Antibody of rori and preparation method and use thereof

Abstract

The invention relates to the technical field of biology, in particular to a ROR1 antibody as well as a preparation method and application thereof. The ROR1 antibody provided by the invention can be highly specifically combined with an antigen, and has rapid and efficient internalization ability. Besides, the anti-ROR1 antibody provided by the invention has high selectivity and high biological activity, not only can be used as an anti-tumor drug or an antibody coupling drug, but also can be used as a diagnostic tool to detect ROR expression in ROR related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to antibodies of ROR1 and methods of making and uses thereof. BACKGROUND

[0002] Receptor tyrosine kinase-like orphan receptor 1 (ROR1), also known as receptor-related neurotrophic tyrosine kinase 1 (NTRKR1), was first discovered in human neuroblastoma cell lines in 1992. It is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family and belongs to the type I receptor tyrosine kinase (RTK) family. The amino acid sequence homology of ROR1 and ROR2 in the ROR family is 58%. ROR1 and ROR2 participate in the non-canonical Wnt signaling pathway by binding to the FZD domain and the ligand Wnt5a. ROR1 can inhibit apoptosis, enhance EGFR signaling, and induce epithelial-mesenchymal transition (EMT).

[0003] The extracellular domain of ROR1 is composed of an immunoglobulin-like domain (Ig) and two cysteine-rich domains (FZD domain and KRD domain), and the intracellular domain is composed of a tyrosine kinase domain, two serine or threonine-rich domains, and a proline-rich domain. ROR1 is a conserved embryonic protein, and its expression gradually decreases with embryonic development. It is almost non-existent or lowly expressed in most adult tissues, but more and more literature has found that ROR1 is expressed in various cancer cells, such as B-cell chronic lymphocytic leukemia (CLL) and other hematological malignancies, solid tumors, and is widely expressed in tumor entities. The expression level in cancer tissues is significantly higher than that in adjacent normal tissues, including melanoma, colon, pancreatic, lung, and breast cancer. Therefore, as a cancer marker, ROR1 has become an ideal drug target for cancer treatment.

[0004] NBE-002 is a ROR1 ADC “NBE-002” acquired by Boehringer Ingelheim for $1.45 billion from NBE Therapeutics. PNU-159682 is site-specifically and quantitatively conjugated to the C-terminus of the heavy and light chains of the antibody via a specific reaction mediated by the transpeptidase Sortase A, resulting in a purer and more uniform product, DAR 4. The monoclonal antibody (HuXBR1-402) was screened from a rabbit antibody library by phage display and binds to the Ig / Fz domain of ROR1 with high affinity (Kd = 5.8 nM). After humanization, the affinity did not decrease. NBE-002 can be called an immunostimulatory ADC because of the target. After the Wnt5 / ROR1 pathway is inhibited, T cell infiltration of the tumor is increased, and “cold” tumors are converted to “hot” tumors. NBE-002 is currently undergoing a 1 / 2 phase clinical trial (NCT04441099) to evaluate the safety and tolerability of patients with advanced solid tumors, especially triple-negative breast cancer.

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

[0006] 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.

[0007] As a pan-cancer tumor marker, the development of high-quality anti-ROR1 antibodies is beneficial for the further development of cancer treatment strategies. Furthermore, anti-ROR1 antibodies can also be used as diagnostic tools to detect ROR1 expression in ROR1-related diseases. In addition, given the promising prospects of ADCs in the field of cancer treatment, there remains an urgent need for ROR1-containing ADCs with effective therapeutic effects. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide an antibody for ROR1, a method for its preparation, and its uses:

[0009] The ROR1 antibody provided by this invention,

[0010] Its heavy chain CDR1, CDR2 or CDR3 has an amino acid sequence as shown in any one of SEQ ID NO: 1~3, 7~8, 11~13, 17~19, 23~25, 29~31, 35~37; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO: 1~3, 7~8, 11~13, 17~19, 23~25, 29~31, 35~37 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: 1~3, 7~8, 11~13, 17~19, 23~25, 29~31, 35~37.

[0011] Its light chain CDR1, CDR2 or CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:4~6, 9~10, 14~16, 20~22, 26~28, 32~34, 38~40; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO:4~6, 9~10, 14~16, 20~22, 26~28, 32~34, 38~40, 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:4~6, 9~10, 14~16, 20~22, 26~28, 32~34, 38~40.

[0012] The anti-ROR1 antibody provided by this invention is obtained through mouse immunization, can bind to the antigen with high specificity, and has rapid and efficient internalization ability. Furthermore, the anti-ROR1 antibody provided by this invention has high selectivity and high biological activity, and can be used not only as an anti-tumor drug or antibody-drug conjugate, but also as a diagnostic tool to detect ROR1 expression in ROR1-related diseases.

[0013] The ROR1 antibody provided by this invention:

[0014] Its heavy chain CDR1 has an amino acid sequence as shown in any one of SEQ ID NO: 1, 7, 11, 17, 23, 29 or 35;

[0015] Its heavy chain CDR2 has an amino acid sequence as shown in any one of SEQ ID NO:2, 8, 12, 18, 24, 30 or 36;

[0016] Its heavy chain CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:3, 13, 19, 25, 31 or 37;

[0017] Its light chain CDR1 has an amino acid sequence as shown in any one of SEQ ID NO:4, 9, 14, 20, 26, 32 or 38;

[0018] Its light chain CDR2 has an amino acid sequence as shown in any one of SEQ ID NO:5, 15, 21, 27, 33 or 39;

[0019] Its light chain CDR3 has an amino acid sequence as shown in any one of SEQ ID NO: 6, 10, 16, 22, 28, 34 or 40;

[0020] 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.

[0021] In some embodiments, the ROR1 antibody:

[0022] The amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:1-3, or in SEQ ID NO:7, 8, 3, or in SEQ ID NO:11-13, or in SEQ ID NO:17-19, or in SEQ ID NO:23-25, or in SEQ ID NO:29-31, or in SEQ ID NO:35-37.

[0023] In some embodiments, the ROR1 antibody:

[0024] The amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:4-6, or in SEQ ID NO:9, 5, 10, or in SEQ ID NO:14-16, or in SEQ ID NO:20-22, or in SEQ ID NO:26-28, or in SEQ ID NO:32-34, or in SEQ ID NO:38-40.

[0025] In some specific embodiments, the ROR1 antibody:

[0026] The amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:1-3, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:4-6, respectively.

[0027] Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:7, 8, 3, respectively, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:9, 5, 10, respectively;

[0028] Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:11-13, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:14-16, respectively;

[0029] Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:23-25, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:26-28, respectively;

[0030] Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:29-31, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:32-34, respectively;

[0031] Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:35-37, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:38-40, respectively;

[0032] Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:17-19, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:20-22, respectively;

[0033] In some specific embodiments, the heavy chain CDR1-3 of the antibody are SYGMS (SEQ ID NO:17), TIPSRGSSTYYPDSVKG (SEQ ID NO:18, X is S), and HLYYYGSSDYAMDY (SEQ ID NO:19), respectively, and the light chain CDR1-3 are KASQDLNSYLS (SEQ ID NO:20, X is S), RANRLED (SEQ ID NO:21, X is D), and LQYDEFPYT (SEQ ID NO:22), respectively.

[0034] In other specific embodiments, the heavy chain CDR1-3 of the antibody are SYGMS (SEQ ID NO:17), TIPSRGSSTYYPDTVKG (SEQ ID NO:18, X is T), and HLYYYGSSDYAMDY (SEQ ID NO:19), respectively, and the light chain CDR1-3 are KASQDLNYYLS (SEQ ID NO:20, X is Y), RANRLEE (SEQ ID NO:21, X is E), and LQYDEFPYT (SEQ ID NO:22), respectively.

[0035] In other specific embodiments, the heavy chain CDR1-3 of the antibody are SYGMS (SEQ ID NO:17), TIPSRGSSTYYPDSVKG (SEQ ID NO:18, X is S), and HLYYYGSSDYAMDY (SEQ ID NO:19), respectively, and the light chain CDR1-3 are KASQDLNAYLSS (SEQ ID NO:20, X is A), RANRLED (SEQ ID NO:21, X is D), and LQYDEFPYT (SEQ ID NO:22), respectively.

[0036] This invention obtains mouse-derived antibodies, and then prepares chimeric antibodies:

[0037] At least one of its heavy chains FR1-4 has an amino acid sequence as shown in any one of SEQ ID NO:41-44, 49-50, 52-54, 59-61, 63-65, 70-72, 76-79; or has a sequence based on an amino acid sequence shown in any one of SEQ ID NO:41-44, 49-50, 52-54, 59-61, 63-65, 70-72, 76-79, 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:41-44, 49-50, 52-54, 59-61, 63-65, 70-72, 76-79.

[0038] At least one of its light chains FR1-4 has an amino acid sequence as shown in any one of SEQ ID NO:45-48, 51, 55-58, 62, 66-69, 73-75, 80-82; or has a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in any one of SEQ ID NO:45-48, 51, 55-58, 62, 66-69, 73-75, 80-82; or has a sequence that is more than 80% homologous (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to the amino acid sequence shown in any one of SEQ ID NO:45-48, 51, 55-58, 62, 66-69, 73-75, 80-82.

[0039] In some embodiments, the ROR1 chimeric antibody provided by the present invention:

[0040] Its heavy chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:41, 49, 52, 59, 63, 70 or 76;

[0041] Its heavy chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:42, 53, 60, 64, 71 or 77;

[0042] Its heavy chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:43, 50, 54, 61, 65, 72 or 78;

[0043] Its heavy chain FR4 has an amino acid sequence as shown in either SEQ ID NO:44 or 79;

[0044] Its light chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:45, 55, 66, 73 or 80;

[0045] Its light chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:46, 51, 56, 67, 74 or 81;

[0046] Its light chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:47, 57, 62, 68, 75 or 82;

[0047] Its light chain FR4 has an amino acid sequence as shown in any one of SEQ ID NO:48, 58 or 69;

[0048] 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.

[0049] In some specific embodiments, the ROR1 chimeric antibody provided by the present invention:

[0050] The amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:41-44, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:45-48, respectively.

[0051] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:49, 42, 50, 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:45, 51, 47, 48, respectively;

[0052] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:52, 53, 54, 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:55-58, respectively;

[0053] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:59-61, 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:45, 51, 62, 48, respectively;

[0054] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:63-65 and 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:66-69, respectively;

[0055] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:70-72 and 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:73-75 and 48, respectively;

[0056] Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:76-79, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:80-82 and 48, respectively.

[0057] More specifically, the ROR1 chimeric antibody provided by this invention,

[0058] Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:88, 90, 92, 94, 96, 98 or 100;

[0059] Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

[0060] For example:

[0061] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:88, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

[0062] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:90, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

[0063] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:92, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

[0064] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:94, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

[0065] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:96, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

[0066] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:98, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

[0067] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:100, and the amino acid sequence of its light chain variable region is shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

[0068] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:88, 90, 92, 94, 96, 98 or 100, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:89.

[0069] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:88, 90, 92, 94, 96, 98 or 100, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:91.

[0070] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:88, 90, 92, 94, 96, 98 or 100, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:93.

[0071] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:88, 90, 92, 94, 96, 98 or 100, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:95.

[0072] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:88, 90, 92, 94, 96, 98 or 100, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:97.

[0073] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:88, 90, 92, 94, 96, 98 or 100, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:99.

[0074] The amino acid sequence of the heavy chain variable region of the ROR1 chimeric antibody is shown in SEQ ID NO:88, 90, 92, 94, 96, 98 or 100, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:101.

[0075] The ROR1 chimeric antibody of this invention has a light chain constant region of the κ subtype and a heavy chain constant region of IgG1. In some specific embodiments, the constant region is derived from the PTT5 vector.

[0076] In some embodiments, the present invention provides a humanized antibody against ROR1:

[0077] At least one of its heavy chains FR1-4 has an amino acid sequence as shown in any one of SEQ ID NO:42, 83-87; or has a sequence based on the amino acid sequence shown in any one of SEQ ID NO:42, 83-87 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 the amino acid sequence shown in any one of SEQ ID NO:42, 83-87.

[0078] At least one of its light chains FR1 to 4 has an amino acid sequence as shown in any one of SEQ ID NO:45, 47 to 48, 51; or has a sequence based on the amino acid sequence shown in any one of SEQ ID NO:45, 47 to 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 the amino acid sequence shown in any one of SEQ ID NO:45, 47 to 48, 51.

[0079] In some specific embodiments, the humanized antibody of ROR1 is:

[0080] Its heavy chain FR1 has the amino acid sequence shown in SEQ ID NO:83;

[0081] Its heavy chain FR2 has the amino acid sequence shown in SEQ ID NO:42;

[0082] Its heavy chain FR3 has an amino acid sequence as shown in SEQ ID NO:84 or 86;

[0083] Its heavy chain FR4 has an amino acid sequence as shown in SEQ ID NO:85 or 87;

[0084] Its light chain FR1 has the amino acid sequence shown in SEQ ID NO:45;

[0085] Its light chain FR2 has the amino acid sequence shown in SEQ ID NO:51;

[0086] Its light chain FR3 has the amino acid sequence shown in SEQ ID NO:47;

[0087] Its light chain FR4 has the amino acid sequence shown in SEQ ID NO:48;

[0088] Alternatively, its FR region has a sequence based on the amino acid sequence shown above, with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence shown above.

[0089] Specifically, the ROR1 humanized antibody,

[0090] Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:102 or 103;

[0091] Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:104 to 107.

[0092] For example,

[0093] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:102, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:104.

[0094] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:102, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:105.

[0095] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:102, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:106.

[0096] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:102, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:107.

[0097] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:103, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:104.

[0098] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:103, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:105.

[0099] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:103, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:106.

[0100] The amino acid sequence of the antibody heavy chain variable region is shown in SEQ ID NO:103, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:107.

[0101] In some specific embodiments, the ROR1 humanized antibody has a light chain constant region of the κ subtype and a heavy chain constant region of IgG1. In some specific embodiments, the constant region is derived from the PTT5 vector.

[0102] Furthermore, the present invention also provides the following biomaterials:

[0103] A) Nucleic acid encoding the ROR1 antibody as described above.

[0104] 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.

[0105] B) A plasmid vector containing the nucleic acid described in A);

[0106] In this invention, the backbone vector of the plasmid vector is a mammalian cell expression vector, for example, the backbone vector is pCDNA5.

[0107] C) The host whose genome integrates the nucleic acid described in A), or is transformed or transfected with the plasmid vector described in B).

[0108] In this invention, the host is a mammalian cell, such as human embryonic kidney HEK293 cells or Chinese hamster ovary cells CHOK1.

[0109] Furthermore, the present invention also provides a method for preparing the ROR1 antibody as described above, which includes: culturing the host as described above to obtain a culture containing the antibody or antigen-binding molecule.

[0110] In some embodiments, the preparation method further includes steps of enriching, extracting, and / or purifying the culture.

[0111] Furthermore, the present invention also provides a labeled antibody obtained by labeling the ROR1 antibody, wherein the labeling is a chemical label or a biological label.

[0112] In this invention, the chemical label is a fluorescent indicator, a chemiluminescent indicator, an isotope and / or a colloidal indicator; the biolabel is a biotin, avidin or an enzyme label.

[0113] In some embodiments,

[0114] 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.

[0115] 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.

[0116] 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;

[0117] The isotopes are selected from one or more of 125I, 131I, 124I, 3H, 14C, 111In, 89Zr or 32P;

[0118] Colloidal indicators are selected from one or more of colloidal gold, colloidal carbon, or colloidal selenium.

[0119] Furthermore, the present invention also provides a conjugate obtained by binding the ROR1 antibody to a medium.

[0120] In some embodiments, the medium is selected from colloidal gold, ELISA plates, magnetic beads, or latex microspheres.

[0121] In this invention, the antibody and the medium can be linked by an amide bond or by a streptavidin-biotin system; this invention does not limit the specific link.

[0122] Furthermore, the present invention also provides antibody-drug conjugates comprising the ROR1 antibody and the drug as described above.

[0123] In some embodiments, the drug is an antitumor drug selected from at least one of MMAE, MMAF, or DXD.

[0124] In this invention, the drug and antibody are directly linked by an amide bond in the antibody-drug conjugate, or they can be linked by a linker; this invention does not limit the specific linker.

[0125] In this invention, the molar ratio of drug to antibody in the antibody-drug 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.

[0126] Furthermore, the present invention provides the use of any one of the following I) to V) in the preparation of a medicament for the prevention and treatment of ROR1-related diseases:

[0127] I) The ROR1 antibody as described above;

[0128] II) Biomaterials as described above;

[0129] III) Cultures prepared by the methods described above or antibodies obtained through purification;

[0130] IV) Labeled antibodies as described above;

[0131] V), as described above, is a coupling agent.

[0132] In this invention, the disease associated with ROR1 is a tumor.

[0133] In some embodiments, the tumor is lymphoma, lung cancer, colon cancer, gastric cancer, lung adenocarcinoma, breast cancer, pancreatic cancer, ovarian cancer, multiple myeloma, or mantle cell lymphoma.

[0134] The present invention also provides a drug comprising any one of the following I) to V);

[0135] I) The ROR1 antibody as described above;

[0136] II) Biomaterials as described above;

[0137] III) Cultures prepared by the methods described above or antibodies obtained through purification;

[0138] IV) Labeled antibodies as described above;

[0139] V), as described above, is a coupling agent.

[0140] In this invention, the drug also includes pharmaceutically acceptable excipients.

[0141] 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.

[0142] Furthermore, the present invention also provides a pharmaceutical composition comprising the aforementioned drug and other antitumor drugs.

[0143] In some embodiments, the antitumor drugs include, but are not limited to, at least one of: cisplatin, carboplatin, oxaliplatin, paclitaxel, vinorelbine, gefitinib, fluorouracil, doxorubicin, erlotinib, rituximab, trastuzumab, pertuzumab, nivolumab, pembrolizumab, atezolizumab, olaparib, niraparib, etoposide, tamoxifen, toremifene, fulvestrant, or letrozole.

[0144] 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.

[0145] The present invention also provides a method for treating ROR1-related diseases, comprising administering the drug or pharmaceutical composition described herein.

[0146] 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.

[0147] Furthermore, the present invention also provides the use of any one of the following i) to vi) in the preparation of ROR1 detection reagents, tumor diagnostic reagents, and / or tumor imaging reagents:

[0148] i) The ROR1 antibody as described above;

[0149] ii) Biomaterials as described above;

[0150] iii) Cultures prepared by the methods described above or antibodies obtained through purification;

[0151] iv) Labeled antibodies as described above;

[0152] v) The combination as described above.

[0153] vi) As described above, couplings.

[0154] In this invention, the tumor is a tumor expressing ROR1.

[0155] The present invention also provides a reagent comprising any one of the following i) to vii):

[0156] i) The ROR1 antibody as described above;

[0157] ii) Biomaterials as described above;

[0158] iii) Cultures prepared by the methods described above or antibodies obtained through purification;

[0159] iv) Labeled antibodies as described above;

[0160] v) The combination as described above.

[0161] vi) As described above, couplings.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] The ROR1 antibody provided by this invention can bind to the antigen with high specificity and has rapid and efficient internalization ability. Furthermore, the anti-ROR1 antibody provided by this invention has high selectivity and high biological activity, and can be used not only as an anti-tumor drug or antibody-drug conjugate, but also as a diagnostic tool to detect ROR1 expression in ROR1-related diseases. Attached Figure Description

[0166] Figure 1 This indicates the binding of antibodies to overexpressing cell lines;

[0167] 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.

[0168] Figure 3 The results of FACS assays show the biological binding of the anti-human ROR1 chimeric antibody to the naturally expressed cell line.

[0169] Figure 4 The study demonstrated the endocytic activity of the anti-human ROR1 chimeric antibody against naturally expressed cell lines A549 and HT29.

[0170] Figure 5 In vitro cell biology and FACS assays show the effects of humanized anti-human ROR1 antibodies on six types of tumor cells.

[0171] Figure 6 The results of the nonspecific binding assay of the anti-human ROR1 humanized antibody are shown.

[0172] 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.

[0173] Figure 8 This demonstrates the in vitro killing effect of humanized anti-ROR1 antibody on overexpressing cells;

[0174] Figure 9 The drug metabolism of the humanized ROR1 antibody in mice was demonstrated.

[0175] Figure 10 The in vitro killing effect of humanized anti-ROR1 antibody on overexpressing cells after conjugation with a small molecule drug was demonstrated. Detailed Implementation

[0176] This invention provides antibodies against ROR1, their preparation methods, and applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. 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. Those skilled in the art can clearly 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.

[0177] 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.

[0178] 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.

[0179] 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…”.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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 the regions in the antibody heavy and light chains other than the CDRs. Although FRs are not directly involved in antigen binding, they significantly influence the conformation of the CDRs and the specificity of antigen binding.

[0185] In this application, "antibody" also includes antibodies that do not contain a light chain or antibodies that do not contain a constant region.

[0186] 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).

[0187] 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.

[0188] 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.

[0189] In this application, "Kabat numbering system" generally refers to the immunoglobulin alignment 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).

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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′.

[0195] 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 2 101 823B1).

[0196] 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".

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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 age, size, weight, and physical condition of the patient being prevented or treated, 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.

[0202] 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.

[0203] 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.

[0204] 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:

[0205] I. The amino acid sequences of the light and heavy chains CDR, variable region, and constant region of murine / chimeric / human antibody molecules, along with their corresponding nucleotide sequences, and optimized CDR amino acid mutation points, are as follows:

[0206] The variable region sequence of the mouse monoclonal antibody is shown below.

[0207] 1. Murine chimeric antibody

[0208] chT7 heavy chain variable region amino acid sequence

[0209] EVMLVESGGGFVRPGGSLTLSCAASGFTFR SYAMS WVRQTPEKRLEWVA TIRYGV DNTYYPDNMKG RFTISRDTAKNTLYLQMSSLRSEDTALYYCTR HSPLLLRSHWYFDV W GTGTTVTVSS(SEQ ID NO:88)

[0210] 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.

[0211] chT7 light chain variable region amino acid sequence

[0212] DIKMTQSPSSMYASLGERVTITC KASQDINNYLS WFQQKPGKSPKTLIC RANRLVD GVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC LQYDEFPWT FGGGTKLEIK(SEQ ID NO:89)

[0213] chT9 heavy chain variable region amino acid sequence

[0214] EVMLVESGGGFVRPGGSLTLSCAASRFTFR NSTMS WVRQTPEKRLEWVATIRGNN SNTFYPDSVKG RFTISRDTAKNTLYLQMSSLRSEDTALYYCAR HSPLLLRSHWYFDV WG TGTTVTVSS(SEQ ID NO:90)

[0215] chT9 light chain variable region amino acid sequence

[0216] DIKMTQSPSSMYASLGERVTITC KASQDINNYLN WFQQKPGKSPKTLIY RANRLVD GVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC LQYDEFPWT FGGGTKLEIK(SEQ ID NO:91)

[0217] chT11 heavy chain variable region amino acid sequence

[0218] QVQLQQPGAELVKPGASVKLSCKASGYTFT SYWMH WVKQRPGQGLEWIG MIHPN SGSSNFNEKFNI KATLTVDKSSSTAYMQLTSLTSEDSAVYYCAR YDYGSQTFDY WGQG TTLTVSS(SEQ ID NO:92)

[0219] chT11 light chain variable region amino acid sequence

[0220] ETTVTQSPASLSVATGEKVTISC KTSSDIDEHMN WYQQKPGEPPKLLIS EGNTVRP G VPSRFSSSGYGTDFVFTIENTLSEDVADYYC LQSDNMPLT FGAGTKLDLK(SEQ ID NO:93)

[0221] chT16 heavy chain variable region amino acid sequence

[0222] EVQLVESGGDLVKPGGSLKLSCAASGFTFS SYGMS WVRQTPDKRLEWVA TIPSRGS STYYPDSVKG RFTISRDNARNTLYLQMSSLKSEDTAMYYCTR HLYYYGSSDYAMDY W GQGTSVTVSS(SEQ ID NO:94)

[0223] chT16 light chain variable region amino acid sequence

[0224] DIKMTQSPSSMYASLGERVTITC KASQDLNSYLSWFQQKPGKSPKTLIY RANRLED GVPSRFSGSGSEQDYSLTISSLEYEDMGIYYC LQYDEFPYT FGGGTKLEIK(SEQ ID NO:95)

[0225] chT24 heavy chain variable region amino acid sequence

[0226] EVQLQQSGPELVQPGASVKMSCKASGYTFT DYNIH WVKQSHGKRLEWIG YIKPTS GDINYNQNFQG KATLTVDKSSSTAYMELRSLTSGESAVYYCAR RGYGNSYAMDY WGQ GTSVTVSS (SEQ ID NO:96)

[0227] chT24 light chain variable region amino acid sequence

[0228] DVLMTQTPLSLTVSLGDQASISC RSSRTIVHSNGNTYLE WYLQKPGQSPKLLIY KVS NRFS GVPDRVSGSGSGTDFTLKISRVEAEDMGVYFC FQGSLFPYT FGGGTKLEIR (SEQ ID NO:97)

[0229] chT26 heavy chain variable region amino acid sequence

[0230] QVQLQQSGAELVRPGASVTLSKASGYTFT DYEMH WVKQTPVHGLEWIG AIDPET GGTDYNQKFKG KAILTADKSSSTAYMELRRSLTSEDSAVYYCTR DYGIFDY WGQGTTLT VSS (SEQ ID NO:98)

[0231] chT26 light chain variable region amino acid sequence

[0232] DIVMTQSQKFMSTSVGDRVSVTC KASQNVGTNVA WYQQKPGQSPKALIY SASYRY S GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQYNSYPLFT FGSGTKLEIK(SEQ ID NO:99)

[0233] chT29 heavy chain variable region amino acid sequence

[0234] QVQLQQSGAELARPGASVKLSCKASGYNFT SYGIT WVKQRTGQGLEWIG EIYPRSG NTYYNEKFKG KATLTADKSSNTAYMELRRSLTSEDSAVYFCAR EGQHFDY WGQGSTLTV SS(SEQ ID NO:100)

[0235] chT29 light chain variable region amino acid sequence

[0236] NIVMTQSPKSMSMSVGERVTLNC KASENVDSYVS WYQQKPEQSPKLLIY GTSNRY T GVPDRFTGSGSATDFTLTISGVQAEDLADYHC GQIYNYPFT FGSGTKLEIK(SEQ ID NO:101)

[0237] 2. List of light and heavy chain nucleotide sequences for each chimeric antibody:

[0238]

[0239]

[0240]

[0241] 3. Humanized antibodies

[0242] T16 humanized molecular heavy chain variable region amino acid sequence

[0243] >T16-H0 heavy chain variable region amino acid sequence

[0244] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLEWVA TIPSRG SSTYYPDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR HLYYYGSSDYAMDY W GQGTTVTVSS(SEQ ID NO:102)

[0245] >T16-H1 heavy chain variable region amino acid sequence

[0246] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYGMS WVRQAPGKGLEWVA TIPSRG SSTYYPDTVKG RFTISRDNAKSSLYLQMSSLRAEDTAVYYCTR HLYYYGSSDYAMDYW GQGTTVTVSS(SEQ ID NO:103)

[0247] T16 humanized molecule light chain variable region amino acid sequence

[0248] >T16-L1 light chain variable region amino acid sequence

[0249] DIQMTQSPSSLSASVGDRVTITC KASQDLNSYLS WFQQKPGKAPKSLIY RANRLED GVPSRFSGSGSETDYTLTISSLQPEDFATYYC LQYDEFPYT FGGGTKVEIK(SEQ ID NO:104)

[0250] >T16-L4 light chain variable region amino acid sequence

[0251] DIQMTQSPSSLSASVGDRVTITC KASQDLNSYLS WFQQKPGKAPKSLIY RANRLEE GVPSRFSGSGSETDYTLTISSLQPEDFATYYC LQYDEFPYT FGGGTKVEIK (SEQ ID NO:105)

[0252] >T16-L5 light chain variable region amino acid sequence

[0253] DIQMTQSPSSLSASVGDRVTITC KASQDLNYYLS WFQQKPGKAPKSLIY RANRLEE GVPSRFSGSGSETDYTLTISSLQPEDFATYYC LQYDEFPYT FGGGTKVEIK(SEQ ID NO:106)

[0254] >T16-L6 light chain variable region amino acid sequence

[0255] DIQMTQSPSSLSASVGDRVTITC KASQDLNAYLS WFQQKPGKAPKSLIY RANRLEE GVPSRFSGSGSETDYTLTISSLQPEDFATYYC LQYDEFPYT FGGGTKVEIK(SEQ ID NO:107)

[0256] 4. CDR regions and sequence numbers of each antibody

[0257]

[0258]

[0259] 5. FR region and sequence number of chimeric antibody

[0260]

[0261]

[0262] 6. FR region and sequence number of humanized antibody

[0263]

[0264]

[0265] II. Sequence Synthesis and Vector Construction of Tool Antibodies

[0266] 1. The naked antibody molecule UC961 corresponding to the ADC drug Zilovertamab vedotin developed by VelosBio (acquired by MSD).

[0267] UC961 heavy chain variable region amino acid sequence:

[0268] LVTVSS(SEQ ID NO:122)

[0269] UC961 light chain variable region amino acid sequence:

[0270] DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGI PPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK(SEQ ID NO:123)

[0271] 2. The naked antibody XBR1-402 corresponding to NBE Therapeutics' ADC drug NBE-002

[0272] XBR1-402 heavy chain variable region amino acid sequence:

[0273] QEQQKESGGGLFKPTDTLTLTCTASGFDISSYYMSWVRQAPGNGLEWIGAIGISGN AYYASWAKSRSTITRNTNLNTVTLKMTSLTAADTATYFCARDHPTYGMDLWGPGTLV TVSS(SEQ ID NO:124)

[0274] XBR1-402 light chain variable region amino acid sequence (light chain is lambda chain):

[0275] SYELTQLPSVSVSLGQTARITCEGNNIGSKAVHWYQQKPGLAPGLLIYDDDERPSG VPDRFSGSNSGDTATLTISGAQAGDEADYYCQVWDSSAYVFGGGTQLTVTG(SEQ ID NO:125)

[0276] 3. The naked anti-C2E3 heavy chain variable region amino acid sequence of LCB71, an ADC drug jointly developed by LegoChem Biosciences and ABL Bio:

[0277] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISHNS GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFISARKSLGRSYSNG MDVWGQGTLVTVSS(SEQ ID NO:126)

[0278] C2E3 light chain variable region amino acid sequence (light chain is lambda chain):

[0279] QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNDVTWYQQLPGTAPKLLIYADSKRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDYSLSGYVFGGGTKLTVLG(SEQ ID NO:127)

[0280] 4. Maiwei Biotechnology's self-produced Isotype control (SH-NC)

[0281] SH-NC heavy chain variable region amino acid sequence:

[0282] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPIFG SSNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAESPLGGGSGYSVSWFDPWG QGTLVTVSS(SEQ ID NO:128)

[0283] SH-NC light chain variable region amino acid sequence:

[0284] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATG IPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYSNWPPWTFGQGTKVEIK(SEQ ID NO:129)

[0285] 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:

[0286] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:130)

[0287] The nucleotide sequence of the IgG1 heavy chain constant region G1m3 of the PTT5 vector used in the protein expression system:

[0288] gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa(SEQ IDNO:131)

[0289] 6. The Km3 amino acid sequence of the IgG1 light chain constant region of the PTT5 vector used in the protein expression system:

[0290] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:132)

[0291] 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:

[0292] Cgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatccccgcgaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggag agtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaccgcggagagtgt(SEQ ID NO:133)

[0293] 7. Amino acid sequence of the lambda constant region of the IgG1 light chain in the PTT5 vector used in the protein expression system:

[0294] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:134)

[0295] The nucleotide sequence of the IgG1 light chain lambda constant region of the PTT5 vector used in the protein expression system is as follows:

[0296] Ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtgga gaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca(SEQ ID NO:135)

[0297] III. Purchasing antigens and verifying antigen binding activity

[0298] 1. ROR1-related recombinant protein antigen information

[0299] Purchase the following commercial reagents:

[0300] Name Manufacturer 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

[0301] 2. Construction of cell lines with different species antigens:

[0302] The amino acid sequence of human ROR1 protein (hROR1 for short) is as follows:

[0303] >ROR1_HUMAN Q01973

[0304] MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL(SEQ ID NO:136)

[0305] The nucleotide sequence of human ROR1 protein (abbreviated as hROR1) is as follows:

[0306] atgcaccggccgcgccgccgcgggacgcgcccgccgctcctggcgctgctggccgcgctgctgctggccgcacgcggggct

[0307] gctgcccaagaaacagagctgtcagtcagtgctgaattagtgcctacctcatcatggaacatctcaagtgaactcaacaaagattcttacctg

[0308] accctcgatgaaccaatgaataacatcaccacgtctctgggccagacagcagaactgcactgcaaagtctctgggaatccacctcccacca

[0309] tccgctggttcaaaaatgatgctcctgtggtccaggagccccggaggctctcctttcggtccaccatctatggctctcggctgcggattagaa

[0310] acctcgacaccacagacacaggctacttccagtgcgtggcaacaaacggcaaggaggtggtttcttccactggagtcttgtttgtcaagtttg

[0311] gcccccctcccactgcaagtccaggatactcagatgagtatgaagaagatggattctgtcagccatacagagggattgcatgtgcaagattt

[0312] attggcaaccgcaccgtctatatggagtctttgcacatgcaaggggaaatagaaaatcagatcacagctgccttcactatgattggcacttcc

[0313] agtcacttatctgataagtgttctcagttcgccattccttccctgtgccactatgccttcccgtactgcgatgaaacttcatccgtcccaaagccc

[0314] cgtgacttgtgtcgcgatgaatgtgaaatcctggagaatgtcctgtgtcaaacagagtacatttttgcaagatcaaatcccatgattctgatgag

[0315] gctgaaactgccaaactgtgaagatctcccccagccagagagcccagaagctgcgaactgtatccggattggaattcccatggcagatcct

[0316] ataaataaaaaatcacaaggtgttataacagcacaggtgtggactaccgggggaccgtcagtgtgaccaaatcagggcgccagtgccagcca

[0317] tggaattcccagtatccccacacacacactttcaccgcccttcgtttcccagagctgaatggaggccattcctactgccgcaacccagggaat

[0318] caaaaggaagctccctggtgcttcaccttggatgaaaactttaagtctgatctgtgtgacatcccagcgtgcgattcaaaggattccaaggag

[0319] aagaataaaatggaaatcctgtacatactagtgccaagtgtggccattcccctggccattgctttactcttcttcttcatttgcgtctgtcggaata

[0320] accagaagtcatcgtcggcaccagtccagaggcaaccaaaacacgtcagaggtcaaaatgtagagatgtcaatgctgaatgcatataaac

[0321] ccaagagcaaggctaaagagctacctctttctgctgtacgctttatggaagaattgggtgagtgtgcctttggaaaaatctataaaaggccatct

[0322] ctatctcccaggcatggaccatgctcagctggttgctatcaagaccttgaaagactataacaacccccagcaatggacggaatttcaacaag

[0323] aagcctccctaatggcagaactgcaccaccccaatattgtctgccttctaggtgccgtcactcaggaacaacctgtgtgcatgctttttgagtat

[0324] attaatcagggggatctccatgagttcctcatcatgagatccccacactctgatgttggctgcagcagtgatgaagatgggactgtgaaatcc

[0325] agcctggaccacggagattttctgcacattgcaattcagattgcagctggcatggaatacctgtctagtcacttctttgtccacaaggaccttgc

[0326] agctcgcaatattttaatcggagagcaacttcatgtaaagatttcagacttggggctttccagagaaatttactccgctgattactacagggtcc

[0327] agagtaagtccttgctgcccattcgctggatgccccctgaagccatcatgtatggcaaattctcttctgattcagatatctggtcctttggggttg

[0328] tcttgtgggagattttcagttttggactccagccatattatggattcagtaaccaggaagtgattgagatggtgagaaaacggcagctcttacca

[0329] tgctctgaagactgcccacccagaatgtacagcctcatgacagagtgctggaatgagattccttctaggagaccaagatttaaagatattcac

[0330] gtccggcttcggtcctgggagggactctcaagtcacacaagctctactactccttcagggggaaatgccaccacacagacaacctccctca

[0331] gtgccagcccagtgagtaatctcagtaaccccagatatcctaattacatgttcccgagccagggtattacaccacagggccagattgctggtt

[0332] tcattggcccgccaatacctcagaaccagcgattcattcccatcaatggatacccaatacctcctggatatgcagcgtttccagctgcccacta

[0333] ccagccaacaggtcctcccagagtgattcagcactgcccacctcccaagagtcggtccccaagcagtgccagtgggtcgactagcactgg

[0334] ccatgtgactagcttgccctcatcaggatccaatcaggaagcaaatattcctttactaccacacatgtcaattccaaatcatcctggtggaatgg

[0335] gtatcaccgtttttggcaacaaatctcaaaaaccctacaaaattgactcaaagcaagcatctttactaggagacgccaatattcatggacacac

[0336] cgaatctatgatttctgcagaactgtaa(SEQ ID NO:137)

[0337] The amino acid sequence of cynomolgus monkey ROR1 protein (abbreviated as cynoROR1) is as follows:

[0338] >ROR1_Cynomolgus monkey A0A2K5WTX7

[0339] MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSPPVQRQPKHVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYMNQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYIFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDAKQASLLGDANIHGHTESMISAEL(SEQ ID NO:138)

[0340] The nucleotide sequence of cynomolgus monkey ROR1 protein (abbreviated as cynoROR1) is as follows:

[0341] atgcaccggccgcgccgccgcgggacgcgcccgccgctcctggcgctgctggccgcgcttctgctggccgcacgcggggctg

[0342] ctgcccaagaaacagagctgtcagtcagtgctgaattagtgcctacctcatcatggaacatttcaagtgaactcaacaaagattcttacctgac

[0343] ccttgatgaaccaatgaataacatcaccacatccctgggccagacagcagaactgcactgcaaagtctctgggaatccacctcccaccatc

[0344] cgctggttcaaaaatgatgctcctgtggtccaggagccccggaggctctcctttcgatccaccatctatggctctcggctgcggattagaaac

[0345] ctcgacaccacagacacgggctacttccagtgcgtggcaacaaacggcaaggaggtggtttcttccactggagtcttgtttgtcaagtttggc

[0346] ccccctcccactgcaagtccaggatactcagatgagtatgaagaagatggattctgtcagccatacagagggattgcatgtgcaagatttatt

[0347] ggcaaccgcaccgtctacatggagtctttgcacatgcaaggggaaatagaaaatcagatcacagctgccttcactatgattggcacttccagt

[0348] cacttatctgataagtgttctcagttcgccattccttccctgtgccactatgccttcccgtactgtgatgaaacttcatccgtcccaaagccccgtg

[0349] acttgtgtcgcgatgaatgtgaaatcctggaaaatgtcctgtgtcaaacagagtacatttttgcaagatcaaatcccatgattctgatgaggctg

[0350] aaactgccaaactgtgaagatctcccccagccagagagcccagaagctgcaaactgtatccggattggaattcccatggcagaccctataa

[0351] ataaaaatcacaagtgttataacagcacaggtgtggactaccggggaaccgtcagcgtgaccaaatcagggcgccagtgccagccgtgg

[0352] aattcccagtatccacacacacacaccttcaccgcacttcgtttcccagagctgaatggaggccattcctactgccgcaacccagggaatca

[0353] gaaggaagctccctggtgcttcaccttggatgaaaactttaagtctgatctgtgtgacatcccagcttgcgattcaaaggattccaaggagaa

[0354] gaataaaatggaaatcttgtacatactcgtgccaagtgttgccattcccctggccattgctttactcttcttcttcatttgtgtctgtcgcaataacc

[0355] agaagtcatcatcaccaccagtccagaggcaaccaaaacacgtcagaggtcaaaatgtagagatgtcaatgctgaatgcatataaacccaa

[0356] gagcaaggctaaagagctgcctctttctgctgtacgctttatggaagaattgggtgaatgtgcctttggaaaaatctataaaggccatctctatc

[0357] tcccaggcatggaccatgctcagctggttgctatcaagaccttgaaagattataacaatccccagcaatggacagaatttcaacaggaagcc

[0358] tccctaatggcagaactgcaccaccccaatattgtctgcctgctaggtgccgtcactcaggaacaacctgtgtgcatgctttttgagtatatgaa

[0359] tcagggggatctccatgagttcctcatcatgcgatccccacactctgatgttggctgcagtagtgatgaagatgggactgtgaaatccagcct

[0360] ggaccatggagattttctgcacattgcaattcagattgcagccggcatggaatacctgtctagtcacttctttgtccacaaggatcttgcagctc

[0361] gcaatattttaatcggagagcaacttcatgtaaaaatttcagacttggggctttccagagaaatttactccgctgattactacagggtccagagt

[0362] aagtccttgctgcccattcgctggatgccgcctgaagccatcatgtatggcaaattctcttccgattcagatatctggtcctttggggttgtcctg

[0363] tgggagattttcagttttggactccagccatattatggattcagtaaccaggaagtgattgagatggtgagaaaacggcagctcttaccatgct

[0364] ctgaagactgcccacccagaatgtacagcctcatgacagagtgctggaatgagattccttccaggagaccaagatttaaagatattcacgtc

[0365] cggcttcggtcctgggagggactgtcaagtcacaccagctctactactccttcggggggaaatgccaccacacagacaacctccctcagtg

[0366] ccagcccagtgagtaatctcagtaaccccagatatcctaattacatattcccgagccagggtattacaccacagggccagattgctggtttcat

[0367] tggcccgccaatacctcagaaccagcgattcattcccatcaatggatacccaatacctcctggatatgcagcgtttccagctgcccactacca

[0368] gccaacaggtcctcccagagtgattcagcactgcccgcctcccaagagtcggtccccaagcagtgccagtgggtcgactagcactggcca

[0369] tgtgactagcttgccctcatcaggatccaatcaggaagcaaatattcctttactaccacacatgtcaattccaaatcatcctggtggaatgggta

[0370] tcaccgtttttggcaacaaatctcaaaaaccgtacaaaattgatgcaaagcaagcatctttgctaggagatgccaatattcatggacacaccga

[0371] atctatgatttctgcagaactgtaa(SEQ ID NO:139)

[0372] The amino acid sequence of mouse ROR1 protein (abbreviated as mROR1) is as follows:

[0373] >ROR1_MOUSE Q9Z139

[0374] MHRPRRRGTRPPPLALLAALLLAARGADAQETELSVSAELVPTSSWNTSSEIDKGSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPSIRWFKNDAPVVQEPRRISFRATNYGSRLRIRNLDTTDTGYFQCVATNGKKVVSTTGVLFVKFGPPPTASPGSSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEVLENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHSFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIAFLFFFICVCRNNQKSSSPPVQRQPKPVRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECTFGKIYKGHLYLPGMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYMNQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSSLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPRFPNYMFPSQGITPQGQIAGFIGPAIPQNQRFIPINGYPIPPGYAAFPAAHYQPAGPPRVIQHCPPPKSRSPSSASGSTSTGHVASLPSSGSNQEANVPLLPHMSIPNHPGGMGITVFGNKSQKPYKIDSKQSSLLGDSHIHGHTESMISAEV(SEQ ID NO:140)

[0375] The nucleotide sequence of mouse ROR1 protein (abbreviated as mROR1) is as follows:

[0376] atgcaccggccgcgccgccgcgggacgcgcccgccaccgctggcgctgctggccgcgctgctgctggccgcacgcggggct

[0377] gatgcccaagaaacagagttgtcagtcagtgctgagctggtgcctacctcgtcctggaacacttcaagtgaaatcgacaaaggttcttactta

[0378] acccttgatgagccgatgaataacatcacaacgtccctggggcagactgcagaactgcactgcaaagtgtctgggaatccacctcccagta

[0379] tccgctggttcaagaatgatgcacctgtggtccaagaacctcggagaatctccttccgggcaaccaactatggctctcggctgcggattaga

[0380] aaccttgacaccacagacactggttacttccagtgtgtggcaacaaatggcaagaaagtggtgtctaccactggtgtcctgtttgtcaaatttg

[0381] ggcctcctccgaccgcaagcccaggatcctcagatgagtatgaagaagatggattctgtcagccgtaccgaggcattgcatgtgcacgattt

[0382] attggcaaccgcactgtgtatatggagtctttgcatatgcagggggaaatagaaaatcagatcacagctgccttcaccatgattggcacctcc

[0383] agccatttatctgataagtgctctcagttcgccatcccttccttgtgccactacgccttcccgtactgtgacgaaacctcatctgtcccaaagccc

[0384] cgtgacttgtgtcgtgatgaatgtgaagtgctggagaatgtcctgtgtcagacagagtcatttttgccagatcaaatcccatgattttgatgag

[0385] gctgaagttaccaaactgtgaggatctcccccagccagagagcccggaagctgcaaactgcatacggattggcattcccatggcggatcct

[0386] ataaataaaaaatcacaaatgctacaatagcacgggtgtagactaccggggaaccgtcagtgtgaccaagtctggacgccagtgccagccat

[0387] ggaattctcagtacccacacacacacagcttcactgctctgcgctttccggagctcaacggaggccactcctactgccgcaaccctggcaa

[0388] ccagaaggaagctccctggtgctttaccttggatgaaaactttaagtctgacctgtgtgacatcccagcatgtgattccaaagattccaaagag

[0389] aagaataaaatggaaatcttgtacattctggtgccaagtgtggccattcccctggctatcgccttcctcttcttcatctgtgtgtgccgcaata

[0390] accagaagtcttcatcaccaccagtccagaggcagccaaaacccgtcagaggacagaatgtggagatgtccatgctcaatgcatacaagc

[0391] ccaagagcaaggctaaagagctgcctctttccgctgtgcgtttcatggaagaattgggtgaatgtacctttggaaaaatctataagggccatct

[0392] ctacctcccaggcatggaccatgctcagctggtggctatcaaaaccttgaaagactataacaacccccagcagtggacagaatttcaacag

[0393] gaagcctctctcatggctgaactacaccaccccaatattgtatgcctcctcggagccgtcacccaggaacaacctgtgtgtatgctctttgagt

[0394] atatgaaccagggagacctccacgagttcctcatcatgcgatccccgcattccgatgtcggctgtagcagtgatgaagatgggacggtcaa

[0395] atccagcctggaccatggtgatttcttacacatagcaattcagatcgcagctggcatggagtacctgtctagtcacttcttcgtgcacaaggac

[0396] cttgcagctcgcaacattttaattggagagcaactgcatgtaaaaatttcagatcttgggctttccagagaaatttactctgctgattactatagg

[0397] gtacagagtaagtcttcactgcccatccgctggatgccccctgaggccatcatgtacggcaaattctcctccgattccgatatctggtctttcg

[0398] gggttgtgttgtgggagattttcagctttggactccagccatattatgggtttagtaatcaggaagtgattgaaatggtgcggaagcggcagct

[0399] cttaccatgttctgaagactgcccgccgcgcatgtacagcctcatgaccgagtgctggaatgagataccttccaggagaccacgctttaaag

[0400] acatccacgtccggcttcgatcctgggagggcctctcaagtcacaccagctctaccaccccctcgggtggaaatgccaccacgcagacca

[0401] cttccctcagtgccagccctgtgagtaacctcagcaacccccgatttcccaattacatgttcccgagccaagggattacaccccagggtcag

[0402] atcgctggtttcattggcccagcgatacctcagaaccagcgcttcatccccatcaatggatacccaatacctcctggctatgcagcctttccag

[0403] ctgcccactaccagcctgcagggcctcccagggtgattcagcactgcccacctccgaagagtcggtccccaagcagcgccagcggatcg

[0404] accagcactggccatgtggccagcttgccctcatcaggatccaatcaggaagcaaacgttcctttgctaccccacatgtcaattccaaatcac

[0405] cctggtggaatgggtatcactgtttttggcaacaaatctcaaaaaccgtacaaaatagactcaaaacaatcgtctttgcttggggactcccatat

[0406] ccatgggcacaccgaatctatgatttctgcagaagtgtaa(SEQ ID NO:141)

[0407] Construction of cell lines of proteins from the same family

[0408] The amino acid sequence of human ROR2 protein (abbreviated as hROR2) is as follows:

[0409] MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLDGQDGPIPTL

[0410] KGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLKNDAPVVQEPRRIIIRKTEYGS

[0411] RLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHSPNHNFQDDYHEDGFCQPY

[0412] RGIACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLC

[0413] DARSRTPKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAAN

[0414] CMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPEL

[0415] GGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCSPRDSSKMGILYILVPSIAIPLVI

[0416] ACLFFLVCMCRNKQKASASTPQRRQLMASPSQDMEMPLINQHKQAKLKEISLSAVRFME

[0417] ELGEDRFGKVYKGHLFGPAPGEQTQAVAIKTLKDKAEGPLREEFRHEAMLRARLQHPNV

[0418] VCLLGVVTKDQPLSMIFSYCSHGDLHEFLVMRSPHSDVGSTDDDRTVKSALEPPDFVHL

[0419] VAQIAAGMEYLSSHHVVHKDLATRNVLVYDKLNVKISDLGLFREVYAADYYKLLGNSL

[0420] LPIRWMAPEAIMYGKFSIDSDIWSYGVVLWEVFSYGLQPYCGYSNQDVVEMIRNRQVLP

[0421] CPDDCPAWVYALMIECWNEFPSRRPRFKDIHSRLRAWGNLSNYNSSAQTSGASNTTQTS

[0422] SLSTSPVSNVSNARYVGPKQKAPPFPQPQFIPMKGQIRPMVPPPQLYVPVNGYQPVPAYG

[0423] AYLPNFYPVQIPMQMAPQQVPPQMVPKPSSHHSGSGSTSTGYVTTAPSNTSMADRAALL

[0424] SEGADDTQNAPEDGAQSTVQEAEEEEEGSVPETELLGDCDTLQVDEAQVQLEA(SEQ ID NO:142)

[0425] The nucleotide sequence of human ROR2 protein is as follows:

[0426] [[ID=1,4]]atggcccggggctcggcgctcccgcggcggccgctgctgtgcatcccggccgtctgggcggccgccgcgcttctgctctcagtg

[0427] tcccggacttcaggtgaagtggaggttctggatccgaacgaccctttaggaccccttgatgggcaggacggcccgattccaactctgaaag

[0428] gttactttctgaattttctggagccagtaaacaatatcaccattgtccaaggccagacggcaattctgcactgcaaggtggcaggaaacccac

[0429] cccctaacgtgcggtggctaaagaatgatgccccggtggtgcaggagccgcggcggatcatcatccggaagacagaatatggttcacga

[0430] ctgcgaatccaggacctggacacgacagacactggctactaccagtgcgtggccaccaacgggatgaagaccattaccgccactggcgt

[0431] cctgtttgtgcggctgggtccaacgcacagcccaaatcataactttcaggatgattaccacgaggatgggttctgccagccttaccggggaa

[0432] ttgcctgtgcacgcttcattggcaaccggaccatttatgtggactcgcttcagatgcagggggagattgaaaaccgaatcacagcggccttc

[0433] accatgatcggcacgtctacgcacctgtcggaccagtgctcacagttcgccatcccatccttctgccacttcgtgtttcctctgtgcgacgcgc

[0434] gctcccggacacccaagccgcgtgagctgtgccgcgacgagtgcgaggtgctggagagcgacctgtgccgccaggagtacaccatcgc

[0435] ccgctccaacccgctcatcctcatgcggcttcagctgcccaagtgtgaggcgctgcccatgcctgagagccccgacgctgccaactgcat

[0436] gcgcattggcatcccagccgagaggctgggccgctaccatcagtgctataacggctcaggcatggattacagaggaacggcaagcacca

[0437] ccaagtcaggccaccagtgccagccgtgggccctgcagcacccccacagccaccacctgtccagcacagacttccctgagcttggaggg

[0438] gggcacgcctactgccggaaccccggaggccagatggagggcccctggtgctttacgcagaataaaaacgtacgcatggaactgtgtga

[0439] cgtaccctcgtgtagtccccgagacagcagcaagatggggattctgtacatcttggtccccagcatcgcaattccactggtcatcgcttgcctt

[0440] ttcttcttggtttgcatgtgccggaataagcagaaggcatctgcgtccacaccgcagcggcgacagctgatggcctcgcccagccaagaca

[0441] tggaaatgcccctcattaaccagcacaaacaggccaaactcaaagagatcagcctgtctgcggtgaggttcatggaggagctgggagagg

[0442] accggtttgggaaagtctacaaaggtcacctgttcggccctgccccgggggagcagacccaggctgtggccatcaaaacgctgaaggac

[0443] aaagcggaggggcccctgcgggaggagttccggcatgaggctatgctgcgagcacggctgcaacaccccaacgtcgtctgcctgctgg

[0444] gcgtggtgaccaaggaccagcccctgagcatgatcttcagctactgttcgcacggcgacctccacgaattcctggtcatgcgctcgccgca

[0445] ctcggacgtgggcagcaccgatgatgaccgcacggtgaagtccgccctggagccccccgacttcgtgcaccttgtggcacagatcgcgg

[0446] cggggatggagtacctatccagccaccacgtggttcacaaggacctggccacccgcaatgtgctagtgtacgacaagctgaacgtgaaga

[0447] tctcagacttgggcctcttccgagaggtgtatgccgccgattactacaagctgctggggaactcgctgctgcctatccgctggatggcccca

[0448] gaggccatcatgtacggcaagttctccatcgactcagacatctggtcctacggtgtggtcctgtgggaggtcttcagctacggcctgcagcc

[0449] ctactgcgggtactccaaccaggatgtggtggagatgatccggaaccggcaggtgctgccttgccccgatgactgtcccgcctgggtgtat

[0450] gccctcatgatcgagtgctggaacgagttccccagccggcggccccgcttcaaggacatccacagccggctccgagcctggggcaacct

[0451] ttccaactacaacagctcggcgcagacctcgggggccagcaacaccacgcagaccagctccctgagcaccagcccagtgagcaatgtg

[0452] agcaacgcccgctacgtggggcccaagcagaaggccccgcccttcccacagccccagttcatccccatgaagggccagatcagaccca

[0453] tggtgcccccgccgcagctctacgtccccgtcaacggctaccagccggtgccggcctatggggcctacctgcccaacttctacccggtgc

[0454] agatcccaatgcagatggccccgcagcaggtgcctcctcagatggtccccaagcccagctcacaccacagtggcagtggctccaccagc

[0455] acaggctacgtcaccacggccccctccaacacatccatggcagacagggcagccctgctctcagagggcgctgatgacacacagaacg

[0456] ccccagaagatggggcccagagcaccgtgcaggaagcagaggaggaggaggaaggctctgtcccagagactgagctgctgggggac

[0457] tgtgacactctgcaggtggacgaggcccaagtccagctggaagcttga (SEQ ID NO: 143)

[0458] In addition, the antigen cell lines involved in the examples are from the following sources:

[0459] Natural tumor cells expressing human ROR1:

[0460] A549 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60084.

[0461] Jeko-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60275.

[0462] HT-29 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60011

[0463] NCI-H1975 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60121.

[0464] Mino was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60677.

[0465] NCI-N87 was purchased from Nanjing Kebai Biotechnology Co., Ltd., product number CBP60491.

[0466] 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:

[0467] Example 1

[0468] 1. Construction of cell lines with different species antigens

[0469] 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.

[0470] 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.

[0471] 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.

[0472] 2. Construction of cell lines containing proteins of the same family

[0473] The nucleotide sequence of hROR2 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 hROR2 was inserted. The vector sequence information was then confirmed by sequencing. After successful plasmid construction, it was transfected into CHOK1 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.

[0474] 3. Facs assay to determine the affinity between antigen cells and tool antibodies.

[0475] Experimental reagents and materials:

[0476]

[0477]

[0478] Experimental procedure:

[0479] 1) Cell collection and seeding plate

[0480] a) Harvest cells in the logarithmic growth phase, ensuring cell viability is above 90%.

[0481] b) Centrifuge at 1000 r / min for 5 min, then discard the supernatant;

[0482] c) Wash the cells once with PBS;

[0483] d) Resuspend the cells using FACS Buffer and count them;

[0484] e) Prepare a solution with a density of 2×10 using FACS Buffer. 6 Cell suspension with cells / mL;

[0485] f) Add 50 μL of cell suspension to each well of a 96-well plate;

[0486] 2) Antibody incubation and detection

[0487] 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;

[0488] b) After mixing, incubate at 4°C in the dark overnight;

[0489] c) Wash cells once with 200 μL of FACS Buffer, centrifuge at 1000 r / min for 5 min, and discard the supernatant;

[0490] 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;

[0491] e) After mixing, incubate at 4°C in the dark for 40 minutes;

[0492] 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;

[0493] g) Detect RL-1 MFI readings using an Intellicyte plus flow cytometer (Excitation Laser: 488nm Blue Laser).

[0494] 3) Data processing

[0495] Analyze FACS data using Prism software.

[0496] Data Analysis Figure 1 Using 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.

[0497] Example 2

[0498] 1. Animal immunization

[0499] Balb / c mice aged 8-10 weeks were immunized using two methods: Freund's adjuvant and water-soluble adjuvant. Freund's adjuvant was administered intraperitoneally twice on days 0 and 14, while water-soluble adjuvant was administered intramuscularly twice on days 0 and 21. The immunogenicity was recombinant human ROR1 protein, recombinantly expressed in HEK293 cells by Acro Biosytem (catalog number RO1-H5250). The first immunization dose was 50 μg, and the second dose was 25 μg. Serum was collected before immunization as a negative control. For Freund's adjuvant immunization, blood was collected via tail vein on day 28 after the initial immunization; for water-soluble adjuvant immunization, blood was collected on day 35 after the initial immunization. Serum titers were measured using an ELISA method on a 96-well microplate coated with recombinant human ROR1 protein. Mice with serum titers meeting the fusion requirements were boosted with a 25 μg antigen diluted to 500 μl with D-PBS and administered intraperitoneally. Spleen cells from mice with high serum titers were collected 18-38 days after the initial immunization for the next step of cell fusion.

[0500] 2. Cell fusion and hybridoma preparation

[0501] 2.1 Preparation of myeloma cells

[0502] The required myeloma cells P3X63Ag8.653 were cultured in 500ml Erlenmeyer flasks until the cell density reached 0.8-1.0E+6, then the medium was changed to complete hybridoma medium for later use.

[0503] 2.2 Preparation of lymph nodes and B lymphocytes

[0504] Mice with titers meeting the requirements were selected, their eyeballs were removed to collect blood, and the serum was separated to serve as a positive control serum for antibody detection. The spleen of the mice was aseptically removed, and a suspension of B lymphocytes was prepared according to conventional methods.

[0505] 2.3 Electro-Cell Fusion (ECF): Myeloma cells P3X63Ag8.653 and B lymphocytes were mixed at a ratio of 4:1 and then fused by electrofusion. After electrofusion, the cells were placed in a 37°C CO2 incubator and allowed to stand for 30 minutes. They were then centrifuged at 1000 RPM at room temperature for 10 minutes. The cells were resuspended in 360 ml of hybridoma selection medium and seeded into 384-well plates, ensuring a seeding density of 8000–20000 cells / well. The medium was changed once every 2–3 days, and positive hybridomas were screened on days 7–10.

[0506] 3. B cell culture and identification

[0507] The fused hybridoma cells were cultured in 384-well plates, and the supernatant was analyzed by FACS to detect antibodies secreted by the hybridoma cells. Several clones were obtained through screening. These clones could bind to human ROR1-overexpressing strains but not CHO-blank.

[0508] Cells. The selected clones were single-celled using a limiting dilution method, and after three rounds, each hybridoma cell clone secreted only one antibody.

[0509] 4. Cloning of the gene encoding mouse monoclonal antibodies

[0510] After expanding the culture of hybridoma cells secreting anti-human ROR1 antibodies, total RNA was extracted from the cells according to the instructions of the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.). The total RNA from the hybridoma cells was reverse transcribed into cDNA using 5×PrimeScript RT Master Mix (Takara). The antibody light chain variable region IgVL(κ) and heavy chain variable region VH sequences were amplified using degenerate primers (Anke Krebber.1997) and Extaq PCR reagent (Takara). The PCR amplification products were purified using a PCR clean-up gel extraction kit (Macherey-Nagel). The amplified PCR products were ligated into a T vector and transformed into E. coli competent cells according to the instructions of the pClone007 SimpleVector Kit (Qingke Biotechnology Co., Ltd.). After amplification and plasmid extraction, DNA sequencing was performed to obtain the monoclonal antibody variable region sequence.

[0511] Example 3

[0512] 1. Preparation of anti-human ROR1 chimeric antibody

[0513] The heavy chain variable region sequence of a murine anti-human ROR1 monoclonal antibody and the published heavy chain constant region sequence of a human monoclonal antibody IgG1 subclass were spliced ​​together and constructed into a mammalian cell expression vector. Similarly, the light chain variable region sequence of a murine anti-human ROR1 monoclonal antibody and the published light chain constant region sequence of a 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.

[0514] 2. In vitro cell binding assay of anti-human ROR1 chimeric antibody to overexpressing cell lines

[0515] 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 overexpressing human ROR1 (CHO-hROR1), mouse ROR1 (CHO-mROR1), human ROR2 (CHO-hROR2), and CHOK1 empty cells were collected by centrifugation at 100g for 5 minutes at room temperature. 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 4-parameter fitting curves or bar charts were analyzed using Prism software.

[0516] Results Analysis Figure 2 In vitro cell biology specific binding FACS assays were performed on chimeric antibodies using the overexpressing cell line CHO-hROR1. Cross-binding properties of the chimeric antibodies with mouse proteins were tested using the overexpressing cell line CHO-mROR1. Cross-binding properties of the chimeric antibodies with family proteins were tested using CHO-hROR2 cells overexpressing human ROR2. Non-specific binding properties of the chimeric antibodies with empty CHOK1 cells were tested. The data showed that several molecules with good binding signals were obtained through screening. chT11, chT16, and chT26 showed comparable or better activity to the control antibody UC961. chT24 showed cross-binding with mouse ROR1. chT26 showed cross-binding properties with the family protein ROR2. Except for chT29, which showed a relatively obvious signal, the other chimeric antibodies showed no significant non-specific binding signals with CHOK1 empty cells.

[0517] 3. In vitro cell binding assay of anti-human ROR1 chimeric antibody to naturally expressed cell lines

[0518] 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.

[0519] analyze( Figure 3 In vitro cell biology binding FACS tests were conducted on chimeric antibodies using Mino, A549 and HT29 cell lines that naturally express human ROR1. The data showed that the screened chT11 and chT16 had comparable activity to the control antibody UC961.

[0520] 4. In vitro binding affinity and kinetics of anti-human ROR1 chimeric antibodies

[0521] Antibody affinity was determined using the anti-human antibody capture method with 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 binding reactions with human ROR1 protein and mouse FAP protein (purchased from ACRObiosystem) for 600 s. Afterward, the AHC probe was transferred to PBS for dissociation reaction for 600 s. After the experiment, the blank control response value was subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting.

[0522] Analysis: Based on in vitro kinetic binding activity analysis, the binding kinetic constant of the chimeric antibody with recombinant human ROR1 protein was in the range of 10⁻⁸ to 10⁻⁹. In comparison, the binding kinetic constant of the target antibody UC961 was in the range of 10⁻⁸, and the binding kinetic constant of the target antibody XBR1-402 was in the range of 10⁻⁹. The obtained chimeric antibody showed a better kinetic binding signal with recombinant human ROR1 protein.

[0523] Table 1: In vitro kinetic binding activity of chimeric antibodies to recombinant human ROR1 protein:

[0524] Antibody Response value KD (M) kon (1 / Ms) kdis (1 / s) chT9 0.2746 6.95E-09 2.03E+05 1.41E-03 chT11 0.3355 1.26E-08 5.72E+05 7.21E-03 chT16 0.3999 1.35E-09 3.00E+05 4.05E-04 chT24 0.2349 3.81E-08 1.86E+05 7.09E-03 chT26 0.2291 3.07E-08 7.95E+05 2.44E-02 chT29 0.2883 7.68E-09 1.85E+05 1.42E-03 UC961 0.3561 1.41E-08 4.46E+05 6.27E-03 XBR1-402 0.3903 7.24E-09 4.40E+05 3.19E-03

[0525] 5. Cellular endocytosis assay of anti-human ROR1 chimeric antibody

[0526] Methods and Procedures: First, prepare cells naturally expressing ROR1, such as human non-small cell lung cancer A549 cells and human colon cancer HT29 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 the antibody and endocytosis reagent to 2 μg / mL at a 1:1 mass ratio using cell culture medium and incubate at 37°C for 15 minutes. Add an equal volume of the labeled antibody, reagent, and marker to the prepared cells and incubate at 37°C for endocytosis for 1-26 hours. Remove the culture plate and directly analyze the fluorescence value using the RL-1 channel of a flow cytometer. The obtained MFI is fitted using Prism software.

[0527] analyze( Figure 4 The endocytic activity of chimeric antibodies against human ROR1 was investigated using naturally expressed cell lines HT29 and A549. The results showed that the chimeric antibodies could undergo endocytosis relatively quickly, which was comparable to or better than the control antibody UC961.

[0528] Table 2: Fluorescence values ​​of endocytosis in naturally expressed cell lines A549 and HT29

[0529]

[0530] Example 4

[0531] 1. Humanization of anti-human ROR1 mouse antibody

[0532] 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 murine 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 murine antibody, such as IGHV1|IGHJ4*01, was selected. Its antibody framework region sequence was used as a template to bind the murine 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 murine 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 murine. The reversion mutation sites were determined by: firstly, comparing the designed humanized antibody sequence with the original murine 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 to 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.

[0533] 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.

[0534] 2. In vitro binding affinity and kinetics experiments of anti-human ROR1 humanized antibody

[0535] 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.

[0536] 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 -9 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.

[0537] In vitro kinetic binding profile of humanized antibody to recombinant human ROR1 protein:

[0538] Table 3: In vitro kinetic binding activity of humanized antibodies to recombinant human ROR1 protein:

[0539]

[0540]

[0541] 3. In vitro cell binding assay of anti-human ROR1 humanized antibody

[0542] Humanized anti-human ROR1 antibody was serially diluted 4-fold starting from an initial 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, 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. 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. A 4-parameter fitting curve was performed using Prism software, and the EC50 and the top value of fluorescence intensity were calculated.

[0543] analyze( Figure 5 In vitro cell biology binding FACS tests were conducted on the selected humanized antibody using six naturally expressed human ROR1 cell lines: Mino cells, A549 cells, HT29 cells, NCI-N87 cells, NCI-H1975 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.

[0544] Table 4: Top values ​​of EC50 and fluorescence intensity for in vitro flow cytometry binding of humanized antibodies to naturally expressed ROR1 cells:

[0545]

[0546] 4. In vitro cell nonspecific binding assay of anti-human ROR1 humanized antibody

[0547] The anti-human ROR1 affinity maturation conjugate antibody was initially diluted 4-fold in eight steps at a concentration of 2 μg / mL. 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 × 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 four-parameter fitting curve was generated using Prism software.

[0548] Results Analysis Figure 6 Non-specific binding experiments were conducted on the anti-human ROR1 humanized antibody with the cell line CHO-hROR2, which overexpresses the ROR2 family protein, and the Chinese hamster ovary cell line CHOK1, which is commonly used to express the protein. The results showed that the control antibody UC961 and the anti-human ROR1 humanized antibody molecule T16-H1L6 did not show any non-specific binding signal with the family protein or empty cells used for expression.

[0549] 5. In vitro cell binding assays of different species of anti-human ROR1 humanized antibodies

[0550] 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. 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.

[0551] 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 the control antibody UC961 and the preferred humanized anti-ROR1 antibody T16-H1L6 both showed good binding signals with CHO-cynoROR1 cell lines overexpressing monkey ROR1, while the control antibody C2E3 showed a binding signal with CHO-mROR1 cell lines overexpressing mouse ROR1.

[0552] 6. In vitro killing assay of anti-human ROR1 humanized antibody

[0553] In vitro cell killing assays were performed using the overexpressing cell line 293-hROR1. 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 plates. The small molecule toxin conjugate 20ADCαHFc-CL-MMAE (purchased from Moradec, catalog number AH-102AE-50) was diluted to 2 μg / mL using appropriate cell culture medium. The humanized antibody was diluted to 2.5 μg / mL using the diluted small molecule toxin conjugate, and then 3-fold diluted 10 times. 50 μl / well of the diluted series of antibodies was added to the cell-seeded plates. 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 the cell plate. After vortexing for 5 minutes, the chemiluminescence was read using a microplate reader. The killing curve was fitted using Prism software with four parameters.

[0554] Results Analysis Figure 8In vitro cell killing experiments were conducted using the ROR1-overexpressing cell line 293-hROR1. The results showed that the preferred humanized anti-human ROR1 antibody molecule T16-H1L6 had in vitro cell killing activity, and its killing level was similar to or better than that of the control antibody UC961.

[0555] 7. Physical characterization and monomer yield analysis of anti-human ROR1 humanized antibody molecules

[0556] Experimental apparatus: UPLC CLASS ACQUITYH (WATERS)

[0557] Analytical column: TSKgel G3000SWXL 7.8*300 (TOSHI, Cat No. 003C03326C)

[0558] Analytical solution: 200 mM K₂HPO₄, 250 mM KCl, pH adjusted to 6.2 with HCl.

[0559] 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.

[0560] 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 95%, indicating good monomer content properties.

[0561] Table 5: Antibody peak retention time and monomer yield

[0562] Antibody Retention time of main peak (min) Monomer rate SEC (%) UC961 10.588 98.69 T16-H0L4 10.936 95.98 T16-H0L5 10.988 95.19 T16-H0L6 10.944 96.10 T16-H1L4 10.931 98.69 T16-H1L5 10.979 98.45 T16-H1L6 10.932 98.61

[0563] 8. Analysis of the hydrophobic properties of humanized antibodies

[0564] Experimental apparatus: ARC (Waters)

[0565] Analytical column used in the experiment: TSKgel Butyl-NPR (4.6mm × 3.5cm, Cat No. 14947)

[0566] Analytical solution: A. 20 mM Histidine, pH 6.0;

[0567] B.20mM Histidine, 1.6M(NH4)2SO4

[0568] Analytical method: The hydrophobic properties of the antibody were analyzed according to the instructions for use of the hydrophobic chromatography column.

[0569] 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.

[0570] Table 6: Hydrophobic properties of humanized antibodies

[0571]

[0572] 9. Drug metabolism analysis in humanized antibody mice

[0573] 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).

[0574] Experimental methods:

[0575] Serum collection:

[0576] 1) Female Balb / C mice, 3 mice / group, were administered 200 μg / mouse via tail vein or intraperitoneal injection;

[0577] 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.

[0578] 3) The last serum collection should be frozen at -20°C for at least 24 hours.

[0579] Detection method:

[0580] 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;

[0581] 2) Prepare the required reagents:

[0582] Blocking solution: 5% BSA + PBS

[0583] Antibody dilution solution: 5% BSA + PBS + 20% blank mouse serum

[0584] ELISA plate washing buffer: 0.1% Tween + PBS

[0585] 3) Wash the coated ELISA plate three times with PBS, 300 μl / well;

[0586] 4) Add blocking solution, 200 μL / well, and seal at 37°C for 1 hour;

[0587] 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.

[0588] 6) Dilute the antibody standard curve with antibody dilution buffer. 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).

[0589] 7) Discard the blocking solution, add diluted antibody and test serum to the enzyme-linked plate for the two coating methods respectively, 100 μl / well, and incubate at 37℃ for 1 h;

[0590] 8) Wash the plate three times with PBST;

[0591] 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;

[0592] 10) Wash the plate three times with PBST;

[0593] 11) TMB color development, 100 μl / well, protected from light for 10 min;

[0594] 12) Add 50 μL of 2 M HCl to terminate the reaction, and take a reading at 450 nm.

[0595] Data Analysis: In vivo drug metabolism experiments were conducted on the preferred molecule T16-H1L6 and the control molecule UC961 in mice. Three female Balb / C mice were used for each sample. Blood samples were collected at different time points after tail vein injection. The antibody content in mouse serum was measured using a fully human antibody detection method. The results showed that the metabolic levels of each molecule differed in mice. T16-H1L6 was metabolized slowly in vivo, with a long half-life of 218-297 hours; the control antibody UC961 was metabolized in vivo for 155-220 hours, with a slightly shorter half-life. The preferred molecules all had longer half-lives than the control molecules. Figure 9 ).

[0596] Table 7. Drug metabolism properties in mice

[0597]

[0598] Example 5

[0599] 1. Humanized antibody-drug conjugate

[0600] 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.

[0601] After conjugation, ensure the drug / antibody conjugation ratio is 4.

[0602] 2. In vitro cytotoxic activity of antibody-drug conjugates

[0603] 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.

[0604] Data Analysis Figure 10 In vitro cell killing experiments were conducted using the ROR1-overexpressing cell line 293-hROR1 to conjugate the antibody with the drug. After 4 days of incubation, the results showed that the selected humanized anti-human ROR1 antibody, after conjugation with the small molecule drug, had significant in vitro cell killing activity, while the negative antibody did not show any in vitro killing activity.

[0605] 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. ROR1 antibody, Its heavy chain CDR1 has an amino acid sequence as shown in any one of SEQ ID NO: 1, 7, 11, 17, 23, 29 or 35; Its heavy chain CDR2 has an amino acid sequence as shown in any one of SEQ ID NO:2, 8, 12, 18, 24, 30 or 36; Its heavy chain CDR3 has an amino acid sequence as shown in any one of SEQ ID NO:3, 13, 19, 25, 31 or 37; Its light chain CDR1 has an amino acid sequence as shown in any one of SEQ ID NO:4, 9, 14, 20, 26, 32 or 38; Its light chain CDR2 has an amino acid sequence as shown in any one of SEQ ID NO:5, 15, 21, 27, 33 or 39; Its light chain CDR3 has an amino acid sequence as shown in any one of SEQ ID NO: 6, 10, 16, 22, 28, 34 or 40; Alternatively, its CDR region has a sequence based on the amino acid sequence shown above, with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence shown above.

2. The ROR1 antibody according to claim 1, characterized in that, The amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:1-3, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:4-6, respectively. Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:7, 8, 3, respectively, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:9, 5, 10, respectively; Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:11-13, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:14-16, respectively; Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:17-19, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:20-22, respectively; Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:23-25, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:26-28, respectively; Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:29-31, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:32-34, respectively; Or the amino acid sequences of its heavy chain CDR1-3 are shown in SEQ ID NO:35-37, and the amino acid sequences of its light chain CDR1-3 are shown in SEQ ID NO:38-40.

3. The ROR1 antibody according to claim 1 or 2, characterized in that, It is a chimeric antibody: Its heavy chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:41, 49, 52, 59, 63, 70 or 76; Its heavy chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:42, 53, 60, 64, 71 or 77; Its heavy chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:43, 50, 54, 61, 65, 72 or 78; Its heavy chain FR4 has an amino acid sequence as shown in either SEQ ID NO:44 or 79; Its light chain FR1 has an amino acid sequence as shown in any one of SEQ ID NO:45, 55, 66, 73 or 80; Its light chain FR2 has an amino acid sequence as shown in any one of SEQ ID NO:46, 51, 56, 67, 74 or 81; Its light chain FR3 has an amino acid sequence as shown in any one of SEQ ID NO:47, 57, 62, 68, 75 or 82; Its light chain FR4 has an amino acid sequence as shown in any one of SEQ ID NO:48, 58 or 69; Alternatively, its FR region has a sequence based on the amino acid sequence shown above, with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence shown above.

4. The ROR1 antibody according to claim 3, characterized in that, The amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:41-44, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:45-48, respectively. Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:49, 42, 50, 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:45, 51, 47, 48, respectively; Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:52, 53, 54, 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:55-58, respectively; Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:59-61, 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:45, 51, 62, 48, respectively; Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:63-65 and 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:66-69, respectively; Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:70-72 and 44, respectively, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:73-75 and 48, respectively; Or the amino acid sequences of its heavy chain FR1-4 are shown in SEQ ID NO:76-79, and the amino acid sequences of its light chain FR1-4 are shown in SEQ ID NO:80-82 and 48, respectively.

5. The ROR1 antibody according to any one of claims 1 to 4, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:88, 90, 92, 94, 96, 98 or 100; Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:89, 91, 93, 95, 97, 99 or 101.

6. The ROR1 antibody according to any one of claims 1 to 5, characterized in that, Its light chain constant region is the κ subtype, and its heavy chain constant region is IgG1.

7. The ROR1 antibody according to claim 1 or 2, characterized in that, Its humanized antibody: Its heavy chain FR1 has the amino acid sequence shown in SEQ ID NO:83; Its heavy chain FR2 has the amino acid sequence shown in SEQ ID NO:42; Its heavy chain FR3 has an amino acid sequence as shown in SEQ ID NO:84 or 86; Its heavy chain FR4 has an amino acid sequence as shown in SEQ ID NO:85 or 87; Its light chain FR1 has the amino acid sequence shown in SEQ ID NO:45; Its light chain FR2 has the amino acid sequence shown in SEQ ID NO:51; Its light chain FR3 has the amino acid sequence shown in SEQ ID NO:47; Its light chain FR4 has the amino acid sequence shown in SEQ ID NO:48; Alternatively, its FR region has a sequence based on the amino acid sequence shown above, with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence shown above.

8. The ROR1 antibody according to claim 1, 2, or 7, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:102 or 103; Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:104 to 107.

9. The antibody or antigen-binding molecule according to any one of claims 1, 2, 7-8, characterized in that, Its light chain constant region is the κ subtype, and its heavy chain constant region is IgG1.

10. Biological materials, including at least one of the following A) to C): A) Nucleic acid encoding the ROR1 antibody according to any one of claims 1 to 9; B) A plasmid vector containing the nucleic acid described in A); C) The host whose genome integrates the nucleic acid described in A), or is transformed or transfected with the plasmid vector described in B).

11. A method for preparing the ROR1 antibody according to any one of claims 1 to 9, comprising: Culture the host as described in claim 10 to obtain a culture containing ROR1 antibody.

12. The labeled antibody obtained by labeling the ROR1 antibody according to any one of claims 1 to 9, wherein the labeling is a chemical label or a biological label.

13. The labeled antibody according to claim 12, characterized in that, The chemical label is a fluorescent indicator, a chemiluminescent indicator, an isotope and / or a colloidal indicator; The biomarker is biotin, avidin, or an enzyme label.

14. The labeled antibody according to claim 13, characterized in that, 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. 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, OregonGreen488, Pacific Blue dye, Pacific Orange dye, Texas Red, or PerCP dye; 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. The isotopes are selected from one or more of 125I, 131I, 124I, 3H, 14C, 111In, 89Zr or 32P; Colloidal indicators are selected from one or more of colloidal gold, colloidal carbon, or colloidal selenium.

15. The conjugate obtained by binding the ROR1 antibody according to any one of claims 1 to 9 to a medium.

16. The combination according to claim 15, characterized in that, The medium is selected from colloidal gold, enzyme-labeled plates, magnetic beads, or latex microspheres.

17. An antibody-drug conjugate comprising the ROR1 antibody and drug as described in any one of claims 1 to 9.

18. The antibody-drug conjugate according to claim 17, characterized in that, The drug is an anti-tumor drug, selected from at least one of MMAE, MMAF, or DXD.

19. Any one of the following (I) to (V) in the preparation of drugs for the prevention and treatment of ROR1-related diseases: I) The ROR1 antibody as described in any one of claims 1 to 9; II) The biomaterial as described in claim 10; III) The culture prepared by the preparation method of claim 11 or the antibody obtained by purification; IV) The labeled antibody according to any one of claims 12 to 14; V) The coupling as described in claim 17 or 18.

20. The application according to claim 19, characterized in that, The disease associated with ROR1 is cancer.

21. The application according to claim 20, 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.

22. The use 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) The ROR1 antibody as described in any one of claims 1 to 9; ii) The biomaterial according to claim 10; iii) The culture obtained by the preparation method of claim 11 or the antibody obtained by purification; iv) The labeled antibody according to any one of claims 12 to 14; v) The combination as described in claim 15 or 16; vi) The coupling as described in claim 17 or 18.

23. The application according to claim 24, characterized in that, The tumor is a tumor that expresses ROR1.

24. A drug, comprising any one of the following: (I) through (VII); I) The ROR1 antibody as described in any one of claims 1 to 9; II) The biomaterial as described in claim 10; III) The culture prepared by the preparation method of claim 11 or the antibody obtained by purification; IV) The labeled antibody according to any one of claims 12 to 14; V) The coupling as described in claim 17 or 18.

25. A pharmaceutical composition comprising the drug of claim 24 and other antitumor drugs.

26. A reagent, comprising any one of the following i) to vii): i) The ROR1 antibody as described in any one of claims 1 to 9; ii) The biomaterial according to claim 10; iii) The culture obtained by the preparation method of claim 11 or the antibody obtained by purification; iv) The labeled antibody according to any one of claims 12 to 14; v) The combination as described in claim 15 or 16; vi) The coupling as described in claim 17 or 18.

27. A method for detecting ROR1 or tumor cells expressing ROR1, comprising detecting a sample with the reagent of claim 26.

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