Anti-ROR1 antibody and its drug conjugate

Abstract

Anti-ROR1 antibodies and multispecific antibodies prepared based on the anti-ROR1 antibody are provided. Furthermore, antibody-drug conjugates containing the anti-ROR1 antibody or the multispecific antibody are provided.

Description

[Technical Field]

[0001] The present invention relates to the field of antibody drugs, and more specifically to anti-ROR1 antibodies and drug conjugates thereof, as well as to the use of said anti-ROR1 antibodies and drug conjugates. [Background technology]

[0002] ROR1 is a transmembrane receptor tyrosine kinase protein and a member of the type I receptor tyrosine kinase (RTK) family. Human ROR1 consists of one extracellular immunoglobulin-like domain (Ig), two cysteine-rich domains (FZD), a near-membrane kringle domain, a single transmembrane domain, one intracellular tyrosine kinase domain (TKD), two serine / threonine-rich domains (S / TRD), and one proline-rich domain (PRD). ROR1 is highly expressed during embryonic and infant development and plays a crucial role in various physiological processes, specifically regulating cell division, proliferation, migration, and chemotaxis, and is essential for the development of nerve, skeletal, and vascular organs. ROR1 expression is significantly reduced during the following developmental stages. In normal adult tissue, ROR1 expression is insufficient, except in B lymphocyte progenitor cells. However, ROR1 is highly expressed in numerous hematological and stereotumorous tumors. Hematological malignancies with high ROR1 expression include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), and myeloid hematological malignancies. Stereotumors expressing ROR1 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 shown that ROR1 can be involved in the non-classical Wnt signaling pathway mediated by Wnt5a, where Wnt5a activates the receptor ROR1 or FZD5, leading to Dvl2 / 3 activation and Akt phosphorylation. Subsequently, Akt promotes IKKα phosphorylation, activating the IKK complex, which in turn promotes phosphorylation of the NF-κB subunit p65 while degrading IκBα. Phosphorylated p65 translocates to the cell nucleus and promotes the transcriptional expression of target genes, including Wnt5a. Wnt5a secretion also promotes a positive feedback loop in a new cycle. Activation of the positive feedback loop ROR1 / Akt / p65 signaling pathway further promotes the secretion of pro-inflammatory factors (e.g., IL-6) and chemotactic factors (e.g., CCL2).Furthermore, some studies claim that ROR1 is related to EMT in tumor cells or to the activation of YAP / TAZ transcription, and that it further enhances tumor development and chemotherapy resistance. [Overview of the project]

[0003] The present invention aims to provide an anti-ROR1 antibody and an antibody-drug conjugate (ADC) containing the anti-ROR1 antibody. The invention also provides for the use of the anti-ROR1 antibody or antibody-drug conjugate in the treatment of tumors or cancer.

[0004] According to a first aspect of the present invention, an anti-ROR1 antibody is provided, which comprises a heavy chain variable region and a light chain variable region, specifically, (Z1) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:61, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region includes LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50, (Z2) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:66, and the light chain variable region includes LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50. (Z3) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region includes LCDR1 represented by SEQ ID NO:67, LCDR2 represented by SEQ ID NO:68, and LCDR3 represented by SEQ ID NO:50. (Z4) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region includes LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:69, and LCDR3 represented by SEQ ID NO:50. (Z5) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region includes LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50. (Z6) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:32, and HCDR3 represented by SEQ ID NO:19, and the light chain variable region includes LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10, or, (Z7) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:18, and HCDR3 represented by SEQ ID NO:19, and the light chain variable region includes LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10.

[0005] In some embodiments, the anti-ROR1 antibody described above comprises a heavy chain variable region and a light chain variable region, specifically, (Z1) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:61, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region includes LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50, or, (Z6) The heavy chain variable region includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:32, and HCDR3 represented by SEQ ID NO:19, and the light chain variable region includes LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10.

[0006] In some embodiments, the CDR is defined by the Kabat numbering rules using the anti-ROR1 antibody described in any one of the above sections.

[0007] In some embodiments, the anti-ROR1 antibody is one of the anti-ROR1 antibodies described in any one of the above paragraphs, and the anti-ROR1 antibody is a mouse-derived antibody, a chimeric antibody, or a humanized antibody.

[0008] In some embodiments, the anti-ROR1 antibody described in any one of the above clauses is a humanized antibody and includes the FR region of a human antibody. In some embodiments, the anti-ROR1 antibody described above includes a heavy chain variable region containing a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:72, and the light chain variable region containing a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:71, or The heavy chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:73, and the light chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO:71, or, The heavy chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:74, and the light chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:75, or, The heavy chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:74, and the light chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:76, or, The heavy chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:74, and the light chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:71, or, The heavy chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:22, and the light chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:23, or, The heavy chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:38, and the light chain variable region includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:23.

[0009] In some embodiments, the anti-ROR1 antibody described above is used, The heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:72, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:71, or, The heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:73, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:71, or, The heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:74, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:75, or, The heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:74, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:76, or, The heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:74, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:71, or, The heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:22, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:23, or, The heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:38, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:23.

[0010] In some embodiments, the anti-ROR1 antibody described above causes the heavy chain variable region to include an amino acid sequence represented by SEQ ID NO:72, the light chain variable region to include an amino acid sequence represented by SEQ ID NO:71, or The heavy chain variable region includes an amino acid sequence represented by SEQ ID NO:38, and the light chain variable region includes an amino acid sequence represented by SEQ ID NO:23.

[0011] In some embodiments, an anti-ROR1 antibody comprising a heavy chain variable region CDR and a light chain variable region CDR selected from any one of (Z1) to (Z5) above binds to an epitope located at amino acids 70 to 130 of the ROR1 protein, and an anti-ROR1 antibody comprising (Z6) or (Z7) above binds to an epitope located at amino acids 130 to 165 of the ROR1 protein, and the amino acid sequence of the ROR1 protein is represented by SEQ ID NO:87.

[0012] In some embodiments, the anti-ROR1 antibody includes a heavy chain constant region. In some embodiments, the heavy chain constant region is an IgG1 or IgG4 subtype.

[0013] In some embodiments, the anti-ROR1 antibody includes a light chain constant region. In some embodiments, the light chain constant region is of the κ type.

[0014] In some embodiments, the anti-ROR1 antibody 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 represented by SEQ ID NO:11 or a variant thereof, and / or the light chain constant region comprises an amino acid sequence represented by SEQ ID NO:12 or a variant thereof. In some embodiments, both the variant of the heavy chain constant region and the variant of the light chain constant region are ordinary variants.

[0015] In some embodiments, the anti-ROR1 antibody is A heavy chain whose amino acid sequence is represented by SEQ ID NO:77 and a light chain whose amino acid sequence is represented by SEQ ID NO:78, or A heavy chain whose amino acid sequence is represented by SEQ ID NO:30 and a light chain whose amino acid sequence is represented by SEQ ID NO:31, or It contains a heavy chain whose amino acid sequence is represented by SEQ ID NO:44 and a light chain whose amino acid sequence is represented by SEQ ID NO:31.

[0016] In some embodiments, the anti-ROR1 antibody described above is an antibody fragment. In some embodiments, the antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, scFv, dsFv, and dAb.

[0017] A second aspect of the present invention provides a multispecific antibody comprising the anti-ROR1 antibody described in the first aspect of the present invention.

[0018] In some embodiments, the multispecific antibody simultaneously targets the ROR1 protein and other proteins, or simultaneously targets multiple different epitopes on the ROR1 protein.

[0019] In some embodiments, the proteins other than ROR1 include tumor-associated antigens.

[0020] In some embodiments, the multispecific antibody is a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first and second antigen-binding regions bind to different epitopes on human ROR1. In some embodiments, the first antigen-binding region does not block the binding of the second antigen-binding region to human ROR1. In some embodiments, the first antigen-binding region binds to amino acids 130-165 of human ROR1, and the second antigen-binding region binds to amino acids 70-130 of human ROR1, and the amino acid sequence of the ROR1 protein is represented by SEQ ID NO:87.

[0021] In some embodiments, the first antigen-binding region includes a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding region includes a heavy chain variable region VH2 and a light chain variable region VL2, specifically, The heavy chain variable region VH1 includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:32, and HCDR3 represented by SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10, or, The heavy chain variable region VH1 includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:18, and HCDR3 represented by SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10. The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO: 61, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO: 50, or, The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:66, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50, or, The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO:67, LCDR2 represented by SEQ ID NO:68, and LCDR3 represented by SEQ ID NO:50, or, The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:69, and LCDR3 represented by SEQ ID NO:50, or, The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50.

[0022] In some embodiments, the bispecific antibody described above comprises the first antigen-binding region VH1 including the heavy chain variable region VH1 which includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:32, and HCDR3 represented by SEQ ID NO:19, and the light chain variable region VL1 which includes LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10. The second antigen-binding region includes the heavy chain variable region VH2, which contains HCDR1 represented by SEQ ID NO:61, HCDR2 represented by SEQ ID NO:46, and HCDR3 represented by SEQ ID NO:65, and the light chain variable region VL2, which contains LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50.

[0023] In some embodiments, the first antigen-binding region is formed by the bispecific antibody described above. The heavy chain variable region VH1 includes the amino acid sequence represented by SEQ ID NO:38, and the light chain variable region VL1 includes the amino acid sequence represented by SEQ ID NO:23, or The heavy chain variable region VH1 includes an amino acid sequence represented by SEQ ID NO:22, and the light chain variable region VL1 includes an amino acid sequence represented by SEQ ID NO:23. The second antigen-binding region is, The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO:72, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO:71, or The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO:73, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO:71, or The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO:74, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO:75, or The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO:74, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO:76, or The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO:74, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO:71.

[0024] In some embodiments, the bispecific antibody described above comprises, in which the first antigen-binding region includes a heavy chain variable region VH1 represented by SEQ ID NO:38 and a light chain variable region VL1 represented by SEQ ID NO:23, and the second antigen-binding region includes a heavy chain variable region VH2 represented by SEQ ID NO:72 and a light chain variable region VL2 represented by SEQ ID NO:71.

[0025] In some embodiments, the bispecific antibody comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 that can associate with each other, and each of Fc1 and Fc2 independently has one or more amino acid substitutions that reduce homodimerization of the Fc region.

[0026] In some embodiments, the bispecific antibody described above causes Fc1 to have a knob-into-hole projection structure (knob) and Fc2 to have a hole structure (hole) using the knob-into-hole technique.

[0027] In some embodiments, a bispecific antibody as described in any one of the above paragraphs comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 that can associate with each other, the Fc1 having a projection structure by knob-into-hole technology, and the Fc2 having a hole structure by knob-into-hole technology. In some embodiments, the 366th amino acid of the Fc1 is W, the 366th amino acid of the Fc2 is S, the 368th amino acid is A, and the 407th amino acid is V, with the numbering following the EU index.

[0028] In some embodiments, a bispecific antibody as described in any one of the above items comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 that can associate with each other, the Fc1 comprising an amino acid sequence represented by SEQ ID NO:79, and the Fc2 comprising an amino acid sequence represented by SEQ ID NO:80.

[0029] In some embodiments, the bispecific antibody described in any one of the above paragraphs results in the bispecific antibody having four chains as shown in (a) to (d) below, i.e., (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) Including [light chain variable region VL2]-[CL2], or, The aforementioned bispecific antibody has four chains as shown in (e), (b), (f), and (d) below, namely, (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) Including the [light chain variable region VL2]-[CL2], Here, 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 constant region of the antibody's light chain, and their amino acid sequences may be the same or different, while CH1 is the first part of the constant region of the antibody's heavy chain.

[0030] In some embodiments, the bispecific antibody described above allows the heavy chain variable region VH1 to contain an amino acid sequence represented by SEQ ID NO:38, the light chain variable region VL1 to contain an amino acid sequence represented by SEQ ID NO:23, the heavy chain variable region VH2 to contain an amino acid sequence represented by SEQ ID NO:72, and the light chain variable region VL2 to contain an amino acid sequence represented by SEQ ID NO:71.

[0031] In some embodiments, the CH1 is the CH1 sequence of IgG, as determined by the bispecific antibody described in any one of the above clauses. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 includes the amino acid sequence represented by SEQ ID NO:88.

[0032] In some embodiments, CL1 and CL2 are constant regions of the light chain of the bispecific antibody described in any one of the above clauses. In some embodiments, CL1 or CL2 are constant regions of the light chain of kappa or lamada, provided that the bispecific antibody described in any one of the above clauses is used. In some embodiments, CL1 and / or CL2 include an amino acid sequence represented by SEQ ID NO:12.

[0033] In some embodiments, the bispecific antibody described above results in the following four chains: Chain 1: Contains the amino acid sequence represented by SEQ ID NO: 81. Chain 2: Contains the amino acid sequence represented by SEQ ID NO: 82. Chain 3: Containing the amino acid sequence represented by SEQ ID NO: 83, and Chain 4: Includes amino acid sequences represented by SEQ ID NO: 84.

[0034] A third aspect of the present invention provides a nucleic acid molecule encoding an anti-ROR1 antibody as described in the first aspect of the present invention, or a multispecific antibody as described in the second aspect of the present invention.

[0035] The preparation method for nucleic acid molecules described in the present invention is a common preparation method in the art, and preferably includes the following preparation methods, namely, a method for obtaining the nucleic acid molecule encoding the monoclone antibody by gene cloning technology such as PCR, or a method for obtaining the nucleic acid molecule encoding the monoclone antibody by artificially synthesizing the entire sequence.

[0036] Those skilled in the art know that substitutions, deletions, alterations, insertions, or additions can be appropriately introduced into nucleic acid molecules encoding the amino acid sequence of an antibody that binds to the human ROR1, in order to provide homologues of a multimeric polynucleotide. Homotoues of the multimeric polynucleotide in the present invention can be obtained by substitutions, deletions, or additions to one or more bases encoding the antibody gene that binds to the human ROR1, within a range that maintains antibody activity.

[0037] A fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention.

[0038] In some embodiments, the vector is an expression vector. In some embodiments, the expression vector includes eukaryotic expression vectors, prokaryotic expression vectors, and viral vectors. In one embodiment, the expression vector is a conventional expression vector in the art and refers to an expression vector comprising appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other appropriate sequences. In some embodiments, the expression vector may be a virus or plasmid, such as an appropriate phage or phagemid. For more technical details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989. For many known techniques and schemes for nucleic acid manipulation, see Current Protocols in Molecular Biology, 2nd edition, edited by Ausubel et al.

[0039] A fifth aspect of the present invention provides a host cell containing a nucleic acid molecule described in the third aspect of the present invention or a vector described in the fourth aspect of the present invention.

[0040] In some embodiments, the host cells include eukaryotic and prokaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. In some embodiments, the host cells are COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5α, BL21(DE3), or TG1 cells.

[0041] In some embodiments, the host cells are immune cells, such as T cells and NK cells.

[0042] A sixth aspect of the present invention provides an antibody-drug conjugate represented by the following formula or a pharmaceutically acceptable salt thereof, [ka] In the formula, Ab is an antibody containing the anti-ROR1 antibody described in the first aspect of the present invention or the multispecific antibody described in the second aspect of the present invention, L is a linker, D is a drug, and n is an integer or decimal number from 1 to 10.

[0043] In some embodiments, the L is -L a -L b -L c -L d has a structure of -, where L a is linked to the antibody, and L d is linked to the drug. Specifically, L a is

Chemical formula

[0044] In some embodiments, L has a structure selected from the following group: i)L a teeth [ka] L b is -C(O)- or -C(O)-NH-(CH2)2-C(O)-, L c-glycine-glycine-phenylalanine-glycine-, -valine-citrulline-, -glycine- or bonded, L d teeth [ka] -NH-CH2-O-CH2-C(O)- or a bond, or ii) L a teeth [ka] L b (CH2) m -C(O)-(m is 2 or 5), L c is -valine-citrulline- or -glycine-, L d teeth [ka] And, or, iii) L a teeth [ka] L b -NH-(CH2-CH2-O)4-(CH2)2-C(O)-, -NH-(CH2)2-C(O)- or bond, L c -valine-citrulline-, -glycine-, -glycine-glycine-phenylalanine-glycine- or bonded, L d teeth [ka] or -NH-CH2-O-CH2-C(O)- and, iiii)L a teeth [ka] L b is -(CH2)3-C(O)-, L cvaline-citrulline-, L d teeth [ka] And, In the formula, the wavy line ~ indicates the connection point with Ab, and * represents L b The connection point is shown, a * indicates the connection point with Lc, and b * This indicates the site of attachment to the drug.

[0045] In some embodiments, L has the following structure: L a teeth [ka] L b is -C(O)-, L c valine-citrulline-, L d teeth [ka] That is the case.

[0046] In some embodiments, L has the following structure: [ka] In the JPEG2026515756000015.jpg104169 formula, 1 indicates the binding site with antibody Ab, and 2 indicates the binding site with toxin D.

[0047] In some embodiments, the drug is selected from the group consisting of cytotoxic compounds, immunomodulators, enzymes, and hormone inhibitors.

[0048] In some embodiments, the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), eribulin, exatecan, maytansine, SN-38, or combinations thereof.

[0049] In some embodiments, the drug is eribulin.

[0050] In some embodiments, the antibody-drug conjugate has the following structure: [ka] In some embodiments, Ab comprises a heavy chain represented by SEQ ID NO:77 and a light chain represented by SEQ ID NO:78, or Ab comprises the following four polypeptide chains, i.e. Chain 1: Contains the amino acid sequence represented by SEQ ID NO: 81. Chain 2: Contains the amino acid sequence represented by SEQ ID NO: 82. Chain 3: Containing the amino acid sequence represented by SEQ ID NO: 83, and Chain 4: Contains amino acid sequences represented by SEQ ID NO: 84, n is between 3.5 and 4.5.

[0051] A seventh aspect of the present invention provides a drug composition comprising (a) an anti-ROR1 antibody as described in the first aspect of the present invention, or a multispecific antibody as described in the second aspect of the present invention, or an antibody-drug conjugate as described in the third aspect of the present invention or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier.

[0052] An eighth aspect of the present invention provides a method for preparing an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in the sixth aspect of the present invention, which is: (1) A step of providing, as the antibody portion, an anti-ROR1 antibody according to the first aspect of the present invention, or a multispecific antibody according to the second aspect of the present invention, (2) A step of binding the antibody portion in step (1) to a linker-drug compound to obtain the antibody-drug complex.

[0053] In some embodiments, the linker-drug compound is selected from the group consisting of LD-1 to LD-17 described in the present invention.

[0054] In some embodiments, the linker-drug compound is [ka] It is selected from the group consisting of [the specified characters].

[0055] In a ninth aspect of the present invention, the use of an anti-ROR1 antibody according to the first aspect of the present invention, a multispecific antibody according to the second aspect of the present invention, an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to the sixth aspect of the present invention, or a drug composition according to the seventh aspect of the present invention is provided for the preparation of a tumor or cancer therapeutic agent.

[0056] A tenth aspect of the present invention provides a method for treating a tumor or cancer, which comprises administering to a subject of interest an anti-ROR1 antibody as described in the first aspect of the present invention, a multispecific antibody as described in the second aspect of the present invention, an antibody-drug conjugate as described in the sixth aspect of the present invention or a pharmaceutically acceptable salt thereof, or a drug composition as described in the seventh aspect of the present invention.

[0057] In some embodiments, the required subjects include humans or non-human mammals.

[0058] In an eleventh aspect of the present invention, it is provided that an anti-ROR1 antibody described in the first aspect of the present invention, or a multispecific antibody described in the second aspect of the present invention, or an antibody-drug conjugate described in the sixth aspect of the present invention or a pharmaceutically acceptable salt thereof, or a drug composition described in the seventh aspect of the present invention be used as a drug. In some embodiments, it is used as a drug for treating tumors or cancer. In some embodiments, the tumor or cancer is a tumor or cancer that expresses the ROR1 protein.

[0059] In a twelfth aspect of the present invention, the use of an anti-ROR1 antibody described in the first aspect of the present invention, or a multispecific antibody described in the second aspect of the present invention, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof described in the sixth aspect of the present invention, or a drug composition described in the seventh aspect of the present invention, as a drug (i.e., therapeutic use) is provided.

[0060] In some embodiments, the tumor or cancer includes a physical tumor and a hematological tumor.

[0061] In some embodiments, the tumor or cancer is selected from the group consisting of breast cancer, pancreatic cancer, lung cancer, esophageal cancer, non-small cell lung cancer, laryngeal tumor, sarcoma, pharyngeal tumor, oral tumor, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colon cancer, colorectal cancer, urothelial carcinoma, cervical cancer, lymphoma, and leukemia.

[0062] In some embodiments, the hematological malignancies include B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), and myeloid hematological malignancies.

[0063] In some embodiments, the leukemia includes B-cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), and non-Hodgkin lymphoma (NHL).

[0064] In some embodiments, the tumor or cancer is selected from breast cancer and hematological malignancies.

[0065] In some embodiments, the lymphomas and leukemias include numerous subtypes of pre-B cell acute lymphoblastic leukemia (B-ALL), B cell chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL), such as diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL).

[0066] Within the scope of the present invention, it should be understood that each of the technical features described above can be combined with each of the technical features specifically described below (for example, in the examples) to constitute a novel or preferred technical embodiment. Due to space limitations, each will not be described individually here. [Brief explanation of the drawing]

[0067] [Figure 1] This diagram shows the tumor suppressor activity of ADC in an HCC1187 breast cancer model in SCID female mice. [Figure 2] This diagram shows the tumor suppressor activity of ADCs in a Jeko-1 mantle cell lymphoma model using SCID female mice. [Figure 3] This diagram shows the tumor suppressor activity of ADCs in a mouse model of ovarian cancer cells transplanted with PA-1. [Modes for carrying out the invention]

[0068] The inventors, after extensive and in-depth research, have designed and constructed a series of anti-ROR1 antibodies that possess a novel CDR sequence and bind specifically to the human ROR1 protein with high affinity. These anti-ROR1 antibodies can specifically bind to epitopes located at amino acids 130-165 or 70-130 of the ROR1 protein. The anti-ROR1 antibodies prepared in this invention can be used to prepare multispecific antibodies containing the ROR1 protein. Specifically, this invention provides a bi-epitope antibody targeting the ROR1 protein, which has a significantly higher ROR1 antigen recognition ability than a mono-antibody. Furthermore, this invention also provides a conjugate of a mono-antibody with a drug, and a conjugate of a bi-epitope antibody targeting the ROR1 protein with a drug. In vitro experiments have shown that the antibody-drug conjugate according to the present invention exhibits remarkable tumor-suppressing effects on cell lines derived from various cancers or tumors (e.g., breast cancer, ovarian cancer, and lymphocyte cancer) and tumor-transplanted mouse models, and is superior to positive controls. Therefore, it has been declared that the antibody-drug conjugate according to the present invention can be applied to the treatment of tumors or cancer as a therapeutic agent for various stereotumors and hematological malignancies.

[0069] Based on the above, the present invention was completed.

[0070] term To better understand the present invention, the following terms are defined.

[0071] Unless otherwise specified, all singular terms include their plural forms, active tense, and past tense.

[0072] Unless otherwise specified in the context, the term "approximately" includes values ​​within the standard deviation range of the related values.

[0073] Unless otherwise specified in the context, terms such as "contains," "possesses," and "includes" throughout the specification and claims should be understood to have a comprehensive meaning, i.e., "includes, but is not limited to," rather than an exclusive or exhaustive meaning. Unless otherwise specified, "includes" shall include "consisting of." For example, HCDR1 containing the amino acid sequence represented by SEQ ID NO:45 shall be defined as the amino acid sequence clearly covering HCDR1 represented by SEQ ID NO:45.

[0074] In the present invention, "subject" or "patient" refers to an animal, including a human patient requiring anti-cancer treatment or therapy. In some embodiments, the present invention can also be applied to any mammal or other animal requiring such ROR1-targeted anti-cancer therapy in veterinary practice. Examples include non-human primates, dogs, felines, pigs, horses, and any other animals requiring ROR1-targeted anti-cancer therapy.

[0075] The term "antibody" is used in its broadest sense, covering a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, single-specificity antibodies, multispecificity antibodies (e.g., bispecificity 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 native IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy region or heavy chain variable region, followed by three constant regions (CH1, CH2, CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain region or light chain variable region, followed by one constant light region (light chain constant region, CL). The light chains are divided into κ or λ. The heavy chain is divided into γ, μ, α, δ, or ε, and the isotypes of antibodies are defined as IgG, IgM, IgA, IgD, and IgE, respectively. Within 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 contains a "D" region of approximately 10 or more amino acids (generally, one can refer to "Basic Immunology" (Paul, W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7), and all of its contents are incorporated by reference for various purposes).

[0076] The term "antibody fragment" refers to a molecule distinct from a complete antibody, containing a portion of the complete antibody, while maintaining 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-region antibodies, single-chain Fab (scFab), diabodies, linear antibodies, and single-chain antibody molecules (e.g., scFv). Here, (i) Fab fragment: has VL, CL, VH and CH1 domains and one disulfide bond between the heavy and light chains; (ii) Fab' fragment: a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragment: has VH and CH1 domains; (iv) Fd' fragment: has VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragment: has VL and VH domains in one arm of the antibody; (vi) dAb fragment: consists of a VH domain; (vii) hinge deletion type antibody: has at least VL, VH, CL and CH1 domains and the hinge region is deleted; (viii) F(ab' (ix) Single-chain antibody molecule (e.g., single-chain Fv, scFv); (x) "Diabodies": Contains a heavy chain variable region (VH) and a light chain variable region (VL) having two antigen-binding sites and linked together in the same polypeptide chain; (xi) "Linear antibody": Contains a pair of Fd fragments (VH-CH1-VH-CH1) linked in series, forming a pair of antigen-binding regions together with a complementary light chain polypeptide; (xii) dsFv: A fragment in which polypeptides in which one amino acid residue each of VH and VL is replaced with a cysteine ​​residue are linked via SS bonds between the cysteine ​​residues.

[0077] Monoclonal antibodies typically require isolation and purification. This means that the purity of interfering proteins and other contaminants produced by the antibody during manufacturing or purification is usually at least 50%, although the possibility of the monoclonal antibody binding to excessive pharmaceutically acceptable carriers or other carriers intended to facilitate its use is not ruled out. In some cases, the purity of monoclonal antibodies is at least 60%, 70%, 80%, 90%, 95%, or 99% w / w of interfering proteins and contaminants produced during manufacturing or purification.

[0078] The term "bispecific antibody" refers to an antibody (including an 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. Conventional technology has already disclosed bispecific antibodies with various structures. They can be classified into IgG-like bispecific antibodies and antibody-fragment type bispecific antibodies based on the integrity of the IgG molecule, into bivalent, trivalent, tetravalent, or higher-valence bispecific antibodies based on the number of antigen-binding regions, and into symmetrical bispecific antibodies and asymmetrical bispecific antibodies based on whether the structure is symmetrical or not. In this context, bispecific antibodies based on antibody fragments, such as Fab fragments lacking an Fc fragment, form a bispecific antibody by binding two or more Fab fragments to a single molecule. These antibodies have low immunogenicity, a small molecular weight, and high tumor tissue penetration. Representative antibody structures of this type include, for example, F(ab)2, scFv-Fab, and (scFv)2-Fab. For IgG-like bispecific antibodies (e.g., those with an Fc fragment), such antibodies have a large relative molecular weight, and the Fc fragment is useful for antibody purification, solubility, and stability improvement. The Fc portion may also bind to the receptor FcRn, potentially increasing the antibody serum half-life. Representative structural models of bispecific antibodies include, for example, KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, and 1Fab-IgG. Examples include 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, and F(ab)4-CrossMAb (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).

[0079] The specific binding of an antibody to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 or 10 10 M -1 This means that the antibody has affinity for a particular target. Specific binding is more detectable on a larger scale and differs from non-specific binding, which involves binding to at least one unrelated target. Specific binding can result from the formation of bonds between specific functional groups or a specific spatial fit (e.g., lock-and-key type), while non-specific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that a monoclonal antibody has and only one target to bind to.

[0080] The term "variable domain" or "variable region" refers to the antigen-binding region within an antigen-binding molecule. VH and VL each contain four conservative framework regions (FRs) and three complementarity-determining regions (CDRs). Here, the term "complementarity-determining region" or "CDR" refers to the region in the variable domain that is primarily responsible for promoting antigen binding. "Framework" or "FR" refers to variable domain residues other than CDR residues. VH contains three CDR regions, namely HCDR1, HCDR2, and HCDR3, and VL contains three CDR regions, namely LCDR1, LCDR2, and LCDR3. Each VH and VL contains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 sequentially from the N-terminus to the C-terminus. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0081] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering system (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 (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). The correspondences between various numbering systems are well known to those skilled in the art. Exemplary numbering systems of the present invention are shown in the table below. [Table 1]

[0082] It should be understood by those skilled in the art that, even under a particular numbering rule, the positional numbers of the CDRs of a particular antibody may differ at several amino acids. For example, under the Kabat rule, there may be antibodies where the LCDR1 position is 24-33 and the HCDR1 position is 98-106. Unless otherwise specified, the variable domains and CDR sequences in the examples of the present invention all apply the "Kabat" numbering rule. In specific embodiments, a certain numbering system (e.g., Kabat) is used to define amino acid residues, but techniques and schemes corresponding to other numbering systems should be considered equivalent.

[0083] The terms “Fc region” or “fragment crystallizable region” are used to define the C-terminal region of an antibody heavy chain, and include native and modified Fc regions. In some embodiments, the Fc region includes two identical or different subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as the region extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Preferred native sequence Fc regions used in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise specified, the numbering convention for Fc regions is the EU index. The C-terminus of the Fc region may be a complete C-terminus ending with the amino acid residue PGK, or it may be a cleaved C-terminus, for example, with one or two C-terminal amino acid residues removed. In one preferred embodiment, the C-terminus of the heavy chain is a cleaved C-terminus ending with PG. Therefore, in some embodiments, the complete antibody composition may include an antibody population from which all K447 residues and / or G446+K447 residues have been removed. In some embodiments, the complete antibody composition may include an antibody population from which K447 residues and / or G446+K447 residues have not been removed. In some embodiments, the complete antibody composition includes an antibody population of an antibody mixture that may or may not have K447 residues and / or G446+K447 residues.

[0084] In some embodiments, the Fc region of the present invention includes modification by knob-into-hole (KIH) technology, which involves introducing a knob structure at the interface of a first subunit and a hole structure at the interface of a second subunit. This positions the knob structure into the hole structure, promoting the formation of heterodimers while suppressing the formation of homodimers. The knob structure is constructed by substituting a smaller amino acid side chain from the interface of the first subunit with a larger side chain (e.g., tyrosine or tryptophan). The hole structure, on the other hand, is constructed at the interface of the second subunit by substituting a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The knob and hole structures are prepared by altering the nucleic acid encoding the polypeptide, and the selectable amino acid substitutions are shown in the table below. [Table 2]

[0085] In addition to the knob-into-hole technique, other techniques for modifying the heavy chain CH3 domain 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.

[0086] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed by juxtaposed adjacent or non-adjacent amino acids through the tertiary folding of one or more proteins. Epitopes formed from adjacent amino acids are usually retained in denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. A single epitope typically contains at least three, more commonly at least five or eight to ten, amino acids to form a unique spatial structure. Methods for determining the spatial structure of an epitope 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).

[0087] Antibodies that recognize the same or overlapping epitopes can be measured by a simple immunoassay, that is, by measuring the competitive ability of one antibody to bind to a target antigen. Antigen-binding epitopes on antibodies can also be measured by X-ray crystallography to identify the interaction residues. Alternatively, if all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody similarly reduce or eliminate the binding of another antibody, then the two antibodies are considered to have the same epitope. If some amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then the two antibodies are considered to have overlapping epitopes.

[0088] Antibody competition can be measured by experiments in which the test antibody inhibits the specific binding of the reference antibody to a common antigen (see, for example, Junghans et al., Cancer Res. 50:1495, 1990). In competitive binding experiments, when the test antibody is in excess of the reference antibody (e.g., at least 2, 5, 10, 20, or 100 times), the test antibody competes with the reference antibody and inhibits its binding by at least 50%, but preferably 75%, 90%, or 99% in competitive binding experiments. Antibodies that can be identified by competitive experiments (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to epitopes adjacent to the reference antibody.

[0089] To distinguish between conservative and non-conservative amino acid substitutions, amino acids are classified as follows: Class I (hydrophobic side chains): Met, Ala, Val, Leu, Ile; Class II (neutral hydrophilic side chains): Cys, Ser, Thr; Class III (acidic side chains): Asp, Glu; Class IV (basic side chains): Asn, Gln, His, Lys, Arg; Class V (residues affecting chain orientation): Gly, Pro; Class 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.

[0090] The term "sequence identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions when two sequences are optimally aligned. During the alignment process, gaps may be introduced as needed to obtain the maximum sequence identity percentage; however, any conservative substitutions cannot be considered part of the sequence identity. Alignment can be performed by techniques known in the art to measure the sequence identity percentage, and disclosed computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software can be used. Those skilled in the art can determine the parameters to be applied to the alignment measurement, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences to be aligned.

[0091] Antibody effector function refers to the function produced by the Fc domain of Ig. These functions may include, for example, antibody-dependent cytotoxicity, antibody-dependent cytophagocytosis, or complement-dependent cytotoxicity. For example, the Fc effector domain can bind to Fc receptors on immune cells with phagocytic or degrading activity, or to components of the complement system, thereby producing effector function. Typically, actions mediated by Fc-binding cells or complement components result in the suppression of proliferation and / or apoptosis of ROR1-expressing target cells. The Fc region of an antibody can attract Fc receptor (FcR)-expressing cells and draw them to the vicinity of antibody-bound target cells. Cells on which FcR is membrane-expressed include FcγRIII (CD16), FcγRII (CD32), and FcγRIII (CD64), which can be effector cells that kill IgG-binding cells. These effector cells include mononuclear cells, macrophages, spontaneous killer cells, neutrophils, and eosinophils. Upon contact of IgG with FcγR, antibody-dependent cytotoxicity (ADCC) or antibody-dependent cytophagocytosis (ADCP) is activated. ADCC is triggered by the secretion of membrane pore-forming proteins and proteases, which stimulates CD16 sup. +It is mediated by effector cells, while phagocytosis is mediated by CD32.sup. + and CD64.sup. + This is mediated by effector cells (see Basic Immunology, 4th edition, Paul ed., Lippincott-Raven, New York, 1997, Chapters 3, 17, 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 cell-binding antibodies can also activate the classical complement pathway to induce complement-dependent cytotoxicity (CDC). When an antibody forms a complex with an antigen, C1q of the complement system binds to the Fc region of the antibody. When C1q binds to a cell-binding antibody, it can trigger a cascade reaction including the activation of C4 and C2 proteolytic enzymes, generating C3 convertase. The degradation of C3 to C3b by C3 convertases enables the activation of terminal complement components, including C5b, C6, C7, C8, and C9. Collectively, these proteins form membrane invasion complex pores on antibody-coated cells. These pores disrupt the integrity of the cell membrane and kill target cells (see Chapter 2 of *Immunobiology*, 6th edition, Janeway et al., Garland Science, New York, 2005).

[0092] The term "antibody-dependent cytotoxicity" or ADCC refers to a cell death induction mechanism that relies on the interaction between antibody-coated target cells and immune cells (sometimes called effector cells) that possess lytic activity. These effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells attach to the Fc effector domain of Ig, and IgG binds to target cells via its antigen-binding site. Effector cells exert activity that induces the death of antibody-coated target cells.

[0093] The term "antibody-dependent cell phagocytosis" or ADCP refers to the process by which antibody-coated cells are partially or completely migrated to the internal region by phagocytic immune cells (e.g., macrophages, neutrophils, dendritic cells), and these cells bind to the Fc effector domain of Ig.

[0094] The term "complement-dependent cytotoxicity" or CDC refers to a cell death induction mechanism in which the Fc effector domain of an antibody bound to a target cell activates a series of enzymatic reactions, ultimately forming pores in the target cell membrane. Typically, an antigen-antibody complex, such as one formed when an antibody coats a target cell, binds to and activates complement component C1q, thereby activating the complement cascade and leading to target cell death. Complement activation may also lead to the accumulation of complement components on the target cell surface and their binding to complement receptors (e.g., CR3) on leukocytes, thereby promoting ADCC.

[0095] "Cytotoxic effect" means the depletion, elimination, and / or killing of target cells. "Cytotoxic preparation" refers to a preparation that has a cytotoxic effect on cells. Cytotoxic preparations can be administered conjugated to an antibody or in combination with an antibody.

[0096] "Cell suppression" refers to the inhibition of cell proliferation. "Cell suppressants" are drugs that have a cell suppression effect on cells in order to inhibit the growth and / or amplification of specific cell subgroups. Cell suppressants can be administered conjugated to antibodies or in combination with antibodies.

[0097] The term "pharmaceutically acceptable" refers to a substance for animal use, particularly for human use, that is approved or eligible for approval by a supervisory authority, or is listed in a pharmacopoeia or other generally accepted pharmacopoeias. The term "pharmaceutically compatible component" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or carrier that binds to an anti-ROR1 antibody or anti-ROR1 antibody-drug conjugate.

[0098] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of an anti-ROR1 antibody or its complex, or a drug administered in conjunction with an anti-ROR1 antibody. Examples of salts include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, superphosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, sugars, methanes, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamonates (i.e., 1,1′methylene-bis-(2-hydroxy-3-naphthoate) salts). A pharmaceutically acceptable salt may contain other molecules, such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have one or more charged atoms in its structure. An example of a pharmaceutically acceptable salt containing multiple charged atoms may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0099] 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, and the offspring of such cells are also included in these terms. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the passage number. Offspring may not have nucleic acid contents that are exactly the same as those of the parent cells and may contain mutations. In this specification, mutant offspring that have the same function or biological activity as those screened or selected in primary transformed cells are included in these terms. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include, but are not limited to, human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including 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., HepG2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, and Pichia piperi. Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia yeast, Saccharomyces cerevisiae, Saccharomyces yeast, Hansenula polymorpha, Cliveromyces yeast, Cliveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysoporium lachnowens Examples include Fusarium species (Fusarium lucknowense), Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrela patens, and Neurospora crassa.Yeasts of the genus Pichia, any yeast of the genus Saccharomyces, Hansenula polymorpha, any yeast of the genus Cliveromyces, Candida albicans, any species of Aspergillus, Trichoderma reesei, Chrysoporium lucknowense, any species of Fusarium, Yarrowia lipolytica, and Neurospora crassa. The host cells under this patent do not include any subject matter that is not protected under patent law.

[0100] According to one aspect of the present invention, an anti-ROR1 antibody is provided.

[0101] In one embodiment of the present invention, the anti-ROR1 antibody targets an epitope located at amino acids 70-130 of the ROR1 protein (whose amino acid sequence is represented by SEQ ID NO:87), Specifically, the antibody includes HCDR1 represented by SEQ ID NO:61, HCDR2 represented by SEQ ID NO:46, HCDR3 represented by SEQ ID NO:65, and LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50. Preferably, the antibody comprises a heavy chain variable region of the amino acid sequence represented by SEQ ID NO:72 or a variant thereof, and a light chain variable region of the amino acid sequence represented by SEQ ID NO:71 or a variant thereof, or The aforementioned antibody includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, HCDR3 represented by SEQ ID NO:66, and LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50. Preferably, the antibody comprises a heavy chain variable region of the amino acid sequence represented by SEQ ID NO:73 or a variant thereof, and a light chain variable region of the amino acid sequence represented by SEQ ID NO:71 or a variant thereof, or The aforementioned antibody includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, HCDR3 represented by SEQ ID NO:65, and LCDR1 represented by SEQ ID NO:67, LCDR2 represented by SEQ ID NO:68, and LCDR3 represented by SEQ ID NO:50. Preferably, the antibody comprises a heavy chain variable region of the amino acid sequence represented by SEQ ID NO:74 or a variant thereof, and a light chain variable region of the amino acid sequence represented by SEQ ID NO:75 or a variant thereof, or The aforementioned antibody includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, HCDR3 represented by SEQ ID NO:65, and LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:69, and LCDR3 represented by SEQ ID NO:50. Preferably, the antibody comprises a heavy chain variable region of the amino acid sequence represented by SEQ ID NO:74 or a variant thereof, and a light chain variable region of the amino acid sequence represented by SEQ ID NO:76 or a variant thereof, or The aforementioned antibody includes HCDR1 represented by SEQ ID NO:45, HCDR2 represented by SEQ ID NO:46, HCDR3 represented by SEQ ID NO:65, and LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50. Preferably, the antibody comprises a heavy chain variable region of the amino acid sequence represented by SEQ ID NO:74 or a variant thereof, and a light chain variable region of the amino acid sequence represented by SEQ ID NO:71 or a variant thereof.

[0102] In one specific embodiment of the present invention, the antibody comprises a heavy chain whose amino acid sequence is represented by SEQ ID NO:77 and a light chain whose amino acid sequence is represented by SEQ ID NO:78.

[0103] In another embodiment of the present invention, the anti-ROR1 antibody targets an epitope located at amino acids 130-165 of the ROR1 protein (whose amino acid sequence is represented by SEQ ID NO:87), Specifically, the antibody includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:32, HCDR3 represented by SEQ ID NO:19, and LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10. Preferably, the antibody comprises a heavy chain variable region of the amino acid sequence represented by SEQ ID NO:38 or a variant thereof, and a light chain variable region of the amino acid sequence represented by SEQ ID NO:23 or a variant thereof, or The aforementioned antibody includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:18, HCDR3 represented by SEQ ID NO:19, and LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10. Preferably, the antibody comprises a heavy chain variable region of the amino acid sequence represented by SEQ ID NO:22 or a variant thereof, and a light chain variable region of the amino acid sequence represented by SEQ ID NO:23 or a variant thereof.

[0104] In one specific embodiment of the present invention, the antibody comprises a heavy chain whose amino acid sequence is represented by SEQ ID NO:44 and a light chain whose amino acid sequence is represented by SEQ ID NO:31, or It contains a heavy chain whose amino acid sequence is represented by SEQ ID NO:30 and a light chain whose amino acid sequence is represented by SEQ ID NO:31.

[0105] The CDR sequence of the antibody according to the present invention is defined by the Kabat numbering rules. However, those skilled in the art can also obtain the CDR sequence of the antibody according to the present invention using other known numbering rules (e.g., IMGT, Chothia, etc.).

[0106] In another aspect of the present invention, a multispecific antibody comprising the anti-ROR1 antibody described in the present invention is provided.

[0107] In one embodiment of the present invention, the multispecific antibody simultaneously targets the ROR1 protein and proteins other than ROR1, and the proteins other than ROR1 include tumor-associated antigens. The multispecific antibody includes an anti-ROR1 antibody as described in any one of the present invention as the antibody portion that targets the ROR1 protein. Preferably, the present invention includes an anti-ROR1 antibody comprising the following anti-ROR1 antibodies, namely HCDR1 represented by SEQ ID NO: 61, HCDR2 represented by SEQ ID NO: 46, HCDR3 represented by SEQ ID NO: 65, and LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO: 50, or an anti-ROR1 antibody comprising HCDR1 represented by SEQ ID NO: 17, HCDR2 represented by SEQ ID NO: 32, HCDR3 represented by SEQ ID NO: 19, and LCDR1 represented by SEQ ID NO: 20, LCDR2 represented by SEQ ID NO: 21, and LCDR3 represented by SEQ ID NO: 10.

[0108] In another embodiment of the present invention, the multispecific antibody is a bispecific antibody (bi-epitope antibody) targeting the ROR1 protein, the bispecific antibody comprising a first antigen-binding region that targets a first epitope (located at amino acids 130-165 of the ROR1 protein) and a second antigen-binding region that targets a second epitope (located at amino acids 70-130 of the ROR1 protein), where the amino acid sequence of the ROR1 protein is represented by SEQ ID NO:87. Preferably, the first antigen-binding region includes HCDR1 represented by SEQ ID NO:17, HCDR2 represented by SEQ ID NO:32, HCDR3 represented by SEQ ID NO:19, and LCDR1 represented by SEQ ID NO:20, LCDR2 represented by SEQ ID NO:21, and LCDR3 represented by SEQ ID NO:10, and the second antigen-binding region includes HCDR1 represented by SEQ ID NO:61, HCDR2 represented by SEQ ID NO:46, HCDR3 represented by SEQ ID NO:65, and LCDR1 represented by SEQ ID NO:63, LCDR2 represented by SEQ ID NO:64, and LCDR3 represented by SEQ ID NO:50.

[0109] The bispecific antibody according to the present invention is a heterodimer formed by the binding of a first Fc mutant (Fc1) and a second Fc mutant (Fc2), both designed with Fc mutations, in a "knob-in-hole" configuration. The technology for designing Fc mutant mutations is widely applied in this field to the preparation of bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include "Knob-into-Hole" provided to Cater et al. (Protein Engineering vol.9 no.7, pp617-621, 1996); Fc-containing heterodimer form (US20100286374A1) formed by electrostatic induction by Amgen's technical staff; heterodimers (SEED bodies) formed by IgG / IgA chain exchange, provided to Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.1-8, 2010); bispecific molecules formed by Genmab's platform technology DuoBody (Science, 2007. 317, (5844)); heterodimer proteins (mAbs3:6) formed by Xencor's technical staff by combining structural calculations and Fc amino acid mutations, and combining different modes of action. 546-557; November / December 2011); Heterodimeric proteins obtained by the charge network-based Fc modification method (CN201110459100.7) of Suzhou Corning Jerry; and other genetic engineering methods that form heterodimeric functional proteins by Fc amino acid modification or functional modification methods. Selecting a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the scope of those skilled in the art. Preferably, the Fc regions of human antibodies IgG1, IgG2, IgG3 and IgG4, more preferably the Fc regions of human antibodies IgG1 and IgG4. One of the first or second Fc variants was randomly selected and subjected to a knob mutation, while the other was subjected to a hole mutation. In the examples, the first Fc variant was subjected to a knob mutation, and the second Fc variant was subjected to a hole mutation.

[0110] In one specific embodiment of the present invention, the bispecific antibody comprises four polypeptide chains, namely, chain 1: containing an amino acid sequence represented by SEQ ID NO: 81, chain 2: containing an amino acid sequence represented by SEQ ID NO: 82, chain 3: containing an amino acid sequence represented by SEQ ID NO: 83, and chain 4: containing an amino acid sequence represented by SEQ ID NO: 84, where chain 1 contains an Fc knob and chain 3 contains an Fc hole, and chains 1 and 3 are linked in the form of a "knob-in-hole" to form a heterodimer.

[0111] The present invention further provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0112] The term “antibody-drug conjugate (ADC)” means that a monoclonal antibody or antibody fragment is conjugated to a biologically active toxic drug via a conjugation unit. The antibody or antibody fragment described herein can be conjugated to an effector molecule in any way. For example, the antibody or antibody fragment can be attached to a toxic drug by chemical or recombinant means. Chemical means for preparing the fusion or conjugate are known in the art. The method for conjugating the antibody or antibody fragment to the drug must be able to conjugate the antibody and the toxic drug without interfering with the antibody's ability to bind to the target molecule.

[0113] The drug may have any cytotoxicity and may be a drug that inhibits cell growth or an immunosuppressant. For example, in the embodiment, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino group, carboxyl group, mercapto group, hydroxyl group, or ketone group that can form a bond with the linker. When the drug is directly linked to the linker, the drug has an active group that reacts before linking to the antibody. Examples of useful drugs include antitubulin drugs, DNA suppository linking reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids.

[0114] Cytotoxic drugs are substances that inhibit or prevent cell function and / or cause cell death or destruction. While cytotoxic drugs can, in principle, kill tumor cells at sufficiently high concentrations, their lack of specificity can lead to serious side effects, such as killing tumor cells while simultaneously inducing apoptosis in normal cells. Examples of cytotoxic drugs include toxins such as small molecule toxins and enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 This includes radioactive isotopes of Lu, chemotherapeutic agents, antibiotics, and nucleases.

[0115] The antibody according to the present invention and the cytotoxic drug can be conjugated via a coupling agent. The coupling agent may be one or more selected from, for example, a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, or a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that conjugates and links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl group-utilizing coupling agent may be one or more selected from cis-aconitic anhydride coupling agents (e.g., cis-aconitic anhydride) or acylhydrazone coupling agents (the binding site is an acylhydrazone).

[0116] Some residues on antibodies (e.g., Cys and Lys) are used for linking to many functional groups, such as imaging reagents (e.g., chromogenic groups and fluorescent groups), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be bound to functional agents to form antibody-functional agent complexes. Functional agents (e.g., drugs, detection reagents, stabilizers) are bound (covalently linked) to antibodies. Functional agents can be linked to antibodies directly or indirectly via linkers.

[0117] Antibodies can bind to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs include a linker (or binding site, sometimes called a linker) located between the drug and the antibody. The term “linker unit” or “binding fragment” refers to a chemical structural fragment or binding that is bound at one end to an antibody or its antigen-binding fragment and at the other end to a drug, which may be bound to another linker before being bound to the drug. Linkers can be degradable or indegradable. Degradable linkers are typically easily degraded in the intracellular environment, for example, by degradation at a target site, releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers such as peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases and endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidases. Examples of peptidyl linkers include dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine; 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., hydrazone linkers, which are hydrolyzed when the pH falls below 5.5) and linkers that are degradable under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by a protease.

[0118] Before linking to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and linking is achieved by this active reactive group. Mercapto-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodized, bromide, or chlorided), halogenated esters (e.g., iodized, bromide, or chlorided), halogenated methyl ketones (e.g., iodized, bromide, or chlorided), benzyl halides (e.g., iodized, bromide, or chlorided), vinyl sulfones, pyridyl disulfide, mercury derivatives (e.g., 3,6-di-(mercurymethyl)dioxane, whose counterion is acetate, chloride, or nitrate), and polymethylenedimethyl sulfide thiosulfonates. An example of a linker is a maleimide linked to an antibody via thiosuccinimide.

[0119] In this invention, a drug linker compound can form an ADC in a single, simple step. In another embodiment, a bifunctional linker compound can form an ADC in a two-step or multi-step method. For example, in the first step, a cysteine ​​residue reacts with the reactive moiety of the linker, and in the next step, the functional group on the linker reacts with the drug, thereby forming an ADC.

[0120] Typically, a functional group on the linker is selected to react specifically with an appropriate reactive group on the drug moiety. As a non-limiting example, the azi compound-based moiety can react specifically with a reactive alkynyl group on the drug moiety. The drug is conjugated to the linker by addition via a 1,3-dipole between the azi and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azi groups), 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 methods are well known to those skilled in the art, as described in *Biological Linking Techniques* (Second Edition (Elsevier)). Those skilled in the art should understand that, regarding the selective reaction between the drug portion and the linker, if complementary reactive functional groups are selected, then either of these complementary groups can be used as both the linker and the drug.

[0121] The present invention further provides a method for preparing ADCs, which includes linking an antibody to a drug linker compound (or drug binding compound (LD), such as LD-1 to LD-17 shown in the present invention) under conditions sufficient to form an antibody complex (ADC).

[0122] In some embodiments, the method according to the present invention includes linking an antibody to a linker compound under conditions sufficient to form an antibody-linker complex. In these embodiments, the method according to the present invention further includes linking the antibody-linker complex to a drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0123] The drug load is sometimes referred to as the drug-to-antibody ratio (DAR), i.e., the average number of drugs bound to each antibody in the ADC. For example, approximately 1 to 10 drugs may be bound to each antibody. In some embodiments, approximately 1 to 8 drugs may be bound to each antibody, preferably in 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 load may be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The general formula of the ADC according to the present invention includes the set of antibody-drug conjugates within the predetermined ranges described above. In embodiments of the present invention, the drug load is represented by n, which is a decimal or an integer. The drug load can be measured by common methods such as UV / visible light spectroscopy, mass spectrometry, ELISA, and HPLC.

[0124] In one embodiment of the present invention, a cytotoxic drug is bound to an antibody via a linking unit.

[0125] The amount of ligand-drug conjugates loaded can be controlled by the following non-limiting methods, specifically: (1) Controlling the molar ratio of drug binding fragments to single antibodies, (2) Controlling the reaction time and temperature, (3) Selecting different reaction reagents, including

[0126] The present invention also provides compositions. Preferably, the composition is a drug composition containing the antibody or its active fragment or its fusion protein as described above, and a pharmaceutically acceptable carrier. Typically, these substances can be prepared in a non-toxic, inert, and pharmaceutically acceptable aqueous carrying medium, where the pH is usually about 5 to 8, preferably about 6 to 8, and the pH value can be varied depending on the properties of the substance being prepared and the disease being treated. The prepared drug composition can be administered by a normal route, specifically including, but not limited to, intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. In the present invention, the term “drug composition” means that the bispecific antibody according to the present invention can be combined with a pharmaceutically acceptable carrier to constitute a drug formulation composition in order to exert a more stable therapeutic effect, and these formulations can ensure the structural integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention and protect the polyfunctional groups of the protein to prevent its degradation (including, but not limited to, aggregation, deammonia, and oxidation). The drug composition according to the present invention contains the above-mentioned anti-ROR1 antibody, bispecific antibody, antibody-drug conjugate, and pharmaceutically acceptable carrier or excipient in a safe and effective amount (e.g., 0.001 to 99 wt%, preferably 0.01 to 90 wt%, more preferably 0.1 to 80 wt%). Such carriers include, but are not limited to, saline solution, buffer solution, glucose, water, glycerin, ethanol, and combinations thereof. The drug formulation must be compatible with the method of administration. The drug composition according to the present invention can be prepared in needle form, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other supplements. Drug compositions such as needles and solutions are preferably prepared under sterile conditions. The dose of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kilogram body weight to about 50 milligrams / kilogram body weight per day. Furthermore, the drug composition according to the present invention can also be used in combination with other therapeutic agents.

[0127] When using a drug composition, a safe and effective dose of antibody or multispecific antibody or its immune complex is administered to a mammal. This safe and effective dose is usually at least about 10 micrograms / kilogram body weight, and in most cases less than about 50 milligrams / kilogram body weight. Preferably, this dose is about 10 micrograms / kilogram body weight to about 10 milligrams / kilogram body weight. Of course, the specific dose should also take into account factors such as the means of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0128] The term "bonding" refers to a situation where this particular group is absent, and the groups on either side of it are directly linked to form a bond.

[0129] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group having a straight or branched chain and containing 1 to 20 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) (i.e., C 1~ C 20 (Alkyl alkyl group). The alkyl group is preferably an alkyl group having 1 to 12 carbon atoms (i.e., C 1~ C 12 Alkyl alkyl groups), more preferably alkyl groups having 1 to 6 carbon atoms (i.e., C 1~(C6 alkyl group). Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl Examples include the n-octyl group, 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched isomers thereof. The alkyl group may be substituted or unsubstituted, and if it is substituted, it may be substituted at any available linking site, and the substituent is preferably one or more selected from a D atom, halogen, alkoxy group, halogenated alkyl group, halogenated alkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0130] The term "alkoxy group" refers to an -O-(alkyl group), and the definition of the alkyl group is as described above. Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy groups. The alkoxy group may be substituted or unsubstituted, and if it is substituted, it may be substituted at any available linking site, and the substituent is preferably one or more selected from a D atom, halogen, alkoxy group, halogenated alkyl group, halogenated alkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0131] The term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic whole-carbocyclic (i.e., monocyclic cycloalkyl group) or polycyclic (i.e., polycyclic cycloalkyl group) 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., a 3 to 20-membered cycloalkyl group). The cycloalkyl group is preferably a cycloalkyl group having 3 to 12 ring atoms (i.e., a 3 to 12-membered cycloalkyl group), more preferably a cycloalkyl group having 3 to 8 ring atoms (i.e., a 3 to 8-membered cycloalkyl group, e.g., C 3~ C7 cycloalkyl group), most preferably a cycloalkyl group having 3 to 6 ring atoms (i.e., a 3 to 6 membered cycloalkyl group, for example C 3~ It is a C6 cycloalkyl group.

[0132] The term "aryl group" refers to a monocyclic all-carbon aromatic ring (i.e., a monocyclic aryl group) or a polycyclic aromatic ring system (i.e., a polycyclic aryl group) having a conjugated π-electron system, and having 6 to 14 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 6- to 14-membered aryl group). The aryl group is preferably an aryl group having 6 to 10 ring atoms (i.e., a 6- to 10-membered aryl group). An example of the monocyclic aryl group is the phenyl group. Non-limiting examples of the polycyclic aryl group include the naphthyl group, anthryl group, and phenyl group. The aforementioned polycyclic aryl group includes the condensation of a phenyl group with one or more heterocyclyl groups or cycloalkyl groups, or the condensation of a naphthyl group with one or more heterocyclyl groups or cycloalkyl groups, where the linking site is on a phenyl group or naphthyl group, in which case the number of ring atoms is followed by the number of ring atoms in the polycyclic aromatic ring system, and non-limiting examples include: [ka] These are some examples.

[0133] The aryl group may be substituted or unsubstituted, and if substituted, it may be substituted at any available linking site, and the substituent is preferably one or more selected from a D atom, halogen, alkyl group, alkoxy group, halogenated alkyl group, halogenated alkoxy group, cycloalkyloxy group, heterocyclyloxy group, hydroxy group, hydroxyalkyl group, oxo group, cyano group, amino group, nitro group, cycloalkyl group, heterocyclyl group, aryl group, and heteroaryl group.

[0134] The term "heteroaryl group" refers to a monocyclic heteroaromatic ring (i.e., a monocyclic heteroaryl group) or a polycyclic heteroaromatic ring system (i.e., a polycyclic heteroaryl group) having a conjugated π-electron system, wherein the ring contains at least one heteroatom (e.g., 1, 2, 3, or 4) selected from nitrogen, oxygen, and sulfur (the nitrogen may be selectively oxidized, i.e., to form a nitrogen oxide; the sulfur may be selectively oxygenated, i.e., to form a sulfoxide or sulfone, but without -OO-, -OS-, or -SS-), and has 5 to 14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) ring atoms (i.e., a 5- to 14-membered heteroaryl group). The heteroaryl group is preferably a heteroaryl group having 5 to 10 ring atoms (i.e., a 5- to 10-membered heteroaryl group), more preferably a heteroaryl group having 5 or 6 ring atoms (i.e., a 5- or 6-membered heteroaryl group).

[0135] The term "cycloalkyloxy group" refers to a cycloalkyl-O- group, and the cycloalkyl group is defined as described above.

[0136] The term "heterocyclyloxy group" refers to a heterocyclyl-O- group, which is defined as described above.

[0137] The term "aryloxy group" refers to an alkyl-O- group, and the alkyl group is defined as described above.

[0138] The term "heteroaryloxy group" refers to a heteroaryl-O- group, and this heteroaryl group is defined as described above.

[0139] The term "alkylthio group" refers to an alkyl-S- group, and the alkyl group is defined as described above.

[0140] The term "halogenated alkyl group" refers to a group in which one or more halogens are substituted for an alkyl group, and the alkyl group is as defined above.

[0141] The term "deuterated alkyl group" refers to an alkyl group in which one or more deuterium atoms are substituted, and the alkyl group is as defined above.

[0142] The term "halogenated alkoxy group" refers to an alkoxy group that has been substituted with one or more halogens, and the alkoxy group is as defined above.

[0143] The term "hydroxyalkyl group" refers to an alkyl group that has been substituted with one or more hydroxyl groups, and the alkyl group is as defined above.

[0144] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0145] The term "hydroxyl group" refers to the -OH group.

[0146] The term "amino group" refers to -NH2.

[0147] The term "cyano group" refers to -CN.

[0148] The term "oxo group" refers to =O.

[0149] N-ethyldiisopropylamine is abbreviated as DIEA.

[0150] N,N-dimethylformamide is abbreviated as DMF.

[0151] O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylureahexafluoroline is abbreviated as HATU.

[0152] 1-Hydroxybenzotriazole is abbreviated as HOBt.

[0153] "Substituting" means that one or more hydrogen atoms in a group, preferably 1 to 6, more preferably 1 to 3, are independently substituted by the corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions with little effort (experimentally or theoretically). For example, an amino group or hydroxyl group with free hydrogen may become unstable when bonded to a carbon atom with an unsaturated (e.g., olefin) bond.

[0154] The present invention will be described below with reference to specific examples. Those skilled in the art will understand that these examples are merely for illustrative purposes and do not limit the scope of the present invention in any way.

[0155] The experimental methods in the following examples are standard procedures unless otherwise specified. The raw materials and reagents used in the following examples are commercially available unless otherwise specified. Example 1: Humanization and expression of D10 antibody The mouse-derived anti-ROR1 single-antibody D10 was modified to humanize it as follows.

[0156] Humanization modifications were performed on mouse-derived antibodies using the CDR transplantation method. By transplanting the CDR region of the heavy chain into the matching heavy chain variable region gene and light chain variable region framework sequence, the heavy chain variable region sequence (represented by SEQ ID NO:1) and light chain variable region sequence (represented by SEQ ID NO:2) of the humanized antibody BRHu-3 were obtained.

[0157] [Table 3] [Table 4]

[0158] The VH and VL regions of the antibody BRHu-3 and the VH and VL regions of monoantibody D10 were combined with the human IgG1 heavy chain constant region and the κ light chain constant region, respectively, to obtain the humanized antibody BRHu-3 and the chimeric antibody ch-D10. IgG1 heavy chain constant region (SEQ ID NO:11): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Kappa light chain constant region (SEQ ID NO:12): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0159] Chimeric antibody ch-D10 (heavy chain amino acid sequence represented by SEQ ID NO: 15, light chain amino acid sequence by SEQ ID NO: 16) and humanized antibody BRHu-3 (heavy chain amino acid sequence represented by SEQ ID NO: 13, light chain amino acid sequence by SEQ ID NO: 14) were expressed in CHO-K1 cells and purified. First, a vector containing the antibody heavy chain and light chain coding sequences was electroporated into CHO-K1 cells. After culturing at 37°C under 5% CO2 conditions for 4 days, the cell culture supernatant was centrifuged at 3000 rpm for 10 minutes. The supernatant was collected and purified with protein A to achieve antibody purity >95%. BRHu-3 heavy chain sequence (SEQ ID NO:13): QVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGVHWIRQPPGKGLEWLGVIWAGGFTNYNSALKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCARRGSSYSMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK BRHu-3 light chain sequence (SEQ ID NO:14): EIVLTQSPATLSLSPGERATLSCASSNVSYIHWYQQKPGQAPRPWIYEISKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNYPLITFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence of chimeric antibody ch-D10 (SEQ ID NO: 15): QVQLKESGPGLVAPSQTLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGFTNYNSALKSRLSISKDNSKSQVLLKMTSLQTDDTAMYYCARRGSSYSMDYWGQGT SVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain sequence of chimeric antibody ch-D10 (SEQ ID NO: 16): EIVLSQSPAITAASLGQKVTITCSASSNVSYIHWYQQRSGTSPRPWIYEISKLASGVPVRFSGSGSGTSYSLTISSMEAEDAAIYYCQQWNYPLITFGSGTKLEIQ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0160] Example 2: Affinity maturation of the humanized antibody BRHu-3 Affinity maturation for the humanized antibody BRHu-3 was performed using the method described below.

[0161] Humanized antibody BRHu-3 was used as a mutation template, primers were designed, and random mutations were introduced into six CDR regions of BRHu-3 by PCR. The mutant DNA was electroporated into SU320+ Helper phage hypersensitive cells and cultured overnight at 37°C. The mutants were isolated and purified by PEG8000 / NaCl precipitation to obtain a phage display mutation library.

[0162] Biopanning was performed on the phage display mutation library to enrich positive monoclones. Antigen ROR2 was coated onto Maxi-sorp plates at 500 ng / well and left overnight at 4°C. The Maxi-sorp plates were blocked with 1% PVA and left at room temperature for 2 hours. Subsequently, the plates were incubated with the phages from the mutation library at 4°C for 2 hours. The supernatant was collected and incubated with negative cells CHO-K1 at 4°C for 2 hours. The supernatant was collected after centrifugation and incubated with CHO-K1-hROR1 cells (a stable metastatic cell line overexpressing human ROR1; refer to SEQ ID NO:87 for the human ROR1 sequence) at 4°C for 2 hours. After adding PBS buffer (PBST) containing 0.05% Tween20 pre-cooled to 4°C and washing 6-8 times, the phages were infected with 1 mL of NEBalpha5F′ cells (purchased from NEB) with an OD600 of 0.8, shaken at 37°C for 1 hour, then the auxiliary phage M13K07 (purchased from NEB) was added, shaken at 37°C for 1 hour, and the mixture was transferred to 40 mL of 2YT / Carb / Kan medium and cultured overnight at 37°C. The mixture was plated, and the enrichment was statistically assessed on day 3. The phages were isolated and purified by PEG 8000 / NaCl precipitation, and the panning process was initiated.

[0163] Following the above procedure, five rounds of biopanning were performed to obtain a successfully enriched phage clone.

[0164] Phage ELISA experiments were performed by randomly selecting monoclones from concentrated phage clones. Recombinant proteins ROR1 (purchased from Kactus Biosystems) and ROR2 (purchased from Kactus Biosystems) were coated onto ELISA plates at 100 ng / well, left overnight at 4°C, and then blocked with 1% PVA for 2 hours at room temperature. On the same day as coating, monoclonal phages were selected, shaken in deep-well plates, and incubated overnight at 37°C. After centrifugation of the deep-well plates, 50 μL of the supernatant was transferred to an ELISA plate and incubated at room temperature for 2 hours. The ELISA plate was washed with PBST, 100 μL of Anti-M13 HRP antibody (purchased from Sino Biological) was added, and incubated at room temperature for 1 hour. Subsequently, the ELISA plate was washed with PBST, then with PBS, 100 μL of TMB was added, and it was left at 37°C for 5-10 minutes. The reaction was stopped by adding 50 μL of 1M phosphate. Absorbance at 450 nm was measured using a microplate reader. Five clones with high affinity were selected and sequenced; these five clones are YR-4, YR-8, YR-10, YR-11, and YR-21. The variable region and CDR region of the antibody are shown below. [Table 5] [Table 6]

[0165] The above VH and VL were combined with the human IgG1 heavy chain constant region and λ light chain constant region (as shown in Example 1), respectively, to obtain the full-length sequence of the antibody. The above antibody was expressed and purified by the method described in Example 1. The heavy chain and light chain of the exemplary antibody YR-8 are represented by SEQ ID NO:30 and SEQ ID NO:31, respectively. YR-8 heavy chain sequence (SEQ ID NO:30): QVQLQESGPGLVKPSETLSLTTCTVSGFSLTRYGVHWIRQPPGKGLEWLGVIWAGGFTNYNSDLKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCARRGDSYSMKYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK YR-8 light chain sequence (SEQ ID NO:31): EIVLTQSPATLSLSPGERATLSCSASSQVSYIHWYQQKPGQAPRPWIYETSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNYPLITFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0166] Example 3: Modification of the affinity mature antibody YR-8 molecule Amino acid mutations were performed on the N at position 60 and S at position 61 of the heavy chain of the YR-8 molecule (the amino acid positions were obtained by Kabat numbering). The mutation scheme included mutating the N at position 60 to E, or mutating the S at position 61 to A, V, T, L, or I. The resulting molecules 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 antibodies obtained after mutation are shown below. [Table 7]

[0167] The VH sequence of the antibody after mutation is as follows: [Table 8]

[0168] The above-mentioned mutant VH was combined with YR-8-VL (SEQ ID NO:23) and the human IgG1 heavy chain constant region and κ light chain constant region (as shown in Example 1) to obtain a complete antibody. The above mutant was expressed and purified by the method described in Example 1. The heavy chain and light chain of the exemplary antibody YR-8-ES obtained in this invention are represented by SEQ ID NO:44 and SEQ ID NO:31, respectively. Heavy chain sequence of YR-8-ES (SEQ ID NO: 44): QVQLQESGPGLVKPSETLSLTTCTVSGFSLTRYGVHWIRQPPGKGLEWLGVIWAGGFTNYESDLKSRLTISKDNSKNQVSLKLSSVTAADTAVYYCARRGDSYSMKYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain sequence of YR-8-ES (SEQ ID NO:31): EIVLTQSPATLSLSPGERATLSCSASSQVSYIHWYQQKPGQAPRPWIYETSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWNYPLITFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0169] Example 4: Humanization of antibody H10 Humanization modifications were performed on mouse-derived anti-ROR1 monoanti-H10 antibodies using the CDR transplantation method. By transplanting the CDR region of the heavy chain into a matching heavy chain variable region gene and a framework sequence of the light chain variable region, 16 human molecules with distinct sequences were obtained. The sequences of the variable regions and CDR regions of the humanized antibodies are shown below. [Table 9]

Table 10

[0170] The variable region of the humanized antibody H10 was combined with the human IgG1 heavy-chain constant region and the λ light-chain constant region (as shown in Example 1) to obtain a complete antibody. The specific antibodies are as follows.

Table 11

[0171] Example 5: Affinity Maturation of Humanized Antibody H10 Saturation mutagenesis was performed on the six CDR regions of the above H10-hVH4-hVL4. Vectors containing the mutated antibody heavy-chain and light-chain coding sequences were electroporated into CHO-K1 cells. The cells were cultured for 4 days under the conditions of 37 °C and 5% CO2. The supernatant was collected and added to a microplate coated with ROR1 (manufacturer: Kactus Biosystems, product number: ROR-HM401), and incubated at 37 °C for 1 hour. The plate was washed, and HRP-labeled goat anti-human IgG (manufacturer: Jackson, 109-005-008) was added and reacted at 37 °C for 1 hour. After washing the plate, TMB solution was added and the reaction was allowed to proceed in the dark at room temperature for 15 minutes, and then the ELISA stop solution was added to stop the reaction. The absorbance at a wavelength of 450 nm was measured using a microplate reader. Six clones with high affinity were selected for sequencing, and the six clones obtained were AM3-ZH1, AM3-ZH3, AM3-ZH4, AM3-ZH5, AM3-ZH6, and AM3-ZH7, respectively. The related antibody sequences are as follows.

Table 12

Table 13

[0172] The above variable region was combined with the human IgG1 heavy chain constant region and κ light chain constant region (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 invention are represented by SEQ ID NO:77 and SEQ ID NO:78, respectively. Heavy chain sequence of AM3-ZH3 (SEQ ID NO:77): EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSSISTGASAYFPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARITTSTWYMDVWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK AM3-ZH3 light chain sequence (SEQ ID NO:78): DIQMTQSPSAMSASVGDRVTITCKASQDIYSYLSWFQQKPGKVPKTLIYRANRLVRGVPSRFSGSGSGQEYTLTISSLQPEDMATYYCLQYDEFPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0173] Example 6: Construction of a biepitope antibody targeting ROR1 In this embodiment, a bispecific antibody targeting ROR1 is constructed using a ROR1 antibody derived from D10 and a ROR1 antibody derived from H10, and the ROR1 antibody derived from D10 and the ROR1 antibody derived from H10 each bind to different epitopes on ROR1.

[0174] In this example, a biepitope antibody with a KIH structure was constructed, and an exemplary structure is shown below.

[0175] Structure 1: The following four chains, namely, Chain 1: YR8VH-CH1-Fcknob, Chain 2: YR8VL-CL, Chain 3: ZH3VH-CH1-FcHole, and Chain 4: Contains ZH3VL-CL; Structure 2: The following four chains, namely, Chain 1: YR8VH-CH1-Fchole, Chain 2: YR8VL-CL, Chain 3: ZH3VH-CH1-Fcknob, and Chain 4: Contains ZH3VL-CL. [Table 14]

[0176] Furthermore, in order to prepare antibodies that bind to different epitopes on ROR1, the present invention also prepared YR-8-ES mutant 1, based on the YR-8-ES antibody, which contains only the Knob (T366W) mutation in the Fc region, and AM3-ZH3 mutant 1, based on the AM3-ZH3 antibody, which contains only the Hole (T366S, L368A, Y407V) mutation in the Fc region. The sequences are shown below. [Table 15]

[0177] For example, YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were each dissolved in PBS at pH 7.2, mixed in a molar ratio of 1:1, 2 mM EDTA was added, and the antibodies were 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, the mixture was eluted with G25 resin, desalted and purified, and filtered through a 0.2 μm filter under sterile conditions to obtain the ROR1-targeting biepitope antibody KIH YR8 / ZH3. Its sequence is shown below. [Table 16]

[0178] Furthermore, in this invention, UC961 is used as a positive control, and its sequence is as shown below. UC961 heavy chain (SEQ ID NO: 85): QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISKDTSKNQVVLTMTNMPDPVDTATYYCARGWYYFDYWGHGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK UC961 light chain (SEQ ID NO: 86): DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Amino acid sequence of human ROR1 (SEQ ID NO:87): MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLR IRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPY CDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALR FPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKEL PLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKS SLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVI EMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPI NGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL

[0179] Example 7: Preparation of Linker-Drug Linker-drug (LD) compounds LD-1 to LD-17 were prepared by pre-binding toxins and linkers for the preparation of ADCs. Exemplary toxins included monomethyl auristatin E (MMAE), eribulin, and exatecan.

[0180] In this embodiment, experimental methods for which specific conditions are not explicitly stated generally follow general conditions or conditions recommended by the raw material or product manufacturers. Reagents for which the specific source is not specified were ordinary reagents purchased from the market.

[0181] The structure of the compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR displacement (δ) is 10 -6 It is given in units of (ppm). 1 A Bruker AVANCE-400 nuclear magnetometer was used for the 1H NMR measurements, with deuterated dimethyl sulfoxide as the measurement solvent and tetramethylsilane (TMS) as the internal standard.

[0182] For the MS measurement, a Shimadzu LCMS-2020 single quadrupole liquid chromatography-mass spectrometer (manufacturer: Shimadzu Corporation, MS model number: 2020 Single Quadrupole MS) was used, the chromatography column was a Phenomenex Gemini NX 5μ, C18, 110Å, 50 x 4.6 mm, the mobile phase was a 0.1% methane acid aqueous solution / 0.1% methane acid in acetonitrile (ACN) solution, and the flow rate was 1 mL / min.

[0183] For RP-HPLC, a Shimadzu Nexera liquid phase preparation system was used, along with a Phenomenex Gemini NX 5μ, C18, 110Å, 150 x 50 mm chromatography column, a 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid in acetonitrile (ACN) solution as the mobile phase, and a flow rate of 50 mL / min.

[0184] 7.1 Preparation of LD-1 [ka] To a 3 mL solution of anhydrous tetrahydrofuran containing 2-tert-butylhydrazine-1,2-dicarboxylate (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 stopped with 1 mL of water containing 60 μL of AcOH. The mixture was then purified by RP-HPLC. After lyophilization of the pure components, 204 mg of white solid A1 was obtained. MS m / z:373.6[M+H] + .

[0185] [ka] To an 8.0 mL solution of acetic acid (AcOH) containing 2-[[tert-butoxycarbonyl-(tert-butoxycarbonylamino)amino]methyl]propyl-2-acrylate tert-butyl (A1) (204 mg, 0.55 mmol), 3,4-dibromofuran-2,5-diketone (140 mg, 0.55 mmol) was added. The mixture was stirred under reflux under an argon atmosphere for 11 days, then concentrated to 3 mL and purified by RP-HPLC to obtain 43 mg of white solid A2 (yield: 22%). MS m / z:354.8 [M+H] + .

[0186] [ka] To a stirred solution of anhydrous dichloromethane (10 mL) containing 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (A2, 350 mg), N-hydroxysuccinimide (230 mg) was added, followed by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (400 mg). The mixture was stirred at room temperature for 30 minutes, and the reaction product was concentrated under reduced pressure until dry. The residue was directly purified by RP-HPLC, and after lyophilization, compound 4 was obtained as a white solid (337 mg). MS m / z:452.0 [M+H] + .

[0187] [ka] 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 directly purified by RP-HPLC and, after lyophilization, a white solid compound 8 (17 mg), i.e., LD-1, was obtained. MS m / z:1243.6 [M+H] + .

[0188] 7.2 Preparation of LD-2 [ka] To an acetonitrile / water (6 / 4, v / v, 2 mL) solution containing compound 6 (20 mg), saturated NaHCO3 (0.2 mL) and anhydrous bromoacetic acid (9 mg) were added. The reaction mixture was stirred at room temperature (22°C) for 10 minutes. The reaction mixture was then directly purified by RP-HPLC and lyophilized to obtain compound 7 (17 mg), i.e., LD-2, as a white solid. MS m / z:1243.6 [M+H] + .

[0189] 7.3 Preparation of LD-3 [ka] To a solution of compound 6 (62 mg) and Fmoc-NH-PEG4-COOH (compound 9, 25 mg, purchased from PurePEG, catalog number 433704-1H) in anhydrous DMF (1 mL), DIEA (0.025 mL) was added, 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 mixture was reacted for another 30 minutes. The crude reaction mixture was directly purified by RP-HPLC, and after lyophilization, compound 10 (61 mg, TFA salt) was obtained as a white solid.

[0190] To an acetonitrile / water (6 / 4, v / v, 2 mL) solution containing compound 10 (37 mg), saturated NaHCO3 (0.03 mL) and anhydrous bromoacetic acid (7 mg) were added. The reaction mixture was stirred at room temperature for 10 minutes, and the crude mixture was purified by RP-HPLC. After lyophilization, compound 11 (32 mg), i.e., LD-3, was obtained as a white solid. MS m / z:491.0 [M+H] + .

[0191] 7.4 Preparation of LD-4 [ka] To a 2 mL solution of anhydrous DMF containing compound 6 (62 mg, TFA salt), maleimidohexanoic acid (compound 18, 12 mg) was added, followed by 0.02 mL of DIEA and 20 mg of HATU. The reaction mixture was stirred at room temperature (22°C). After 15 minutes, the crude reaction mixture was directly purified by RP-HPLC and, after lyophilization, obtained compound 19 (62 mg, TFA salt), i.e., LD-4, as a white solid. MS m / z:1316.6 [M+H] + .

[0192] 7.5 Preparation of LD-5 [ka] To a solution of anhydrous DMF (2 mL) containing compound 12 (65 mg, prepared according to WO2022026915) and MMAE (72 mg), DIEA (0.002 mL) was added, 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 ether (40 mL), the organic phase was dried over NaSO4, concentrated under reduced pressure until dry, and finally crude compound 13 was obtained dissolved in methanol (2 mL). Zinc powder (200 mg) was added to the methanol solution containing compound 13, followed by methaneic acid (0.2 mL). The reaction mixture was stirred at room temperature for 30 minutes. The filtrate, from which the solid had been removed by filtration, was directly purified by RP-HPLC, and after lyophilization, compound 14 (72 mg) was obtained as a white solid. To a solution of anhydrous DMF (2 mL) containing compound 14 (TFA salt, 66 mg) and Fmoc-NH-PEG4-COOH (purchased from PurePEG, catalog number 433704, 25 mg), DIEA (0.025 mL) was added, followed by HATU (20 mg). The mixture was stirred at room temperature. After 16 hours, the crude mixture was purified by RP-HPLC, and after lyophilization, compound 15 (72 mg) was obtained as a white powder.

[0193] Compound 15 (70 mg) was dissolved in acetonitrile / water (6 / 4, v / v, 3 mL) solution, 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 mixture was reacted for 0.5 hours. The crude reaction mixture was directly purified by RP-HPLC, and after lyophilization, compound 17 (46 mg), i.e., LD-5, was obtained as a white solid. MS m / z:1426.7 [M+H] + .

[0194] 7.6 Preparation of LD-6 [ka] To a solution of anhydrous N,N-dimethylformamide (2 mL) containing compound 20 (60 mg, purchased from InnoPep) and exatecan mesylate (compound 21, 53 mg, purchased from Advance ChemBlocks, catalog number 10484), N-ethyldiisopropylamine (0.05 mL) was added, followed by O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylureahexafluoroline (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, after lyophilization, compound 22 (67 mg, trifluoroacetate) was obtained as a yellow powder.

[0195] Compound 4 (25 mg, synthesized according to the method reported in PCT / US2022 / 078563) was added to a solution of N,N-dimethylformamide (2 mL) containing compound 22 (44 mg), followed by the addition of 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, after lyophilization, compound 23, i.e., LD-6, was obtained as a yellow solid (42 mg). MS m / z:1090.2 [M+H] + .

[0196] 7.7 Preparation of LD-14 [ka] To a solution of anhydrous N,N-dimethylformamide (2 mL) containing compound 1 (77 mg, purchased from MedChemExpress, HY-41189), eribulin (compound 2, mesylate, 82 mg, purchased from MedChemExpress, HY-13442) was added, followed by the addition of 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 mixture was stirred at room temperature for 15 minutes. The reaction mixture was directly purified by RP-HPLC, and after lyophilization, compound 3 (110 mg, trifluoroacetate) was obtained as a white solid.

[0197] 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 the addition of 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, after lyophilization, compound 5 (43 mg), i.e., LD-14, was obtained as a white solid. MS m / z:1471.6 [M+H] + . 1HNMR(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).

[0198] 7.8 Preparation of LD-7 [ka] Compound 4 (12 mg) was added to a 1 mL solution of N,N-dimethylformamide containing compound 24 (21 mg, synthesized according to the process reported in US10155821B2), followed by the addition of diisopropylethylamine (10 μL). The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP-HPLC and, after lyophilization, yielded compound 25 (i.e., LD-7, 23 mg) as a yellow solid. MS m / z:1177.4 [M+H] + .

[0199] 7.9 Preparation of LD-8 [ka] To a dimethylformamide (3 mL) solution containing compound 27 (92 mg, 0.2 mmol, purchased from ChemScene, CS-0105172) and Fmoc-Gly-OH (90 mg, purchased from Combi-Blocks, San Diego), diisopropylethylamine (0.1 mL) was added, followed by 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. After lyophilization, compound 28 (76 mg) was obtained as a white powder.

[0200] Compound 28 (73 mg) was dissolved in anhydrous dimethylformamide (1 mL), bis(4-nitrophenyl carbonate (purchased from 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 reaction product was directly purified by RP-HPLC to obtain compound 29 (75 mg) as a white powder.

[0201] Diisopropylethylamine (0.02 mL) was added to a solution of anhydrous dimethylformamide (1 mL) containing compound 29 (54 mg) and exatecan mesylate (compound 21, exatecan 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), the organic layer was dried over sodium sulfate, and concentrated under reduced pressure until dry. The residue was suspended in acetonitrile / water (2 mL, 6 / 4, v / v). Sodium hydroxide (aqueous solution, 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 reaction product was purified by RP-HPLC to obtain compound 30 (33 mg, TFA salt) as a yellow powder.

[0202] Compound 31 (10 mg, purchased from Ambeed) was added to a dimethylformamide (1 mL) solution containing compound 30 (32 mg), 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 obtain compound 32 (i.e., LD-8, 28 mg) as a yellow solid. MS m / z:985.4 [M+H] + .

[0203] 7.10 Preparation of LD-9 [ka] To an acetonitrile / water (6 / 4, v / v, 1 mL) solution containing compound 24 (21 mg, synthesized according to the process reported in US10155821B2), saturated sodium bicarbonate aqueous solution (0.02 mL) and anhydrous bromoacetic acid (10 mg) were added. The reaction mixture was stirred at room temperature for 15 minutes, and the mixture was directly purified by RP-HPLC to obtain compound 26 (19 mg) as a yellow powder. MS m / z: m / z 961.4 [M+H] + .

[0204] 7.11 Preparation of LD-10 [ka] Compound 4 (12 mg, see preparation process for LD-7) was added to a dimethylformamide (1 mL) solution containing compound 30 (20 mg), 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 obtain compound 33 (i.e., LD-10, 19 mg) as a yellow solid. MS m / z:1170.4 [M+H] + .

[0205] 7.12. Preparation of LD-11 [ka] To an acetonitrile / water (6 / 4, v / v, 1 mL) solution containing compound 30 (19 mg), saturated sodium bicarbonate aqueous solution (0.02 mL) and anhydrous bromoacetic acid (10 mg) were added. The reaction mixture was stirred at room temperature for 15 minutes, and the mixture was directly purified by RP-HPLC to obtain compound 34 (i.e., LD-11, 17 mg) as a yellow powder. MS m / z:954.2 [M+H] + .

[0206] 7.13. Preparation of LD-12 [ka] To a solution of dimethylformamide anhydride (1 mL) containing compound 3 (25 mg), cyclopentanediacitic anhydride (5 mg) was added, followed by diisopropylethylamine (0.01 mL). The reaction mixture was stirred at room temperature for 2 hours and 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 product was concentrated under reduced pressure, and the residue was purified by RP-HPLC to obtain compound 39 (i.e., LD-12, 23 mg) as a white powder. MS m / z:1415.7 [M+H] + .

[0207] 7.14 Preparation of LD-13 [ka] A solution of compound 35 (92 mg, synthesized according to the procedure reported in Bioconjugate Chem., 2006, 17(3), 831 - 840) and eribulin mesylate (2, 82 mg) in anhydrous dimethylformamide (2 mL) was added with diisopropylethylamine (0.04 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), 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 (aqueous solution, 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 reaction product was purified by RP - HPLC to obtain compound 36 (97 mg, TFA salt) as a white powder.

[0208] Compound 4 (6 mg) was added to a solution of compound 36 (for 12 mg) in dimethylformamide (1 mL), and then diisopropylethylamine (5 μL) was added. The reaction mixture was stirred at room temperature. After 1 hour, the crude mixture was purified by RP - HPLC and freeze - dried to obtain compound 37 (i.e., LD - 13, 12 mg) as a white solid. MS m / z: 1478.6 [M + H] + 。

[0209] 7.15, Preparation of LD - 15

Chemical Structure

[0210] 7.16 Preparation of LD-16 [ka] To a dimethylformamide (1 mL) solution containing compound 3 (12 mg), compound 6-maleimidohexanoic acid N-hydroxysuccinimide (5 mg, purchased from Sigma-Aldrich) was added, 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, after lyophilization, compound 41 (i.e., LD-16, 11 mg) was obtained as a white solid. MS m / z:1328.8 [M+H] + .

[0211] 7.17 Preparation of LD-17 [ka] To an acetonitrile / water (6 / 4, v / v, 1 mL) solution containing compound 3 (13 mg), saturated sodium bicarbonate aqueous solution (0.01 mL) and anhydrous bromoacetic acid (5 mg) were added. The reaction mixture was stirred at room temperature for 15 minutes, and the mixture was directly purified by RP-HPLC to obtain compound 42 (i.e., LD-17, 9 mg) as a white powder. MS m / z:1255.6 [M+H] + .

[0212] Example 8: Preparation of ADC An ADC drug is prepared by reacting the antibody according to the present invention with a drug linker (LD), and the DAR value of the prepared ADC is measured.

[0213] Simply put, antibodies were treated with a reducing agent (e.g., tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT)) to reduce some or all of the cysteine ​​disulfide residues, forming highly nucleophilic cysteinethiol groups (-CH2SH). The partially or completely reduced antibodies were then reacted with a drug linker or linker reagent and an electrophilic functional group (e.g., maleimide or bromoacetyl) to finally prepare the ADC.

[0214] The method for measuring the DAR value of the ADC is as follows.

[0215] The drug action rate (DAR) of the ADC according to the present invention was analyzed by hydrophobic interaction chromatography-high-performance liquid chromatography (HIC-HPLC). The ADC was separated in the chromatography column using a MabPac HIC-Butyl column (4.6 x 100 mm, 5 μm, catalog number 088558, purchased from ThermoFisher, USA). Buffer A was defined as 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate, and Buffer B was defined as 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile. The initial conditions were stabilized at 85% A and 15% B. Linear gradient elution was performed using 5% A and 85% B for 30 minutes, followed by elution using 5% A and 95% B for an additional 5 minutes. The flow rate and temperature were set to 0.5 mL / min and 25°C. The drug distribution of the ADC at 214 nm and 280 nm was measured to calculate the DAR value.

[0216] 8.1 Preparation of ADC-1 [ka] The 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) (the molar ratio of tris(2-carboxyethyl(phosphine hydrochloride) to antibody was 2.8:1). After incubation at 37°C for approximately 120 minutes, LD-4 was added to the reducing antibody as a drug linker (the molar ratio of drug linker to antibody was 5:1), and the mixture was eluted with G25 resin at room temperature for 1 hour to purify the desalted product. After filtration through a 0.2 μm filter under sterile conditions, ADC-1 was obtained and stored frozen. Analysis by the above hydrophobic interaction chromatography-high-performance liquid chromatography confirmed that the mean DAR value of ADC-1 was between 3.7 and 4.3.

[0217] 8.1 Preparation of ADC-2 (Preparation of monoclonal antibody and drug conjugate) [ka] The 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 (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody: 10:1). After incubation at 37°C for approximately 120 minutes, LD-14 was added to the reduced antibody as a drug linker (molar ratio of drug linker to antibody: 5:1), and the mixture was eluted with G25 resin at room temperature for 1 hour to purify the desalted product. After filtration through a 0.2 μm filter under sterile conditions, ADC-2 was obtained and stored frozen. Analysis by the above hydrophobic interaction chromatography-high-performance liquid chromatography confirmed that the mean DAR value of ADC-2 was between 3.7 and 4.3.

[0218] 8.2 Preparation of ADC-3 (Preparation of a bispecific antibody-drug conjugate) [ka] YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were each dissolved in PBS at pH 7.2 and first mixed in a molar ratio of 1:1. 2 mM EDTA was added, and the antibodies were reduced with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 10:1). After incubation at 25°C for approximately 16 hours, LD-14 was added as a drug linker to the reducing antibody KIH YR8 / ZH3 (molar ratio of drug linker to antibody KIH YR8 / ZH3 was 5:1). Elution was performed with G25 resin at room temperature for 1 hour, desalting was purified, and after filtration through a 0.2 μm filter under sterile conditions, ADC-3 was obtained and frozen for storage. Analysis by the above hydrophobic interaction chromatography-high-performance liquid chromatography confirmed that the mean DAR value of ADC-3 was between 3.7 and 4.3.

[0219] 8.3 Preparation of ADC-4 [ka] YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were each dissolved in PBS at pH 7.2 and first mixed in a molar ratio of 1:1. 2 mM EDTA was added, and the antibodies were reduced with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 10:1). After incubation at 25°C for approximately 16 hours, LD-1 was added as a drug linker to the reducing antibody KIH YR8 / ZH3 (molar ratio of drug linker to antibody KIH YR8 / ZH3 was 5:1), eluted with G25 resin at room temperature for 1 hour, desalted and purified, and filtered through a 0.2 μm filter under sterile conditions to obtain ADC-4, which was then cryopreserved. Analysis by the above hydrophobic interaction chromatography-high-performance liquid chromatography confirmed that the mean DAR value of ADC-4 was between 3.7 and 4.3.

[0220] 8.5. Preparation of ADC-5 (Preparation of monoclonal antibody and drug conjugate) [ka] The 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 (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody: 10:1). After incubation at 37°C for approximately 120 minutes, 5 molar ratios of LD-1 were added to the reduced antibody as a drug linker (molar ratio of LD-1 to antibody: 5:1). The mixture was eluted with G25 resin at room temperature for 1 hour, desalted and purified, and filtered through a 0.2 μm filter under sterile conditions to obtain ADC-5, which was then cryopreserved. Analysis by the above hydrophobic interaction chromatography-high-performance liquid chromatography confirmed that the mean DAR value of ADC-5 was between 3.7 and 4.3.

[0221] Furthermore, ADCs were prepared using the same method as for ADC-2 and ADC-5, in which negative control IgG and positive control UC961 antibody were conjugated to LD-14 and LD-1, respectively. These were named ADC-6 (UC961 conjugated to LD-14) and ADC-7 (UC961 conjugated to LD-1), and the drug loads of ADC-8 (IgG conjugated to LD-14) and ADC-9 (IgG conjugated to LD-1) were 3.7 to 4.3.

[0222] Exemplary prepared ADCs are summarized in Table 7 below. [Table 17]

[0223] Furthermore, in the present invention, the following ADCs have also been prepared by the ADC preparation method according to Example 8, where Ab is the anti-ROR1 antibody or the bispecific antibody according to the present invention, and n is 1 to 8. Ab-LD-2: [ka] Ab-LD-3:

change

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[0224] Example 9: Measurement of the biological activity of monoclonal antibodies 9.1 Binding activity of monoclonal antibodies to human ROR1 protein For affinity measurement with the ROR1 protein, an Octet R8 (manufacturer: Sartorius) was used as the measuring instrument. Antibodies were captured using an AHC (anti-human IgG Fc capture) biosensor (manufacturer: Sartorius), and then this sensor was immersed in the analyte, the ROR1 antigen. The following five steps were performed in this experiment.

[0225] 1. Baseline (60 seconds); 2. Loading (antibody capture) (150 seconds, 1.5 nm); 3. Baseline (100 seconds); 4. Association (binding to antigen ROR1, 60 seconds); 5. Dissociation (isolation from antigen ROR1, 60 seconds). After measurement, the sensor was regenerated by alternately immersing it in regeneration buffer (glycine, pH 1.5) and neutralization buffer (PBS) for 5 seconds each, for a total of three cycles. PBS was used as the running buffer in this experiment. The results are shown in Tables 8 to 11 below.

[0226] 9.2 Binding activity of monoclonal antibodies to human ROR1-expressing cells The binding of the anti-ROR1 antibody according to the present invention to human ROR1-expressing cells, MDA-MB-231 cells (purchased from the Chinese Academy of Sciences Cell Bank) and Jeko-1 cells (purchased from the Chinese Academy of Sciences Cell Bank), was measured by flow cytometry. MDA-MB-231 cells / Jeko-1 cells were incubated with antibody at different concentrations (starting concentration 66.67 nM, 6-fold dilution, 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 (manufacturer: Invitrogen, catalog number: 12-4998-82, diluted 1:100) was added, and the cells were incubated in the dark at 4°C for 30 minutes. After washing the cells twice with 2% BSA-PBS, the median fluorescence intensity was measured using a flow cytometer BD C6 plus. The results are shown in Tables 8 to 11 below.

[0227] 9.3 Binding activity of monoclonal antibodies to human ROR2 protein The binding of humanized antibodies to ROR2, a homologous protein of ROR1, was measured by ELISA. hROR2 (manufacturer: Kactus Biosystems, catalog number: ROR-HM402) was diluted to a concentration of 1 μg / ml in PBS, and the microplate was coated and incubated at 37°C for 1 hour. Subsequently, the plate was blocked with 5% BSA-PBS blocking solution at 37°C for 1 hour. After washing the plate with PBST, the antibody was diluted to different concentrations (antibody starting concentration 333.33 nM, 3-fold dilution, total of 8 concentrations) and added to the plate, and incubated at 37°C for 1 hour. After washing the plate, HRP-labeled goat anti-human IgG (manufacturer: Sigma-Aldrich, catalog number: A0170, diluted 1:10000) was added and reacted at 37°C for 1 hour. After washing the plate, TMB solution was added and the reaction was allowed to proceed in the dark at room temperature for 15 minutes, after which the reaction was stopped by adding ELISA stop solution. The absorbance at a wavelength of 450 nm was measured using a microplate reader. The results are shown in Tables 8 to 11 below.

[0228] [Table 18] As is clear from the results in Table 8, the humanized antibody BRHu-3 had a similar affinity for the ROR1 protein as the ch-D10 antibody, and its binding ability to MDA-MB-231 cells was similar as that of the ch-D10 antibody. In addition, neither of them bound to ROR2.

[0229] [Table 19] As is clear from the results in Table 9, affinity-mature antibodies YR-4, YR-8, YR-10, YR-11, and YR-21 all exhibit significantly improved affinity for ROR1 compared to the humanized antibody BRHu-3. Regarding binding to MDA-MB-231 and Jeko-1 cells, YR-8, YR-10, and YR-11 all exhibit 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, but YR-8 did not.

[0230] [Table 20] As is clear from the results in Table 10, all mutants except YR-8 and YR-8-ES bound to ROR2. YR-8-ES exhibited comparable affinity for ROR1 and cell-binding activity to YR-8.

[0231] [Table 21] As is clear from the results in Table 11, compared to H10-hVH4-hVL4, the affinity-mature antibodies showed improved affinity to ROR1 and binding to MDA-MB-231 cells, albeit to varying degrees. In particular, AM3-ZH3 showed a more than 9-fold improvement in affinity. None of the molecules other than AM3-ZH1 bound to ROR2.

[0232] Example 10: Antibody binding experiment to ROR1-expressing cells Measuring the specific binding ability of anti-hROR1 diabody IgG antibodies to the hROR1 antigen expressed on the cell surface is extremely important for antibodies, especially therapeutic antibodies in the body. 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, in a 37°C, 5% CO2 culture chamber. The experimental procedure is as follows.

[0233] After dissociating logarithmic growth cells with 0.25% pancreatic enzyme, they were resuspended in staining buffer (purchased from Biolengend) and washed, and the cells were counted, with 4.5 × 10⁶ cells per 100 μL of staining buffer. 5 Individual cells were added to a 96-well plate at 45 μL / well. Furthermore, to prevent nonspecific binding of the antibody to human tumor cells, 5 μL of human TruStain (purchased from BioLegend) receptor blocker was added per well. The test antibody was diluted to a starting concentration of 200 nM, then diluted again in staining buffer, and further diluted threefold to obtain a total of 11 concentrations, including 200 nM and the zero point. The diluted test antibody was added to the 96-well plate at 50 μL per well, with the highest final concentration being 100 nM. After thorough mixing, the cells were reacted at 4°C for 15 minutes. After the reaction, the cells were washed in staining buffer, and then PE-labeled constant region (Fc) specific antibody (rabbit anti-human IgG-PE conjugate, purchased from BioLegend, 410707) was added at 5 μL / 2 × 10⁶ per 100 μl of staining buffer. 5Individual cells were suspended and reacted at 4°C for 15 minutes. After the cell reaction, the cells were washed in staining buffer and resuspended in 100 μL of staining buffer. The indications in the PE channels of single cells were analyzed using a Novocyte 3000 instrument (Agilent). The antibody protein concentration was used as the x-coordinate, and the corresponding indication in the PE channel was used as the y-coordinate. A dose-response curve was plotted using the 4PL (4-parameter logistic) equation from Sigmoidal, and after analysis, the EC (Edible Cell Analysis) was performed. 50 A value was generated. The equation is as follows:

[0234] Y=Bottom+(X^Hillslope)×(Top-Bottom) / (X^HillSlope+EC50^HillSlope), MFI fold = Maximum PE reading of the antibody-treated experimental group / PE reading of the antibody-free group.

[0235] The results of the FACS binding activity of the test substance, the anti-hROR1 antibody, with human ROR1 are shown in Table 12. As is clear from the results, all of the anti-ROR1 antibodies according to the present invention can specifically bind to ROR1-expressing cells PA-1 and Jeko-1, and moreover, their binding ability is high. Furthermore, in the same cell line, the dual epitope antibody can recognize and label a higher total amount of antigen compared to the single antibody, specifically showing a significant increase in the MFI fold ratio. [Table 22]

[0236] Example 11: Cytotoxicity experiment of ADC molecule In this experiment, the inhibitory effects of anti-ROR1 antibody-MMAE conjugate and anti-ROR1 antibody-erythromycin conjugate on the growth of various tumor cell lines were studied. In this experiment, the anti-proliferative effect of the drug was evaluated using CellTiterGlo2 (Promega) reagent. The cell lines used in the experiment were PA-1 (ATCC, CRL-1572, human ovarian teratocarcinoma cells), Jeko-1 (ATCC CRL-3006, human mantle cell lymphoma cells), and HCC-187 (ATCC CRL-2322, human breast cancer cells). The experimental procedure is as follows.

[0237] PA-1 cells were cultured in ATCC EMEM medium containing 10% fetal bovine serum, Jeko-1 cells were cultured in RPMI-1640 medium containing 20% fetal bovine serum, and HCC-187 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Each of the three types of cells was seeded in a 96-well plate at a density of 2×10 3 ~5×10 3 cells per well, at 50 μL / well. After culturing for 24 hours, the anti-ROR1 antibody-MMAE conjugate or anti-ROR1 antibody-erythromycin conjugate diluted with different concentrations of culture medium, or the control antibody-MMAE conjugate or control antibody-erythromycin conjugate, was added at 100 μL / well. While duplicates were set for each concentration, a solvent control and a cell-free medium well corresponding to the concentration were set. After culturing in a 37°C, 5% CO2 incubator for 96 hours (PA-1, Jeko-1) or 144 hours (HCC-1187), 100 μL of CellTiterGlo2 was added per well, and the mixture was placed on an orbital shaker at room temperature and mixed for 15 minutes. The luminescence value was measured, and the IC 50 value (nM) of each cell by the anti-ROR1 antibody-MMAE conjugate or anti-ROR1 antibody-erythromycin conjugate was calculated. The results are as shown in Table 13.

Table 23

[0238] As is clear from Table 13, the anti-ROR1 antibody-MMAE complex or the anti-ROR1 antibody-eribulin complex exhibits significant killing activity against each of the three types of tumor cells with different ROR1 expression levels, and the inhibitory effect is related to the different binding method or different toxin used. This demonstrates that the anti-ROR1 antibody complex according to the present invention can specifically kill ROR1-positive cells via ROR1-mediated endocytosis. Furthermore, since it exhibits significant inhibitory activity against cancer cell lines derived from breast cancer, ovarian cancer, and lymphocyte cancer, it is demonstrated that the obtained ADC has a relatively good inhibitory effect against various types of solid tumors and hematological malignancies.

[0239] Example 12: Measurement of the in vivo efficacy of ADC molecules 12.1 Measurement of ADC efficacy in a mouse model transplanted with breast cancer cell lineage HCC1187 Human ROR1-expressing breast cancer cell lineage HCC1187 (purchased from ATCC) was subcutaneously implanted in severe combined immunodeficiency CB17 SCID female mice (purchased from Charles River Laboratories) at a rate of 1 × 10⁶ 7 Individual cells / animals were transplanted. After transplantation, the average tumor size was 162 mm. 3 On day 0, the mice were grouped and either administered a single intravenous injection of 3.0 mg / kg of ADC1, ADC2, ADC3, ADC4, or ADC5 prepared in Example 8, or administered 1.0 mg / kg of ADC2, ADC3, ADC4, or ADC5 intravenously once every four days for a total of two injections (days 0 and 4, Q4D x 2). In the control group, mice were intravenously injected with 4 mL / kg of PBS. Tumor size and body weight were measured in the mice over the following 48 days, and the tumor suppression rate (TGI) was calculated. The formula for calculating TGI is as follows:

[0240] TGI(%)=1-{(Td-T0) / (Cd-C0)}×100% In the formula, Td and Cd are the average tumor volumes of the treatment group and control group on the day of tumor volume measurement, and T0 and C0 are the average tumor volumes of the treatment group and control group on day 0.

[0241] The results are shown in Figure 1 and Table 14. [Table 24]

[0242] As is clear from the results, the ADC according to the present invention can significantly suppress the growth of HCC1187 transplanted tumors in mice.

[0243] 12.2 Measurement of ADC efficacy in a mouse model transplanted with mantle cell lymphoma cells (Jeko-1). Human ROR1-expressing mantle cell lymphoma cell lineage Jeko-1 (purchased from ATCC) was subcutaneously implanted in severe combined immunodeficiency CB17 SCID female mice (purchased from Charles River Laboratories) at a dose of 1 × 10⁶ 7 Individual cells / animals were transplanted. After transplantation, the average tumor size was 156 mm. 3 Upon reaching this stage (day 0), the mice were grouped and either administered ADC1, ADC2, ADC3, ADC4, or ADC5 prepared in Example 8 as a single intravenous injection at 3.0 mg / kg, or administered ADC2, ADC3, ADC4, or ADC5 at 1.0 mg / kg once every four days for a total of three injections (days 0, 4, and 8, Q4D x 3). In the control group, mice were intravenously injected with PBS at 4 mL / kg. The tumor size and body weight of the mice were measured over the following 51 days, and the tumor suppression rate (TGI) was calculated. The results are shown in Figure 2 and Table 15. [Table 25]

[0244] As is clear from these results, the ADC according to the present invention can significantly suppress the growth of Jeko-1 transplanted tumors in mice.

[0245] 12.3. Measurement of ADC efficacy in a mouse model transplanted with ovarian cancer cell lineage PA-1 Human ROR1-expressing ovarian cancer cell lineage PA-1 (purchased from ATCC) was subcutaneously in 5 × 10⁶ female nude mice (purchased from Charles River Laboratories). 6 Individual cells / animals were transplanted. After transplantation, the average tumor size was 150 mm. 3 On day 0, the mice were grouped and administered either ADC1 or ADC3 prepared in Example 8 as a single intravenous injection at a dose of 1.0 mg / kg or 3.0 mg / kg. In the control group, mice were administered PBS intravenously at a dose of 4 mL / kg. Tumor size and body weight were measured over the following 13 days, and the tumor suppression rate (TGI) was calculated. The results are shown in Figure 3 and Table 16. [Table 26]

[0246] As is clear from these results, the ADC according to the present invention can significantly suppress the growth of PA-1 transplanted tumors in mice.

[0247] All documents referenced in this invention are incorporated by reference in the same manner as each document may be referenced independently. Furthermore, it should be understood that, after reading the above description of this invention, persons skilled in the art may make various changes or amendments to this invention, and that these equivalent forms are also included within the scope defined by the claims attached to this invention.

Claims

1. An anti-ROR1 antibody comprising a heavy chain variable region and a light chain variable region, specifically, (Z1) The heavy chain variable region includes HCDR1 represented by SEQ ID NO: 61, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO:

50. (Z2) The heavy chain variable region includes HCDR1 represented by SEQ ID NO: 45, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 66, and the light chain variable region includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO:

50. (Z3) The heavy chain variable region includes HCDR1 represented by SEQ ID NO: 45, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region includes LCDR1 represented by SEQ ID NO: 67, LCDR2 represented by SEQ ID NO: 68, and LCDR3 represented by SEQ ID NO:

50. (Z4) The heavy chain variable region includes HCDR1 represented by SEQ ID NO: 45, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 69, and LCDR3 represented by SEQ ID NO:

50. (Z5) The heavy chain variable region includes HCDR1 represented by SEQ ID NO: 45, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO:

50. (Z6) The heavy chain variable region includes HCDR1 represented by SEQ ID NO: 17, HCDR2 represented by SEQ ID NO: 32, and HCDR3 represented by SEQ ID NO: 19, and the light chain variable region includes LCDR1 represented by SEQ ID NO: 20, LCDR2 represented by SEQ ID NO: 21, and LCDR3 represented by SEQ ID NO: 10, or, (Z7) The heavy chain variable region includes HCDR1 represented by SEQ ID NO: 17, HCDR2 represented by SEQ ID NO: 18, and HCDR3 represented by SEQ ID NO: 19, and the light chain variable region includes LCDR1 represented by SEQ ID NO: 20, LCDR2 represented by SEQ ID NO: 21, and LCDR3 represented by SEQ ID NO:

10. Anti-ROR1 antibody.

2. The heavy chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 72, and the light chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 71, or, The heavy chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 73, and the light chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 71, or, The heavy chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 74, and the light chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 75, or, The heavy chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 74, and the light chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 76, or, The heavy chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 74, and the light chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 71, or, The heavy chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 22, and the light chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 23, or, The heavy chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO: 38, and the light chain variable region includes a sequence having at least 85% sequence identity with the amino acid sequence represented by SEQ ID NO:

23. The anti-ROR1 antibody according to claim 1.

3. The anti-ROR1 antibody comprises a heavy chain constant region and a light chain constant region. Preferably, the heavy chain constant region comprises an amino acid sequence represented by SEQ ID NO: 11 or a variant thereof, and / or the light chain constant region comprises an amino acid sequence represented by SEQ ID NO: 12 or a variant thereof. The anti-ROR1 antibody according to claim 1 or 2.

4. The aforementioned anti-ROR1 antibody is A heavy chain whose amino acid sequence is represented by SEQ ID NO: 77 and a light chain whose amino acid sequence is represented by SEQ ID NO: 78, or A heavy chain whose amino acid sequence is represented by SEQ ID NO: 30 and a light chain whose amino acid sequence is represented by SEQ ID NO: 31, or It includes a heavy chain whose amino acid sequence is represented by SEQ ID NO: 44 and a light chain whose amino acid sequence is represented by SEQ ID NO:

31. The anti-ROR1 antibody according to any one of claims 1 to 3.

5. The anti-ROR1 antibody is an antibody fragment, preferably the antibody fragment is Fab, Fab', F(ab') 2 It is selected from the group consisting of Fd, Fv, scFv, dsFv, and dAb. The anti-ROR1 antibody according to claim 1 or 2.

6. A multispecific antibody comprising the anti-ROR1 antibody according to any one of claims 1 to 5.

7. The multispecific antibody is a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region and the second antigen-binding region bind to different epitopes on human ROR1. Preferably, the first antigen-binding region binds to amino acids at positions 130-165 on human ROR1, the second antigen-binding region binds to amino acids at positions 70-130 on human ROR1, and the amino acid sequence of the ROR1 protein is represented by SEQ ID NO:

87. The multispecific antibody according to claim 6.

8. The first antigen-binding region includes a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding region includes a heavy chain variable region VH2 and a light chain variable region VL2, specifically, The heavy chain variable region VH1 includes HCDR1 represented by SEQ ID NO: 17, HCDR2 represented by SEQ ID NO: 32, and HCDR3 represented by SEQ ID NO: 19, and the light chain variable region VL1 includes LCDR1 represented by SEQ ID NO: 20, LCDR2 represented by SEQ ID NO: 21, and LCDR3 represented by SEQ ID NO: 10, or, The heavy chain variable region VH1 includes HCDR1 represented by SEQ ID NO: 17, HCDR2 represented by SEQ ID NO: 18, and HCDR3 represented by SEQ ID NO: 19, and the light chain variable region VL1 includes LCDR1 represented by SEQ ID NO: 20, LCDR2 represented by SEQ ID NO: 21, and LCDR3 represented by SEQ ID NO:

10. The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO: 61, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO: 50, or, The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO: 45, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 66, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO: 50, or, The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO: 45, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO: 67, LCDR2 represented by SEQ ID NO: 68, and LCDR3 represented by SEQ ID NO: 50, or, The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO: 45, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 69, and LCDR3 represented by SEQ ID NO: 50, or, The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO: 45, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO:

50. Preferably, the heavy chain variable region VH1 includes HCDR1 represented by SEQ ID NO: 17, HCDR2 represented by SEQ ID NO: 32, and HCDR3 represented by SEQ ID NO: 19, and the light chain variable region VL1 includes LCDR1 represented by SEQ ID NO: 20, LCDR2 represented by SEQ ID NO: 21, and LCDR3 represented by SEQ ID NO:

10. The heavy chain variable region VH2 includes HCDR1 represented by SEQ ID NO: 61, HCDR2 represented by SEQ ID NO: 46, and HCDR3 represented by SEQ ID NO: 65, and the light chain variable region VL2 includes LCDR1 represented by SEQ ID NO: 63, LCDR2 represented by SEQ ID NO: 64, and LCDR3 represented by SEQ ID NO:

50. The multispecific antibody according to claim 7.

9. The aforementioned first antigen-binding region is The heavy chain variable region VH1 includes an amino acid sequence represented by SEQ ID NO: 38, and the light chain variable region VL1 includes an amino acid sequence represented by SEQ ID NO: 23, or The heavy chain variable region VH1 includes an amino acid sequence represented by SEQ ID NO: 22, and the light chain variable region VL1 includes an amino acid sequence represented by SEQ ID NO:

23. The second antigen-binding region is, The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO: 72, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO: 71, or The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO: 73, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO: 71, or The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO: 74, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO: 75, or The heavy chain variable region VH2 includes the amino acid sequence represented by SEQ ID NO: 74, and the light chain variable region VL2 includes the amino acid sequence represented by SEQ ID NO: 76, or The heavy chain variable region VH2 includes an amino acid sequence represented by SEQ ID NO: 74, and the light chain variable region VL2 includes an amino acid sequence represented by SEQ ID NO:

71. Preferably, the first antigen-binding region includes a heavy chain variable region VH1 represented by SEQ ID NO: 38 and a light chain variable region VL1 represented by SEQ ID NO: 23, and the second antigen-binding region includes a heavy chain variable region VH2 represented by SEQ ID NO: 72 and a light chain variable region VL2 represented by SEQ ID NO:

71. The multispecific antibody according to claim 7 or 8.

10. The bispecific antibody comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 that can associate with each other, and each of Fc1 and Fc2 independently has one or more amino acid substitutions that reduce homodimerization of the Fc region. Preferably, Fc1 has a projection structure (knob) using knob-into-hole technology, and Fc2 has a hole structure (hole) using knob-into-hole technology. More preferably, the amino acid at position 366 of Fc1 is W, 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, and the numbering follows the EU index. Most preferably, Fc1 comprises an amino acid sequence represented by SEQ ID NO: 79, and Fc2 comprises an amino acid sequence represented by SEQ ID NO:

80. A multispecific antibody according to any one of claims 7 to 9.

11. The aforementioned bispecific antibody has four chains as shown in (a) to (d) below, i.e., (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) Including [light chain variable region VL2]-[CL2], The aforementioned bispecific antibody has four strands as shown in (e), (b), (f), and (d) below, i.e., (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) Including [light chain variable region VL2]-[CL2], Here, 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 independently the constant region of the antibody's light chain, and CH1 is the first part of the constant region of the antibody's heavy chain. Preferably, the heavy chain variable region VH1 includes an amino acid sequence represented by SEQ ID NO: 38, the light chain variable region VL1 includes an amino acid sequence represented by SEQ ID NO: 23, the heavy chain variable region VH2 includes an amino acid sequence represented by SEQ ID NO: 72, and the light chain variable region VL2 includes an amino acid sequence represented by SEQ ID NO:

71. More preferably, the bispecific antibody has the following four chains, i.e., Chain 1: Contains the amino acid sequence represented by SEQ ID NO:

81. Chain 2: Contains the amino acid sequence represented by SEQ ID NO:

82. Chain 3: Containing the amino acid sequence represented by SEQ ID NO: 83, and Chain 4: Contains amino acid sequences represented by SEQ ID NO: 84, The multispecific antibody according to claim 10.

12. A nucleic acid molecule encoding an anti-ROR1 antibody according to any one of claims 1 to 5, or a multispecific antibody according to any one of claims 6 to 11.

13. (1) A vector containing the nucleic acid molecule described in claim 12, (2) A biomaterial which is a host cell containing the nucleic acid molecule described in claim 12 or the vector described in (1).

14. An antibody-drug conjugate represented by the following formula or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein Ab is an anti-ROR1 antibody according to any one of claims 1 to 5, or an antibody comprising a multispecific antibody according to any one of claims 6 to 11, L is a ligator, D is a drug, and n is an integer or decimal number from 1 to 10.

15. The aforementioned L is -L a -L b -L c -L d - has a structure, where L a L is linked to an antibody. d It is linked to drugs, and specifically, L a teeth 【Chemistry 2】 selected from the group consisting of, wherein the wavy line ~ indicates the linking site with Ab, and * indicates the linking site with L b indicating the linking site with L b ha-(CH 2 )mC(O)-,-NH-(CH 2 -CH 2 -O)p-(CH 2 )sC(O)-, -C(O)-NH-(CH 2 )qC(O)-,-NH-(CH 2 ) is selected from the group consisting of rC(O)- and bonds, where m is an integer from 0 to 10, preferably 0, 2, 3, or 5; p is an integer from 1 to 8; s is an integer from 0 to 6, preferably p is 4 and s is 2; q is an integer from 1 to 5, preferably 2; and r is an integer from 1 to 5, preferably 2. L c This is a peptide residue or linkage consisting of 1 to 7 amino acids, wherein the amino acids are selected from the group consisting of valine, citrulline, glycine, phenylalanine, alanine, proline, isoleucine, lysine, serine, glutamic acid, and aspartic acid, and the amino acids are either unsubstituted or each is independently substituted with one or more substituents, each substituent independently being a halogen, hydroxyl group, cyano group, amino group, or C 1～ C 6 Alkyl alkyl groups, C halogenated compounds 1～ C 6 Alkyl alkyl group, C 1～ C 6 Alkoxy group, and C 3～ C 7 It is selected from the group consisting of cycloalkyl groups, L d is -NH-CH 2 -O-CH 2 -C(O)-, -NH-R a -CH 2 It is selected from the group consisting of -OC(O)- and bonds, where R a is a phenyl group or a 5-6 membered heterocyclyl group, wherein the phenyl group and the 5-6 membered heterocyclyl group are either unsubstituted or each is independently substituted with one or more substituents, and each substituent is independently 【Transformation 3】 Halogen, oxo group, hydroxyl group, cyano group, amino group, C 1～ C 6 Alkyl alkyl groups, C halogenated compounds 1～ C 6 Alkyl alkyl groups, and C 1～ C 6 It is selected from the group consisting of alkoxy groups, where # indicates a linkage site with a phenyl group or a 5-6 membered heterocyclyl group. The antibody-drug conjugate according to claim 14 or a pharmaceutically acceptable salt thereof.

16. The aforementioned L has a structure selected from the following group: i) L a teeth 【Chemistry 4】 L b is -C(O)- or -C(O)-NH-(CH 2 ) 2 -C(O)-, L c -glycine-glycine-phenylalanine-glycine-, -valine-citrulline-, -glycine- or bonded, L d teeth 【Transformation 5】 -NH-CH 2 -O-CH 2 -C(O)- or a bond, or ii) L a teeth 【Transformation 6】 L b (CH 2 ) m -C(O)- (m is 2 or 5), L c is -valine-citrulline- or -glycine-, L d teeth 【Transformation 7】 And, or, iii) L a teeth 【Transformation 8】 L b is -NH-(CH 2 -CH 2 -O) 4 -(CH 2 ) 2 -C(O)-, -NH-(CH 2 ) 2 -C(O)- or bond, L c -valine-citrulline-, -glycine-, -glycine-glycine-phenylalanine-glycine- or bonded, L d teeth 【Chemistry 9】 or -NH-CH 2 -O-CH 2 -C(O)- and, iiii) L a teeth 【Chemistry 10】 L b ha-(CH 2 ) 3 -C(O)-, L c valine-citrulline-, L d teeth 【Chemistry 11】 And, In the formula, the dashed line ~ indicates the connection point with Ab, and * represents L b The connection point is shown, a ＊ indicates the connection point with Lc, and b ＊ This indicates the site of attachment to the drug. Preferably, L a teeth 【Chemistry 12】 L b is -C(O)-, L c valine-citrulline-, L d teeth 【Chemistry 13】 That is, The antibody-drug conjugate according to claim 15 or a pharmaceutically acceptable salt thereof.

17. The aforementioned drug is selected from the group consisting of cytotoxic compounds, immunomodulators, enzymes, and hormone inhibitors. Preferably, the drug is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), eribulin, exatecan, maytansine, SN-38, or combinations thereof. An antibody-drug conjugate according to any one of claims 14 to 16, or a pharmaceutically acceptable salt thereof.

18. It has the following structure: 【Chemistry 14】 In the formula, Ab and n are defined in claim 14, Preferably, Ab comprises a heavy chain represented by SEQ ID NO: 77 and a light chain represented by SEQ ID NO: 78, or Ab comprises the following four polypeptide chains, i.e., Chain 1: Contains the amino acid sequence represented by SEQ ID NO:

81. Chain 2: Contains the amino acid sequence represented by SEQ ID NO:

82. Chain 3: Containing the amino acid sequence represented by SEQ ID NO: 83, and Chain 4: Contains amino acid sequences represented by SEQ ID NO: 84, n is between 3.5 and 4.

5. The antibody-drug conjugate according to claim 17 or a pharmaceutically acceptable salt thereof.

19. (a) an anti-ROR1 antibody according to any one of claims 1 to 5, or a multispecific antibody according to any one of claims 6 to 11, or an antibody-drug conjugate according to any one of claims 14 to 18 or a pharmaceutically acceptable salt thereof, (b) A drug composition containing a pharmaceutically acceptable carrier.

20. The use of an anti-ROR1 antibody according to any one of claims 1 to 5, or a multispecific antibody according to any one of claims 6 to 11, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 14 to 18, or a drug composition according to claim 19, in the preparation of a tumor or cancer therapeutic agent, Preferably, the tumor or cancer is a solid tumor or a hematological tumor. More preferably, the tumor or cancer is selected from the group consisting of breast cancer, pancreatic cancer, lung cancer, esophageal cancer, non-small cell lung cancer, laryngeal tumor, sarcoma, pharyngeal tumor, oral tumor, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colon cancer, colorectal cancer, urothelial carcinoma, cervical cancer, lymphoma, and leukemia. Most preferably, the tumor or cancer expresses ROR1. use.

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