Preparation of LD-8

[0598] Diisopropylethylamine (0.1 mL) was added to a solution of compound 27 (92 mg, 0.2 mmol, purchased from ChemScene, CS-0105172) and Fmoc-Gly-OH (90 mg, purchased from Combi-Blocks, San Diego) in dimethylformamide (3 mL), followed by the addition of HATU (114 mg, 0.3 mmol). The reaction mixture was stirred at room temperature for 6 hours, and the crude product was purified by RP-HPLC and lyophilized to give compound 28 as a white powder (76 mg).

[0599] Compound 28 (73 mg) was dissolved in anhydrous dimethylformamide (1 mL), and bis(4-nitrophenol) carbonate (Sigma-Aldrich, 45 mg, 0.15 mmol) was added, followed by diisopropylethylamine (0.02 mL). The mixture was stirred at room temperature for 16 hours, and the crude reactant was purified directly by RP-HPLC to give compound 29 as a white powder (75 mg).

[0600] Diisopropylethylamine (0.02 mL) was added to anhydrous dimethylformamide (1 mL) containing compound 29 (54 mg) and Exatecan mesylate (compound 21, ethatecan mesylate, 27 mg). The mixture was stirred at room temperature for 3 hours and then diluted with water (20 mL). The mixture was extracted with ethyl acetate (40 mL), and the organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was suspended in acetonitrile / water (2 mL, 6 / 4, v / v). Sodium hydroxide (1 M, 0.35 mL) was added to this solution, and the mixture was stirred at room temperature for 2 hours. Hydrochloric acid (1 N, 0.2 mL) was added, and the reactants were purified by RP-HPLC to give compound 30 (33 mg, TFA salt) as a yellow powder.

[0601] Compound 31 (10 mg, purchased from Ambeed) was added to a solution of compound 30 (32 mg) in dimethylformamide (1 mL), followed by diisopropylethylamine (20 μL). The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP-HPLC and lyophilized to give compound 32 (i.e., LD-8, 28 mg) as a yellow solid.

[0602] MS m / z: 985.4 [M+H] +.

[0603] 7.10 Preparation of LD-9

[0604] A saturated aqueous solution of sodium bicarbonate (0.02 mL) and bromoacetic anhydride (10 mg) were added to an acetonitrile / water (6 / 4, v / v, 1 mL) solution of compound 24 (21 mg, synthesized according to US10155821B2). The reaction mixture was stirred at room temperature for 15 minutes and then purified directly by RP-HPLC to give compound 26 (i.e., LD-9, 19 mg) as a yellow powder.

[0605] MS m / z:m / z 961.4[M+H] + .

[0606] 7.11 Preparation of LD-10

[0607] Compound 4 (12 mg, see the preparation procedure for LD-7) was added to a solution of compound 30 (20 mg) in dimethylformamide (1 mL), followed by diisopropylethylamine (10 μL). The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP-HPLC and lyophilized to give compound 33 (i.e., LD-10, 19 mg) as a yellow solid.

[0608] MS m / z: 1170.4 [M+H] + .

[0609] 7.12 Preparation of LD-11

[0610] A solution of compound 30 (19 mg) in acetonitrile / water (6 / 4, v / v, 1 mL) was added to a saturated aqueous solution of sodium bicarbonate (0.02 mL) and bromoacetic anhydride (10 mg). The reaction mixture was stirred at room temperature for 15 minutes and then purified directly by RP-HPLC to give compound 34 (i.e., LD-11, 17 mg) as a yellow powder.

[0611] MS m / z: 954.2 [M+H] + .

[0612] 7.13 Preparation of LD-12

[0613] Cycloglutaric anhydride (5 mg) was added to compound 3 (25 mg, dissolved in anhydrous dimethylformamide (1 mL) solution), followed by diisopropylethylamine (0.01 mL). The reaction mixture was stirred at room temperature for 2 hours, then diluted with DCM (4 mL). Pentafluorophenol (10 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (20 mg) were added, and the mixture was stirred at room temperature. After 30 minutes, the reaction mixture was concentrated under reduced pressure, and the residue was purified by RP-HPLC to give compound 39 (i.e., LD-12, 23 mg) as a white powder.

[0614] MS m / z: 1415.7 [M+H] + .

[0615] 7.14 Preparation of LD-13

[0616] Diisopropylethylamine (0.04 mL) was added to a solution of compound 35 (92 mg, synthesized according to the procedure reported in Bioconjugate Chem., 2006, 17(3), 831-840) and erybulin methanesulfonate (2, 82 mg) in anhydrous dimethylformamide (2 mL). The mixture was stirred at room temperature for 3 hours and then diluted with water (30 mL). The mixture was extracted with ethyl acetate (50 mL), and the organic layer was dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was dissolved in acetonitrile / water (4 mL, 6 / 4, v / v). Sodium hydroxide (1 M, 0.7 mL) was added to this solution, and the mixture was stirred at room temperature for 2 hours. Hydrochloric acid (1 N, 0.4 mL) was added, and the reactants were purified by RP-HPLC to give compound 36 as a white powder (97 mg, TFA salt).

[0617] Compound 4 (6 mg) was added to a solution of compound 36 (12 mg) in dimethylformamide (1 mL), followed by diisopropylethylamine (5 μL). The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP-HPLC and lyophilized to give compound 37 (i.e., LD-13, 12 mg) as a white solid.

[0618] MS m / z: 1478.6 [M+H] + .

[0619] 7.15 Preparation of LD-15

[0620] A saturated aqueous solution of sodium bicarbonate (0.01 mL) and bromoacetic anhydride (5 mg) were added to an acetonitrile / water (6 / 4, v / v, 1 mL) solution of compound 36 (13 mg). The reaction mixture was stirred at room temperature for 15 minutes and then purified directly by RP-HPLC to give compound 40 (i.e., LD-15) as a white powder (10 mg).

[0621] MS m / z: 1262.2 [M+H] + .

[0622] 7.16 Preparation of LD-16

[0623] Compound 6-maleimide hexanoic acid N-hydroxysuccinimide ester (5 mg, Sigma-Aldrich) was added to a solution of compound 3 (12 mg) in dimethylformamide (1 mL), followed by diisopropylethylamine (5 μL). The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP-HPLC and lyophilized to give compound 41 (i.e., LD-16, 11 mg) as a white solid.

[0624] MS m / z: 1328.8 [M+H] + .

[0625] 7.17 Preparation of LD-17

[0626] A saturated aqueous solution of sodium bicarbonate (0.01 mL) and bromoacetic anhydride (5 mg) were added to an acetonitrile / water (6 / 4, v / v, 1 mL) solution of compound 3 (13 mg). The reaction mixture was stirred at room temperature for 15 minutes and then purified directly by RP-HPLC to give compound 42 (i.e., LD-17, 9 mg) as a white powder.

[0627] MS m / z: 1255.6 [M+H] + .

[0628] Example 8: Preparation of ADC

[0629] The antibody disclosed herein was reacted with a drug-conjugate (LD) to prepare an ADC drug, and the DAR value of the prepared ADC was determined.

[0630] In simple terms, antibodies are treated with reducing agents (such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT)) to reduce some or all of the cysteine ​​disulfide residues, forming highly nucleophilic cysteine ​​thiol groups (-CH2SH). This partially or completely reduced antibody then reacts with a drug conjugate and an electrophilic functional group (such as maleimide or bromoacetyl) to ultimately prepare an ADC.

[0631] Methods for determining the DAR value of ADC:

[0632] The DAR value of the ADC disclosed herein was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The ADC was separated in the column using a MabPac HIC-Butyl analytical column (4.6 x 100 mm, 5 μm, catalog number 088558, Thermo Fisher, USA). Chromatographic conditions: Mobile phase A was 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate, and mobile phase B was 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile. Gradient elution: 0–2 min 15% buffer B, 2–22 min 15%–85% buffer B, 22–25 min 85% buffer B, 25–28 min 85%–15% buffer B, 28–30 min 15% buffer B. Flow rate and temperature were set at 0.5 mL / min and 25 °C. Drug distribution in the ADC was detected at 214 nm and 280 nm to calculate the DAR value.

[0633] 8.1 Preparation of ADC-1

[0634] Antibody UC961 was dissolved in PBS at pH 7.2, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 2.8:1). After incubation at 37°C for approximately 120 minutes, drug linker LD-4 was added to the reduced antibody (molar ratio of drug linker to antibody was 5:1), and incubated at room temperature for 1 hour. The solution was purified and desalted by elution with G25 resin, and filtered through a 0.2 μm filter under sterile conditions to obtain ADC-1, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-1 was between 3.7 and 4.3.

[0635] 8.2 Preparation of ADC-2

[0636] Antibody AM3-ZH3 dissolved in PBS at pH 7.2 was mixed with 2 mM EDTA and reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio 10:1). After incubation at 37°C for approximately 120 minutes, drug linker LD-14 was added to the reduced antibody (drug linker:antibody molar ratio 5:1), and incubated at room temperature for 1 hour. The mixture was purified and desalted by elution with G25 resin, and filtered aseptically through a 0.2 μm filter to obtain ADC-2, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-2 was between 3.7 and 4.3.

[0637] 8.3 Preparation of ADC-3

[0638] YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were dissolved separately in PBS at pH 7.2 and mixed at a 1:1 molar ratio to obtain KIHYR8 / ZH3 antibody. 2 mM EDTA was added, and the KIHYR8 / ZH3 antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio 10:1). After incubation at 25°C for approximately 16 hours, the drug linker LD-14 was added to the reduced antibody KIHYR8 / ZH3 (drug linker:antibody molar ratio 5:1), and incubated at room temperature for 1 hour. The mixture was purified and desalted by elution with G25 resin, and filtered aseptically through a 0.2 μm filter to obtain ADC-3, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography determined that the average DAR value of ADC-3 was between 3.7 and 4.3.

[0639] 8.4 Preparation of ADC-4

[0640] YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were dissolved separately in PBS at pH 7.2 and mixed at a 1:1 molar ratio to obtain KIHYR8 / ZH3 antibody. 2 mM EDTA was added, and the KIHYR8 / ZH3 antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio of 10:1). After incubation at 25°C for approximately 16 hours, drug linker LD-1 was added to the reduced antibody KIHYR8 / ZH3 (drug linker:antibody molar ratio of 5:1), and incubated at room temperature for 1 hour. The mixture was purified and desalted by elution with G25 resin, and filtered aseptically through a 0.2 μm filter to obtain ADC-4, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-4 was between 3.7 and 4.3.

[0641] 8.5 Preparation of ADC-5

[0642] Antibody AM3-ZH3 was dissolved in PBS at pH 7.2, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio 10:1). After incubation at 37°C for approximately 120 minutes, 5 molar ratio of drug linker LD-1 was added to the reduced antibody (LD-1:antibody molar ratio 5:1), and incubated at room temperature for 1 hour. The solution was purified and desalted by elution with G25 resin, and filtered aseptically through a 0.2 μm filter to obtain ADC-5, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-5 was between 3.7 and 4.3.

[0643] In addition, using the same method as that used to prepare ADC-2 and ADC-5, negative control IgG and positive control UC961 antibodies were prepared and conjugated with LD-14 and LD-1, respectively, and named ADC-6 (UC961 conjugated with LD-14) and ADC-7 (UC961 conjugated with LD-1), respectively. The drug loading of ADC-8 (IgG conjugated with LD-14) and ADC-9 (IgG conjugated with LD-1) ranged from 3.7 to 4.3.

[0644] The exemplary ADCs prepared are summarized in Table 9 below:

[0645] Table 9. ADCs disclosed herein

[0646] In addition, this disclosure also uses the ADC preparation method in Example 8 to prepare the following ADC, wherein Ab is the anti-ROR1 antibody of this disclosure, or the bispecific antibody (bi-epitope antibody) of this disclosure, and n is 1-8.

[0647] Ab-LD-2:

[0648] Ab-LD-3:

[0649] Ab-LD-4:

[0650] Ab-LD-5:

[0651] Ab-LD-6:

[0652] Ab-LD-7:

[0653] Ab-LD-8:

[0654] Ab-LD-9:

[0655] Ab-LD-10:

[0656] Ab-LD-11:

[0657] Ab-LD-12:

[0658] Ab-LD-13:

[0659] Ab-LD-15:

[0660] Ab-LD-16:

[0661] Ab-LD-17:

[0662] Example 9: Detection of biological activity of monoclonal antibodies

[0663] 9.1 Binding activity of monoclonal antibodies to human ROR1 protein

[0664] Affinity assays for ROR1 protein were performed using the OctetR8 (Sartorius) instrument. An AHC (Anti-hIgG Fc Capture) biosensor (Sartorius) was used to capture the antibody, which was then immersed in the ROR1 antigen analyte. The experiment consisted of five steps:

[0665] 1. Baseline (60 seconds), 2. Loading (capture antibody) (150 seconds, 1.5nm), 3. Baseline (100 seconds), 4. Association (bind to antigen ROR1, 60 seconds), 5. Dissociation (dissociate antigen ROR1, 60 seconds).

[0666] After the test, the sensor was regenerated by alternating between regeneration buffer (glycine, pH 1.5) and neutralization buffer (PBS) for 5 seconds, for a total of 3 cycles. PBS was used as the running buffer in this experiment.

[0667] The results are shown in Table 10-13 below.

[0668] 9.2 Binding activity of monoclonal antibodies to human ROR1-expressing cells

[0669] Flow cytometry was used to detect the binding of the disclosed anti-ROR1 antibody to human ROR1-expressing cells MDA-MB-231 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) and Jeko-1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences). MDA-MB-231 / Jeko-1 cells were incubated with different concentrations of antibody (starting at 66.67 nM, 6-fold dilution, for a total of 8 concentrations) at 4°C for 30 minutes. After washing the cells twice with 2% BSA-PBS, PE-labeled goat anti-human IgG Fc (Invitrogen, catalog number: 12-4998-82, 1:100 dilution) was added, and the reaction was carried out at 4°C in the dark for 30 minutes. After washing the cells twice with 2% BSA-PBS, the median fluorescence intensity was detected using a BD C6 plus flow cytometer.

[0670] The results are shown in Table 10-13 below.

[0671] 9.3 Binding activity of monoclonal antibodies to human ROR2 protein

[0672] The binding of humanized antibody to ROR1 homolog ROR2 was detected using an ELISA method. hROR2 (manufacturer: Kaika Biotechnology, catalog number: ROR-HM402) was diluted to 1 μg / mL in PBS and coated onto microplates, then incubated at 37°C for 1 hour. The plates were then blocked with 5% BSA-PBS blocking buffer at 37°C for 1 hour. After washing with PBST, the antibody was diluted to different concentrations (starting from 333.33 nM, 3-fold dilutions, for a total of 8 concentrations) and added to the plates, then incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG (manufacturer: Sigma-Aldrich, catalog number A0170, 1:10000 dilution) was added, and the reaction was carried out at 37°C for 1 hour. After washing, TMB solution was added, and the reaction was incubated at room temperature in the dark for 15 minutes, then the reaction was terminated by adding ELISA Stopping Solution. The absorbance was measured at 450 nm using a microplate reader.

[0673] The results are shown in Table 10-13 below.

[0674] Table 10. Results of activity assay for the humanized antibody BRHu-3 at D10

[0675] Table 10 shows that the humanized antibody BRHu-3 and the ch-D10 antibody have similar affinity for ROR1 protein, and similar binding ability to MDA-MB-231 cells, and neither of them binds to ROR2.

[0676] Table 11. Results of BRHu-3 affinity maturation molecular activity assay

[0677] Table 11 shows that, compared with BRHu-3, the affinity maturation antibodies YR-4, YR-8, YR-10, YR-11, and YR-21 all exhibited significantly improved affinity for ROR1. Regarding binding to MDA-MB-231 and Jeko-1 cells, YR-8, YR-10, and YR-11 all demonstrated stronger cell-binding ability than the humanized antibody BRHu-3. Regarding binding to ROR2, YR-4, YR-10, YR-11, and YR-21 all bound to ROR2, while YR-8 did not bind to ROR2.

[0678] Table 12. Results of YR-8 mutant molecule activity assay

[0679] Table 12 shows that, except for YR-8 and YR-8-ES, all other mutants bind to ROR2. Compared with YR-8, YR-8-ES has comparable affinity and cell-binding activity to ROR1.

[0680] Table 13. Results of molecular activity assay for H10-VH4VL4 affinity maturation.

[0681] Table 13 shows that, compared to H10-hVH4-hVL4, the affinity maturation antibodies showed varying degrees of increased affinity for ROR1 and binding to MDA-MB-231 cells, especially AM3-ZH3, whose affinity increased by more than 9 times. Except for AM3-ZH1, none of the other molecules bound to ROR2.

[0682] Example 10: Antibody binding experiment with ROR1-expressing cells

[0683] The cell lines selected for this experiment were: PA-1 (ATCC CRL-1572 human ovarian teratoma cells) and Jeko-1 (ATCC CRL-3006, human mantle cell lymphoma cells). PA-1 cells were cultured in ATCC EMEM medium containing 10% fetal bovine serum, and Jeko-1 cells were cultured in RPMI-1640 medium containing 20% ​​fetal bovine serum at 37°C in a 5% CO2 incubator.

[0684] The experimental procedure is as follows:

[0685] Cells in logarithmic growth phase were lysed with 0.25% trypsin, resuspended in staining buffer (Biolengend), washed, counted, and adjusted to 4.5 × 10⁻⁶ cells / mL. 5Add 45 μL of staining buffer per 100 μL of cells to each well of a 96-well plate. Add 5 μL of human TruStain (BioLegend) receptor blocking agent to each well to prevent non-specific binding of the antibody to human tumor cells. Dilute the test antibody to an initial concentration of 200 nM in staining buffer, then perform 3-fold dilutions to obtain 11 concentration points, including 200 nM and zero. Add 50 μL of the diluted test antibody to each well of the 96-well plate, resulting in a final maximum concentration of 100 nM. Mix well and incubate at 4°C for 15 minutes. After the reaction, wash the cells in staining buffer, then resuspend the PE-labeled constant region (Fc) specific antibody (rabbit anti-human IgG PE conjugate, BioLegend, 410707) in 5 μL / 2×10⁻⁶ cells / well. 5 Cells were incubated at 100 μL staining buffer per cell and reacted at 4 °C for 15 min. After the reaction, cells were washed in staining buffer and resuspended in 100 μL staining buffer. Single-cell readings on the PE channel were analyzed using a Novocyte 3000 (Agilent) instrument. A dose-response curve was plotted using the Sigmoidal,4PL four-parameter equation, with antibody protein concentration on the x-axis and the corresponding PE channel reading on the y-axis. EC was generated after analysis. 50 Value. The equation is:

[0686] Y=Bottom+(X^Hillslope)*(Top-Bottom) / (X^HillSlope+EC 50 ^HillSlope),

[0687] MFI fold = highest PE reading in the antibody-treated experimental group / PE reading in the untreated group;

[0688] The results of FACS binding activity of anti-hROR1 antibody to human ROR1 are shown in Table 14.

[0689] The results showed that the anti-ROR1 antibodies disclosed herein could specifically bind to PA1 and Jeko1 cells expressing ROR1, and the binding ability was high. Furthermore, on the same cell line, the total amount of antigen that the dual-epitope antibodies could recognize and label was higher than that of the single-epitope antibodies, specifically manifested as a significant increase in the MFI fold ratio.

[0690] Table 14. Binding ability of anti-hROR1 antibody to cell lines

[0691] Example 11: Cytotoxicity assay of ADC molecules

[0692] This experiment investigated the inhibitory effects of anti-ROR1 antibody-MMAE and anti-ROR1 antibody-Eribulin conjugates on the proliferation of various tumor cell lines. CellTiterGlo2 (Promega) reagent was used to evaluate the anti-proliferative effects of the drugs. The cell lines used in the experiment were: PA-1 (ATCC, CRL-1572 human ovarian teratoma cells), Jeko-1 (ATCC, CRL-3006, human mantle cell lymphoma cells), and HCC-1187 (ATCC, CRL-2322 human breast cancer cells). The experimental procedure is as follows:

[0693] PA-1 cells were cultured in ATCC EMEM medium containing 10% fetal bovine serum, Jeko-1 cells in RPMI-1640 medium containing 20% ​​fetal bovine serum, and HCC-1187 cells in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The three cell types were cultured at 2 × 10⁻⁶ cells / mL. 3 -5×10 3 Cells were seeded at a density of 50 μL / well in 96-well plates. After culturing for 24 h, 100 μL / well of anti-ROR1 antibody-MMAE, anti-ROR1 antibody-Eribulin conjugate, or control antibody-MMAE or control antibody-Eribulin conjugate diluted with different concentrations of culture medium was added to each well. Each concentration was replicated, and wells with corresponding solvent controls and cell-free culture medium were also included. After culturing at 37°C, 5% CO2 for 96 h (PA-1, Jeko-1) or 144 h (HCC-1187), 100 μL of CellTiterGlo2 was added to each well. The plates were mixed for 15 min at room temperature on a shaker. The luminescence value was measured, and the IC50 of anti-ROR1 antibody-MMAE or anti-ROR1 antibody-Eribulin conjugate against various cell types was calculated. 50 Value (nM). The results are shown in Table 15.

[0694] Table 15. IC50 of ADCs for inhibiting different cell proliferation 50 Value (nM)

[0695] As shown in Table 15, the anti-ROR1 antibody-MMAE or -Eribulin conjugates exhibited significant killing effects on three tumor cell lines with different ROR1 expression levels. The inhibitory effect was related to the different conjugation methods or different toxins used. This indicates that the disclosed anti-ROR1 antibody conjugates can specifically kill ROR1-positive cells through ROR1-mediated endocytosis. Furthermore, they showed significant inhibitory effects on cancer cell lines from breast cancer, ovarian cancer, and lymphoma, respectively, demonstrating that the disclosed ADC has good inhibitory effects on various solid tumors and hematological malignancies.

[0696] Example 12: In vivo efficacy testing of ADC molecules

[0697] 12.1 ADC Efficacy Testing in a Mouse Model Transplanted with HCC1187 Breast Cancer Cell Line

[0698] 1×10 7 One cell / human ROR1-expressing breast cancer cell line HCC1187 (purchased from ATCC) was transplanted subcutaneously into female severely combined immunodeficient CB17 SCID mice (purchased from Charles River Laboratories). Following transplantation, when the tumor size reached an average of 162 mm... 3 On day 0, mice were divided into groups and administered either a single intravenous injection of 3.0 mg / kg ADC-1, ADC-2, ADC-3, ADC-4, or ADC-5 prepared in Example 8, or a total of two intravenous injections of 1.0 mg / kg ADC-2, ADC-3, ADC-4, or ADC-5 every four days (day 0 and day 4, Q4D×2). In the control group, mice were administered 4 mL / kg PBS intravenously. For the next 48 days, tumor size and body weight were measured, and tumor inhibition rate (TGI) was calculated. The TGI was calculated as follows: TGI(%) = 1 - [(Td-T0) / (Cd-C0)] × 100%

[0699] Where Td and Cd are the average tumor volumes of the treatment group and the control group on the day of tumor volume measurement, and T0 and C0 are the average tumor volumes of the treatment group and the control group on day 0.

[0700] The results are shown in Figure 1 and Table 16.

[0701] Table 16. Tumor inhibition rate of different ADCs on subcutaneous tumors in HCC1187 mice

[0702] The results showed that the ADC disclosed in this study could significantly inhibit the growth of HCC1187 xenografts in mice.

[0703] 12.2 ADC Efficacy Testing in a Jeko-1 Cellular Lymphoma Cell Line Transplanted Mouse Model

[0704] 1×10 7 Jeko-1 mantle cell lymphoma cells (purchased from ATCC) expressing only human ROR1 were transplanted subcutaneously into female severely combined immunodeficient CB17 SCID mice (purchased from Charles River Laboratories). Following transplantation, tumors reached an average size of 156 mm. 3On day 0, mice were divided into groups and administered either a single intravenous injection of 3.0 mg / kg ADC-1, ADC-2, ADC-3, ADC-4, or ADC-5 prepared in Example 8, or 1.0 mg / kg ADC-2, ADC-3, ADC-4, or ADC-5 intravenously every four days for a total of three times (day 0, day 4, and day 8, Q4D×3). In the control group, mice were administered 4 mL / kg PBS intravenously. After 51 days, tumor size and body weight were measured in the transplanted mice, and the tumor inhibition rate (TGI) was calculated. The results are shown in Figure 2 and Table 17.

[0705] Table 17. Tumor inhibition rate of different ADCs on subcutaneous tumors in Jeko-1 mice

[0706] The results showed that the ADC disclosed in this study could significantly inhibit the growth of Jeko-1 xenografts in mice.

[0707] 12.3 ADC Efficacy Testing in PA-1 Ovarian Cancer Cell Transplantation Mouse Model

[0708] 5×10 6 One cell / one human ROR1-expressing ovarian cancer cell line PA-1 (purchased from ATCC) was transplanted subcutaneously into female nude mice (purchased from Charles River Laboratories). Following transplantation, when the tumor size reached an average of approximately 150 mm... 3 On day 0, mice were divided into groups and administered a single intravenous injection of 1.0 mg / kg or 3.0 mg / kg ADC1 or ADC3 prepared in Example 8. In the control group, mice were administered 4 mL / kg PBS intravenously. Tumor size and body weight were measured and TGI were calculated over 13 days. Results are shown in Figure 3 and Table 18.

[0709] Table 18. Tumor inhibition rate of different ADCs on subcutaneous tumors in PA-1 mice

[0710] The results showed that the ADC disclosed in this study could significantly inhibit the growth of PA-1 xenografts in mice.

[0711] Example 13: Phagocytic activity of ROR1-ADC combined with anti-SIRPα antibody against cancer cells

[0712] (1) Induction and preparation of macrophage M2

[0713] Fresh human PBMCs (purchased from Shanghai Heyousheng Biotechnology) were centrifuged at 400g for 10 min, and the cell pellet was collected and resuspended in RPMI 1640 basal medium (Sigma, catalog number R8758) to a cell density of 1E8 / mL. The cells were cultured at 37℃ and 5% CO2 for 2 h to allow adhesion. The culture supernatant was removed, and the cells were washed once. The medium was then replaced with complete RPMI 1640 medium containing 80 ng / mL human M-CSF (purchased from MedChemExpress, catalog number HY-P7050) for induction, changing the medium every 3 days. After 7 days of induction, the culture supernatant was removed, and complete RPMI 1640 medium containing 20 ng / mL human IL-10 (purchased from PEPROTech, 200-10-10UG) and 80 ng / mL M-CSF was added. The cells were cultured for another 2 days to induce M2 macrophages. Cells were digested with trypsin and collected, then resuspended in cell culture medium and the cell density was adjusted to 2E6 / mL.

[0714] (2) Preparation and staining of target cells

[0715] Gastric cancer cells (SNU-601), endometrial cancer cells (RL95-2), and non-small cell lung cancer cells (HCC827), all purchased from Nanjing Kebai, were collected. Cells were washed twice with PBS and resuspended in PBS to a cell count of 1E6 / mL. 0.3 μL of 1 mM CFSE (Abcam, catalog number AB113853) was added to each mL of cell suspension, and the mixture was incubated at 37°C for 10 min. The reaction was terminated by adding 5 volumes of complete culture medium. The cells were centrifuged, the supernatant was discarded, and the cells were washed once with culture medium. The cells were then resuspended in cell culture medium, and the cell density was adjusted to 4E6 / mL.

[0716] (3) ADCP experiment

[0717] Dilute ADC-3 to a final working concentration of 0.5 μg / mL with complete RPMI 1640 medium, and dilute anti-SIRPα antibody 14# or isotype control antibody human IgG1 (purchased from Sino Biological Inc., catalog number HG1K) to a final working concentration of 10 μg / mL. Add 25 μL of target cells, 16.7 μL of drug, and 25 μL of macrophages sequentially to 1.5 mL centrifuge tubes. Incubate at 37°C and 5% CO2 for 2 hours, then centrifuge and discard the supernatant. Wash once with flow cytometry staining buffer (purchased from Biolegend, catalog number 420201). Prepare a blocking buffer containing 5 μg / mL IgG1 in 100 μL per tube using flow cytometry staining solution. Incubate on ice for 30 min, then centrifuge and wash once. Add 100 μL of cell staining solution containing 5 μL of APC Anti-huCD14 antibody (Invitrogen, catalog number 2763960) to each tube, incubate on ice for 30 min, centrifuge and wash twice. Resuspend cells in cell staining buffer, analyze by flow cytometry, and calculate the phagocytic rate of macrophages on target cells.

[0718] Phagocytosis (%) = A / (A+B)×100, where A is the number of FITC-positive cells and B is the number of APC- and FITC-co-positive cells.

[0719] The results are shown in Figures 4A-4C.

[0720] The results showed that the combination of ADC-3 and anti-SIRPα antibody 14# significantly improved the phagocytic capacity of macrophages for cancer cells compared with each single-drug group.

[0721] Example 14: Killing of MDA-MB-468 cells by combination of ROR1-ADC and anti-PD-1 / L1 antibody

[0722] (1) Activation of human PBMCs

[0723] CD3 / CD28 antibodies (purchased from Invitrogen, catalog numbers 16-0037-81 and 16-0289-81) were coated onto culture dishes at a concentration of 5 μg / mL one day in advance and incubated overnight at 4°C. Fresh human PBMCs (purchased from Ausen Biotech) were centrifuged, counted, and then analyzed at 1×10⁻⁶. 6 A concentration of 100 cells / mL was prepared in a petri dish and placed in an incubator at 37°C for 3 days for activation.

[0724] (2) Co-culture of PBMCs with tumor cells to kill tumor cells

[0725] MDA-MB-468 cells were digested, centrifuged, counted, and then analyzed at 5 × 10⁻⁶ cells. 350 μL of ADC-3 was added to each well of a 96-well plate, and the next day, the plates were treated with ADC-3. ADC-3 was prepared to a 10× drug concentration, and 10 μL was added to each well of a 96-well plate containing MDA-MB-468 cells. The plates were incubated at 37°C. In the experiment involving ADC-3 in combination with pembrolizumab (purchased from Selleck, catalog number A200506), the final concentration of ADC-3 was 3 nM. In the experiment involving ADC-3 in combination with avirulumab (purchased from Selleck, catalog number A201504), the final concentration of ADC-3 was 0.4 nM.

[0726] After 48 hours of incubation, human PBMCs activated with CD3 / CD28 antibody were centrifuged, counted, and then analyzed at 1×10⁻⁶. 4 Add 30 μL of reagent per well to a 96-well plate, simultaneously administering pembrolizumab and avermectin. Both pembrolizumab and avermectin are prepared at a 10× concentration and added in 10 μL increments to the 96-well plate. The concentration of pembrolizumab is 50 μg / mL, and the concentration of avermectin is 10 μg / mL. Incubate the plate at 37°C.

[0727] After 48 hours of incubation, 100 μL of Cell Titer-Glo reagent (Promega, catalog number G7572) was added to each well. The culture plate was placed on a microplate shaker and mixed for 3 minutes. The plate was then incubated at room temperature for 15 minutes, and the luminescence signal was detected using a microplate reader. The killing rate of ADC-3 combined with pembrolizumab / avirumab against MDA-MB468 cells was calculated. The results are shown in Figures 5A and 5B.

[0728] The results showed that the ADC-3 combined with pembrolizumab significantly enhanced the killing effect on MDA-MB-468 cells compared with the ADC-3 and pembrolizumab monotherapy groups. Similarly, the ADC-3 combined with avelumab group showed a significantly stronger killing effect on MDA-MB-468 cells than the monotherapy groups.

[0729] Example 15: Killing of SNU-601 cells by ROR1-ADC in combination with anti-PD-1 / L1 antibody

[0730] 1. Activation of human PBMCs

[0731] CD3 / CD28 antibodies (purchased from Invitrogen, catalog numbers 16-0037-81 and 16-0289-81) were coated onto culture dishes at a concentration of 5 μg / mL one day in advance and incubated overnight at 4°C. Fresh human PBMCs (purchased from Ausen Biotech) were centrifuged and counted, then analyzed at 1×10⁻⁶. 6A concentration of 100 cells / mL was prepared in a petri dish and placed in an incubator at 37°C for 3 days for activation.

[0732] 2. PBMC co-culture with tumor cells to kill tumor cells

[0733] Human gastric cancer cell line SNU-601 was digested, centrifuged, counted, and then processed at a concentration of 5 × 10⁻⁶ cells. 3 Add 50 μL of ADC-3 to each well of a 96-well plate, and treat with ADC-3 the next day. The final concentration of ADC-3 is 5 nM. Prepare a 10× drug concentration of ADC-3 and add 10 μL to each well of a 96-well plate containing SNU-601 cells. After incubating the culture plate at 37°C for 48 hours, centrifuge and count the CD3 / CD28 antibody-activated human PBMCs, and then perform a 1×10⁻⁶ PCR. 4 30 μL of a 10 μg / mL Avelumab solution was added to each well of a 96-well plate, along with 10 μL of the Avelumab solution. After incubating the plates at 37°C for 48 hours, 100 μL of CellTiter-Glo reagent (Promega, catalog number G7572) was added to each well. The plates were then mixed on a microplate shaker for 3 minutes, followed by incubation at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader. The cytotoxic effect of ADC-3 combined with Avelumab on SNU-601 cells was calculated. The results are shown in Figure 6.

[0734] The results showed that, compared with the ADC-3 and Avelumab monotherapy groups, ADC-3 combined with Avelumab significantly enhanced the killing effect of PBMCs on SNU-601 cells.

[0735] Example 16: The killing effect of ROR1-ADC combined with vincristine on cancer cells

[0736] Breast cancer cells MDA-MB-468 (purchased from ATCC) were cultured in DMEM medium (purchased from Sigma, catalog number D6429) containing 10% fetal bovine serum (purchased from Excel, catalog number FND500) at 37°C in a 5% CO2 incubator. Cells were collected by centrifugation, washed with PBS, stained with trypan blue for counting, and the cell density was adjusted to 3 × 10⁶ cells / year. 3Cells were seeded at a density of 90 μL per well in 96-well plates. After 24 h of culture, 10 μL of different concentrations of ADC-3 or vincristine (purchased from Maclean's, catalog number 57-22-7) or a mixture of both drugs were added to each well. The final working concentrations of ADC-3 ranged from 0.0625 nM to 4 nM, with 2-fold dilutions for a total of 7 concentrations. The final working concentration of vincristine was 0.25 nM. DMEM medium served as a blank control. After culturing at 37°C and 5% CO2 for 120 h, 100 μL of CellTiter-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was then placed on a shaker and mixed for 4 minutes. Subsequently, the culture plate was incubated at room temperature for 20 minutes. The luminescence signal was detected using a microplate reader, and the killing effect on MDA-MB-468 cells was calculated. The results are shown in Figure 7.

[0737] The results showed that both ADC-3 and vincristine had significant killing effects on MDA-MB-468 cells. Compared with the single-drug group, the combined administration group significantly enhanced the killing effect on MDA-MB-468 cells, and the two had a synergistic effect.

[0738] Example 17: The killing effect of ROR1-ADC in combination with chemotherapy drugs on cancer cells

[0739] Jeko-1 (mantle cell lymphoma, Chinese Academy of Sciences Cell Bank) or Ri-1 (diffuse large B-cell lymphoma cell line, purchased from Nanjing Kebai Biotechnology) cells were collected by centrifugation, resuspended in 1640 complete medium, and then stained with trypan blue for counting. The cell density was adjusted to 3.33 × 10⁻⁶ cells / year. 4 Add 90 μL of cell suspension to each well of a 96-well white plate and incubate overnight at 37°C. The next day, add 5 μL of solvent (1640 complete medium) or ADC-3 drug, and 5 μL of solvent (1640 complete medium) or vincristine (purchased from Maclean's) / cisplatin (purchased from Maclean's) / etoposide (purchased from Selleck's), respectively, to each well. The final drug concentrations are shown in Table 19. Incubate the plate at 37°C for 5 days. Remove the plate, allow it to reach room temperature, add 100 μL of CellTiter-Glo detection solution, shake for 2 minutes in a microplate reader, incubate at room temperature in the dark for 10 minutes, then centrifuge the plate at 500g for 3 minutes. Read the data using a Tecan microplate reader and analyze the data using Graphpad software. The experimental results are shown in Figures 8A, 8B, and 8C.

[0740] Table 19. Drug Types and Dosage Concentrations

[0741] The results showed that the combination of ADC-3 and vincristine significantly inhibited the growth of Ri-1 cells compared to either ADC-3 or vincristine alone, and the two drugs exhibited a synergistic inhibitory effect. Similarly, the combination of ADC-3 and cisplatin / etoposide significantly inhibited the growth of Jeko-1 cells compared to either drug alone, and the two drugs also showed a synergistic inhibitory effect. These experiments fully demonstrate that the combined use of ADC-3 and chemotherapy drugs can significantly enhance the killing effect of chemotherapy drugs on tumor cells.

[0742] Example 18: The killing effect of ROR1-ADC combined with BCL-2 inhibitor on cancer cells

[0743] Ri-1 cells (diffuse large B-cell lymphoma cell line, purchased from Nanjing Kebai Biotechnology) were collected by centrifugation, resuspended in 1640 complete medium, and counted using trypan blue staining. The cell density was adjusted to 3.33E4 / mL, and 90 μL of cell suspension was added to each well of a 96-well white plate. The plate was incubated overnight at 37°C. The next day, 5 μL of solvent control (1640 complete medium) or ADC-3 (final concentration 0.14 nM) and 5 μL of solvent control or Veneclair (final concentration 3 nM) were added to each well, and the plate was incubated at 37°C for 5 days. After removing the plate and allowing it to return to room temperature, 100 μL of CellTiter-Glo detection solution was added. The plate was shaken for 2 minutes in a microplate reader, incubated at room temperature in the dark for 10 minutes, and then centrifuged at 500g for 3 minutes. Data were read using a Tecan microplate reader and analyzed using Graphpad software. The results are shown in Figure 9.

[0744] The results showed that the growth inhibition effect of the combination of ADC-3 and Veneclare was significantly better than that of the single-drug group, that is, ADC-3 can enhance the inhibition of target cell growth by Veneclare and the two have a synergistic effect.

[0745] Example 19: The killing effect of ROR1-ADC in combination with HS006 and peglucosamine on cancer cells

[0746] NU-DUL-1 diffuse large B-cell lymphoma cells (purchased from Shanghai Qida) were cultured in RPMI-1640 complete medium (purchased from Gibco, catalog number 11875119) containing 15% fetal bovine serum (purchased from Gibco, catalog number 10099141C) at 37°C in a 5% CO2 incubator. Cells were collected by centrifugation, counted by trypan blue staining, and the cell density was adjusted to 5 × 10⁶ cells / year. 3Cells were seeded at a density of 90 μL per well into 96-well plates. After culturing for 24 h, 10 μL of ADC-3, HS006, pephosphatamide (an active metabolite of cyclophosphamide, purchased from MCE, catalog number HY-117433), or a mixed drug combination of ADC-3, HS006, and pephosphatamide diluted in RPMI-1640 complete medium were added to each well. The working concentrations of ADC-3, HS006, and pephosphatamide were 1.25 nM, 10 μg / mL, and 200 nM, respectively. The solvent medium was a blank control group. After culturing at 37℃ and 5% CO2 for 96 h, 100 μL of Cell Titer-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was then placed on a fixed-track shaker for 4 minutes to mix, followed by incubation at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader, and the cell viability of NU-DUL-1 cells at each drug concentration was calculated to evaluate the in vitro cell-killing ability of the drug. The results are shown in Figure 10.

[0747] The results showed that, compared with the single-drug group, ADC-3 combined with HS006 and cyclophosphamide (the active metabolite of cyclophosphamide) had a significant killing effect on NU-DUL-1 cells.

[0748] Example 20: The killing effect of ROR1-ADC in combination with HS006 and doxorubicin on cancer cells

[0749] NU-DUL-1 diffuse large B-cell lymphoma cells (purchased from Shanghai Qida) were cultured in RPMI-1640 complete medium (purchased from Gibco, catalog number 11875119) containing 15% fetal bovine serum (purchased from Gibco, catalog number 10099141C) at 37°C in a 5% CO2 incubator. Cells were collected by centrifugation, counted by trypan blue staining, and the cell density was adjusted to 5 × 10⁶ cells / year. 3Cells were seeded at a density of 90 μL per well into 96-well plates. After culturing for 24 h, 10 μL of ADC-3, HS006, doxorubicin (purchased from Selleck, catalog number E2561), or a mixture of ADC-3, HS006, and doxorubicin diluted in RPMI-1640 complete medium were added to each well. The working concentrations of ADC-3, HS006, and doxorubicin were 1.25 nM, 10 μg / mL, and 2.634 nM, respectively. The solvent medium was a blank control group. After culturing at 37℃ and 5% CO2 for 96 h, 100 μL of Cell Titer-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was placed on a fixed-track shaker and mixed for 4 minutes. Then, the culture plate was incubated at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader, and the cell viability of NU-DUL-1 cells at each drug concentration was calculated to evaluate the in vitro killing ability of the drug on cells. The results are shown in Figure 11.

[0750] The results showed that ADC-3 and doxorubicin both had significant killing effects on NU-DUL-1 cells. Compared with the single drug group, the combined drug group significantly enhanced the killing effect on NU-DUL-1 cells, and ADC-3 and doxorubicin had a synergistic effect on the killing effect on NU-DUL-1 cells.

[0751] Example 21: The killing effect of ROR1-ADC in combination with HS006 and prednisolone on cancer cells

[0752] NU-DUL-1 diffuse large B-cell lymphoma cells (purchased from Shanghai Qida) were cultured in RPMI-1640 complete medium (purchased from Gibco, catalog number 11875119) containing 15% fetal bovine serum (purchased from Gibco, catalog number 10099141C) at 37°C in a 5% CO2 incubator. Cells were collected by centrifugation, counted by trypan blue staining, and the cell density was adjusted to 5 × 10⁶ cells / year. 3Cells were seeded at a density of 90 μL per well into 96-well plates. After culturing for 24 h, 10 μL of ADC-3, HS006, prednisolone (purchased from Selleck, catalog number S1737), or a mixture of ADC-3, HS006, and prednisolone diluted in RPMI-1640 complete medium were added to each well, so that the working concentration of ADC-3 was 1.25 nM, the working concentration of HS006 was 10 μg / mL, and the working concentration of prednisolone was 100 nM. The solvent medium was used as a blank control group. After culturing at 37℃ and 5% CO2 for 96 h, 100 μL of Cell Titer-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was then placed on a fixed-track shaker for 4 minutes to mix, followed by incubation at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader, and the cell viability of NU-DUL-1 cells at each drug concentration was calculated to evaluate the in vitro cell-killing ability of the drug. The results are shown in Figure 12.

[0753] The results showed that ADC-3 and prednisolone (the active metabolite of prednisolone) had a certain killing effect on NU-DUL-1 cells. Compared with the single drug group, the combined drug group significantly enhanced the killing effect on NU-DUL-1 cells, and ADC-3 and prednisolone had a synergistic effect on the killing effect on NU-DUL-1 cells.

[0754] Example 22: The killing effect of ROR1-ADC combined with gemcitabine on cancer cells

[0755] Triple-negative breast cancer cells MDA-MB-468 (purchased from Nanjing Kebai) were cultured in DMEM medium (purchased from Gibco, catalog number 11995065) containing 10% fetal bovine serum (purchased from Gibco, catalog number 10099141C), i.e., complete DMEM medium, at 37°C in a 5% CO2 incubator. Cells were collected by centrifugation, stained with trypan blue for counting, and the cell density was adjusted to 3 × 10⁶ cells / year. 3Cells were seeded at a density of 90 μL per well in 96-well plates. After 24 h of culture, 10 μL of ADC-3, gemcitabine (purchased from Selleck, catalog number S1714), or a mixture of ADC-3 and gemcitabine diluted in DMEM complete medium were added to each well, bringing the working concentration of ADC-3 to 0.25 nM and the working concentration of gemcitabine to 3.125 nM. The medium was a blank control. After culturing at 37°C and 5% CO2 for 120 h, 100 μL of Cell Titer-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was then placed on a shaker to mix for 4 minutes, followed by incubation at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader, and the cell viability of MDA-MB-468 cells at each drug concentration was calculated to evaluate the in vitro cell-killing ability of the drugs. The results are shown in Figure 13.

[0756] The results showed that ADC-3 and gemcitabine had a certain killing effect on MDA-MB-468 cells. Compared with the single drug group, the combined drug group significantly improved the killing effect on MDA-MB-468 cells, and ADC-3 and gemcitabine had a synergistic effect on the killing effect on NU-DUL-1 cells.

[0757] Example 23: The killing effect of ROR1-ADC combined with paclitaxel on cancer cells

[0758] Triple-negative breast cancer cells MDA-MB-468 (purchased from Nanjing Kebai) were cultured in DMEM medium (purchased from Gibco, catalog number 11995-065) containing 10% fetal bovine serum (purchased from Gibco, catalog number 10099141C), i.e., complete DMEM medium, at 37°C in a 5% CO2 incubator. Cells were collected by centrifugation, counted by trypan blue staining, and the cell density was adjusted to 3 × 10⁶ cells / year. 3 Cells were seeded at a density of 90 μL per well in 96-well plates. After 24 h of culture, 10 μL of ADC-3, paclitaxel (purchased from Selleck, catalog number S1150), or a mixture of ADC-3 and paclitaxel diluted in DMEM complete medium were added to each well, bringing the working concentration of ADC-3 to 0.25 nM and the working concentration of paclitaxel to 20 nM. The medium was a blank control. After culturing at 37°C and 5% CO2 for 96 h, 100 μL of Cell Titer-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was mixed on a shaker for 4 minutes, and then incubated at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader, and the cell viability of MDA-MB-468 cells at each drug concentration was calculated to evaluate the in vitro cell-killing ability of the drugs. The results are shown in Figure 14.

[0759] The results showed that ADC-3 and paclitaxel had a certain killing effect on MDA-MB-468 cells. Compared with the single drug group, the combined drug group significantly improved the killing effect on MDA-MB-468 cells, and ADC-3 and paclitaxel had a synergistic effect on the killing effect on NU-DUL-1 cells.

[0760] Example 24: ROR1-ADC combined with Hi-CHP and R-CHP to kill cancer cells

[0761] NU-DUL-1 diffuse large B-cell lymphoma cells (purchased from Shanghai Qida) were cultured in RPMI-1640 medium (purchased from Gibco, catalog number C22400500BT) containing 10% fetal bovine serum (purchased from Ecosai, catalog number FND500) at 37°C in a 5% CO2 incubator. NU-DUL-1 cells in the logarithmic growth phase were collected, trypsinized, centrifuged, and counted, and then cultured at 6 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 80 μL / well in 96-well plates. The next day, the cells were treated with 10 μL of RPMI solution in each well. ADC-3 and Hi-CHP (where Hi represents zabetumumab, C represents pefosamide, H represents doxorubicin, and P represents prednisolone) or R-CHP (where R represents rituximab, C represents pefosamide, H represents doxorubicin, and P represents prednisolone) combination drugs were diluted in 1640 complete medium. The working concentration of ADC-3 was 1.2 nM, the working concentrations of zabetumumab and rituximab (purchased from Roche, catalog number SH0195) were both 400 nM, the working concentration of pefosamide (purchased from MCE, HY-117433) was 30 nM, the working concentration of doxorubicin (purchased from Selleck, E2516) was 0.5 nM, and the working concentration of prednisolone (purchased from Selleck, S1737) was 100 nM. Each group was configured with 3 replicates. After culturing 96 wells at 37°C in a 5% CO2 incubator for 96 hours, 50 μL of CellCounting-Lite 2.0 reagent (purchased from Vazyme, catalog number DD1101-02) was added to each well. The culture plate was then placed on a microplate shaker and mixed for 5 minutes. Following this, the culture plate was incubated at room temperature for 10 minutes. The luminescence signal was detected using a microplate reader, and the viability of NU-DUL-1 cells after drug treatment in each group was calculated to assess the in vitro killing activity of the drug on cells. The results are shown in Figure 15.

[0762] The results showed that ADC-3, Hi-CHP and R-CHP groups all showed certain killing ability against NU-DUL-1 cells. Compared with ADC-3 alone, Hi-CHP and R-CHP groups, ADC-3 combined with Hi-CHP / R-CHP significantly enhanced the killing effect on NU-DUL-1 cells and showed a synergistic effect.

[0763] Example 25: ROR1-ADC combined with Hi-CHOP and R-CHOP to kill cancer cells

[0764] NU-DUL-1 diffuse large B-cell lymphoma cells (purchased from Shanghai Qida) were cultured in RPMI-1640 medium (purchased from Gibco, catalog number C22400500BT) containing 10% fetal bovine serum (purchased from Ecosai, catalog number FND500) at 37°C in a 5% CO2 incubator. NU-DUL-1 cells in the logarithmic growth phase were collected, trypsinized, centrifuged, and counted, and then cultured at 6 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 80 μL / well in 96-well plates. The next day, the cells were treated with drugs by adding 10 μL of either ADC-3 diluted in RPMI 1640 complete medium or a combination of Hi-CHOP (where Hi represents zabetumumab, C represents pefosamide, H represents doxorubicin, O represents vincristine, and P represents prednisolone) or R-CHOP (where R represents rituximab, C represents pefosamide, H represents doxorubicin, O represents vincristine, and P represents prednisolone) or R-CHOP (where R represents rituximab, C represents pefosamide, H represents doxorubicin, O represents vincristine, and P represents prednisolone) to each well. The working concentration of ADC-3 was 1.2 nM. Zabetumumab and rituximab (purchased from Roche) were also added. The working concentrations of the following drugs were as follows: SH0195 (400 nM), phosphatidylcholine (30 nM, purchased from MCE, catalog number HY-117433), doxorubicin (0.5 nM, purchased from Selleck, catalog number E2516), prednisolone (100 nM, purchased from Selleck, catalog number S1737), and vincristine (0.13 nM, purchased from Selleck, catalog number S1241). Each group was configured with 3 replicates. After culturing 96 wells at 37°C in a 5% CO2 incubator for 96 hours, 50 μL of CellCounting-Lite 2.0 reagent (purchased from Vazyme, catalog number DD1101-02) was added to each well. The culture plate was then placed on a microplate shaker and mixed for 5 minutes. The plate was then incubated at room temperature for 10 minutes. The luminescence signal was detected using a microplate reader, and the viability of NU-DUL-1 cells after drug treatment in each group was calculated to assess the in vitro killing activity of the drug on cells. The results are shown in Figure 16.

[0765] The results showed that ADC-3, Hi-CHOP and R-CHOP groups all showed certain killing ability against NU-DUL-1 cells. Compared with ADC-3 alone, Hi-CHOP and R-CHOP groups, ADC-3 combined with Hi-CHOP / R-CHOP significantly enhanced the killing effect on NU-DUL-1 cells and showed a synergistic effect.

[0766] Example 26: Construction of cell lines resistant to HER2 antagonists

[0767] A cell line resistant to trastuzumab (Enhertu) was constructed using human lung cancer cells NCI-H2170 (purchased from Nanjing Kebai Biotechnology Co., Ltd.).

[0768] The resistance curve of NCI-H2170 to trastuzumab was detected, and IC50 was used. 30 As the initial dosing concentration, it is then increased to the IC50 concentration. 50 As the initial drug concentration, the concentration of trastuzumab was continuously increased, and the cells were cultured for more than 6 months until NCI-H2170 cells, namely NCI-H2170 / Enhertu-R cells, were screened out that could still grow normally at an Enhertu concentration of 13.1 nM. Furthermore, NCI-H2170 / Enhertu-R cells could still maintain drug resistance after continuous passage, cryopreservation and thawing.

[0769] Drug resistance testing:

[0770] NCI-H2170 and NCI-H2170 / Enhertu-R cells were digested with trypsin, counted, and seeded in 96-well plates at a density of 4000 / 5000 cells per well. The next day, the cells were treated with trastuzumab (DS8201, prepared according to WO2015115091A1), trastuzumab (purchased from Selleck, catalog number A2007), and DXd (Exatecan derivative, purchased from Selleck, catalog number E2891). The initial concentration of trastuzumab was 333.3 nM, and the cells were serially diluted 5-fold to obtain 9 spots.

[0771] In NCI-H2170 / Enhertu-R cells with a trastuzumab resistance concentration of 5.23 nM, the initial concentration of trastuzumab was 343.6 nM, and it was serially diluted 5-fold to obtain 9 spots. The initial concentration of DXd was 100 μM, and it was serially diluted 5-fold to obtain 9 spots.

[0772] In NCI-H2170 / Enhertu-R cells with a trastuzumab resistance concentration of 6.54 nM, DXd was initially diluted at a concentration of 10 μM and then serially diluted 5-fold to obtain 9 spots.

[0773] In NCI-H2170 / Enhertu-R cells with a trastuzumab resistance concentration of 13.1 nM, DXd was initially diluted to 100 nM and then serially diluted 3-fold to obtain 9 spots.

[0774] A blank control group was set up, with three replicates per group. After treatment with trastuzumab and DXd for 5-6 days, 100 μL of Cell Titer-Glo reagent (Promega, catalog number G7572) was added to each well. The culture plate was placed on a microplate shaker and mixed for 3 minutes. Then, the culture plate was incubated at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader. In vitro proliferation inhibition curves of trastuzumab, trastuzumab, and DXd on NCI-H2170 and NCI-H2170 / Enhertu-R cells were plotted, and the IC50 was calculated. 50 Values. The results are shown in Table 20 and Figures 17-23.

[0775] Table 20. In vitro cell proliferation inhibition IC50 50 (nM)

[0776] The results showed that trastuzumab significantly inhibited the in vitro proliferation of NCI-H2170 cells, while NCI-H2170 / Enhertu-R resistant cells exhibited consistent and stable resistance to trastuzumab. Simultaneously, the toxin DXd of trastuzumab showed no significant difference in inhibitory effects on the proliferation of NCI-H2170 and NCI-H2170 / Enhertu-R cells, but trastuzumab significantly inhibited the proliferation of NCI-H2170 cells, while having no inhibitory effect on the in vitro proliferation of NCI-H2170 / Enhertu-R cells. This demonstrates that the resistant strain NCI-H2170 / Enhertu-R is resistant to the antibody portion of trastuzumab, but not to its toxin DXd.

[0777] Example 27: Killing of HER2 antagonist-resistant cancer cells by anti-ROR1 antibody-drug conjugate

[0778] NCI-H2170 and NCI-H2170 / Enhertu-R cells were digested with trypsin, counted, and seeded in 96-well plates at a density of 4000 / 5000 cells per well. The next day, the cells were treated with trastuzumab and ADC-3. The initial concentration of trastuzumab was 333.3 nM, and it was serially diluted 5-fold to obtain 9 spots. The administration was carried out as follows:

[0779] In NCI-H2170 / Enhertu-R cells with a trastuzumab resistance concentration of 6.54 nM, the initial concentration of ADC-3 was 50 nM, and 9 spots were obtained by serial dilution of 5-fold.

[0780] In NCI-H2170 / Enhertu-R cells with a trastuzumab resistance concentration of 13.1 nM, the initial ADC-3 concentration was 333.3 nM, and 9 spots were obtained by serial dilution of 5-fold.

[0781] Three auxiliary wells were set up for each group, and a blank control group was also set up. After 5-6 days of treatment with ADC-3 and trastuzumab, 100 μL of CellTiter-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was placed on a fixed-track shaker and mixed for 3 minutes. Then, the culture plate was incubated at room temperature for 15 minutes. The luminescence signal was detected by microplate reader. In vitro proliferation inhibition curves of trastuzumab and ADC-3 on NCI-H2170 / Enhertu-R cells were plotted, and IC50 was calculated. 50 Values. The results are shown in Table 21, Figure 24, and Figure 25.

[0782] Table 21. In vitro proliferation inhibition IC50 of ADC-3 on NCI-H2170 / Enhertu-R cells 50 (nM)

[0783] The results showed that ADC-3 significantly inhibited the proliferation of NCI-H2170 / Enhertu-R cells at both different trastuzumab resistance concentrations. This fully demonstrates that the disclosed anti-ROR1 antibody-drug conjugate can significantly inhibit the proliferative activity of resistant cells even when cells are significantly resistant to both trastuzumab and dexamethasone.

[0784] Example 28: Killing of HER2 antagonist-resistant cancer cells by anti-ROR1 antibody-drug conjugate

[0785] 21.1 Building BT474 / Enhertu-R:

[0786] Referring to Example 26, using an in vitro concentration escalation method, BT474 / Enhertu-R cells resistant to trastuzumab were constructed based on BT474 (breast cancer cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.), with a resistance concentration of 0.52 nM.

[0787] 21.2 Detection of the cytotoxic activity of Enhertu against BT474 / Enhertu-R cells

[0788] BT474 and BT474 / Enhertu-R cells were digested with trypsin, centrifuged, counted, and seeded in 96-well plates at a density of 5000 cells / well. The next day, the cells were treated with trastuzumab. The maximum concentration of trastuzumab was 1000 nM, followed by a 5-fold serial dilution of 300 nM to obtain 8 spots. DXd (exatecan derivative, purchased from Selleck, catalog number E2891) was started at 100 nM and serially diluted 3-fold to obtain 9 spots. A blank control group was set up, and 3 auxiliary wells were set up in each group. Five days after treatment with trastuzumab and DXd, 100 μL of CellTiter-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was placed on a fixed-track shaker and mixed for 3 minutes. Then, the culture plate was incubated at room temperature for 15 minutes. The luminescence signal was detected by a microplate reader. In vitro proliferation inhibition curves of Enhertu / DXd on BT474 and BT474 / Enhertu-R cells were plotted. The results are shown in Figures 26A and 26B.

[0789] The results showed that trastuzumab significantly inhibited the proliferation of BT474 cells. Compared with BT474, trastuzumab did not significantly inhibit the proliferation of the drug-resistant cell line BT474 / Enhertu-R. Even at a concentration of 1000 nM, the inhibition rate of trastuzumab on the proliferation of BT474 / Enhertu-R cells was only 15.6%, indicating that BT474 / Enhertu-R had developed resistance to trastuzumab.

[0790] Meanwhile, DXd inhibited the proliferation of BT474 cells (IC). 50 The value was 22.05 nM, while DXd inhibited the proliferation of BT474 / Enhertu-R cells by IC50. 50 The value was 142.7 nM, and the inhibition rate at the maximum DXd concentration of 100 nM was only 39.65%, which fully demonstrates that BT474 / Enhertu-R also developed resistance to DXd.

[0791] 21.3 Detection of ADC-3 killing activity against BT474 / Enhertu-R cells

[0792] During the construction of the BT474 / Enhertu-R drug-resistant cell line, when the resistance concentration to trastuzumab was 0.2 nM, BT474 cells had developed a certain degree of resistance to trastuzumab, and the killing activity of ADC-3 against it was tested.

[0793] BT474 and BT474 / Enhertu-R cells were digested with trypsin, counted, and seeded at a density of 5000 cells / well in 96-well plates. The next day, the cells were treated with trastuzumab and ADC-3. The initial concentration of trastuzumab was 333.3 nM, serially diluted 5-fold to obtain 9 spots. The initial concentration of ADC-3 was 50 nM, serially diluted 5-fold to obtain 9 spots. Three auxiliary wells were set up for each group, and a blank control group was also included. Five days after treatment with ADC-3 and trastuzumab, 100 μL of CellTiter-Glo reagent (purchased from Promega, catalog number G7572) was added to each well. The culture plate was placed on a shaker and mixed for 3 minutes, then incubated at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader. In vitro proliferation inhibition curves of BT474 / Enhertu-R cells by trastuzumab and ADC-3 were plotted, and the IC50 was calculated. 50 Value. The result is shown in Figure 27.

[0794] The results showed that ADC-3 significantly inhibited the proliferation of BT474 / Enhertu-R cells, and the inhibitory effect of ADC-3 on the proliferation of BT474 / Enhertu-R cells was IC50. 50 The concentration is 1.48 nM. This indicates that the ADC-3 disclosed herein can kill cells resistant to HER2 antagonists and Dxd, and can be used to treat cancers resistant to HER2 antagonists or Dxd.

[0795] Example 29: Killing of HER2 antagonist-resistant cancer cells by anti-ROR1 antibody-drug conjugate

[0796] 22.1 Building HCC1954 / Enhertu-R:

[0797] HCC1954 cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, and incubated at 37°C in a 5% CO2 incubator. HCC1954 cells were digested, counted, and seeded at a density of 5000 cells / well in 96-well plates. The next day, trastuzumab was diluted to different concentrations to treat HCC1954 cells. Starting at 333.3 nM, trastuzumab was serially diluted 5-fold to obtain 9 different concentrations. A blank control group was also included. Each group had 3 replicates. After 6 days of trastuzumab treatment, 100 μL of CellTiter-Glo reagent (Promega, catalog number G7572) was added to each well. The culture plate was mixed on a microplate shaker for 3 minutes, followed by incubation at room temperature for 15 minutes. The luminescence signal was detected using a microplate reader to obtain the in vitro proliferation inhibition curve and IC50 of trastuzumab against HCC1954 cells.50 The value is 0.44nM.

[0798] HCC1954 cells were digested, counted, and then analyzed at a concentration of 7 × 10⁻⁶. 5 Cells were seeded in 10cm culture dishes and cultured overnight. The culture medium was then changed, and different concentrations of trastuzumab were added to treat the cells at an IC50 concentration. 50 The concentration of trastuzumab was gradually increased until the cells could grow normally in culture conditions containing 6.5 nM trastuzumab, thus obtaining the drug-resistant cell line HCC1954 / Enhertu-R.

[0799] 22.2 In vitro proliferation inhibition experiment of trastuzumab on HCC1954 and HCC1954 / Enhertu-R cells

[0800] HCC1954 and HCC1954 / Enhertu-R cells in logarithmic growth phase were trypsinized and counted, then seeded at a density of 2000 cells / well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator. The next day, the cells were treated with trastuzumab and DXd. Trastuzumab and DXd were serially diluted 5-fold starting at 300 nM to obtain 9 concentrations, with each concentration set in 3 replicates. After 5 days of drug treatment, 100 μL of CellTiter-Glo reagent (purchased from Promega, catalog number G7572) was added to each well, and the culture plate was mixed on a microplate shaker for 3 minutes. The culture plate was then incubated at room temperature for 15 minutes, and the luminescence signal was detected using a microplate reader. The in vitro proliferation inhibition curves of trastuzumab and DXd on HCC1954 and HCC1954 / Enhertu-R cells were obtained. The results are shown in Table 22, Figure 28A and Figure 28B.

[0801] Table 22. Inhibitory effects of trastuzumab and DXd on in vitro cell proliferation

[0802] The results showed that HCC1954 / Enhertu-R cells exhibited significant resistance to trastuzumab. Furthermore, DXd showed an IC50 inhibitory effect on the proliferation of HCC1954 and HCC1954 / Enhertu cells. 50 The values ​​were 2.01 nM and 55.51 nM, respectively, indicating that HCC1954 / Enhertu cells developed some resistance to DXd.

[0803] 22.3 Detection of ADC-3 killing activity against HCC1954 / Enhertu-R cells

[0804] HCC1954 / Enhertu-R cells in logarithmic growth phase were trypsinized and counted, then seeded at a density of 2000 cells / well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator. The next day, the cells were treated with ADC-3 and trastuzumab. ADC-3 and trastuzumab were serially diluted 5-fold from a starting concentration of 300 nM to obtain 9 concentrations, with each concentration in triplicate. After 5 days of drug treatment, 100 μL of CellTiter-Glo reagent (Promega, catalog number G7572) was added to each well, and the plate was mixed on a microplate shaker for 3 minutes. The plate was then incubated at room temperature for 15 minutes, and the luminescence signal was detected using a microplate reader. The in vitro proliferation inhibition curves of ADC-3 and trastuzumab on HCC1954 / Enhertu-R cells were obtained. The results are shown in Figure 29.

[0805] The results showed that ADC-3 significantly inhibited the proliferation of HCC1954 / Enhertu-R cells, with an IC50 concentration of 1,000 mg / dL. 50 The concentration is 1.38 nM. This indicates that the ADC-3 of this application can kill cells resistant to HER2 antagonists and DXd, and can be used to treat cancers resistant to HER2 antagonists or DXd.

[0806] All references to this disclosure are incorporated herein by reference as if each document were individually incorporated herein by reference. Furthermore, it should be understood that, after reading the foregoing teachings of this disclosure, those skilled in the art may make various alterations or modifications to this disclosure, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method of treating cancer, comprising administering an anti-ROR1 antibody or a drug conjugate thereof to a subject, wherein the subject has cancer resistant to anticancer therapeutic agents; Preferably, the anti-ROR1 antibody or its drug conjugate comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding domain comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein: The heavy chain variable region VH1 includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:38, and the light chain variable region VL1 includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23; or The heavy chain variable region VH1 includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:22, and the light chain variable region VL1 includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23; and The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:72, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:71; or The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:73, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:71; or The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:74, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:75; or The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:74, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:76; or The heavy chain variable region VH2 includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:74, and the light chain variable region VL2 includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:

71. The amino acid sequences of the HCDR and the LCDR are determined according to the Kabat, Chothia, AbM, or IMGT numbering system; more preferably, The heavy chain variable region VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:38, and the light chain variable region VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:23; and The heavy chain variable region VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:72, and the light chain variable region VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:71; most preferably, The heavy chain variable region VH1 includes HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:10; or The heavy chain variable region VH1 includes HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:18, and HCDR3 as shown in SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:10; and The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:61, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:50; or The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:66, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:50; or The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:67, LCDR2 as shown in SEQ ID NO:68, and LCDR3 as shown in SEQ ID NO:50; or The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:69, and LCDR3 as shown in SEQ ID NO:50; or The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:45, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:50; optionally, The heavy chain variable region VH1 includes HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:10; and The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:61, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:

50.

2. The method of claim 1, wherein the first antigen-binding domain of the anti-ROR1 antibody or its drug conjugate comprises: The heavy chain variable region VH1 contains the amino acid sequence shown in SEQ ID NO:38, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:38, and the light chain variable region VL1 contains the amino acid sequence shown in SEQ ID NO:23, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:23; or The heavy chain variable region VH1 contains an amino acid sequence as shown in SEQ ID NO:22, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:22, and the light chain variable region VL1 contains an amino acid sequence as shown in SEQ ID NO:23, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

23. Furthermore, the second antigen-binding domain of the anti-ROR1 antibody or its drug conjugate comprises: The heavy chain variable region VH2 comprises the amino acid sequence shown in SEQ ID NO:72, or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:72, and the light chain variable region VL2 comprises the amino acid sequence shown in SEQ ID NO:71, or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:71; or The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:73, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:73, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:71, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:71; or The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:74, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:74, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:75, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:75; or The heavy chain variable region VH2 comprises the amino acid sequence shown in SEQ ID NO:74, or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:74, and the light chain variable region VL2 comprises the amino acid sequence shown in SEQ ID NO:76, or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:76; or The heavy chain variable region VH2 contains an amino acid sequence as shown in SEQ ID NO:74, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:74, and the light chain variable region VL2 contains an amino acid sequence as shown in SEQ ID NO:71, or contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

71. Preferably, the first antigen-binding domain comprises: The heavy chain variable region VH1 comprises the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region VL1 comprises the amino acid sequence shown in SEQ ID NO:23; and the second antigen-binding domain comprises: The heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:72, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:

71.

3. The method according to claim 1 or 2, wherein the anti-ROR1 antibody or its drug conjugate comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, wherein Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region. Preferably, Fc1 has a protruding structure (knob) according to the tool-in-hole technique, and Fc2 has a hole structure (hole) according to the tool-in-hole technique; More preferably, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, with the numbering based on the EU index. Most preferably, Fc1 contains the amino acid sequence of SEQ ID NO: 79, and Fc2 contains the amino acid sequence of SEQ ID NO:

80.

4. The method according to any one of claims 1 to 3, wherein the anti-ROR1 antibody or its drug conjugate comprises four chains as shown in (a)-(d) below: (a) [heavy chain variable region VH1]-[CH1]-[Fc1], (b) [Light chain variable region VL1]-[CL1], (c)[heavy chain variable region VH2]-[CH1]-[Fc2], and (d) [Light chain variable region VL2]-[CL2]; or It includes the four chains shown in (e), (b), (f), and (d) below: (e)[heavy chain variable region VH1]-[CH1]-[Fc2], (b) [Light chain variable region VL1]-[CL1], (f)[heavy chain variable region VH2]-[CH1]-[Fc1], and (d)[Light chain variable region VL2]-[CL2]; in, The structures shown in equations (a), (b), (c), (d), (e), and (f) are arranged from the N-terminus to the C-terminus, where CL1 and CL2 are each independently the light chain constant region of the antibody, and CH1 is the first part of the heavy chain constant region of the antibody. Preferably, the heavy chain variable region VH1 contains the amino acid sequence shown in SEQ ID NO:38, the light chain variable region VL1 contains the amino acid sequence shown in SEQ ID NO:23, the heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:72, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:

71. More preferably, the anti-ROR1 antibody or its drug conjugate comprises the four chains described below: Chain 1, comprising the amino acid sequence shown in SEQ ID NO:81; chain 2, comprising the amino acid sequence shown in SEQ ID NO:82; chain 3, comprising the amino acid sequence shown in SEQ ID NO:83; and chain 4, comprising the amino acid sequence shown in SEQ ID NO:

84.

5. The method according to any one of claims 1 to 4, wherein the anti-ROR1 antibody-drug conjugate has the structure shown in the following formula: Where Ab is the anti-ROR1 antibody, L is the linker, D is the drug, and n is an integer or decimal from 1 to 10; Preferably, the L has -L a -L b -L c -L d - structure, where L a Linked to antibodies, L d Related to drugs, including: L a Selected from Among them, the wavy line * indicates the connection point with Ab, and * indicates the connection point with L. b The connection point; L b The components are selected from -(CH2)mC(O)-, -NH-(CH2CH2O)p-(CH2)sC(O)-, -C(O)-NH-(CH2)qC(O)-, -NH-(CH2)rC(O)- and bonds, wherein m is an integer from 0 to 10, preferably m is 0, 2, 3 or 5; p is an integer from 1 to 8, and s is an integer from 0 to 6; preferably p is 4, s is 2; q is an integer from 1 to 5, preferably q is 2; r is an integer from 1 to 5, preferably r is 2; L c It is an oligopeptide composed of 1 to 7 amino acids, or L c The amino acid is selected from valine, citrulline, glycine, phenylalanine, alanine, proline, isoleucine, lysine, serine, glutamic acid, and aspartic acid. The amino acid is either unsubstituted or independently substituted by one or more substituents, wherein each substituent is independently selected from halogen, hydroxyl, cyano, amino, C... 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-7 cycloalkyl; L d Selected from -NH-CH2-O-CH2-C(O)-, -NH-R a -CH2-OC(O)- and bonds, where R a It is a phenyl or a 5-6 membered heterocyclic group, wherein the phenyl and the 5-6 membered heterocyclic group are unsubstituted or independently substituted by one or more substituents, wherein each substituent is independently selected from... Halogen, oxo group, hydroxyl group, cyano group, amino group, C 1-6 Alkyl, Halogenated C 1-6 Alkyl and C 1-6 Alkoxy, where # represents the linking site with a phenyl or 5-6 membered heterocyclic group.

6. The method of claim 5, wherein the L has a structure selected from the group consisting of: i)L a for L b For -C(O)- or -C(O)-NH-(CH2)2-C(O)-, L c It is one of the following oligopeptides: -glycine-glycine-phenylalanine-glycine, -valine-citrulline-, or -glycine-, or L c For key, and L d for -NH-CH2-O-CH2-C(O)- or bond; or ii)L a for L b The expression is -(CH2)mC(O)-, where m is 2 or 5, and L c For -valine-citrulline- or -glycine-, and L d for or iii)L a for L b For -NH-(CH2-CH2-O)4-(CH2)2-C(O)-, -NH-(CH2)2-C(O)- or bonds, L c It is one of the following oligopeptides: -valine-citrulline-, -glycine-, or -glycine-glycine-phenylalanine-glycine-, or L c For key, and L d for Or -NH-CH2-O-CH2-C(O)-; and iiii)L a for L b For -(CH2)3-C(O)-, L c For -valine-citrulline-, and L d for in, wavy lines * indicates the connection point with Ab, and * indicates the connection point with L. b The connection point, a * Indicates with L c The connection point, b * Indicates the connection point with the drug; Preferably: L a for L b For -C(O)-, L c For -valine-citrulline-oligopeptide, and L d for Among them, wavy lines * indicates the connection point with Ab, and * indicates the connection point with L. b The connection point, a * Indicates with L c The connection point, b * Indicates the connection point with the drug.

7. The method according to any one of claims 1 to 6, wherein the drug in the anti-ROR1 antibody-drug conjugate is selected from cytotoxic compounds, immunomodulators, enzymes, and hormone inhibitors; Preferably, the drug is selected from erribulin, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), exatecan, maytansine and its analogues, SN-38 or combinations thereof; more preferably, the anti-ROR1 antibody-drug conjugate has the following structure: in, Ab is an anti-ROR1 antibody, and n is an integer or decimal from 1 to 10; most preferably, the Ab comprises four polypeptide chains as described below: Chain 1, comprising the amino acid sequence shown in SEQ ID NO:81; chain 2, comprising the amino acid sequence shown in SEQ ID NO:82; chain 3, comprising the amino acid sequence shown in SEQ ID NO:83; and chain 4, comprising the amino acid sequence shown in SEQ ID NO:

84. n is 3.5-4.

5.

8. The method according to any one of claims 1 to 7, wherein the anticancer therapeutic agent is: (i) an inhibitor of topoisomerase I or a derivative thereof; or (ii) an antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof; preferably, The topoisomerase I inhibitor is selected from DXd, SN-38, esaxatecan, rubitecan, topotecan, irinotecan, camptothecin, or derivatives thereof; more preferably, the topoisomerase I inhibitor is DXd or derivatives thereof.

9. The method according to claim 8, wherein the antibody comprising an antibody-drug conjugate of a topoisomerase I inhibitor or a derivative thereof is an anti-tumor-associated antigen antibody; preferably, the antibody is an anti-HER2 antibody.

10. The method of claim 9, wherein the anti-HER2 antibody comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 128, HCDR2 shown in SEQ ID NO: 129, and HCDR3 shown in SEQ ID NO: 130, and the light chain variable region includes LCDR1 shown in SEQ ID NO: 131, LCDR2 shown in SEQ ID NO: 132, and LCDR3 shown in SEQ ID NO: 133; preferably, The anti-HER2 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 134 and a light chain variable region as shown in SEQ ID NO: 135; more preferably, The anti-HER2 antibody comprises a heavy chain as shown in SEQ ID NO: 136 and a light chain as shown in SEQ ID NO:

137.

11. The method according to claim 8, wherein the antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof is selected from trastuzumab deruxtecan, trastuzumab rezetecan, JSKN-003 (Anbenitamab repodatecan), IBI-354, TQB-2101, BL-M07D1, BNT-323 (Trastuzumab Pamirtecan), FDA022, GQ1005, DAN-311, T-PL1, PRO1102, and MTX-1000; preferably, the antibody-drug conjugate comprising a topoisomerase I inhibitor or a derivative thereof is trastuzumab deruxtecan.

12. The method according to any one of claims 1 to 7, wherein the anticancer therapeutic agent is a HER2 antagonist; preferably, the HER2 antagonist is an antiHER2 antibody, an antiHER2 antibody-drug conjugate, or a small molecule HER2 inhibitor.

13. The method of claim 12, wherein the anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1 shown in SEQ ID NO: 128, HCDR2 shown in SEQ ID NO: 129, and HCDR3 shown in SEQ ID NO: 130, and the light chain variable region includes LCDR1 shown in SEQ ID NO: 131, LCDR2 shown in SEQ ID NO: 132, and LCDR3 shown in SEQ ID NO: 133; preferably, The anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain variable region as shown in SEQ ID NO: 134 and a light chain variable region as shown in SEQ ID NO: 135; more preferably, The anti-HER2 antibody or anti-HER2 antibody-drug conjugate comprises a heavy chain as shown in SEQ ID NO: 136 and a light chain as shown in SEQ ID NO:

137.

14. The method according to claim 12 or 13, wherein the anticancer therapeutic agent is an anti-HER2 antibody-drug conjugate, and the drug is a microtubule inhibitor or a topoisomerase I inhibitor; preferably, wherein the drug is selected from exatecan, monomethylaurestatin E (MMAE), monomethylaurestatin F (MMAF), maytansine compounds, SN-38, irinotecan, and topotecan.

15. The method according to claim 12, wherein the anti-HER2 antibody is selected from trastuzumab, pertuzumab, zanidatamab, zenocutuzumab, inetetamab, margetuximab, HLX22, IAH0968, BAT1006, B002T, HK001, TrasGEX (timigutuzumab), and FS102; The anti-HER2 antibody-drug conjugates mentioned are selected from trastuzumab (DS-8201), vedicitumab, trastuzumab rezetecan, SYD985 (trastuzumab duocarmazine), trastuzumab botidotin, BAT8001, TAA013, MRG002 (trastuzumab vedotin), LCB14-0110, SYA1501, DB-1303, JSKN-003 (Anbenitamab repodatecan), BL-M07D1, TQB2102, GQ1005, IBI354, NCB001 (anvatabart opadotin), MM-302, and DX126-262. The small molecule HER2 inhibitors mentioned are selected from neratinib and lapatinib. Canertinib, zongertinib, and irbinitinib.

16. The method according to any one of claims 1 to 15, wherein the subject has cancer resistant to anticancer therapeutic agents, wherein the cancer is selected from: breast cancer, gastric cancer, lung cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, endometrial cancer, pancreatic cancer, cervical cancer, squamous cell carcinoma, small cell lung cancer, gastric / esophageal junction (GEJ) adenocarcinoma, esophageal squamous cell carcinoma, esophageal cancer, bladder cancer, salivary gland cancer, biliary tract cancer, Paget's disease, hepatocellular carcinoma, glioblastoma, renal cell carcinoma, gastrointestinal tumors, and colon cancer. Rectal cancer, glioma, mesothelioma, head and neck cancer, skin cancer, uterine cancer, peritoneal cancer, liver cancer, vulvar cancer, melanoma, laryngeal tumors, pharyngeal tumors, oral tumors, urothelial carcinoma, neuroblastoma, lymphoma, leukemia, malignant lymphoma, sarcoma, plasmacytoma, triple-negative breast cancer, triple-positive breast cancer, HER2-positive breast cancer, hormone receptor-positive breast cancer, and multiple myeloma; preferably, the cells of the cancer express ROR1; more preferably, the drug resistance is acquired due to treatment with anticancer agents.

17. The method according to any one of claims 1 to 16, further comprising administering an additional therapeutic agent to the subject; preferably, wherein the additional therapeutic agent is selected from one or more of immune checkpoint inhibitors, epidermal growth factor receptor (EGFR) inhibitors, B-cell antigen inhibitors, BTK inhibitors, BCL-2 inhibitors, CDK4 / 6 inhibitors, and chemotherapeutic agents; more preferably, the additional therapeutic agent is selected from one or more of SIRPα binding antagonists, PD-1 binding antagonists, PD-L1 binding antagonists, CD20 binding antagonists, CD47 binding antagonists, and chemotherapeutic agents.

18. The method for treating cancer according to claim 17, wherein: The SIRPα binding antagonists are selected from: BR105, CC-95251, HCB-101, BI765063, GS-0189, IBI397, BI-770371, APX-700, ES-004, ADU1805, ELA-026, BYON-4228, ALX-148 (evorpacept), timdarpacept, IMM01, TTI-621, TTI-622, JMT601 (CPO107), SL-172154, SIRPα-F8, JMT601 (CPO107), SS002M91, SIRPα-lgG4-Fc-Fc, and hCD172a (SIRPα)-Fc-LIGHT; The PD-1 binding antagonists mentioned are selected from pembrolizumab, zimberelimab, nivolumab, cemiplimab, pidilizumab, AMG-404, MEDI0680, spartalizumab, tislelizumab, toripalimab, genolimzumab, and camrelizumab. mrelizumab, sintilimab, dostarlimab, lambrolizumab, sasanlimab, cetrelimab, serplulimab, retifanlimab, balstilimab, prolgolimab, budigalimab, vopratelimab Retifanlimab, Cadonilimab, BMS-986213 (Relatlimab + Nivolumab), Ivorescimab, Geptanolimab, Iparomlimab, Pucotenlimab, AK-105, CS-1003, BI-754091, LZM-009, Sym-021, BAT-1306, PD1-PIK, Tebo telimab), RO-7247669(PD-1 / LAG-3), FS-118(LAG-3 / PD-L1), RO-7121661(PD-1 / TIM-3), RG7769(PD-1 / TIM-3), PF-06936308(PD-1 / CTLA4), MGD-019(PD-1 / CTLA4), KN-046(PD-1 / CTLA4), XmAb-20717(PD-1 / CTLA4), AK-104(CTLA4 / PD-1) and MEDI-5752(CTLA4 / PD-1); The PD-L1 binding antagonists mentioned are selected from: avelumab, atezolizumab, envafolimab, durvalumab, adebrelimab, BMS-936559 (MDX1105), cosibelimab, lodapolimab, garivulimab, envafolimab, opucolimab, manelimab, CX-072, CBT-502 (TQB2450), and MSB-23.

11. Sugemalimab, A167 (KL-A167), STI-A1015 (IMC-001), FAZ-053, BMS-936559 (MDX1105), INCB086550, GEN-1046 (PD-L1 / 4-1BB), FPT-155 (CTLA4 / PD-L1 / CD28), M7824 (PD-L1 / TGFβ-EC domain), CA-170 (PD-L1 / VISTA), CDX-527 (CD27 / PD-L1), LY-3415244 (TIM-3 / PDL1), INBRX-105 (4-1BB / PDL1), and GNS-1480 (PD-L1 / EGFR); The CD20 binding antagonist is selected from zuberitamab, obinutuzumab, ibritumomab, ofatumumab, tositumomab, ocrelizumab, ubliximab, and rituximab; and The CD47 binding antagonists mentioned are selected from magrolimab, lemzoparlimab, letaplimab, ligufalimab, AO-176, simridarlimab (IBI-322), gentulizumab, ZL-1201, IMC-002, SRF-231, CC-90002 (also known as INBRX-103), NI-1701 (also known as TG-1801), STI-6643, SHR-1603, HLX-24, LQ-001, B6H12, TAY-018, PT-240, 1F8-GMCSF, SY-102, and KD-015.

19. The method for treating cancer according to claim 17, wherein the chemotherapeutic drug is selected from: nitrogen mustard antitumor drugs, anthracycline antitumor drugs, glucocorticoid antitumor drugs, vincristine alkaloid antitumor drugs, platinum antitumor drugs, camptothecin antitumor drugs, taxane antitumor drugs, antimetabolites antitumor drugs, podophyllodoalkaloid antitumor drugs, and combinations thereof; Preferably, the chemotherapy drug is selected from: cyclophosphamide, doxorubicin, prednisone, paclitaxel, albumin-bound paclitaxel, vincristine, vinorelbine tartrate, gemcitabine, etoposide, azacitidine, palbociclib, doxorubicin, cisplatin, carboplatin, prednisolone, irinotecan, topotecan, esaxatecan, rubitecan, and combinations thereof.

20. The method for treating cancer according to claim 17, wherein the BTK inhibitor is selected from ibrutinib, BTKi RN486, netabrutinib, acalabrutinib, zanubrutinib, and orelabrutinib; the BCL-2 inhibitor is selected from veneclade and navicola; and the CDK4 / 6 inhibitor is selected from palbociclib, abeciclib, ribociclib, lerociclib, avozidil, ronizil, pirosiclib, and trilaciclib.

21. The method of claim 17, wherein the additional therapeutic agent is selected from a combination of an anti-CD20 antibody and a chemotherapeutic agent; preferably, the chemotherapeutic agent is selected from one or more of cyclophosphamide, doxorubicin, prednisone / prednisolone, and vincristine; more preferably, the additional therapeutic agent is selected from any one of the following: a) A combination of anti-CD20 antibody and cyclophosphamide; b) A combination of anti-CD20 antibody and doxorubicin; c) A combination of anti-CD20 antibody and prednisone / prednisolone; d) A combination of anti-CD20 antibody and gemcitabine; e) A combination of anti-CD20 antibody and paclitaxel; f) A combination of anti-CD20 antibody, cyclophosphamide, doxorubicin, and prednisone / prednisolone; and g) A combination of anti-CD20 antibody, cyclophosphamide, doxorubicin, vincristine, and prednisone / prednisolone; Most preferably, the anti-CD20 antibody is zabetoumab or rituximab.

22. The method of treating cancer according to any one of claims 17 to 21, wherein the anti-ROR1 antibody or its pharmaceutical conjugate and other therapeutic agents are administered simultaneously, separately or sequentially.

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