ROR1 antibodies or antigen binding molecules

Abstract

The invention relates to the technical field of biology, in particular to ROR1 antibodies or antigen binding molecules. The anti-ROR1 antibody provided by the invention has good targeting property, not only can be used for targeting therapy of ROR1 related diseases such as tumors, but also can be used for preparing antibody drug conjugates, and also can be used as a diagnostic tool for detecting ROR1 expression in the ROR1 related diseases. Experiments show that the antibody has high specificity and biological activity.

Description

[0001] This application claims priority to Chinese Patent Application No. 202411197860.9, filed on August 29, 2024, entitled "ROR1 Antibody or Antigen Binding Molecule", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biotechnology, and more particularly to ROR1 antibodies or antigen-binding molecules. Background Technology

[0003] ROR1, or receptor tyrosine kinase-like orphan receptor 1 (ROR1, also known as receptor-associated neurotrophic tyrosine kinase 1, NTRKR1), consists of an extracellular immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD and KRD domains), and an intracellular domain consisting of a tyrosine kinase domain, two serine or threonine-rich domains, and a proline-rich domain. ROR1 and ROR2 participate in the non-canonical Wnt signaling pathway by binding the ligand Wnt5a through the FZD domain. ROR1 can inhibit apoptosis, enhance EGFR signaling, and induce epithelial-mesenchymal transition (EMT).

[0004] ROR1 is a conserved embryonic protein whose expression gradually decreases during embryonic development. It is almost absent or poorly expressed in most adult tissues. However, an increasing number of studies have found that ROR1 is expressed in a variety of cancer cells, such as B-cell chronic lymphocytic leukemia (CLL) and other hematologic malignancies, as well as in solid tumors. It is widely expressed in tumor solids, and its expression level in cancerous tissues is significantly higher than that in adjacent normal tissues, including melanoma, colon, pancreas, lung cancer, and breast cancer. Therefore, as a cancer biomarker, ROR1 has become an ideal drug target for cancer treatment.

[0005] Zilovertamab vedotin (MK-2140, VLS-101) is an ADC developed by VelosBio (acquired by MSD) for the treatment of hematologic malignancies and solid tumors, with a global potential first-in-class (FIC) status. VLS-101 contains UC-961 (a humanized IgG1 monoclonal antibody, Cirmtuzumab, Kd = 2 nM), with an inductor payload of MC-VC-PAB-MMAE and DAR 4. In a Phase I clinical trial in hematologic malignancies (NCT03833180), VLS-101 at 2.5 mg / kg every 3 weeks demonstrated good tolerability and efficacy against advanced mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). A Phase II clinical trial (NCT04504916) in patients with solid tumors (including breast and lung cancer) is ongoing.

[0006] NBE-002 was acquired by Boehringer Ingelheim for $1.45 billion from NBE Therapeutics and its ROR1 ADC, “NBE-002”. PNU-159682 is specifically and quantitatively conjugated to the C-terminus of both the heavy and light chains of the antibody via a Sortase A-mediated specific reaction, resulting in a purer and more homogeneous product, DAR 4. The monoclonal antibody (HuXBR1-402) was screened from a rabbit antibody library using phage display. It binds to the Ig / Fz domain of ROR1 with high affinity (Kd = 5.8 nM), and its affinity did not decrease after humanization. NBE-002 is classified as an immunostimulatory ADC because of its target-specific nature: inhibition of the Wnt5 / ROR1 pathway increases T cell infiltration into tumors and transforms “cold” tumors into “hot” tumors. NBE-002 is currently undergoing a phase 1 / 2 clinical trial (NCT04441099) to evaluate its safety and tolerability in patients with advanced solid tumors, particularly triple-negative breast cancer.

[0007] LCB71 is another anti-ROR1 ADC, jointly developed by two South Korean biopharmaceutical companies, LegoChem Biosciences and ABL Bio. CStone Pharmaceuticals holds the exclusive rights to develop and commercialize LCB71 outside of South Korea. LCB71 utilizes ConjuALL™ technology, an enzyme-mediated site-specific conjugation technique. This conjugation method employs isoprenyltransferase, an enzyme that naturally catalyzes the isoprenylation of cysteine ​​residues in the CaaX motif, with a DAR value of 2. LCB71 uses prodrug ADC technology (pPBD), with a masking component in the PBD toxin, making it non-toxic in normal tissues. When the ADC is internalized by tumor cells, the masking component is cleaved by beta-glucuronidase, restoring the toxin's toxicity and killing tumor cells. The glucuronide bond in its linker is relatively stable in the bloodstream and can be cleaved by lysosomal β-glucuronidase, which is highly expressed in tumor cells and the tumor microenvironment, selectively releasing PBD, inducing DNA damage in tumor cells, and triggering apoptosis. On March 3, 2022, CStone Pharmaceuticals' clinical trial application for its ROR1 ADC drug LCB71 (CS5001) injection was approved by the NMPA, marking the first such approval. LCB71 demonstrated antitumor activity in both Jeko-1 (human mantle cell lymphoma) and MDA-MB-231 (human breast cancer cell) xenograft models in a dose-dependent manner.

[0008] Although several antibody drugs targeting ROR1 have been disclosed in existing technologies, there remains an urgent need to develop high-quality anti-ROR1 antibodies as a pan-cancer tumor marker. These antibodies could serve as the basis for developing antibody-based targeted therapies for cancers expressing ROR1, and could also be used as diagnostic tools to detect ROR1 expression in ROR1-related diseases. Furthermore, given the promising prospects of ADCs in cancer treatment, there is still a pressing need for ROR1-containing ADCs with effective therapeutic effects. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide ROR1 antibody or antigen-binding molecule.

[0010] One of the objectives of this invention is to provide antibodies that specifically recognize ROR1, and to prepare chimeric antibodies and humanized antibodies from the selected antibodies.

[0011] Another object of the present invention is to provide an antibody-drug conjugate (ADC) targeting ROR1 and a composition containing the molecule.

[0012] Furthermore, the present invention also relates to the therapeutic and diagnostic uses and methods of these antibodies, antibody fragments, and antibody-drug conjugates.

[0013] Therefore, the technical solution provided by the present invention is as follows:

[0014] This invention provides ROR1 antibody or antigen-binding molecule.

[0015] At least one of its heavy chains CDR1 to CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:1 to 3, 7 to 9, 12 to 14, 18 to 20; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO:1 to 3, 7 to 9, 12 to 14, 18 to 20 by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence that is more than 80% homologous (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to an amino acid sequence shown in any one of SEQ ID NO:1 to 3, 7 to 9, 12 to 14, 18 to 20.

[0016] At least one of its light chains CDR1 to CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:4 to 6, 10 to 1, 15 to 17, 21 to 23; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO:4 to 6, 10 to 11, 15 to 17, 21 to 23 by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence that is more than 80% homologous (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to an amino acid sequence shown in any one of SEQ ID NO:4 to 6, 10 to 11, 15 to 17, 21 to 23.

[0017] The anti-ROR1 antibody provided by this invention has highly specific binding and rapid and efficient internalization capabilities. Its high selectivity and high level of biological activity make it suitable as a therapeutic monoclonal antibody or antibody-drug conjugate for small molecule toxins.

[0018] In this embodiment of the invention, the invention provides a ROR1 antibody or antigen-binding molecule.

[0019] Its heavy chain CDR1 has an amino acid sequence as shown in any one of SEQ ID NO:1, 7, 12 or 18;

[0020] Its heavy chain CDR2 has an amino acid sequence as shown in any one of SEQ ID NO:2, 8, 13 or 19;

[0021] Its heavy chain CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:3, 9, 14 or 20;

[0022] Its light chain CDR1 has an amino acid sequence as shown in any one of SEQ ID NO:4, 10, 15 or 21;

[0023] Its light chain CDR2 has an amino acid sequence as shown in any one of SEQ ID NO:5, 16 or 22;

[0024] Its light chain CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:6, 11, 17 or 23;

[0025] Alternatively, its CDR region has a sequence in which one or more amino acids have been substituted, deleted, added and / or replaced based on the amino acid sequence shown above; or has a sequence that is more than 80% (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) homologous to the amino acid sequence shown above.

[0026] In some embodiments, the present invention provides a ROR1 antibody or antigen-binding molecule.

[0027] Its heavy chain CDR1-3 has the amino acid sequences shown in SEQ ID NO:1-3, and its light chain CDR1-3 has the amino acid sequences shown in SEQ ID NO:4-6.

[0028] Or its heavy chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:7-9, and its light chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:5, 10 or 11.

[0029] Or its heavy chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:12-14, and its light chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:15-17;

[0030] Alternatively, its heavy chain CDR1-3 may have the amino acid sequences shown in SEQ ID NO:18-20, and its light chain CDR1-3 may have the amino acid sequences shown in SEQ ID NO:21-23. In some specific embodiments, the present invention provides ROR1 antibodies or antigen-binding molecules.

[0031] The amino acid sequences of its heavy chain CDR1-3 are TYAMS, IISTGGSAYYASWAEG, and ESYNDYGEMDL, respectively, and the amino acid sequences of its light chain CDR1-3 are QSSEDIDSYLA, AASTLAS, and QSAYYSSSFERA, respectively.

[0032] Or the amino acid sequences of its heavy chain CDR1-3 are TYAMS, IISTGGSAYYASWAEG, and ESYNDYGEMDL, respectively, and the amino acid sequences of its light chain CDR1-3 are QSSEDIDDYLA, AASTLAS, and QSAYYSSSFERA, respectively.

[0033] Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:7-9, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:10-12.

[0034] Or, the amino acid sequences of its heavy chain CDR1-3 are SYDMS, VIRLRGNTYHASWAKG, and VGDDDVGYAYL, respectively, and the amino acid sequences of its light chain CDR1-3 are QASQSIFNYLS, RASTLAS, and QSGYYSVTANYGGA.

[0035] Or, the amino acid sequences of its heavy chain CDR1-3 are SYDMS, VIRLRGNTYYASWAKG, VGDDDVGYAYL, and the amino acid sequences of its light chain CDR1-3 are QASQSIFNYLS, RASTLAS, QSGYYSVTANYGGA.

[0036] Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:18-20, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:21-23.

[0037] This invention obtains rabbit-derived antibodies, which are then humanized and chimeric antibodies are prepared.

[0038] In some embodiments, the present invention provides ROR1 antibodies or antigen-binding molecules as chimeric antibodies or murine antibodies:

[0039] At least one of its heavy chains FR1-4 has an amino acid sequence as shown in any one of SEQ ID NO:24-27, 32-35, 39-41, 45-47; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO:24-27, 32-35, 39-41, 45-47 with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to an amino acid sequence shown in any one of SEQ ID NO:24-27, 32-35, 39-41, 45-47.

[0040] At least one of its light chains FR1-4 has an amino acid sequence as shown in any one of SEQ ID NO:28-31, 36-38, 42-44, 48-51; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO:28-31, 36-38, 42-44, 48-51 by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence that is more than 80% homologous (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to an amino acid sequence shown in any one of SEQ ID NO:28-31, 36-38, 42-44, 48-51.

[0041] In some specific embodiments, the present invention provides ROR1 antibodies or antigen-binding molecules.

[0042] Its heavy chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:24, 32, 39 or 45;

[0043] Its heavy chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:25, 33 or 46;

[0044] Its heavy chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:26, 34, 40 or 47;

[0045] Its heavy chain FR4 has an amino acid sequence as shown in any one of SEQ ID NO:27, 35 or 41;

[0046] Its light chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:28, 36, 42 or 48;

[0047] Its light chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:29, 37 or 49;

[0048] Its light chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO: 30, 38, 43 or 50;

[0049] Its light chain FR4 has an amino acid sequence as shown in any one of SEQ ID NO:31, 44 or 51;

[0050] Alternatively, its FR region has a sequence in which one or more amino acids have been substituted, deleted, added and / or replaced based on the amino acid sequence shown above; or has a sequence that is more than 80% (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) homologous to the amino acid sequence shown above.

[0051] Specifically, this invention provides ROR1 antibodies or antigen-binding molecules:

[0052] Its heavy chain FR1-4 has the amino acid sequences shown in SEQ ID NO:24, 25, 26, 27, and its light chain FR1-4 has the amino acid sequences shown in SEQ ID NO:28, 29, 30, 31, respectively.

[0053] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:32, 33, 34, 35, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:36, 37, 38, 31, respectively;

[0054] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:39,25,40,41 in sequence, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:42,29,43,44 in sequence;

[0055] Alternatively, its heavy chain FR1-4 may have the amino acid sequences shown in SEQ ID NO:45, 46, 47, 35, and its light chain FR1-4 may have the amino acid sequences shown in SEQ ID NO:48, 49, 50, 51. More specifically, the present invention provides ROR1 antibodies or antigen-binding molecules.

[0056] The amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:24, 25, 26, 27, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:28, 29, 30, 31, respectively.

[0057] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:32, 33, 34, 35, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:36, 37, 38, 31, respectively;

[0058] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:39, 25, 40, 41, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:42, 29, 43, 44, respectively.

[0059] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:45, 46, 47, 35, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:48, 49, 50, 51, respectively.

[0060] In one specific embodiment, the antibody or antigen-binding molecule,

[0061] Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:68, 70, 72 or 74;

[0062] Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:69, 71, 73 or 75.

[0063] For example:

[0064] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:68, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:69, 71, 73 or 75.

[0065] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:70, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:69, 71, 73 or 75.

[0066] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:72, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:69, 71, 73 or 75.

[0067] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:74, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:69, 71, 73 or 75.

[0068] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in any one of SEQ ID NO:68, 70, 72 or 74, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:69.

[0069] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in any one of SEQ ID NO:68, 70, 72 or 74, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:71.

[0070] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in any one of SEQ ID NO:68, 70, 72 or 74, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:73.

[0071] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in any one of SEQ ID NO: 68, 70, 72 or 74, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO 75.

[0072] In this invention, the antibody or antigen-binding molecule is a chimeric antibody, wherein its light chain constant region is the κ subtype and its heavy chain constant region is IgG1. Specifically, the heavy chain constant region is G1m3 of IgG1. More specifically, the constant region is derived from the PTT5 vector.

[0073] In some embodiments, the present invention provides ROR1 antibodies or antigen-binding molecules as humanized antibodies:

[0074] At least one of its heavy chains FR1-4 has an amino acid sequence as shown in any one of SEQ ID NO:25, 35, 52-54, 58-60, 63, 65; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO:25, 35, 52-54, 58-60, 63, 65, by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence that is more than 80% homologous (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to an amino acid sequence shown in any one of SEQ ID NO:25, 35, 52-54, 58-60, 63, 65.

[0075] At least one of its light chains FR1-4 has an amino acid sequence as shown in any one of SEQ ID NO:51, 55-57, 61-62, 64, 66-67; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO:51, 55-57, 61-62, 64, 66-67 with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to an amino acid sequence shown in any one of SEQ ID NO:51, 55-57, 61-62, 64, 66-67.

[0076] In some specific embodiments, the present invention provides ROR1 antibodies or antigen-binding molecules.

[0077] Its heavy chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:52, 58 or 65;

[0078] Its heavy chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:25, 53 or 59;

[0079] Its heavy chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:54, 60 or 63;

[0080] Its heavy chain FR4 has the amino acid sequence shown in SEQ ID NO:35;

[0081] Its light chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:55, 61 or 66;

[0082] Its light chain FR2 has the amino acid sequence shown in SEQ ID NO:56;

[0083] Its light chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:57, 62, 64 or 67;

[0084] Its light chain FR4 has the amino acid sequence shown in SEQ ID NO:51;

[0085] Alternatively, its FR region has a sequence in which one or more amino acids have been substituted, deleted, added and / or replaced based on the amino acid sequence shown above; or has a sequence that is more than 80% (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) homologous to the amino acid sequence shown above.

[0086] Specifically, this invention provides ROR1 antibodies or antigen-binding molecules.

[0087] Its heavy chain FR1-4 has the amino acid sequences shown in SEQ ID NO:52, 53, 54, 35, and its light chain FR1-4 has the amino acid sequences shown in SEQ ID NO:55, 56, 57, 51, respectively.

[0088] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:58, 59, 60, 35, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:61, 56, 62, 51, respectively.

[0089] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:52, 25, 63, 35 in sequence, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:55, 56, 64, 51 in sequence;

[0090] Or its heavy chain FR1-4 has the amino acid sequence shown in SEQ ID NO:65, 25, 63, 35, and its light chain FR1-4 has the amino acid sequence shown in SEQ ID NO:66, 56, 67, 51.

[0091] In one specific embodiment, the antibody or antigen-binding molecule,

[0092] Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:76, 78, 81 or 83;

[0093] Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:77, 79, 80, 82 or 84.

[0094] For example:

[0095] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:76, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:77, 79, 80, 82 or 84.

[0096] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:78, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:77, 79, 80, 82 or 84.

[0097] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:81, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:77, 79, 80, 82 or 84.

[0098] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:83, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:77, 79, 80, 82 or 84.

[0099] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:76, 78, 81 or 83, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:77.

[0100] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:76, 78, 81 or 83, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:79.

[0101] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:76, 78, 81 or 83, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:80.

[0102] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:76, 78, 81 or 83, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:82.

[0103] The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding molecule is shown in SEQ ID NO:76, 78, 81 or 83, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:84.

[0104] In this invention, the antibody or antigen-binding molecule is a humanized antibody, whose light chain constant region is the κ subtype and whose heavy chain constant region is IgG1. Specifically, the heavy chain constant region is G1m3 of IgG1. More specifically, the constant region is derived from the PTT5 vector.

[0105] Furthermore, the present invention also provides nucleic acids encoding ROR1 antibodies or antigen-binding molecules as described above.

[0106] The nucleic acid described in this invention is a nucleic acid encoding the CDR region of the antibody as described above, or a nucleic acid encoding the variable region, or a nucleic acid encoding the full length of the antibody. This invention does not limit the specific nucleic acid to this type.

[0107] Furthermore, the present invention also provides a plasmid vector containing the nucleic acid as described above.

[0108] In this invention, the backbone vector of the plasmid vector is a mammalian cell expression vector, for example, the backbone vector is pCDNA5.

[0109] Furthermore, the present invention also provides a host whose genome integrates the nucleic acids as described above, or is transformed or transfected with the plasmid vector as described above.

[0110] In this invention, the host is a mammalian cell, such as human embryonic kidney HEK293 cells or Chinese hamster ovary cells CHOK1.

[0111] Furthermore, the present invention also provides a method for preparing the ROR1 antibody or antigen-binding molecule as described above, comprising: culturing the host as described above to obtain a culture containing the antibody or antigen-binding molecule.

[0112] The preparation method further includes steps of enriching, extracting and / or purifying the culture.

[0113] Furthermore, the present invention also provides a labeled antibody comprising the ROR1 antibody or antigen-binding molecule as described above, and a labeling substance.

[0114] In this invention, the labeling substance is a chemical label or a biological label.

[0115] In this embodiment of the invention,

[0116] The chemical label is a fluorescent indicator, a chemiluminescent indicator, an isotope and / or a colloidal indicator;

[0117] The biomarker is biotin, avidin, or an enzyme label.

[0118] In some specific embodiments,

[0119] The enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridge, alkaline phosphatase-antialkaline phosphatase bridge, and β-galactosidase-antiβ-galactosidase bridge.

[0120] The fluorescent indicator is selected from one or more of the following: AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, or PerCP dye;

[0121] The chemiluminescent indicator is selected from one or more of acridine ester, acridine sulfonamide and its derivatives, luminol, isoluminol, isoluminol isothiocyanate and its derivatives, N-(4-aminobutyl)-N-ethyl isoluminol, 4,5-diaminophthalic acid hydrazide or aminobutylethyl benzoyl hydrazide.

[0122] Isotopes selected 125 I, 131 I, 124 I, 3 H, 14 C 111 In、 89 Zr or 32 One or more of P;

[0123] Colloidal indicators are selected from one or more of colloidal gold, colloidal carbon, or colloidal selenium.

[0124] Furthermore, the present invention also provides a conjugate comprising the ROR1 antibody or antigen-binding molecule as described above, and a solid-phase medium or a non-solid-phase medium.

[0125] In this embodiment of the invention, the solid-phase medium or non-solid-phase medium is selected from colloidal gold, ELISA plates, magnetic beads, or latex microspheres. In this invention, the antibody and the medium can be linked via amide bonds or via a streptavidin-biotin system; this invention does not limit the specific method used.

[0126] Furthermore, the present invention also provides a conjugate comprising, as described above, a ROR1 antibody or antigen-binding molecule, and a drug.

[0127] In this invention, the drug is an antitumor drug. Preferably, the drug is an antitumor drug containing amino or carboxyl groups; more preferably, the drug is a protein or amino acid-based antitumor drug, selected from at least one of MMAE, MMAF, or DXD.

[0128] In this invention, the drug and antibody are directly linked by an amide bond in the conjugate, or they can be linked together by a linker; this invention does not limit the specifics.

[0129] In this invention, the molar ratio of drug to antibody in the conjugate is (0.1-20):1, preferably (1-10):1, more preferably (2-6):1. In specific embodiments, the molar ratio of drug to antibody is 2:1, 3:1, 4:1, 5:1 or 6:1.

[0130] Furthermore, the present invention provides the use of any one of the following (I) to (VII) in the preparation of a medicament for the prevention and treatment of ROR1-related diseases:

[0131] I) Antibodies or antigen-binding molecules as described above;

[0132] II) Nucleic acids as described above;

[0133] III) Plasmid vectors as described above;

[0134] IV) The host as described above;

[0135] V) Cultures prepared by the methods described above or antibodies obtained through purification;

[0136] VI) Labeled antibodies as described above;

[0137] VII) Couplings as described above.

[0138] In this invention, the disease associated with ROR1 is a tumor.

[0139] The present invention also provides a medicine comprising any one of the following: I) to VII);

[0140] I) Antibodies or antigen-binding molecules as described above;

[0141] II) Nucleic acids as described above;

[0142] III) Plasmid vectors as described above;

[0143] IV) The host as described above;

[0144] V) Cultures prepared by the methods described above or antibodies obtained through purification;

[0145] VI) Labeled antibodies as described above;

[0146] VII) Couplings as described above.

[0147] In this invention, the drug also includes pharmaceutically acceptable excipients.

[0148] In this invention, the dosage form of the drug includes, but is not limited to, oral preparations, injections, aerosols, or suppositories. Oral preparations include, but are not limited to, tablets, capsules, pills, oral liquids, or dispersants. Injections include, but are not limited to, injectable solutions or powders for injection.

[0149] Furthermore, the present invention also provides a pharmaceutical composition comprising the aforementioned drug and other therapeutic agents.

[0150] The other therapeutic agents are antitumor drugs. As a feasible example, the antitumor drugs include, but are not limited to, at least one of the following: cisplatin, carboplatin, oxaliplatin, paclitaxel, vinorelbine, gefitinib, fluorouracil, doxorubicin, erlotinib, rituximab, trastuzumab, pertuzumab, nivolumab, pembrolizumab, atezolizumab, olaparib, niraparib, etoposide, tamoxifen, toremifene, fulvestrant, or letrozole.

[0151] In the pharmaceutical composition, multiple drugs may exist in a mixture or independently; this invention does not limit this. Multiple drugs may be administered simultaneously or sequentially; this invention also does not limit this.

[0152] The present invention also provides a method for treating ROR1-related diseases, comprising administering the drug or pharmaceutical composition described herein.

[0153] The administration methods of the drug or drug composition include, but are not limited to: oral administration, inhalation spray, buccal administration, nasal administration, vaginal administration, rectal administration, topical administration, and parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or administration via an external implantation device. Oral, intraperitoneal, or intravenous administration is preferred.

[0154] Furthermore, the present invention also provides the use of any one of the following i) to vii) in the preparation of ROR1 detection reagents, tumor diagnostic reagents, and / or tumor imaging reagents:

[0155] i) Antibodies or antigen-binding molecules as described above;

[0156] ii) Nucleic acids as described above;

[0157] iii) Plasmid vectors as described above;

[0158] iv) The host as described above;

[0159] v) Cultures prepared by the methods described above or antibodies obtained through purification;

[0160] vi) Labeled antibodies as described above;

[0161] vii), the combination as described above.

[0162] viii), as described above, are couplings.

[0163] In this invention, the tumor is a tumor expressing ROR1.

[0164] In this embodiment of the invention, the tumor is lymphoma, lung cancer, colon cancer, gastric cancer, lung adenocarcinoma, breast cancer, pancreatic cancer, ovarian cancer, multiple myeloma, or mantle cell lymphoma.

[0165] The present invention also provides a reagent comprising any one of the following i) to vii):

[0166] i) Antibodies or antigen-binding molecules as described above;

[0167] ii) Nucleic acids as described above;

[0168] iii) Plasmid vectors as described above;

[0169] iv) The host as described above;

[0170] v) Cultures prepared by the methods described above or antibodies obtained through purification;

[0171] vi) Labeled antibodies as described above;

[0172] vii), the combination as described above.

[0173] viii), as described above, are couplings.

[0174] The reagents described in this invention also include buffer solutions to facilitate the reaction. Examples include phosphate buffer, carbonate buffer, acetate buffer, Tris-HCl buffer, and HEPES buffer. The reagents also include auxiliaries to maintain reaction stability, such as BSA, DDT, β-mercaptoethanol, glycerol, vitamin C, or Tween.

[0175] This invention provides a method for detecting ROR1 or ROR1-expressing tumor cells, comprising detecting a sample using the aforementioned reagents. The detection method includes, but is not limited to, ELISA, flow cytometry, and / or immunohistochemistry. The samples include, but are not limited to, cells, blood, tissue sections, or tissue homogenates.

[0176] The present invention also provides a method for the diagnosis or imaging of tumors, comprising detecting a sample after processing it with the reagents as described above, or detecting a sample after administering the reagents as described above to a subject. The processing includes, but is not limited to, incubating the sample in contact with the reagents, wherein the sample includes, but is not limited to, cells, blood, sections, or tissue homogenates. The administration to the subject includes, but is not limited to, administration via the gastrointestinal tract, intravenous administration, or intraperitoneal injection, wherein the detection includes, but is not limited to, the detection of fluorescent or radioactive substances.

[0177] This invention provides an anti-ROR1 antibody as described herein, which exhibits excellent targeting properties. It can be used not only for targeted therapies of ROR1-related diseases such as tumors, but also for the preparation of antibody-drug conjugates and as a diagnostic tool to detect ROR1 expression in ROR1-related conditions. Experiments show that this antibody possesses high specificity and biological activity. Attached Figure Description

[0178] Figure 1 This indicates the binding of antibodies to overexpressing cell lines;

[0179] Figure 2 The cell biology binding FACS test results of the chimeric antibody in the CHO-hROR1 cell line overexpressing the cell line are shown.

[0180] Figure 3 In vitro cell binding assays of anti-human ROR1 chimeric antibody with naturally expressed cell lines;

[0181] Figure 4 The study demonstrated the endocytic activity of the anti-human ROR1 chimeric antibody against naturally expressed cell lines A549, HT29, and Mino.

[0182] Figure 5 In vitro cell biology and FACS assays show the effects of humanized anti-human ROR1 antibodies on tumor cells.

[0183] Figure 6The results of nonspecific binding assays of anti-human ROR1 humanized antibodies are shown.

[0184] Figure 7 The results of FACS binding experiments of anti-human ROR1 humanized antibody with cell lines overexpressing ROR1 protein from different species are shown.

[0185] Figure 8 This demonstrates the in vitro killing effect of humanized anti-ROR1 antibody on overexpressing cells;

[0186] Figure 9 This demonstrates the drug metabolism of ROR1 antibody in mice.

[0187] Figure 10 The in vitro killing effect of humanized anti-ROR1 antibody on overexpressing cells after conjugation with a small molecule drug was demonstrated.

[0188] Figure 11 The study demonstrated the tumor-suppressive effect of a humanized anti-human ROR1 antibody conjugated with a small molecule drug on tumor-bearing mice.

[0189] Figure 12 The effect of humanized anti-human ROR1 antibody conjugated with a small molecule drug on tumor weight in tumor-bearing mice. Detailed Implementation

[0190] This invention provides ROR1 antibodies or antigen-binding molecules, which can be implemented by those skilled in the art with appropriate modifications to process parameters, based on the content of this document. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0191] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0192] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0193] In this application, the terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0194] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0195] In this application, "specific binding" refers to the fact that antigen-binding molecules (e.g., antibodies) typically bind antigens specifically to substantially the same antigens with high affinity, but do not bind unrelated antigens with high affinity. Affinity is usually reflected by the equilibrium dissociation constant (KD), where a lower KD indicates higher affinity. For example, for antibodies, high affinity typically refers to a KD of approximately 10. -6 M or lower, approximately 10 -7 M or lower, approximately 10 -8 M or lower, approximately 1×10 -9 M or lower, approximately 1×10 -10 M or lower, 1×10 -11 M or lower or 1×10 -12 M or lower KD. KD is calculated as follows: KD = Kd / Ka, where Kd represents the dissociation rate and Ka represents the binding rate. The equilibrium dissociation constant KD can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0196] In this application, "antigen-binding molecule" refers to a molecule that specifically binds to an antigen. For example, the antigen-binding molecule includes, but is not limited to, antibodies or antibody mimics. "Antibody mimic" refers to a biomolecule that mimics the structure and function of a natural antibody through non-natural synthetic methods. For example, antibody mimics include, but are not limited to, affibody, affitin, affilin, designed ankylosing spondylamine repeat (DARPin), nucleic acid aptamers, or Kunitz-type domain peptides.

[0197] In this application, "antibody" includes a typical "quadruple-chain antibody," which belongs to the immunoglobulin class composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0198] In this application, "antibody" encompasses various forms and structures, including complementary-determining regions (CDRs) and antibody framework regions (FRs). CDRs are hypervariable regions of the antibody, located at the amino termini of the heavy chain (VH) and light chain (VL). These regions contain highly variable amino acid sequences, allowing for precise binding of the antibody to the antigen. The three main CDR regions are CDR-H1, CDR-H2, and CDR-H3 (in the heavy chain) and CDR-L1, CDR-L2, and CDR-L3 (in the light chain). FRs are regions in the antibody heavy and light chains other than the CDRs. While not directly involved in antigen binding, FRs significantly influence the conformation of the CDRs and the specificity of antigen binding.

[0199] In this application, "antibody" also includes antibodies that do not contain a light chain or antibodies that do not contain a constant region.

[0200] In this application, the "antibody" can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0201] In this application, "chimeric antibody" refers to an antibody that has a variable sequence of immunoglobulin derived from one source organism (such as rat, mouse, rabbit, or alpaca) and a constant region of immunoglobulin derived from a different organism (such as human). Chimeric antibodies are typically prepared using genetic engineering, which involves splicing the V region gene of a monoclonal antibody from one source organism with the C region gene of an antibody from another organism to form a chimeric gene, which is then inserted into a vector and transfected into antibody molecules expressed in myeloma tissue.

[0202] In this application, "humanized antibody" refers to a non-human antibody that has been genetically engineered to improve its amino acid sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody originates from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the variable region FR and / or constant region) originates from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to strengthen immune responses.

[0203] In this application, "Kabat numbering system" generally refers to the immunoglobulin matching and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0204] In this application, the "heavy chain constant region" refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in the binding of the antibody to the antigen but exhibits effector functions, such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domains of the antibody. The "heavy chain constant region" can be selected from: the CH1 domain, the hinge region, the CH2 domain, the CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter includes only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from the Fc or CH3 domain.

[0205] In this application, the "light chain constant region" refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0206] In this application, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0207] In this application, "identity" can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. Taking into account the number of vacancies that need to be introduced for optimal alignment of the two sequences and the length of each vacancy, the percentage of identity between the two sequences varies with the common positions of the sequences.

[0208] In this application, "nucleic acid" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′.

[0209] In this application, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, and single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones bonded or chemically modified residues. Nucleic acid molecule also encompasses DNA and RNA molecules suitable as carriers for the direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into the subject to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2101823 B1).

[0210] In this application, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which the vector has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors".

[0211] In this application, "host" or "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be completely identical to parental cells in their nucleic acid contents and may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0212] In this application, "medicine" allows the biologically active ingredients contained therein to exist in an effective form and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the pharmaceutical composition.

[0213] In this application, "prevention and treatment" includes prevention and / or treatment. "Treatment" refers to surgical or therapeutic treatment aimed at preventing, mitigating (reducing) undesirable physiological changes or lesions, such as cancer and tumors, in the treated individual. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as mitigation, reduction, weakening, mitigation, and relief are used, they also include elimination, disappearance, and non-occurrence.

[0214] In this application, "subject" refers to an organism receiving treatment for a specific disease or symptom as described in this invention. Exemplarily, "subject" includes mammals receiving treatment for a disease or symptom, including bovine, equine, sheep, suidae, canine, feline, rodent, and primate animals, with preferred mammals being humans, cats, dogs, or pigs.

[0215] In this application, the terms "therapeutic effective amount" or "preventive effective amount" refer to an amount sufficient, within reasonable medical judgment, to treat or prevent a patient's disease while avoiding serious side effects with a sufficiently low risk-reward ratio. The preventive or therapeutic effective amount of a compound will vary depending on the specific compound chosen (e.g., considering the compound's potency, effectiveness, and half-life), the chosen route of administration, the disease being prevented or treated, the severity of the disease, the patient's age, size, weight, and physical condition, the patient's medical history, the duration of prevention or treatment, the nature of concurrent therapies, the desired preventive or therapeutic effect, etc., but can still be conventionally determined by those skilled in the art. When an active ingredient is administered to an individual alone, the therapeutic effective amount refers to that ingredient alone. When a combination is used, the therapeutic effective amount refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously.

[0216] In this application, "cancer" refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The terms "tumor" or "tumor" herein refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0217] In this application, "imaging" refers to the process of converting certain substances or energy into observable or measurable signals to demonstrate their morphology, properties, structure, or distribution. For example, in this invention, after treating samples with antibodies bearing chemical or biological labels, commonly used medical imaging techniques such as X-rays, MRI, CT scans, and ultrasound are used to distinguish cells expressing ROR1 from other cells, or tissues expressing ROR1 from other tissues.

[0218] The test materials used in this invention are all commercially available products, and the sequences of the antibodies, antigens, and nucleic acids involved are as follows:

[0219] I. The amino acid sequences of the light and heavy chains CDR, variable region, and constant region of rabbit / chimeric / human antibody molecules, their corresponding nucleotide sequences, and the optimized amino acid mutation points of the CDR are as follows;

[0220] The variable region sequence of the mouse monoclonal antibody is shown below.

[0221] 1. Rabbit-derived chimeric antibody

[0222] chrN1 heavy chain variable region amino acid sequence

[0223] QSVEESGGRLVTPGTPLTLTCTVSGFSLS TYAMS WVRQAPGKGLEWIG IISTGGSAYYASWAEG RFTISRTSTTVDLKITSPTTEDTATYFCAR ESYNDYGEMDL WGQGTLVPVSS (SEQ ID NO:68, CDR1~3 are SEQ ID NO:1~3 respectively, FR1~4 are SEQ ID NO:24~27 respectively)

[0224] Note: Underlined text indicates CDR. The CDR notation method is based on the Kabat antibody encoding scheme, and the subsequent sequence encoding method is the same.

[0225] chrN1 light chain variable region amino acid sequence

[0226] AIEMTQTPSSVSAAVGGTVTINC QSSEDIDSYLA WYQQKPGQPPKLLIY AASTLAS GVPSRFKGSGSWTEFTLTISDLECADAATYYC QSAYYSSSFERA FGGGTEVVVK(SEQ ID NO:69, CDR1~3 are SEQ ID NO:4~6 respectively, FR1~4 are SEQ ID NO:28~31 respectively)

[0227] chrN2 heavy chain variable region amino acid sequence

[0228] QSVEESGGRLVTPGTPLTLTCTVSGFSLN TYAMA WVRQAPGKGLEYIG IINTGGYAYYANWAKG RFTISRTSTTVDLKMTSLTTEDTATYFCAR ADYNDYGDFDL WGQGTLVTVSS (SEQ ID NO:70, CDR1~3 are SEQ ID NO:7~9 respectively, FR1~4 are SEQ ID NO:32~35 respectively)

[0229] chrN2 light chain variable region amino acid sequence

[0230] AIEMTQTPSFVSADVGGTVTINC QASEDIDDYLA WYQQKPGQPPNLLIY AASTLAS GVPSRFKGSGSGTEYTLTISDVQCDDAATYYC QSAYYSGSIDFT FGGGTEVVVK(SEQ ID NO:71, CDR1~3 are SEQ ID NO:10, 5, 11 respectively, FR1~4 are SEQ ID NO:36~38 and 31 respectively)

[0231] chrN25 heavy chain variable region amino acid sequence

[0232] QSLEESGGRLVTPGTPLTLTCKVSGFSLS SYDMS WVRQAPGKGLEWIG VIRLRGNTYHASWAKG RFTISKTSTTVDLSITSPTIEDTATYFCAR VGDDDVGYAYL WGQGTLVTISS (SEQ ID NO:72, CDR1~3 are SEQ ID NO:12~14 respectively, FR1~4 are SEQ ID NO:39,25,40~41 respectively)

[0233] chrN25 light chain variable region amino acid sequence

[0234] AEVVMTQTPASVEAAVGGTVTIKC QASQSIFNYLS WYQQKPGQPPKLLIY RASTLAS GVPSRFKGSGSGTEYTLTISDLECADAATYYC QSGYYSVTANYGGA FGGGTEVAVK (SEQ ID NO:73, CDR1~3 are SEQ ID NO:15~17 respectively, FR1~4 are SEQ ID NO:42, 29, 43~44 respectively)

[0235] chrN27 heavy chain variable region amino acid sequence

[0236] QSVEESGGRLVTPGTPLTLTCTASGFTIS RYHVN WVRQAPGRGLEWIG MIYGGSSDTDYASWAKG RFTISKTSTTVDLQITSPTTEDTATYFCAR SYLHYSHYPHL WGQGTLVTVSS (SEQ ID NO:74, CDR1~3 are SEQ ID NO:18~20 respectively, FR1~4 are SEQ ID NO:45~47 and 35 respectively)

[0237] chrN27 light chain variable region amino acid sequence

[0238] AQVLTQTPSSVSAAVGGTVTINC QSSQSVYSNNYLA WYQQKPGQPPKLLIF DASTLAS GVPSRFKGSGSGTQFTLTINGVQCDDAATYYC QGAYESADYYNV FGGGTKVEIK (SEQ ID NO:75, CDR1~3 are SEQ ID NO:21~23 respectively, FR1~4 are SEQ ID NO:48~51 respectively)

[0239] 2. Humanized antibodies

[0240] N1 humanized molecular heavy chain variable region amino acid sequence

[0241] >N1-H1 variable region amino acid sequence

[0242] EVQLVESGGGLVQPGGSLRLSCAASGFTLS TYAMS WVRQAPGKGLEWVG IISTGGSAYYASWAEG RFTISRDTSKSITYLQMSSLRAEDTAVYYCAR ESYNDYGEMDL WGQGTLVTVSS (SEQ ID NO:76, CDR1~3 are SEQ ID NO:1~3 respectively, FR1~4 are SEQ ID NO:52~54 and 35 respectively)

[0243] N1 humanized molecule light chain variable region amino acid sequence

[0244] >N1-L2 variable region amino acid sequence

[0245] DIQMTQSPSSVSASVGDRVTITC QSSEDIDDYLA WYQQKPGKAPKLLIY AASTLASGVPSRFSGSGSWTDFTLTISSLQPEDFATYYC QSAYYSSSFERA FGGGTKVEIK (SEQ ID NO:77, CDR1~3 are SEQ ID NO:4~6 respectively, FR1~4 are SEQ ID NO:55~57 and 51 respectively)

[0246] N2 humanized molecular heavy chain variable region amino acid sequence

[0247] >N2-H5 variable region amino acid sequence

[0248] EVQLVESGGGLVQPGGSLRLSCSASGFTFS TYAMA WVRQAPGKGLEYVG IINTGGYAYYANWAKG RFTISRDTSKNTLYLQMSSLRAEDTAVYYCAR ADYNDYGDFDL WGQGTLVTVSS (SEQ ID NO:78, CDR1~3 are SEQ ID NO:7~9 respectively, FR1~4 are SEQ ID NO:58~60 and 35 respectively)

[0249] N2 humanized molecule light chain variable region amino acid sequence

[0250] >N2-L0 variable region amino acid sequence

[0251] DIQMTQSPSTLSASVGDRVTITC QASEDIDDYLA WYQQKPGKAPKLLIY AASTLAS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QSAYYSGSIDFT FGGGTKVEIK (SEQ ID NO:79, CDR1-3 are SEQ ID NO:10, 5 and 11 respectively, FR1-4 are SEQ ID NO:61, 56, 62 and 51 respectively)

[0252] >N2-L1 variable region amino acid sequence

[0253] DIQMTQSPSTLSASVGDRVTITC QASEDIDDYLA WYQQKPGKAPKLLIY AASTLAS GVPSRFSGSGSGTEYTLTISSLQPDDFATYYC QSAYYSGSIDFT FGGGTKVEIK (SEQ ID NO: 80, CDR1-3 are SEQ ID NO: 10, 5 and 11 respectively, FR1-4 are SEQ ID NO: 61, 56, 62 and 51 respectively)

[0254] N25 humanized molecular heavy chain variable region amino acid sequence

[0255] >N25-H2 variable region amino acid sequence

[0256] EVQLVESGGGLVQPGGSLRLSCAASGFTLS SYDMS WVRQAPGKGLEWIG VIRLRGNTYYASWAKG RFTISKDTKSIVYLQMSSLRAEDTAVYYCAR VGDDDVGYAYL WGQGTLVTVSS (SEQ ID NO:81, CDR1-3 are SEQ ID NO:12-14 respectively, FR1-4 are SEQ ID NO:52, 25, 63 and 35 respectively)

[0257] N25 humanized molecule light chain variable region amino acid sequence

[0258] >N25-L1 variable region amino acid sequence

[0259] DIQMTQSPSSVSASVGDRVTITC QASQSIFNYLS WYQQKPGKAPKLLIY RASTLAS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QSGYYSVTANYGGA FGGGTKVEIK (SEQ ID NO: 82, CDR1-3 are SEQ ID NO: 15-17 respectively, FR1-4 are SEQ ID NO: 55, 56, 64 and 51 respectively)

[0260] N27 humanized molecular heavy chain variable region amino acid sequence

[0261] >N27-H2 variable region amino acid sequence

[0262] EVQLVESGGGLVQPGGSLRLSCAASGFTIS RYHVN WVRQAPGKGLEWIG MIYGGSSDTDYASWAKG RFTISKDTKSIVYLQMSSLRAEDTAVYYCAR SYLHYSHYPHL WGQGTLVTVSS (SEQ ID NO:83, CDR1-3 are SEQ ID NO:18-20 respectively, FR1-4 are SEQ ID NO:65, 25, 63 and 35 respectively)

[0263] N27 humanized molecule light chain variable region amino acid sequence

[0264] >N27-L0 variable region amino acid sequence

[0265] AIQLTQSPSSLSASVGDRVTITC QSSQSVYSNNYLA WYQQKPGKAPKLLIY DASTLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QGAYESADYYNV FGGGTKVEIK (SEQ ID NO: 84, CDR1-3 are SEQ ID NO: 21-23 respectively, FR1-4 are SEQ ID NO: 66, 56, 67 and 51 respectively)

[0266] 3. Number the CDR sequences in the antibody:

[0267]

[0268]

[0269] 4. Number the FR sequence in the chimeric antibody.

[0270]

[0271]

[0272] 5. Number the FR sequence in the humanized antibody.

[0273]

[0274]

[0275] II. Sequence Synthesis and Vector Construction of Tool Antibodies

[0276] 1. The naked antibody molecule UC961 corresponding to the ADC drug Zilovertamab vedotin developed by VelosBio (acquired by MSD).

[0277] UC961 heavy chain variable region amino acid sequence:

[0278] QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS

[0279] UC961 light chain variable region amino acid sequence:

[0280] DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK

[0281] 2. The naked antibody XBR1-402 corresponding to NBE Therapeutics' ADC drug NBE-002.

[0282] XBR1-402 heavy chain variable region amino acid sequence:

[0283] QEQQKESGGGLFKPTDTLTLTCTASGFDISSYYMSWVRQAPGNGLEWIGAIGISGNAYYASWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFCARDHPTYGMDLWGPGTLVTVSS

[0284] XBR1-402 light chain variable region amino acid sequence (light chain is lambda chain):

[0285] SYELTQLPSVSVSLGQTARITCEGNNIGSKAVHWYQQKPGLAPGLLIYDDDERPSGVPDRFSGSNSGDTATLTISGAQAGDEADYYCQVWDSSAYVFGGGTQLTVTG

[0286] 3. The naked anti-C2E3 C2E3 heavy chain variable region amino acid sequence of LCB71, an ADC drug jointly developed by LegoChem Biosciences and ABL Bio:

[0287] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISHNSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFISARKSLGRSYSNGMDVWGQGTLVTVSS

[0288] C2E3 light chain variable region amino acid sequence (light chain is lambda chain):

[0289] QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNDVTWYQQLPGTAPKLLIYADSKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDYSLSGYVFGGGTKLTVLG

[0290] 4. Maiwei Biotechnology's self-produced Isotype control (SH-NC)

[0291] SH-NC heavy chain variable region amino acid sequence:

[0292] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPIFGSSNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAESPLGGGSGYSVSWFDPWGQGTLVTVSS

[0293] SH-NC light chain variable region amino acid sequence:

[0294] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSNWPPWTFGQGTKVEIK

[0295] 5. The amino acid sequence of the G1m3 constant region of the IgG1 heavy chain in the PTT5 vector used in the protein expression system:

[0296] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0297] The nucleotide sequence of the IgG1 heavy chain constant region G1m3 of the PTT5 vector used in the protein expression system:

[0298] gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa

[0299] 6. The Km3 amino acid sequence of the IgG1 light chain constant region of the PTT5 vector used in the protein expression system:

[0300] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0301] The nucleotide sequence of the Km3 nucleotide region of the IgG1 light chain constant region of the PTT5 vector used in the protein expression system:

[0302] Cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatccccgcgaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccagg agagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaccgcggagagtgt

[0303] 7. Amino acid sequence of the lambda constant region of the IgG1 light chain in the PTT5 vector used in the protein expression system:

[0304] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0305] The nucleotide sequence of the IgG1 light chain lambda constant region of the PTT5 vector used in the protein expression system is as follows:

[0306] ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtg gagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca

[0307] III. Purchasing antigens and verifying antigen binding activity

[0308] 1. ROR1-related recombinant protein antigen information

[0309] Purchase the following commercial reagents:

[0310] name factory model Human ROR1-His protein AcroBiosystems RO1-H522y Human ROR1-hFc protein AcroBiosystems RO1-H5250 Mouse ROR1-His protein AcroBiosystems RO1-M5221 Human ROR2-His protein AcroBiosystems RO2-H52E5

[0311] 2. Construction of cell lines containing antigens from different species:

[0312] The amino acid sequence of human ROR1 protein (hROR1 for short) is as follows:

[0313] >ROR1_HUMAN Q01973

[0314] MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL

[0315] The nucleotide sequence of human ROR1 protein (abbreviated as hROR1) is as follows:

[0316]

[0317] The amino acid sequence of the monkey ROR1 protein (cynoROR1 for short) is as follows:

[0318] >ROR1_Cynomolgus monkeyA0A2K5WTX7

[0319] MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSPPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYMNQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYIFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDAKQASLLGDANIHGHTESMISAEL

[0320] The nucleotide sequence of cynomolgus ROR1 protein (abbreviated as cynoROR1) is as follows:

[0321]

[0322] The amino acid sequence of mouse ROR1 protein (mROR1) is as follows:

[0323] >ROR1_MOUSE Q9Z139MHRPRRRGTRPPPLALLAALLLAARGADAQETELSVSAELVPTSSWNTSSEIDKGSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPSIRWFKNDAPVVQEPRRISFRATNYGSRLRIRNLDTTDTGYFQCVATNGKKVVSTTGVLFVKFGPPPTASPGSSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEVLENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHSFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIAFLFFFICVCRNNQKSSSPPVQRQPKPVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECTFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYMNQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSSLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRFPNYMFPSQGITPQGQIAGFIGPAIPQNQRFIPINGYPIPPGYAAFPAAHYQPAGPPRVIQHCPPPKSRSPSSASGSTSTGHVASLPSSGSNQEANVPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQSSLLGDSHIHGHTESMISAEV

[0324] The nucleotide sequence of the mouse ROR1 protein (mROR1) is as follows:

[0325]

[0326] The amino acid sequence of human ROR2 protein (hROR2) is as follows:

[0327] MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLDGQDGPIPTLKGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLKNDAPVVQEPRRIIIRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCDARSRTPKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAANCMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCSPRDSSKMGILYILVPSIAIPLVIACLFFLVCMCRNKQKASASTPQRRQLMASPSQDMEMPLINQHKQAKLKEISLSAVRFMEELGEDRFGKVYKGHLFGPAPGEQTQAVAIKTLKDKAEGPLREEFRHEAMLRARLQHPNVVCLLGVVTKDQPLSMIFSYCSHGDLHEFLVMRSPHSDVGSTDDDRTVKSALEPPDFVHLVAQIAAGMEYLSSHHVVHKDLATRNVLVYDKLNVKISDLGLFREVYAADYYKLLGNSLLPIRWMAPEAIMYGKFSIDSDIWSYGVVLWEVFSYGLQPYCGYSNQDVVEMIRNRQVLPCPDDCPAWVYALMIECWNEFPSRRPRFKDIHSRLRAWGNLSNYNSSAQTSGASNTTQTSSLSTSPVSNVSNARYVGPKQKAPPFPQPQFIPMKGQIRPMVPPPQLYVPVNGYQPVPAYGAYLPNFYPVQIPMQMAPQQVPPQMVPKPSSHHSGSGSTSTGYVTTAPSNTSMADRAALLSEGADDTQNAPEDGAQSTVQEAEEEEEGSVPETELLGDCDTLQVDEAQVQLEA

[0328] The nucleotide sequence of human ROR2 protein is as follows:

[0329]

[0330] In addition, the antigen cell lines involved in the examples are from the following sources:

[0331] Natural tumor cells expressing human ROR1:

[0332] A549 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60084.

[0333] Jeko-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60275.

[0334] HT-29 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60011

[0335] NCI-H1975 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60121.

[0336] Mino was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60677.

[0337] NCI-N87 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60491.

[0338] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0339] Example 1

[0340] 1. Construction of cell lines with different species antigens

[0341] The nucleotide sequence of hROR1 was constructed into the commercially available pCDNA5 vector from Invitrogen. Specifically, the pCDNA5 vector was digested with NheI and PmeI, and the nucleotide sequence of hROR1 was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 using lipofectamine 3000. Overexpressing cell lines were then selected using the antibiotic Hygromycin. The successfully constructed cell lines were named CHO-hROR1 and 293-hROR1 cells, respectively.

[0342] The nucleotide sequence of cynoROR1 was constructed into the commercially available pCDNA5 vector from Invitrogen. Specifically, the pCDNA5 vector was double-digested with NheI and PmeI, and the nucleotide sequence of cynoROR1 was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 using lipofectamine 3000, respectively. Overexpressing cell lines were then screened using the antibiotic Hygromycin. Through monoclonalization, the monoclonal overexpressing cell line CHO-cynoROR1 was finally obtained.

[0343] The nucleotide sequence of mROR1 was constructed into the commercially available pCDNA5 vector from Invitrogen. Specifically, the pCDNA5 vector was digested with NheI and PmeI, and the nucleotide sequence of mROR1 was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into human embryonic kidney HEK293 cells and Chinese hamster ovary cells CHOK1 using lipofectamine 3000. Overexpressing cell lines were then screened using the antibiotic Hygromycin. Through monoclonalization, the monoclonal overexpressing cell line CHO-mROR1 was finally obtained.

[0344] 2. Construction of cell lines containing proteins of the same family

[0345] The hROR2 nucleotide sequence was constructed into the Invitrogen-modified pCDNA5 vector. Specifically, the pCDNA5 vector was digested with NheI and PmeI, and the hROR2 nucleotide sequence was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into CHOK1 Chinese hamster ovary cells using lipofectamine 3000. Overexpressing cell lines were then screened using the antibiotic Hygromycin. Through monoclonalization, the monoclonal overexpressing cell line CHO-hROR2 was finally obtained.

[0346] Facs assay for the affinity between antigen cells and tool antibodies

[0347] Experimental reagents and materials:

[0348]

[0349] Experimental procedure:

[0350] 1) Cell collection and seeding plate

[0351] a) Harvest cells in the logarithmic growth phase, ensuring cell viability is above 90%.

[0352] b) Centrifuge at 1000 r / min for 5 min, then discard the supernatant;

[0353] c) Wash the cells once with PBS;

[0354] d) Resuspend the cells using FACS Buffer and count them;

[0355] e) Prepare a solution with a density of 2×10 using FACS Buffer. 6 Cell suspension with cells / mL;

[0356] f) Add 50 μL of cell suspension to each well of a 96-well plate;

[0357] 2) Antibody incubation and detection

[0358] a) Add 50 μL of test samples of different concentrations to the experimental group, starting with a sample concentration of 20 ug / ml and setting up 3 dilution gradient points;

[0359] b) After mixing, incubate at 4°C in the dark overnight;

[0360] c) Wash cells once with 200 μL of FACS Buffer, centrifuge at 1000 r / min for 5 min, and discard the supernatant;

[0361] d) Add APC-labeled secondary antibody (1:1500 dilution) to the 96-well plate, and add an equal volume of FACSBuffer to the blank control group;

[0362] e) After mixing, incubate at 4°C in the dark for 40 minutes;

[0363] f) Wash cells once with 200 μL of FACS Buffer, centrifuge at 1000 r / min for 5 min, and finally resuspend cells with 100 μL of FACS Buffer;

[0364] g) Detect RL-1 MFI readings using an Intellicyte plus flow cytometer (Excitation Laser: 488nm Blue Laser).

[0365] 3) Data processing

[0366] Analyze FACS data using Prism software.

[0367] Data Analysis ( Figure 1Using the overexpression cell line CHO-hROR1, in vitro cell biology combined with FACS testing showed that all three control antibodies bound to the overexpression cell lines. Among them, UC961 had a higher binding signal, while XBR1-402 and C2E3 had slightly lower binding signals.

[0368] Example 2

[0369] Production of rabbit-derived anti-human ROR1 monoclonal antibody

[0370] 2.1 Animal Immunization

[0371] To obtain rabbit monoclonal antibodies recognizing the human ROR1 antigen, this invention selected New Zealand white rabbits for immunization. The initial immunization consisted of 200 μg of antigen, emulsified with an equal volume of Freund's complete adjuvant and the antigen (purchased from AcroBiosystems, catalog number RO1-H5250), and administered via multiple injections at the back. Booster immunizations were performed every two weeks, using 100 μg of antigen, emulsified with an equal volume of Freund's incomplete adjuvant and administered via multiple injections at the back. After five immunizations, the serum titer was measured using standard ELISA methods. Rabbits with high titers were selected and given a single pulse immunization three days prior to antibody selection, via intraperitoneal injection of 50 μg of protein. This pulse immunization did not require adjuvant emulsification, and PBS was used as the buffer. Spleens were harvested three days later.

[0372] 2.2 Splenic cell isolation

[0373] Rabbit spleens were surgically removed and placed in sterile cell culture dishes. The spleens were rinsed with DPBS containing 100 U / ml penicillin and 100 μg / ml streptomycin. The spleens were then minced with surgical scissors and gently ground into single cells using a syringe core. The cell suspension was filtered through a 100 μm cell sieve, and the single-cell filtrate was collected. The cells were centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in RPMI-1640 containing 5% fetal bovine serum.

[0374] 2.3B Cell Culture and Identification

[0375] Rabbit memory B cells were obtained by co-incubating biotin-labeled ROR1 protein with successfully immunized rabbit lymphocytes. These cells were then cultured in 96-well cell culture plates at 37°C and 5% CO2 using B cell culture medium. After 10-14 days of culture, the binding activity of the clonal supernatant was detected at the protein level using ELISA; clones with a protein binding activity greater than 5 times the background level were considered positive. The ELISA-positive supernatant was then analyzed by FACS to detect the binding of the supernatant to stable transfected CHO-hROR1 and CHO-mROR1 cells, and FACS-positive single clones were finally selected.

[0376] 2.4 Cloning of the gene encoding rabbit monoclonal antibody

[0377] B-cell positive clones were collected. Total RNA was extracted from a subset of positive clones using RNAiso Plus and reverse transcribed into cDNA. The light and heavy chain variable regions were amplified by PCR and constructed into a PTT5 expression vector containing the corresponding heavy and light chain constant regions for sequencing. The correct sequences were obtained. The sequencing results were analyzed using VBASE2 (http: / / www.vbase2.org / vbscAb.php) to obtain the light and heavy chain variable region sequences of the antibody.

[0378] Example 3

[0379] 1. Preparation of anti-human ROR1 chimeric antibody

[0380] The heavy chain variable region sequence of the rabbit-derived anti-human ROR1 monoclonal antibody and the published heavy chain constant region sequence of the human monoclonal antibody IgG1 subclass were spliced ​​together and constructed into a mammalian cell expression vector. Similarly, the light chain variable region sequence of the rabbit-derived anti-human ROR1 monoclonal antibody and the published light chain constant region sequence of the human monoclonal antibody κ subclass were spliced ​​together and constructed into a mammalian cell expression vector. The constructed heavy chain and light chain vectors of the anti-human ROR1 chimeric antibody were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After approximately 7 days, the cell supernatant was collected and purified using Mabselect to obtain the anti-human ROR1 chimeric antibody protein.

[0381] 2. In vitro cell binding assay of anti-human ROR1 chimeric antibody to overexpressing cell lines

[0382] Starting with an initial concentration of 10 μg / mL, the anti-human ROR1 chimeric antibody was serially diluted 2.5-fold or 4-fold, resulting in 12 or 8 concentration points. 50 μL of antibody from each concentration point was added to a 96-well plate. CHO-hROR1 cells with high human ROR1 expression were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended to a cell density of approximately 2 × 10⁶ cells / well. 6 50 μL of cells per milliliter was added to the wells of a 96-well plate containing antibody. After incubation at 4°C for 1 hour, APC-labeled goat anti-human IgG secondary antibody was added. After further incubation at 4°C for 1 hour, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and a 4-parameter fitting curve was generated using Prism software.

[0383] Results Analysis Figure 2In vitro cell biology binding FACS tests were conducted on chimeric antibodies using the overexpressing cell line CHO-hROR1. The data showed that several molecules with good binding signals were screened and had comparable activity to the control antibody UC961, such as chrN2, chrN25, chrN1, and chrN27.

[0384] 3. In vitro cell binding assay of anti-human ROR1 chimeric antibody to naturally expressed cell lines

[0385] The anti-human ROR1 chimeric antibody was serially diluted 4-fold starting from an initial concentration of 10 μg / mL, resulting in 8 concentration points. 50 μL of antibody from each concentration point was added to a 96-well plate. Cells naturally expressing human ROR1 were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended to a cell density of approximately 2 × 10⁶ cells / well. 6 50 μL of cells per milliliter was added to the wells of a 96-well plate containing antibody. After incubation at 4°C for 1 hour, APC-labeled goat anti-human IgG secondary antibody was added. After further incubation at 4°C for 1 hour, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and a 4-parameter fitting curve was generated using Prism software.

[0386] analyze( Figure 3 In vitro cell biology binding FACS tests were conducted on chimeric antibodies using Mino and HT29 cell lines that naturally express human ROR1. The data showed that the selected chrN2, chrN25, chrN1, and chrN27 antibodies had comparable activity to the control antibody UC961.

[0387] 4. In vitro binding affinity and kinetics experiments of anti-human ROR1 chimeric antibodies

[0388] Antibody affinity was determined using the anti-human antibody capture method with a Fortebio (BLITZ pro1.1.0.28) instrument. For the assay, the capture antibody (AHC) bioprobe (purchased from SARTORIUS, catalog number 18-5060) of the Fc fragment of the anti-human antibody was immersed in PBS for 10 min. 200 μl of diluted antibody sample (including the chimeric antibody of this invention and the control antibody; working antibody concentration was 15 μg / mL) was loaded onto the AHC bioprobe, and then equilibrated in PBS for 100 s. The AHC probe was then further subjected to a binding reaction with human ROR1 protein (purchased from ACRO biosystem, catalog number RO1-H522y) for 600 s. Afterward, the AHC probe was transferred to PBS for dissociation for 600 s. After the experiment, the blank control response value was subtracted, and the kinetic constant of antigen-antibody binding was calculated using a 1:1 Langmuir binding model fitting.

[0389] Analysis: Based on in vitro kinetic binding activity analysis, the binding kinetic constant between the chimeric antibody and recombinant human ROR1 protein is within 10. -9 Up to 10 -11 In contrast, the binding kinetic constant of the target antibody UC961 is at a level of 10. -8 At the level, the binding kinetic constant of the target antibody XBR1-402 is at 10. -9 At the level of [unclear], the obtained chimeric antibody showed a good kinetic binding signal to recombinant human ROR1 protein.

[0390] Table 1: In vitro kinetic binding activity of chimeric antibodies to recombinant human ROR1 protein:

[0391] Antibody Response value KD(M) kon(1 / Ms) kdis(1 / s) chrN1 0.4028 1.49E-09 2.97E+05 4.41E-04 chrN2 0.3625 5.67E-11 3.96E+05 2.24E-05 chrN25 0.3339 3.81E-09 1.89E+05 7.20E-04 chrN27 0.4028 1.49E-09 2.97E+05 4.41E-04 XBR1-402 0.3726 6.96E-09 4.17E+05 2.91E-03 UC961 0.3269 1.07E-08 4.67E+05 4.98E-03

[0392] 5. Cellular endocytosis assay of anti-human ROR1 chimeric antibody

[0393] Methods and Procedures: First, prepare cells naturally expressing ROR1, such as human non-small cell lung cancer A549 cells, human colon cancer HT29 cells, and mantle cell lymphoma Mino cells. Digest and count the cells using trypsin-free digestion solution, resuspend them in culture medium, and adjust the cell density to 2E6 / mL. Reconstitute the endocytosis reagent (purchased from Sartorius, catalog number 90565) with sterile water to a final concentration of 100 μg / mL. The molecular weight of the endocytosis reagent is approximately 1 / 3 of the antibody molecular weight, so the molar ratio of reagent to antibody during incubation is 3:1. Dilute both the antibody and endocytosis reagent to 2 μg / mL using cell culture medium at a 1:1 mass ratio and incubate at 37°C for 15 minutes. Serially dilute the labeled antibody reagent to 7 spots using a 2-fold serial dilution. Add the diluted antibody reagent to the prepared cells and incubate at 37°C for endocytosis for 5 hours. Remove the culture plate and directly analyze the fluorescence value using the RL-1 channel of a flow cytometer. The obtained MFI curve is fitted using the four-parameter method in Prism software.

[0394] analyze( Figure 4 The endocytic activity of the chimeric anti-human ROR1 antibody was investigated using naturally expressed cell lines A549, HT29, and Mino. The results showed that the chimeric antibody could undergo endocytosis relatively quickly, which was comparable to or better than the control antibody UC961.

[0395] Example 4

[0396] 1. Humanization of anti-human ROR1 rabbit antibody

[0397] Based on the antibody coding schemes of Kabat and Chothia, the amino acid sequence regions of the six antigen complementarity determinants (CDRs) of the heavy and light chains of rabbit antibodies, as well as the framework region supporting the conserved three-dimensional conformation of the antibody, were determined. Subsequently, by analyzing and searching known human antibody sequences, the variable region sequence of the heavy chain of a human antibody most similar to that of the rabbit antibody, such as IGHV1|IGHJ4*01, was selected. Its antibody framework region sequence was used as a template to bind the rabbit antibody heavy chain CDR to the human antibody framework region, ultimately generating the humanized antibody heavy chain variable region sequence. The same process was used to generate the humanized antibody light chain variable region sequence. Antibodies with rabbit antibody CDRs directly transplanted into the human framework region often exhibit a sharp decrease in binding activity; therefore, it is necessary to revert individual amino acids in the framework region from human to rabbit. The reversion mutation sites were determined by: firstly, comparing the designed humanized antibody sequence with the original rabbit antibody sequence to check which amino acids differ; and secondly, checking whether these amino acids play an important role in supporting the antibody structure or in binding with the antigen. While designing humanized sequences, it is also necessary to check for potential post-translational modification sites, such as N (asparagine) glycosylation sites, N deamidation sites, and D (aspartic acid) isomerization sites.

[0398] The variable region heavy chain gene of the humanized antibody was constructed into a mammalian cell expression vector containing the heavy chain constant region gene of the human monoclonal antibody IgG1 subclass; the light chain gene was constructed into a mammalian cell expression vector containing the light chain constant region gene of the human monoclonal antibody κ subclass. The constructed heavy chain and light chain vectors of the anti-human ROR1 humanized antibody were paired and mixed, and HEK293 cells were transfected with polyethyleneimine (PEI). After about 7 days, the cell supernatant was collected and purified using Mabselect to obtain the anti-human ROR1 humanized antibody protein.

[0399] 2. In vitro binding affinity and kinetics experiments of anti-human ROR1 humanized antibody

[0400] Antibody affinity was determined using the anti-human antibody capture method on a Fortebio (BLITZ pro1.1.0.28) instrument. During the assay, the capture antibody (AHC) bioprobe of the Fc fragment of the anti-human antibody was immersed in PBS for 10 min. 200 μl of diluted antibody sample (including the chimeric antibody of this invention and the control antibody; the working antibody concentration was 15 μg / mL) was loaded onto the AHC bioprobe, and then equilibrated in PBS for 100 s. The AHC probe was then further subjected to a binding reaction with human ROR1 protein diluted to 100 nM (purchased from ACRObiosystem) for 600 s. Afterward, the AHC probe was transferred to PBS for a dissociation reaction for 600 s. After the experiment, the blank control response value was subtracted, and the antigen-antibody binding kinetic constant was calculated using software to fit a 1:1 Langmuir binding pattern. The instrument's built-in software was then used for curve processing and fitting.

[0401] Data analysis: Analysis of in vitro kinetic binding activity showed that the binding kinetic constant between the anti-human ROR1 humanized antibody and recombinant human ROR1 protein remained at 10. -8 -10 -10 Although the binding kinetic constant of the target antibody UC961 was slightly lower than that of the chimeric antibody, it was still within 10. -8 High level. The modified humanized antibodies exhibit good kinetic binding signals.

[0402] In vitro kinetic binding profile of humanized antibody to recombinant human ROR1 protein:

[0403] Table 2: In vitro kinetic binding activity of humanized antibodies to recombinant human ROR1 protein:

[0404]

[0405]

[0406] 3. In vitro cell binding assay of anti-human ROR1 humanized antibody

[0407] Humanized anti-human ROR1 antibody was serially diluted 4-fold starting at a concentration of 2 μg / mL, resulting in 8 concentration points. 50 μL of antibody from each concentration point was added to a 96-well plate. Mino, A549, HT29, NCI-N87, NCI-H1975, RPMI8226, and Jeko-1 cells, all naturally expressing human ROR1, were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended to a cell density of approximately 2 × 10⁶ cells / well. 6 50 μL of cells per milliliter was added to the wells of a 96-well plate containing antibody. After incubation at 4°C for 1 hour, APC-labeled goat anti-human IgG secondary antibody was added. After further incubation at 4°C for 1 hour, the mean fluorescence reading of the cell population was analyzed by flow cytometry. A 4-parameter fitting curve was performed using Prism software, and the EC50 and the top value of fluorescence intensity were calculated.

[0408] Analysis (e.g.) Figure 5 In vitro cell biology binding FACS tests were conducted on the selected humanized antibody using seven naturally expressed human ROR1 cell lines: Mino cells, A549 cells, HT29 cells, NCI-N87 cells, NCI-H1975 cells, RPMI8226 cells, and Jeko-1 cells. The data showed that the selected humanized antibody had high in vitro cell binding activity, which was comparable to or better than that of the control antibody UC961.

[0409] Table 3: Top values ​​of EC50 and fluorescence intensity of humanized antibody binding to naturally expressed ROR1 cells in vitro via flow cytometry.

[0410]

[0411]

[0412] 4. In vitro cell nonspecific binding assay of anti-human ROR1 humanized antibody

[0413] Anti-human ROR1 affinity maturation conjugate antibody was initially diluted 4-fold at a concentration of 20 μg / mL in four gradients. 50 μL of each diluted antibody was added to a 96-well plate. CHO-hROR2 cells overexpressing human ROR2 and CHOK1 hamster ovary cells were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended to a cell density of approximately 2 × 10⁻⁶ cells / well. 650 μL of cells per milliliter was added to the wells of a 96-well plate containing antibody. After incubation at 4°C for 1 hour, APC-labeled goat anti-human IgG secondary antibody was added. After further incubation at 4°C for 1 hour, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and the results were visualized as bar charts using Prism software.

[0414] Results Analysis Figure 6 Non-specific binding experiments were conducted on humanized anti-human ROR1 antibodies against the cell line CHO-hROR2, which overexpresses the ROR2 family protein, and the Chinese hamster ovary cell line CHOK1, which is commonly used for protein expression. The results showed that the control antibodies XBR1-402 and C2E3 had strong non-specific binding signals with CHOK1 and CHO-hROR2, while the control antibody UC961 and various humanized anti-human ROR1 antibody molecules did not have non-specific binding signals with the family protein or empty cells used for expression.

[0415] 5. In vitro cell binding assays of different species of anti-human ROR1 humanized antibodies

[0416] Anti-human ROR1 affinity maturation conjugate antibody was initially diluted 4-fold at a concentration of 20 μg / mL, and 50 μL of each diluted antibody was added to a 96-well plate. CHO-hROR2 cells overexpressing human ROR2 and CHOK1 hamster ovary cells were collected by centrifugation at 100g for 5 minutes at room temperature. The cells were washed once with PBS containing 0.5% BSA, centrifuged at 100g for 5 minutes at room temperature, and resuspended at a density of approximately 2 x 10⁶ cells / mL. 50 μL of this resuspended cell line was added to the wells of the antibody-added 96-well plate. After incubation at 4°C for 1 hour, APC-labeled goat anti-human IgG secondary antibody was added. After further incubation at 4°C for 1 hour, the mean fluorescence reading of the cell population was analyzed by flow cytometry, and a 4-parameter curve was generated using Prism software.

[0417] Results Analysis Figure 7 In vitro cell FACS binding experiments were conducted on humanized anti-human ROR1 antibodies against CHO-mROR1 and CHO-cynoROR1 cell lines overexpressing ROR1 protein from different species. The results showed that all control antibodies and the selected humanized anti-ROR1 antibody had good binding signals with CHO-cynoROR1 cell line overexpressing monkey ROR1, while only the control antibody C2E3 showed a binding signal with CHO-mROR1 cell line overexpressing mouse ROR1.

[0418] 6. In vitro killing assay of anti-human ROR1 humanized antibody

[0419] In vitro killing assays were performed using the 293-hROR1 overexpressing cell line. Cells were digested and counted, and the cell density was adjusted to 5E4 / mL using appropriate culture medium. 50 μL / well was seeded into 96-well white plates. Small molecule toxin conjugate reagent 2... 0 ADCαHFc-CL-MMAE (purchased from Moradec, catalog number AH-102AE-50) was diluted to 2 μg / mL using the corresponding cell culture medium; the humanized antibody was diluted to 2.5 μg / mL using the diluted small molecule toxin conjugation reagent, and then 3-fold diluted to set 10 gradients; 50 μl / well of the diluted series of antibodies was added to a white plate containing cells; the plate was incubated at 37°C for 4 days; CellTiter-Glo (purchased from Promega, catalog number G7573) was prepared according to the reagent instructions, 100 μl / well was added to the cell plate, and the mixture was shaken for 5 minutes. The chemiluminescence was read using a microplate reader; the killing curve was fitted using Prism software with four parameters.

[0420] Results Analysis Figure 8 In vitro cell killing experiments were conducted using the ROR1-overexpressing cell line 293-hROR1. The results showed that all the selected humanized anti-human ROR1 antibody molecules had in vitro cell killing activity, which was similar to or better than the control antibody UC961.

[0421] 7. Physical characterization and monomer rate analysis of anti-human ROR1 humanized antibody molecules

[0422] Experimental apparatus: UPLC CLASS ACQUITYH (WATERS)

[0423] Analytical column: TSKgel G3000SWXL 7.8*300 (TOSHI, CatNo 003C03326C)

[0424] Analytical solution: 200 mM K₂HPO₄, 250 mM KCl, pH adjusted to 6.2 with HCl.

[0425] Analytical method: 50 μl of antibody with a concentration of 1 mg / ml was injected into a pre-equilibrated chromatography column and flowed at room temperature and a flow rate of 0.75 ml / min for 45 min. The absorbance of the A280 instrument was measured at the same time. The monomer content and ratio of the antibody were determined based on the peak time and peak volume.

[0426] Results analysis: The monomer content of the selected anti-human ROR1 humanized antibody molecules was analyzed. The results showed that the monomer content of each molecule was above 97%, indicating good monomer content properties.

[0427] Table 4: Retention time of the main peak and monomer yield of the antibody are shown in the table below.

[0428] Antibody Retention time of the main peak (min) Monomer percentage (SEC) UC961 10.588 98.69 N1-H1L2 10.226 98.48 N2-H5L0 10.355 99.55 N2-H5L1 10.36 99.29 N25-H2L1 11.381 99.87 N27-H2L0 10.912 97.15

[0429] 8. Analysis of the hydrophobic properties of humanized antibodies

[0430] Experimental apparatus: ARC (Waters)

[0431] Analytical column used in the experiment: TSKgel Butyl-NPR (4.6mm x 3.5cm, Cat No. 14947)

[0432] Analytical solution: A. 20 mM Histidine, pH 6.0;

[0433] B.20mM Histidine, 1.6M(NH4)2SO4

[0434] Analytical method: The hydrophobic properties of the antibody were analyzed according to the instructions for use of the hydrophobic chromatography column.

[0435] Results analysis: Hydrophobic properties analysis of the selected anti-human ROR1 humanized antibody molecules showed that the hydrophobicity HIC values ​​of each selected molecule were greater than 0.7, indicating good hydrophobic properties.

[0436] Table 5: Hydrophobic properties of humanized antibodies

[0437] Antibody Retention time of the main peak (min) HIC UC961 16.080 0.825 XBR1-402 14.556 0.979 C2E3 14.671 0.967 hzN1-H1L2 15.402 0.894 hzN2-H5L0 16.529 0.780 hzN2-H5L1 16.613 0.771 hzN25-H2L1 15.288 0.905 hzN27-H2L0 14.320 1.003

[0438] 9. Drug metabolism analysis in humanized antibody mice

[0439] Experimental materials: humanized antibody, mouse serum collected at different time points, antibody to be tested binding antigen, anti-huIgG Fab monoclonal antibody (Sigma, I5260-1ML), HRP-labeled goat anti-human IgG secondary antibody (Jackson, code: 109-035-098).

[0440] Experimental methods:

[0441] Serum collection:

[0442] 1) Female Balb / C mice, 3 mice / group, were administered 200 μg / mouse via tail vein or intraperitoneal injection;

[0443] 2) Collect blood from the tail vein at the time points specified in the experimental design, keep the blood samples at room temperature for more than 30 minutes, collect serum at 4000 rpm for 15 minutes, and store at -20℃. To prevent serum evaporation, the final serum collection volume should be greater than 20 μL.

[0444] 3) The last serum collection should be frozen at -20°C for at least 24 hours.

[0445] Detection method:

[0446] 1) Coat the binding antigen and anti-IgG Fab monoclonal antibody separately with PBS in 96-well ELISA plates, 0.2 μg / ml, 100 μl / well, and incubate overnight at 4°C;

[0447] 2) Prepare the required reagents:

[0448] Blocking solution 5% BSA + PBS

[0449] Antibody dilution solution: 5% BSA + PBS + 20% blank mouse serum

[0450] ELISA plate washing buffer: 0.1% Tween + PBS

[0451] 3) Wash the coated ELISA plate three times with PBS, 300 μl / well;

[0452] 4) Add blocking solution, 200 μL / well, and seal at 37°C for 1 hour;

[0453] 5) Dilute the starting serum to a suitable concentration using blocking buffer, and then dilute it to a suitable concentration range using antibody diluent containing the same serum concentration. The specific dilution factor needs to be adjusted based on the preliminary experiment. In principle, the final colorimetric value of the serum to be tested should be within the range of the standard curve colorimetric value.

[0454] 6) Dilute the antibody standard curve with antibody diluent. The dilution of the standard curve should still be adjusted according to the preliminary experiment to make the standard curve fit a linear curve (if suitable software is available, an S-shaped curve can also be fitted).

[0455] 7) Discard the blocking solution, add diluted antibody and test serum to the enzyme-linked plates of the two coating methods respectively, 100 μl / well, and incubate at 37℃ for 1 h;

[0456] 8) Wash the plate three times with PBST;

[0457] 9) Dilute the secondary antibody 1:5000, add it to the washed ELISA plate, 100 μL / well, and incubate at 37°C for 40 min;

[0458] 10) Wash the plate three times with PBST;

[0459] 11) TMB color development, 100 μl / well, protected from light for 10 min;

[0460] 12) Add 50 μL of 2 M HCl to terminate the reaction, and take a reading at 450 nm.

[0461] Data Analysis ( Figure 9In vivo drug metabolism experiments were conducted on four samples: three preferred molecules (N27-H2L0, N2-H5L0, and N1-H2L1) and the control molecule UC961. Three female Balb / C mice were used for each sample. Blood samples were collected at different time points after tail vein injection, and the antibody levels in mouse serum were measured using a fully human antibody detection method. The results showed that the metabolic levels of each molecule differed in mice. N1-H2L1 was metabolized slowly with a long half-life of 289-343 hours; N27-H2L0 and N2-H5L0 were also metabolized slowly, with a metabolic time of 195-289 hours; and the control antibody UC961 had a slightly shorter half-life, with a metabolic time of 155-220 hours. The preferred molecules all had longer half-lives than the control molecule.

[0462] Table 6. Drug metabolism properties in mice

[0463]

[0464]

[0465] Example 4

[0466] 1. Humanized antibody-drug conjugate

[0467] Conjugation was conducted using the company's in-house ADC conjugation platform. The conjugated drug was Monomethyl auristatin E (MMAE; SGD-1010), a synthetic derivative of sarsaparilla toxin 10, which effectively inhibits mitosis by suppressing microtubule polymerization. MMAE is widely used as a cytotoxic component in the production of antibody-drug conjugates (ADCs) for cancer treatment.

[0468] After conjugation, ensure the drug / antibody conjugation ratio is 4:1.

[0469] 2. In vitro cytotoxic activity of antibody-drug conjugates

[0470] The in vitro cytotoxic activity assay of the antibody-drug conjugate was performed using the 293-hROR1 overexpressing cell line. Cells were digested and counted, and the cell density was adjusted to 1E5 / mL using the appropriate culture medium. 50 μl / well was seeded into 96-well plates. The antibody-drug conjugate was diluted to 10 μg / mL using the cell-specific culture medium, and then three-fold diluted 10 times. 50 μl / well of the diluted antibody was added to each well of the cell-coated plate. The plates were incubated at 37°C for 4 days. CellTiter-Glo (purchased from Promega, catalog number G7573) was prepared according to the reagent instructions, and 100 μl / well was added to each cell plate. After vortexing for 5 minutes, the chemiluminescence was read using a microplate reader. The cytotoxicity curve was fitted using Prism software with four parameters.

[0471] Data Analysis ( Figure 10 In vitro cell killing experiments were conducted using the ROR1-overexpressing cell line 293-hROR1 to conjugate antibody-drug mixtures. After 4 days of incubation, the results showed that each of the selected humanized anti-human ROR1 antibodies, after conjugation with small molecule drugs, had significant in vitro cell killing activity, similar to or better than the control antibody UC961, while the negative control antibody showed no in vitro killing activity.

[0472] 3. In vivo efficacy of antibody-drug conjugates

[0473] To evaluate and compare the efficacy of N1-H1L2-MMAE-D4, N27-H2L0-MMAE-D4, UC961-MMAE-D4 and N2-H5L0-MMAE-D4 against subcutaneous xenografts of human mantle cell lymphoma in JeKo-1 mice.

[0474] A human mantle cell lymphoma JeKo-1 subcutaneous xenograft model was established in 5-6 week old female NOD-Scid mice by subcutaneous inoculation with JeKo-1 cells. The tumors were allowed to grow to 100-150 mm. 3 Subsequently, animals were grouped according to tumor volume into a negative control group (saline) and groups receiving 1 or 3 mg / kg of each drug. Ten animals were in the negative control group, and six animals were in each of the other groups. The drugs were administered intravenously (IV) once a week for a total of two weeks, with an injection volume of 0.1 mL / 10 g. Efficacy was evaluated based on tumor growth inhibition rate (TGI), and safety was evaluated based on changes in animal weight and mortality.

[0475] At the end of the trial (day 14 after drug administration), the mean tumor volume in the negative control group was 2921.5 mm. 3 The mean tumor volumes in the N1H1L2-MMAE-D41 and 3 mg / kg groups were 2693.3 and 711.8 mm, respectively. 3 The mean tumor volumes in the N27H2L0-MMAE-D41 and 3 mg / kg groups were 2227.7 mm and 912.0 mm, respectively. 3 The mean tumor volumes in the UC961-MMAE-D41 and 3 mg / kg groups were 2699.3 mm and 930.4 mm, respectively. 3 The mean tumor volumes in the N2 H5L0-MMAE-D41 and 3 mg / kg groups were 2287.7 mm and 935.1 mm, respectively. 3Compared with the negative control group, the high-dose groups (3 mg / kg) showed significant differences (P < 0.001). All of the above drugs significantly inhibited the growth of subcutaneous xenografts in JeKo-1 mice in a dose-dependent manner. The TGI of N1 H1L2-MMAE-D41 at 3 mg / kg was 8% and 79%, respectively; the TGI of N27H2L0-MMAE-D41 at 3 mg / kg was 25% and 71%, respectively; the TGI of UC961-MMAE-D41 at 3 mg / kg was 8% and 71%, respectively; and the TGI of N2 H5L0-MMAE-D41 at 3 mg / kg was 23% and 71%, respectively.

[0476] like Figures 11-12 At the end of the experiment, as shown in Tables 7 and 8, the average tumor weight was 3.19 g in the negative control group, 2.71 g and 0.79 g in the N1 H1L2-MMAE-D41 and 3 mg / kg groups, 2.58 g and 0.99 g in the N27 H2L0-MMAE-D41 and 3 mg / kg groups, 2.98 g and 0.86 g in the UC961-MMAE-D41 and 3 mg / kg groups, and 2.62 g and 0.96 g in the N2H5L0-MMAE-D41 and 3 mg / kg groups. The TGI values ​​for the N1 H1L2-MMAE-D41 and 3 mg / kg groups were 15.0% and 75.4%, respectively. The tumor weight gain rates (TGIs) for the H2L0-MMAE-D41 and 3 mg / kg groups were 18.9% and 69.1%, respectively; for the UC961-MMAE-D41 and 3 mg / kg groups, the TGIs were 6.4% and 73.0%, respectively; and for the N2 H5L0-MMAE-D41 and 3 mg / kg groups, the TGIs were 17.8% and 70.0%, respectively. Compared with the negative control group, the tumor weight in the high-dose groups (3 mg / kg) was significantly different (P < 0.05). # All <0.001).

[0477] No animals died in any group, and no drug-related significant weight loss or other symptoms occurred. The tumor-bearing mice tolerated the drugs very well.

[0478] Table 7. Pharmacodynamic analysis of tumor volume in different groups of the JeKo-1 mouse subcutaneous xenograft model of human mantle cell lymphoma.

[0479]

[0480] Note: The first administration time is D0; P indicates comparison with the saline group; - indicates not applicable.

[0481] Table 8. Drug efficacy analysis of tumor weight in different groups in the JeKo-1 mouse subcutaneous xenograft model of human mantle cell lymphoma.

[0482]

[0483] Note: P # This indicates a comparison with the saline group; - indicates that it is not applicable.

[0484] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A ROR1 antibody or antigen-binding molecule, characterized in that, Its heavy chain CDR1-3 has the amino acid sequences shown in SEQ ID NO:1-3, and its light chain CDR1-3 has the amino acid sequences shown in SEQ ID NO:4-6. Or its heavy chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:7-9, and its light chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:5, 10 or 11. Or its heavy chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:12-14, and its light chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:15-17; Or its heavy chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:18-20, and its light chain CDR1-3 has the amino acid sequence shown in SEQ ID NO:21-23.

2. The antibody or antigen-binding molecule according to claim 1, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:68, 70, 72 or 74; Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:69, 71, 73 or 75.

3. The antibody or antigen-binding molecule according to claim 1, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:76, 78, 81 or 83; Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:77, 79, 80, 82 or 84.

4. Biomaterials, including at least one of the following: a) Nucleic acid encoding the ROR1 antibody or antigen-binding molecule as described in any one of claims 1 to 3; b) A plasmid vector containing the nucleic acid described in a); c) A host whose genome integrates the nucleic acid described in a), or is transformed or transfected with the plasmid vector described in b); d) A conjugate comprising the ROR1 antibody or antigen-binding molecule as described in any one of claims 1 to 3, and a drug, preferably an antitumor drug selected from at least one of MMAE, MMAF, or DXD.

5. Any one of the following (I) to (VI) in the preparation of drugs for the prevention and treatment of ROR1-related diseases: I) The antibody or antigen-binding molecule as described in any one of claims 1 to 3; II) The nucleic acid described in claim 4; III) The plasmid vector as described in claim 4; IV) The host as described in claim 4; V) Cultures obtained from the host described in claim 4 or purified antibodies; VI) The coupling compound as described in claim 4.

6. The application according to claim 5, characterized in that, The disease associated with ROR1 is cancer.

7. The application of any one of the following i) to vi) in the preparation of ROR1 detection reagents, tumor diagnostic reagents and / or tumor imaging reagents: i) An antibody or antigen-binding molecule as described in any one of claims 1 to 3; ii) The nucleic acid as described in claim 4; iii) The plasmid vector as described in claim 4; iv) The host as described in claim 4; v) Cultivating the culture obtained from the host described in claim 4 or the purified antibody; vi) The coupling compound as described in claim 4.

8. The application according to claim 7, characterized in that, The tumor is a tumor that expresses ROR1.

9. The application according to claim 8, characterized in that, The tumors are lymphoma, lung cancer, colon cancer, stomach cancer, lung adenocarcinoma, multiple myeloma, breast cancer, pancreatic cancer, ovarian cancer, or mantle cell lymphoma.

10. A medicine, comprising any one of the following (I) to (VI); I) The antibody or antigen-binding molecule as described in any one of claims 1 to 3; II) The nucleic acid described in claim 4; III) The plasmid vector as described in claim 4; IV) The host as described in claim 4; V) Cultures obtained from the host described in claim 4 or purified antibodies; VI) The coupling compound as described in claim 4.

11. A pharmaceutical composition comprising the medicament of claim 10 and other therapeutic agents.

12. A reagent, comprising any one of the following i) to vi): i) An antibody or antigen-binding molecule as described in any one of claims 1 to 3; ii) The nucleic acid as described in claim 4; iii) The plasmid vector as described in claim 4; iv) The host as described in claim 4; v) Cultivating the culture obtained from the host described in claim 4 or the purified antibody; vi) The coupling compound as described in claim 4.

13. A method for detecting ROR1 or tumor cells expressing ROR1, comprising detecting a sample with the reagent of claim 12.

Images