Anti-ROR1 antibody, immunoconjugate thereof and use thereof

Abstract

The present invention relates to an anti-ROR1 antibody, an immunoconjugate thereof and the use thereof. Provided in the present invention are an anti-ROR1 antibody, an immunoconjugate containing the anti-ROR1 antibody, and the use in the preparation of a drug for treating and / or preventing diseases, particularly diseases related to the overexpression of ROR1, such as tumors.

Description

Anti-ror1 antibodies, immunoconjugates thereof, and uses thereof

[0001] This application claims priority to the application with the title of "Anti-ror1 antibodies, immunoconjugates thereof, and uses thereof", filed on April 18, 2024, with the application number of CN 202410470842.7, the entire content of which is incorporated herein. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and more specifically, the present application relates to anti-ROR1 antibodies, immunoconjugates and uses thereof. BACKGROUND

[0003] Triple negative breast cancer (TNBC) refers to breast cancer with negative expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, accounting for about 20% of all breast cancer types. At present, the treatment of TNBC mainly relies on surgery and radiotherapy and chemotherapy, and there is a lack of targeted therapy and high response therapy. Compared with other types of breast cancer, TNBC is more likely to spread beyond the breast, and is more likely to relapse after treatment. TNBC is more invasive and has a poorer prognosis. TNBC is one of the most deadly diseases for women, and there is an urgent clinical need and huge market potential.

[0004] Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that play important roles in a variety of cellular processes, including growth, motility, differentiation, and metabolism. Therefore, dysregulation of RTK signaling can lead to a variety of diseases, such as cancer. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is one of the members of the receptor RTKs family, with a molecular weight of about 105 kDa. Human ROR1 is composed of an extracellular immunoglobulin-like domain (Ig), two cysteine-rich domains (FZD), a juxtamembrane kringle domain (KRD), a single-pass transmembrane domain, and an intracellular tyrosine kinase domain (TKD), two serine / threonine-rich domains (S / TRD), and a proline-rich domain (PRD).

[0005] During early embryonic development, ROR1 regulates cell division, proliferation, migration, and cell chemotaxis by mediating Wnt signaling pathway signaling, and has physiological effects on embryonic neural, auditory, skeletal, and vascular organogenesis. The expression of ROR1 gradually decreases during embryonic development, and is very low in most children and adult normal tissues, but is highly up-regulated in cancerous tissues. ROR1 is highly expressed in various hematological tumors and solid tumors, including B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid hematological cancers; and solid tumors including TNBC, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, melanoma, etc. ROR1 in tumor cells binds Wnt5a and activates the non-canonical Wnt signaling pathway, promoting cell migration, invasion, EMT, and metastasis, etc. In addition, MET can phosphorylate ROR1 and regulate tumor cell growth. It has been reported that the expression of ROR1 is associated with the activation of YAP / TAZ transcription, thereby enhancing tumorigenesis and chemoresistance. ROR1 expression is up-regulated in HER2+ breast cancer patients treated with T-DM1 and leads to drug resistance, and ROR1 highly-expressed tumor cells have increased stemness and self-renewal capacity. The expression of ROR1 is also closely related to disease progression and treatment effect, and high ROR1 expression is associated with poor prognosis of TNBC. Compared with patients with lower ROR1 expression, TNBC patients with higher ROR1 expression have significantly lower disease-free survival and overall survival. Therefore, ROR1 is an ideal anti-tumor target and tumor diagnostic marker.

[0006] Therefore, there is a need in the art to develop antibodies and immunoconjugates targeting ROR1 for use in the treatment of tumors. SUMMARY

[0007] The present application aims to provide an anti-ROR1 antibody, and an immunoconjugate comprising the anti-ROR1 antibody, and use in the manufacture of a medicament for treating and / or preventing a disease, a disease associated with overexpression of ROR1, such as a tumor. In a first aspect of the present application, an anti-ROR1 antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof comprises: an immunoglobulin heavy chain variable region (VH) comprising a heavy chain HCDR1, a HCDR2 and a HCDR3, wherein the HCDR1 is or comprises an amino acid sequence as set forth in SEQ ID NO: 1, the HCDR2 is or comprises an amino acid sequence as set forth in SEQ ID NO: 2, and the HCDR3 is or comprises an amino acid sequence as set forth in SEQ ID NO: 3; and an immunoglobulin light chain variable region (VL) comprising a light chain LCDR1, a LCDR2 and a LCDR3, wherein the LCDR1 is or comprises an amino acid sequence as set forth in SEQ ID NO: 4, the LCDR2 is or comprises an amino acid sequence as set forth in SEQ ID NO: 5, and the LCDR3 is or comprises an amino acid sequence as set forth in SEQ ID NO: 6.

[0008] In one or more embodiments, the anti-ROR1 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 7, 9 or 10, or at least 85% identical to the amino acid sequence as set forth in SEQ ID NO: 7, 9 or 10, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 8, 11, 12, 13 or 14, or at least 85% identical to the amino acid sequence as set forth in SEQ ID NO: 8, 11, 12, 13 or 14.

[0009] In one or more embodiments, the anti-ROR1 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 7, and a light chain variable region comprising an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 8; or

[0010] the anti-ROR1 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region comprising an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 11; or

[0011] the anti-ROR1 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region comprising an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 12; or

[0012] the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 13; or

[0013] the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 14; or

[0014] the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 11 ; or

[0015] the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 12; or

[0016] the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 13; or

[0017] the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to or having at least about 85% identity to SEQ ID NO: 14.

[0018] In one or more embodiments, the anti-RORl antibody or antigen-binding fragment thereof comprises: a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:

[0019] the VH region and the VL region are or comprise the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or

[0020] the VH region and the VL region are or comprise the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 11, respectively; or

[0021] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 9 and SEQ ID NO: 12, respectively; or

[0022] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 9 and SEQ ID NO: 13, respectively; or

[0023] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 9 and SEQ ID NO: 14, respectively; or

[0024] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 11, respectively; or

[0025] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 12, respectively; or

[0026] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 13, respectively; or

[0027] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 14, respectively.

[0028] In one or more embodiments, the anti-RORl antibody or antigen-binding fragment thereof is or comprises a heavy chain as set forth in an amino acid sequence of SEQ ID NO: 18, 20, or 25 or at least about 85% identical thereto and a light chain as set forth in an amino acid sequence of SEQ ID NO: 19, 21, 22, 23, or 24 or at least about 85% identical thereto.

[0029] In one or more embodiments, the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain (HC) and a light chain (LC), wherein: the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 18; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 19; or

[0030] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 21; or

[0031] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 22; or

[0032] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 23; or

[0033] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 24; or

[0034] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 21; or

[0035] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 22; or

[0036] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 23; or

[0037] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 24.

[0038] In one or more embodiments, the anti-RORl antibody or antigen-binding fragment thereof is a murine, humanized, chimeric or fully human antibody.

[0039] In a second aspect of the present application, there is provided a polynucleotide encoding an anti-RORl antibody or antigen-binding fragment thereof as described in any one of the embodiments of the present application.

[0040] In a third aspect of the present application, there is provided an immunoconjugate comprising: an antibody as described in any one of the embodiments of the present application; and a drug linked thereto.

[0041] In one or more embodiments, the immunoconjugate has the formula Ab-(L-D)y, wherein: Ab is an antibody or antigen-binding fragment thereof as described in any one of the embodiments of the present application; L is a linker; D is a drug; and y is a number from 1 to 10, either as a decimal or an integer; more preferably, y is an integer or decimal from 1 to 8, more preferably an integer or decimal from 7 to 8.

[0042] In one or more embodiments, the immunoconjugate has a structure according to Formula I:

[0043] wherein Ab is an antibody or antigen-binding fragment thereof according to any of the embodiments described herein, and y is an integer or decimal number from 1 to 8, more preferably an integer or decimal number from 7 to 8.

[0044] In one or more embodiments, the drug comprises a molecule that inhibits a tumor, a molecule that targets a tumor surface marker, a molecule that targets a surface marker of an immune cell, a radioactive moiety, a detectable label, or a combination thereof.

[0045] In one or more embodiments, the molecule that targets a tumor surface marker is an antibody or ligand that binds to a tumor surface marker; or the molecule that inhibits a tumor is an anti-tumor cytokine or an anti-tumor toxin.

[0046] In one or more embodiments, the antibody that binds to a tumor surface marker is an antibody that recognizes an antigen other than ROR1, including EGFR, EGFRvIII, mesothelin, HER2, EphA2, Her3, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, ASGPR1, or CDH16.

[0047] In one or more embodiments, the anti-tumor cytokine comprises IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, or a variant thereof.

[0048] In one or more embodiments, the antitumor toxin comprises: a toxin acting on tubulin; a toxin acting on DNA or derivatives thereof; a compound acting on intracellular metabolism, transcription, translation or signal transduction or derivatives thereof; more preferably CPT2C, camptothecin, 20-deoxycamptothecin, 10-methoxyamptothecin, 9-methoxycamptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, exatecan, deruxtecan (Dxd), irinotecan (DX-8951f), lurtotecan (GG-211), topotecan, irinotecan (CPT11), simmitecan, sinotecan, lipotecan (TLC388), gimmitecan (ST1481), diflomotecan (BN-80915), CZ48, Camptothecin chloroacetate (DLSFFZ93W4), Camptothecin lysinate (NSC610457), HM910, SN38, GI-149893, cositecan (karenitecin), Elomotecan (BN-80927), 11-cyanocamptothecin (NSC609951), 7-acetyl-Camptothecin (SCHEMBL6851483), 9-Glycineamidocamptothecin HCl (MLS000756803), 7-hydroxymethylcamptothecin, 9-Glycineamidocamptothecin HCl (MLS000756803), 10-amino-11-hydroxy-camptothecin (BDBM50285230), diflomotecan, 9-nitrocamptothecin (9-NC), 9-aminocamptothecin (9-AC), 10-aminocamptothecin (10-AC), kanitecan (BNP-1350) or 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (AR-67); a maytansinoid; monomethyl auristatin DE and DF (MMAE and MMAF); dolastatin;Calicheamicin or a derivative thereof; an anthracycline, doxorubicin or daunorubicin; methotrexate; vindesine; a taxane, docetaxel, paclitaxel, larotaxel, tesetaxel or ortataxel; a trichothecene; a duocarmycin; a pyrrolobenzodiazepine (PBD); SN-38; GX-2; CC1065.

[0049] In one or more embodiments, the CPT2C has a structure as shown in Formula II:

[0050] In one or more embodiments, in the immunoconjugate, the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region and the VL region are or comprise the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 12, respectively; or the VH region and the VL region are or comprise the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 13, respectively.

[0051] In one or more embodiments, in the immunoconjugate, the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein: the heavy chain is or comprises the amino acid sequence set forth in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence set forth in SEQ ID NO: 22; or the heavy chain is or comprises the amino acid sequence set forth in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0052] In one or more embodiments, the linker is a cleavable linker; preferably, the linker is a tumor-specific cleavable linker.

[0053] In one or more embodiments, the linker is an acid-labile linker, a peptidase-sensitive linker, a photo-labile linker, a dimethyl linker, or a disulfide-containing linker.

[0054] In one or more embodiments, the linker comprises a peptide moiety, a reactive functional group, a benzoic acid or benzyloxy carbonyl group, or any combination thereof; wherein the peptide moiety comprises SC, VC, VA, FK, EVC, GGFG; the reactive functional group comprises MC, SMCC, sulfo-SMCC, SPP, SPDP; the benzoic acid or benzyloxy carbonyl group comprises PABA, PAB, GABA.

[0055] In one or more embodiments, the linker comprises MC-VA, MC-VC, SC-VC-PAB, SPDP, SMCC, SuO-VA-PAB, SuO-VC-PAB, MC-VA-PAB, MC-VC-PAB, MC-GGFG.

[0056] In one or more embodiments, the (L-D) moiety comprises: MC-GGFG-CPT2C, MC-VC-PAB-MMAE, MC-GGFG-Dxd, MC-VA-MMAE, MC-VC-MMAE, MC-VA-PAB-MMAE, MC-GGFG-MMAE, SC-VC-PAB-MMAE, MC-VA-MMAF, MC-VC-MMAF, MC-VA-PAB-MMAF, MC-GGFG-MMAF, SC-VC-PAB-MMAF, MC-VC-PAB-MMAF, MC-GGFG-camptothecin, MC-GGFG-20-deoxycamptothecin, MC-GGFG-10-methoxyamptothecin, MC-GGFG-9-methoxycamptothecin, MC-GGFG-10-hydroxycamptothecin, MC-GGFG-7-ethyl-10-hydroxycamptothecin, MC-GGFG-exatecan, MC-GGFG-DX-8951f, MC-GGFG-GG-211, MC-GGFG-topotecan, MC-GGFG-CPT11, MC-GGFG-simmitecan, MC-GGFG-sinotecan, MC-GGFG-TLC388, MC-GGFG-ST1481, MC-GGFG-BN-80915, MC-GGFG-CZ48, MC-GGFG-DLSFFZ93W4, MC-GGFG-NSC610457, MC-GGFG-HM910, MC-GGFG-SN38, MC-GGFG-GI-149893, MC-GGFG-cositecan, MC-GGFG-BN-80927, MC-GGFG-NSC609951, MC-GGFG-7-acetyl-Camptothecin, MC-GGFG-9-Glycineamidocamptothecin HCl, MC-GGFG-7-hydroxymethylcamptothecin, MC-GGFG-9-Glycineamidocamptothecin HCl, MC-GGFG-10-amino-11-hydroxy-camptothecin, MC-GGFG-diflomotecan, MC-GGFG-9-NC, MC-GGFG-9-AC, MC-GGFG-10-AC, MC-GGFG-BNP-1350, MC-GGFG-AR-67, CZY-8.

[0057] In a fourth aspect of the application, there is provided a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof as described in any embodiment of the application, or an immunoconjugate as described in any embodiment of the application.

[0058] In a fifth aspect of the application, there is provided use of an antibody or antigen-binding fragment thereof as described in any embodiment of the application, an immunoconjugate as described in any embodiment of the application, or a pharmaceutical composition as described in any embodiment of the application, in the manufacture of a medicament, kit or pharmaceutical pack for the diagnosis or treatment of a tumour.

[0059] In one or more embodiments, the tumour is a ROR1 -expressing tumour.

[0060] In one or more embodiments, the tumour comprises a primary tumour, a locally advanced tumour, a metastatic tumour; and / or, the tumour comprises: a lymphoma, a breast cancer, a lung cancer, an ovarian cancer, a neuroblastoma, a renal cell carcinoma, a colon cancer, a colorectal cancer, an epidermoid carcinoma, a melanoma, a myeloma, a gastric cancer, a brain cancer, a pancreatic cancer, a cervical cancer, a liver cancer, a bladder cancer, a prostate cancer, a testicular cancer, a thyroid cancer, a uterine cancer, an adrenal cancer, a head and neck cancer, and combinations thereof.

[0061] In one or more embodiments, the lymphoma comprises a B-cell leukemia, a B-cell chronic lymphocytic leukemia, an acute myeloid leukemia, an acute lymphoblastic leukemia, a Burkitt's lymphoma, a mantle cell lymphoma; the breast cancer comprises a breast ductal carcinoma, a triple negative breast cancer; the lung cancer comprises a non-small cell lung cancer, a small cell lung cancer; more preferably, the non-small cell lung cancer comprises a lung adenocarcinoma, a lung squamous cell carcinoma, a lung large cell neuroendocrine carcinoma, a lung lymphoepithelioma-like carcinoma.

[0062] In a sixth aspect of the application, there is provided a kit or pharmaceutical pack comprising: an antibody or antigen-binding fragment thereof as described in any embodiment of the application, or a polynucleotide as described in any embodiment of the application, or an immunoconjugate as described in any embodiment of the application, or a pharmaceutical composition as described in any embodiment of the application.

[0063] Other aspects of the application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1, ELISA detection of hROR1 -Fc antigen activity.

[0065] Figure 2, Serum titers of immunized mice.

[0066] Figure 3, Binding curve of humanized anti-RORl antibodies (Ab0~4, positive control antibody, negative control antibody) to MDA-MB-231 cells.

[0067] Figure 4, Binding curve of humanized anti-RORl antibodies (Ab0, Ab5~8, positive control antibody, negative control antibody) to MDA-MB-231 cells.

[0068] Figure 5, Binding curve of humanized anti-RORl antibodies (Ab0~4, positive control antibody, negative control antibody) to JeKo-1 cells.

[0069] Figure 6, Binding curve of humanized anti-RORl antibodies (Ab0, Ab5~8, positive control antibody, negative control antibody) to JeKo-1 cells.

[0070] Figure 7, Binding curve of humanized anti-RORl antibodies (Ab0~4, positive control antibody, negative control antibody) to A549 cells.

[0071] Figure 8, Binding curve of humanized anti-RORl antibodies (Ab0, Ab5~8, positive control antibody, negative control antibody) to A549 cells.

[0072] Figure 9, Binding curve of humanized anti-RORl antibodies (Ab0, Ab1~8, positive control antibody, negative control antibody) to human ROR2 antigen.

[0073] Figure 10, Endocytosis activity detection of humanized anti-RORl antibodies (Ab0, Ab2, Ab7, positive control antibody, negative control antibody) in CHOK1 / hRORl cells.

[0074] Figure 11, Anti-tumor efficacy detection of Ab2-ADC7 on NOG mouse subcutaneously transplanted JeKo-1 cell animal model.

[0075] Figure 12, Body weight change of Ab2-ADC7 on NOG mouse subcutaneously transplanted JeKo-1 cell animal model.

[0076] Figure 13, Anti-tumor efficacy detection of Ab2-ADC7 on NOG mouse subcutaneously transplanted HCC70 cell animal model.

[0077] Figure 14, Body weight change of Ab2-ADC7 on NOG mouse subcutaneously transplanted HCC70 cell animal model.

[0078] Figure 15, Anti-tumor efficacy detection of Ab7-ADC6, Ab7-ADC7 on BALB / c nude mouse subcutaneously transplanted MDA-MB-231 cell animal model.

[0079] Figure 16, Body weight changes of Ab7-ADC6, Ab7-ADC7 in the animal model of BALB / c nude mice subcutaneously transplanted with MDA-MB-231 cells.

[0080] Figure 17, Anti-tumor efficacy of Ab2-ADC7 in the animal model of BALB / c nude mice subcutaneously transplanted with MDA-MB-231 cells.

[0081] Figure 18, Body weight changes of Ab2-ADC7 in the animal model of BALB / c nude mice subcutaneously transplanted with MDA-MB-231 cells.

[0082] Figure 19, Anti-tumor efficacy of Ab2-ADC7 in the animal model of NCG mice subcutaneously transplanted with breast cancer PDX animal model LD1-2009-362153.

[0083] Figure 20, Body weight changes of Ab2-ADC7 in the animal model of NCG mice subcutaneously transplanted with breast cancer PDX animal model LD1-2009-362153.

[0084] Figure 21, Anti-tumor efficacy of Ab2-ADC7 in the animal model of Nu / Nu mice subcutaneously transplanted with breast cancer PDX animal model LD1-2009-361973.

[0085] Figure 22, Body weight changes of Ab2-ADC7 in the animal model of Nu / Nu mice subcutaneously transplanted with breast cancer PDX animal model LD1-2009-361973.

[0086] Figure 23, Anti-tumor efficacy of Ab2-ADC7 in the animal model of NU / NU mice subcutaneously transplanted with lymphoma PDX animal model LD1-0026-410827.

[0087] Figure 24, Body weight changes of Ab2-ADC7 in the animal model of NU / NU mice subcutaneously transplanted with lymphoma PDX animal model LD1-0026-410827.

[0088] Figure 25, Anti-tumor efficacy of Ab2-ADC7 in the animal model of BALB / c nude mice subcutaneously transplanted with lung cancer PDX animal model LU-01-1621.

[0089] Figure 26, Body weight changes of Ab2-ADC7 in the animal model of BALB / c nude mice subcutaneously transplanted with lung cancer PDX animal model LU-01-1621. DETAILED DESCRIPTION

[0090] The present inventors have made intensive studies and provided an anti-ROR1 antibody, and an immunoconjugate comprising the anti-ROR1 antibody, and use in the preparation of a medicament for treating and / or preventing a disease, a disease associated with overexpression of ROR1, such as a tumor.

[0091] Definitions

[0092] The term "ROR1" or "Receptor tyrosine kinase-like orphan receptor 1" refers to Receptor tyrosine kinase-like orphan receptor family 1 (mRNA: NM_005012.4 (transcript variant 1), mRNA: NM_001083592.2 (transcript variant 2), Protein: NP_001077061.1).

[0093] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen recognition site within a variable region of the immunoglobulin molecule. As used herein, "antibody" includes intact monoclonal antibodies, intact polyclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single domain antibodies (VHH), single chain Fv (scFv), multispecific antibodies (e.g., bispecific antibodies that bind to at least two different epitopes), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen recognition site of an antibody, and any other modified configuration of the immunoglobulin molecule that exhibits the desired biological activity, so long as the antibody exhibits the desired biological activity. An antibody can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy chain constant regions, designated alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. An antibody can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0094] The sequences of about 110 amino acids near the N-terminus of both the heavy and light chains are highly variable and form the variable region (V region); the remaining amino acids are relatively stable and form the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity-determining region (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of three CDR regions and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal. The three CDR regions of the light chain refer to light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), and light chain complementarity determining region 3 (LCDR3); the three CDR regions of the heavy chain refer to heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3).

[0095] The CDRs of an antibody can be determined by a variety of numbering systems, such as CCG, Kabat, Chothia, IMGT, a combination of Kabat / Chothia, etc. These numbering systems are known in the art, and can be found, for example, at www.bioinf.org.uk / abs / index.html#kabatnum. For example, as used herein with respect to an antibody, the CDRs of the antibody can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0096] As used herein, an "antigen binding fragment" of an antibody or immunoglobulin chain (heavy or light) includes a portion of an antibody that lacks some amino acids compared to a full-length chain, but which can specifically bind to an antigen. Such a fragment can be considered biologically active in that it can specifically bind to a target antigen, or can compete with other antibodies or antigen binding fragments for binding to a particular epitope. In some embodiments, such a fragment comprises at least one CDR present in a full-length light or heavy chain, and in some examples, it includes a short chain heavy and / or light chain or portions thereof. Such biologically active fragments can be produced by recombinant DNA techniques or can be produced, for example, by enzymatic or chemical cleavage of intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFv, scFv-Fc, diabodies, minibodies, scAb, and dAbs, and can be derived from any mammal, including but not limited to humans, mice, rats, camels, or rabbits. Functional portions of antibodies (such as one or more CDRs as disclosed herein) can be linked by covalent bonds to secondary proteins or small molecule compounds, and thereby used as targeted therapeutics for specific targets.

[0097] Methods for producing and purifying antibodies and antigen binding fragments are well known in the art and can be found, for example, in Current Protocols in Immunology, Chapters 5-8 and 15. For example, mice can be immunized with human ROR1 or fragments thereof, the resulting antibodies can be renatured, purified, and can be subjected to amino acid sequencing using conventional methods. Antigen binding fragments can likewise be prepared using conventional methods. The antibodies or antigen binding fragments described herein have one or more human FR regions added to the CDR regions of non-human origin using genetic engineering methods. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) at http: / / imgt.cines.fr by alignment of the IMGT human antibody variable germline genes database and MOE software, or from The Immunoglobulin Journal, 2001 ISBN 012441351.

[0098] The term "anti-RORl antibody" or "antibody that binds to RORl" means an antibody that is capable of binding RORl with sufficient affinity that the antibody is useful as a diagnostic and / or therapeutic agent. An anti-RORl antibody binds to a non- related, non-RORl protein (e.g., ROR2) to a lesser extent, e.g., by about 10% less, as measured by a radioimmunoassay (RIA) or surface plasmon resonance (SPR) technique, than the extent of binding of the antibody to RORl.

[0099] The term "Kon" or "Ka" refers to the association rate constant, a constant used to assess how quickly an antibody and antigen combine to form an antigen-antibody complex. The term "Koff" or "Kd" refers to the dissociation rate constant, a constant used to assess how quickly an antigen-antibody complex dissociates back into an antibody and an antigen. The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Generally, the antibodies of the present application bind ROR1 with a dissociation equilibrium constant (KD) of less than about 10"7M, for example, less than about 10"8M, 10"9M, or 10"10M or less.

[0100] The term "humanized antibody" also known as CDR-grafted antibody refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. This can overcome the heterogeneity reaction induced by chimeric antibodies due to carrying a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. The germline DNA sequences of human heavy and light chain variable regions can be found in the "VBase" human germline sequence database (available on the internet at www.mrccpe.com.ac.uk / vbase), as well as in Kabat, E. A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th Ed. To avoid the decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal back or reverse mutations to maintain activity.

[0101] A "humanized antibody" can retain the antigenic specificity of the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies can minimally contain a chimeric antibody that includes sequences derived from non-human immunoglobulin. In some cases, CDR region residues in the human immunoglobulin (recipient antibody) are replaced by CDR region residues from a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, that have the desired properties, affinities, and / or abilities. In some cases, FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain amino acid modifications not found in the recipient or donor antibodies. These modifications can be made to further improve the properties of the antibody, such as binding affinity.

[0102] The term "antibody fragment" means a portion of an intact antibody and includes antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single-chain antibodies, and multi-specific antibodies formed from antibody fragments.

[0103] "Sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are the same between the sequences. "Sequence similarity" represents the percentage of amino acids that are either the same or represent a conservative amino acid substitution. Methods for assessing the degree of sequence identity or sequence similarity between amino acids or nucleotides are known to those of skill in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.

[0104] An "effective amount" of an agent refers to the amount necessary to effect a desired physiological change in the cells or tissues receiving its administration.

[0105] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents adverse effects of a disease.

[0106] The term "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0107] The term "pharmaceutical composition" refers to a preparation which is in such form as to be effective for use in therapy, and which contains at least one active ingredient. A composition of the present application can be formulated to be compatible with its intended route of administration.

[0108] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is not toxic to the subject to which the composition is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0109] The term "treatment / prevention" (and grammatical variations thereof) refers to attempting to alter the natural course of a disease in an individual, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of disease, alleviating symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, ameliorating or eliminating symptoms of disease states, and improving prognosis. In some embodiments, the antibodies of the present application are used to delay development of a disease or to slow the progression of a disorder.

[0110] The term "detectable label" refers to a marker that can be attached to an antibody for determining the presence or absence of a particular target in a subject to be tested and the amount of presence. The "detectable label" can be, but is not limited to, an enzyme, a fluorescent label, a radionuclide, a quantum dot, colloidal gold, etc. More specifically, for example, it can be selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0111] In the present application, the tumor is a ROR1-expressing tumor; preferably, the ROR1-expressing tumor is a B-cell related tumor. The tumor includes, but is not limited to, lymphoma (including but not limited to B-cell leukemia, B-cell chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, Burkitt's lymphoma, mantle cell lymphoma), breast cancer (including but not limited to, for example, breast ductal carcinoma, triple negative breast cancer), lung cancer (including but not limited to non-small cell lung cancer, for example, lung adenocarcinoma, lung squamous cell carcinoma, lung large cell neuroendocrine carcinoma, lung lymphoepithelioma-like carcinoma, etc., small cell lung cancer), ovarian cancer, neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, squamous cell carcinoma of the epithelium, melanoma, myeloma, gastric cancer, brain cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer, head and neck cancer, and combinations thereof. The triple negative breast cancer refers to breast cancer that is negative for the expression of the genes of estrogen receptor (ER), progesterone receptor (PR) or Her2 / neu.

[0112] Anti-ROR1 antibody

[0113] The present application provides an anti-ROR1 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises: an immunoglobulin heavy chain variable region (VH) comprising a heavy chain HCDR1, a HCDR2 and a HCDR3, wherein the HCDR1 is or comprises an amino acid sequence as set forth in SEQ ID NO: 1, the HCDR2 is or comprises an amino acid sequence as set forth in SEQ ID NO: 2, and the HCDR3 is or comprises an amino acid sequence as set forth in SEQ ID NO: 3; and an immunoglobulin light chain variable region (VL) comprising a heavy chain LCDR1, a LCDR2 and a LCDR3, wherein the LCDR1 is or comprises an amino acid sequence as set forth in SEQ ID NO: 4, the LCDR2 is or comprises an amino acid sequence as set forth in SEQ ID NO: 5, and the LCDR3 is or comprises an amino acid sequence as set forth in SEQ ID NO: 6.

[0114] The present application provides an anti-ROR1 antibody, which comprises a heavy chain variable region of an amino acid sequence as shown in SEQ ID NO: 7, 9 or 10, and a light chain variable region of an amino acid sequence as shown in SEQ ID NO: 8, 11, 12, 13 or 14. The present application also includes an antibody with a heavy chain variable region amino acid sequence having 85% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identity to the sequence shown in SEQ ID NO: 7, 9 or 10, a light chain variable region amino acid sequence having 85% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identity to the sequence shown in SEQ ID NO: 8, 11, 12, 13 or 14, and having the same function as the antibody described in the examples of the present application; preferably, the CDRs in the heavy chain variable region / light chain variable region of the humanized antibody are conserved.

[0115] The present application provides an anti-ROR1 antibody, which comprises HCDR1, HCDR2 and HCDR3 contained in an amino acid sequence as shown in SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 contained in an amino acid sequence as shown in SEQ ID NO: 8; or

[0116] The present application provides an anti-ROR1 antibody, which comprises HCDR1, HCDR2 and HCDR3 contained in an amino acid sequence as shown in SEQ ID NO: 9, and LCDR1, LCDR2 and LCDR3 contained in an amino acid sequence as shown in SEQ ID NO: 11; or

[0117] The present application provides an anti-ROR1 antibody, which comprises HCDR1, HCDR2 and HCDR3 contained in an amino acid sequence as shown in SEQ ID NO: 9, and LCDR1, LCDR2 and LCDR3 contained in an amino acid sequence as shown in SEQ ID NO: 12; or

[0118] The present application provides an anti-ROR1 antibody, which comprises HCDR1, HCDR2 and HCDR3 contained in an amino acid sequence as shown in SEQ ID NO: 9, and LCDR1, LCDR2 and LCDR3 contained in an amino acid sequence as shown in SEQ ID NO: 13; or

[0119] the anti-RORl antibody comprises HCDR1, HCDR2, and HCDR3 contained within the amino acid sequence of SEQ ID NO: 9, and LCDR1, LCDR2, and LCDR3 contained within the amino acid sequence of SEQ ID NO: 14; or

[0120] the anti-RORl antibody comprises HCDR1, HCDR2, and HCDR3 contained within the amino acid sequence of SEQ ID NO: 10, and LCDR1, LCDR2, and LCDR3 contained within the amino acid sequence of SEQ ID NO: 11; or

[0121] the anti-RORl antibody comprises HCDR1, HCDR2, and HCDR3 contained within the amino acid sequence of SEQ ID NO: 10, and LCDR1, LCDR2, and LCDR3 contained within the amino acid sequence of SEQ ID NO: 12; or

[0122] the anti-RORl antibody comprises HCDR1, HCDR2, and HCDR3 contained within the amino acid sequence of SEQ ID NO: 10, and LCDR1, LCDR2, and LCDR3 contained within the amino acid sequence of SEQ ID NO: 13; or

[0123] the anti-RORl antibody comprises HCDR1, HCDR2, and HCDR3 contained within the amino acid sequence of SEQ ID NO: 10, and LCDR1, LCDR2, and LCDR3 contained within the amino acid sequence of SEQ ID NO: 14.

[0124] The present application provides an anti-RORl antibody, which comprises HCDR1, HCDR2, and HCDR3 contained within the amino acid sequence of SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 contained within the amino acid sequence of SEQ ID NO: 8; and

[0125] the anti-RORl antibody comprises an amino acid mutation at position 44 and / or position 49 of the amino acid sequence of SEQ ID NO: 7, preferably the mutation is G44R, S49A; and / or

[0126] the anti-RORl antibody comprises an amino acid mutation at one, two, three, or all of positions 2, 49, 64, and 67 of the amino acid sequence of SEQ ID NO: 8, preferably the mutation is I2T, Y49D, G64S, S67Y; and / or

[0127] the anti-RORl antibody comprises an amino acid mutation at one, two, or all of positions 4, 22, and 73 of the amino acid sequence set forth in SEQ ID NO: 8, preferably the mutation is M4V, T22R, L73F; and / or

[0128] the anti-RORl antibody comprises an amino acid mutation at position 13 of the amino acid sequence set forth in SEQ ID NO: 8, preferably the mutation is A13M.

[0129] The present application provides an anti-RORl antibody comprising a heavy chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7, and a light chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8; or

[0130] the anti-RORl antibody comprises a heavy chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11 ; or

[0131] the anti-RORl antibody comprises a heavy chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12; or

[0132] the anti-RORl antibody comprises a heavy chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13; or

[0133] the anti-RORl antibody comprises a heavy chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14; or

[0134] the anti-RORl antibody comprises a heavy chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10, and a light chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11 ; or

[0135] the anti-RORl antibody comprises a heavy chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10, and a light chain variable region of an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12; or

[0136] the anti-RORl antibody comprises a heavy chain variable region of an amino acid sequence identical to or having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to or having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13; or

[0137] the anti-RORl antibody comprises a heavy chain variable region of an amino acid sequence identical to or having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to or having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14.

[0138] The present disclosure provides an anti-RORl antibody comprising: a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:

[0139] the VH region and the VL region are or comprise an amino acid sequence as set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or

[0140] the VH region and the VL region are or comprise an amino acid sequence as set forth in SEQ ID NO: 9 and SEQ ID NO: 11, respectively; or

[0141] the VH region and the VL region are or comprise an amino acid sequence as set forth in SEQ ID NO: 9 and SEQ ID NO: 12, respectively; or

[0142] the VH region and the VL region are or comprise an amino acid sequence as set forth in SEQ ID NO: 9 and SEQ ID NO: 13, respectively; or

[0143] the VH region and the VL region are or comprise an amino acid sequence as set forth in SEQ ID NO: 9 and SEQ ID NO: 14, respectively; or

[0144] the VH region and the VL region are or comprise an amino acid sequence as set forth in SEQ ID NO: 10 and SEQ ID NO: 11, respectively; or

[0145] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 12, respectively; or

[0146] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 13, respectively; or

[0147] the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 14, respectively.

[0148] The present disclosure provides an anti-RORl antibody comprising a heavy chain (HC) having an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18, 20, or 25, and a light chain (LC) having an amino acid sequence that is identical to or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to 19, 21, 22, 23, or 24. The present disclosure provides an anti-RORl antibody comprising: a heavy chain (HC) and a light chain (LC), wherein:

[0149] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 18, 20, or 25;

[0150] the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 19, 21, 22, 23, or 24.

[0151] The present disclosure provides an anti-RORl antibody comprising: a heavy chain (HC) and a light chain (LC), wherein:

[0152] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 18; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 19; or

[0153] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 21; or

[0154] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 22; or

[0155] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 23; or

[0156] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 24; or

[0157] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 21; or

[0158] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 22; or

[0159] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 23; or

[0160] the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 24.

[0161] The anti-ROR1 antibody provided by the present application can specifically bind to human ROR1 efficiently, can block the action of ROR1 efficiently, and can carry another substance coupled thereto to a target cell efficiently. The anti-ROR1 antibody provided by the present application can include, but is not limited to, a monoclonal antibody, a bispecific antibody, a diabody, a multispecific antibody, a multibody, a minibody, a domain antibody, an antibody mimetic (or a synthetic antibody), a chimeric antibody, or an antibody fusion (or an antibody conjugate) and a fragment thereof, and includes various forms of antibodies disclosed herein. The antibody fragment of the antibody disclosed in the present application can include Fab, Fab', F(ab')2, scFab, Fv, dsFv, scFv, scFv-Fc, a minibody, a bifunctional antibody, scAb, or dAb.

[0162] The antibody disclosed in the present application can consist of a polypeptide of only a heavy chain comprising a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 7, 9, or 10, or only a light chain comprising a light chain variable region of an amino acid sequence as set forth in SEQ ID NO: 8, 11, 12, 13, or 14.

[0163] An antibody disclosed in the present disclosure can share specific regions or sequences with another antibody disclosed in the present disclosure. In some embodiments, it can share the constant region of an antibody or antigen binding fragment. In some embodiments, it can share the Fc region. In some embodiments, it can share the framework of the variable region.

[0164] In some embodiments, the antibody has the typical structure of an antibody found in nature. Camelids produce antibodies consisting of a single heavy chain, but the structural unit of such antibodies typically comprises a tetrameric polypeptide, wherein the tetramer comprises a pair of two polypeptide chain bodies consisting of different 2 polypeptide chains. In a typical antibody, the pair of polypeptide chain bodies comprises one full-length light chain (about 25 kDa) and one full-length heavy chain (about 50 to 70 kDa). Each chain shows a characteristic folding pattern and is composed of several immunoglobulin domains, which are composed of about 90 to 110 amino acids. These domains are the basic units that make up the antibody polypeptide. The amino-terminal portion of each chain typically comprises a portion called the variable region or V region, which recognizes the antigen. The carboxy-terminal portion is more evolutionarily conserved than the amino-terminal and includes a portion called the constant region or C region. Human light chains are typically classified as kappa (K) or lambda (λ) light chains, and these respectively comprise one variable region and one constant region.

[0165] Heavy chains are typically classified as mu (μ), delta (δ), gamma (γ), alpha (α) or epsilon (ε) chains, and these are respectively defined as IgM, IgD, IgG, IgA and IgE isotypes. IgG has various subtypes, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM subtypes include IgM and IgM2. IgA subtypes include IgA1 and IgA2. In humans, IgA and IgD isotypes comprise 4 heavy chains and 4 light chains.

[0166] IgG and IgE isotypes comprise 2 heavy chains and 2 light chains, and IgM isotypes comprise 5 heavy chains and 5 light chains. The heavy chain constant region typically includes at least one domain that exhibits effector function. The number of heavy chain constant regions varies depending on the isotype. IgG heavy chains, for example, comprise 3 C regions called CH1, CH2 and CH3, respectively. The antibodies disclosed in the present disclosure can be any of these isotypes and subtypes. In some embodiments, the antibody is an IgG1, IgG2a, IgG2b, IgG3 or IgG4 subtype. In some embodiments, the antibody of the present disclosure is an IgG1 type or an IgG2 type. In another embodiment, the antibody of the present disclosure is an IgG1 type.

[0167] The heavy chain variable region and the light chain variable region according to the present application can be linked to at least a portion of a human constant region. The choice of constant region can be determined in part by whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity is desired. For example, human isotypes IgGl and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not. In addition, human IgGl and IgG3 induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be lambda or kappa.

[0168] In some embodiments, the heavy chain and light chain variable regions disclosed herein can be freely combined to make various forms of antibodies, and for example, can form single antibodies, such as ScFv or domain antibodies, or full length antibodies.

[0169] Each of the heavy chain and light chain variable regions disclosed herein can be combined with various target heavy and light chain constant regions to form the heavy and light chains of a complete antibody, respectively. In addition, the respective heavy and light chain sequences combined with the constant regions can be further combined to form a complete antibody structure.

[0170] Any of the variable regions of the heavy or light chains of the antibodies according to the present application can be linked to at least a portion of a constant region. The constant region can be chosen according to whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity, etc. is desired. Those skilled in the art will be able to know that other variable regions, including IgGl, IgG2, IgG3, or IgG4 heavy chain constant regions, any kappa or lambda light chain constant regions, or other modified variable regions can be combined with the variable regions disclosed herein to achieve a target characteristic (e.g. stability, expression, manufacturability, or other).

[0171] One or more of the residues in the above disclosed heavy chain, light chain, variable region, or CDR sequences of the antibodies of the present application can comprise conservative amino acid substitutions. Conservative amino acid substitutions refer to substitutions that do not substantially affect the activity of the polypeptide or its antigenicity. In some embodiments, conservative amino acid substitutions refer to substitution with another residue belonging to the same class in the following classification of amino acids. Naturally occurring amino acids can be classified according to common side-chain properties as follows: 1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He; 2) neutral hydrophilic: Cys, Ser, Thr, Gin; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe. Conservative amino acid substitutions can also include non-naturally occurring amino acid residues, such as peptidomimetics, and such residues are typically introduced via chemical synthesis rather than cellularly. Non-conservative substitutions include substitutions with residues from other classes in the above classification. Such substitutions can be made in regions of an antibody that are homologous to antibodies of the present application or in regions that are not homologous thereto.

[0172] The present application also includes a fusion protein of an anti-ROR1 antibody, which includes a first domain of an antibody or antibody fragment as described in the present application and a second domain for prolonging in vivo half-life and / or having conjugation with effector cells. The fusion protein can be a binding molecule capable of specifically binding to cells expressing ROR1. In the second domain, the fragment for prolonging in vivo half-life can include serum albumin or a fragment thereof, polyethylene glycol, a serum albumin-binding domain (such as an anti-serum albumin antibody), a polyethylene glycol-liposome complex, etc. In the second domain, the fragment for having conjugation with effector cells can include an immunoglobulin Fc region, etc., preferably selected from a human immunoglobulin Fc region. The human immunoglobulin Fc region includes a mutation for changing Fc-mediated effector functions, including one or more of a combination of CDC activity, ADCC activity, and ADCP activity. The immunoglobulin can be selected from one or more of a combination of IgG, IgA1, IgA2, IgD, IgE, IgM, etc., and the IgG can be specifically selected from one or more of a combination of IgG1, IgG2, IgG3, or IgG4 subtypes, etc. The immunoglobulin Fc region included in the antibody fusion protein can allow the fusion protein to form a dimer, while prolonging the in vivo half-life of the fusion protein and increasing Fc-mediated related activities. In the present application, the immunoglobulin Fc region can be an Fc region of human IgG1, and more specifically can be a wild-type IgG1 Fc sequence, which can be introduced with a mutation for changing Fc-mediated effector functions, for example, a) a mutation for changing Fc-mediated CDC activity; b) a mutation for changing Fc-mediated ADCC activity; or c) a mutation for changing Fc-mediated ADCP activity.

[0173] In the fusion protein of the anti-ROR1 antibody according to the present application, a linker peptide can be provided between the first domain and the second domain. The linker peptide can be a flexible polypeptide chain composed of alanine (A) and / or serine (S) and / or glycine (G), and the length of the linker peptide can be 3 to 30 amino acids, preferably 3 to 9, 9 to 12, 12 to 16, 16 to 20, for example, the length of the linker peptide can be 8 or 15.

[0174] In a specific embodiment of the present application, the present inventors conjugated the humanized anti-ROR1 antibody with a drug to prepare an antibody-drug conjugate, and as a result, it was shown to have a significant killing effect on tumor cells.

[0175] The present application also provides an isolated polynucleotide encoding the antibody of the present application, or encoding the fusion protein. The polynucleotide can be RNA, DNA or cDNA, etc. The method for providing the isolated polynucleotide should be known to one skilled in the art, for example, can be prepared by automatic DNA synthesis and / or recombinant DNA technology, etc., can also be isolated from suitable natural sources.

[0176] pharmaceutical agent

[0177] The present application also provides a pharmaceutical agent capable of conjugating with the antibody of the present application to form an immunoconjugate (e.g. antibody-drug conjugate). The pharmaceutical agent includes (but is not limited to) a molecule inhibiting tumor, a molecule targeting tumor surface marker, a molecule targeting surface marker of immune cell, a radioactive group, a detectable label, or a combination thereof.

[0178] In some embodiments, the drug of the present application can be a tumor-inhibiting molecule. Specifically, the tumor-inhibiting molecule includes, but is not limited to, camptothecin (CPT), such as, but not limited to, exatecan and its derivatives deruxtecan (Dxd), irinotecan (DX-8951f), lurtotecan (GG-211), CZ48, HM910, sinotecan, lipotecan (TLC388), 9-nitrocamptothecin (9-NC), 9-aminocamptothecin (9-AC), kanitecan (BNP-1350), gimitecan (ST1481), difloretcan (BN-80915), 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (AR-67), CPT2C; maytansinoid; auristatin, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF); dolastatin; dolastatin; calicheamicin or its derivatives; anthracycline, such as daunorubicin or doxorubicin; methotrexate; vindesine; taxane, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecene; CC1065; cytokine, such as IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, etc. In terms of mechanism of action, the tumor-inhibiting molecule can be an anti-tumor toxin, including toxins acting on microtubulin proteins, such as monomethyl auristatin (MMAE) related compounds and their derivatives, maytansinoid related compounds and their derivatives; toxins acting on DNA, such as duocarmycin, calicheamicin, pyrrolobenzodiazepines (PBDs), SN-38, Dxd, CPT2C, etc. related compounds and their derivatives; and related compounds and their derivatives acting on other functions within cells, such as metabolism, transcription, translation, signal transduction, etc. The present application also includes analogs, isomers, precursors, etc. of these small molecule compounds as toxins.

[0179] As a preferred mode of the present application, the drug described in the present application is a camptothecin (CPT) molecule, such as but not limited to: camptothecin, 20-deoxycamptothecin, 10-methoxyamptothecin, 9-methoxycamptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, exatecan, deruxtecan (Dxd), DX-8951f, lurtotecan (GG-211), topotecan, irinotecan (CPT11), simmitecan, sinotecan, lipotecan (TLC388), simmitecan (ST1481), diflomotecan (BN-80915), CZ48, Camptothecin chloroacetate (DLSFFZ93W4), Camptothecin lysinate (NSC610457), HM910, SN38, GI-149893, cositecan (karenitecin), Elomotecan (BN-80927), 11-cyanocamptothecin (NSC609951), 7-acetyl-Camptothecin (SCHEMBL6851483), 9-Glycineamidocamptothecin HCl (MLS000756803), 7-hydroxymethylcamptothecin, 9-Glycineamidocamptothecin HCl (MLS000756803), 10-amino-11-hydroxy-camptothecin (BDBM50285230), diflomotecan, 9-nitrocamptothecin (9-NC), 9-aminocamptothecin (9-AC), 10-aminocamptothecin (10-AC), kanitecan (BNP-1350), 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (AR-67), GX-2 (see CN117164601A for details, the relevant contents of CN117164601A are hereby incorporated in their entirety), CPT2C.

[0180] As a preferred mode of the present application, the CPT2C of the present application has a structure as shown in Formula II.

[0181] In some embodiments, the agent of the present application can also be an enzyme active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the enediyne antitumor antibiotics.

[0182] In some embodiments, the agent can be at least one molecule targeting a tumor surface marker, which can work synergistically with the antibody of the present application to more precisely target tumor cells. In other embodiments, the agent can also be a molecule targeting a surface marker of immune cells. The molecule targeting a surface marker of immune cells can recognize immune cells, which carry the antibody of the present application to the immune cells, while the antibody of the present application can target the immune cells to tumor cells, thereby inducing immune cells to specifically kill tumors while utilizing the killing effect of the antibody of the present application.

[0183] For therapeutic purposes, the agent can be a radioactive moiety, i.e., a moiety consisting of or including a radioisotope or radionuclide (e.g., 3H, 14C, 15N, 33P, 35S, 90Y, 99Tc, 111In, 123I, 125I, 131I, 201TI, 213Bi), a toxin, or a cytotoxic moiety such as a cytostatic agent.

[0184] For detection purposes, the agent can be a detectable label. Detectable labels include, but are not limited to, fluorescent labels, chromogenic labels, protein tags; such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase), prosthetic groups, fluorescent materials (e.g., fluorescent proteins such as GFP, RFP, etc., dyes, rhodamine, fluorescein and its derivatives such as FITC, cyanine dyes), luminescent materials, bioluminescent materials, radioactive materials, chemiluminescent moieties, biotin moieties, metallic particles (e.g., gold particles), magnetic particles (e.g., having a core containing magnetite (Fe3O4) and / or maghemite (Fe2O3)), predetermined polypeptide moieties, etc., and can comprise more than one label. The label used to label the antibody for detection and / or analysis and / or diagnostic purposes depends on the particular detection / analysis / diagnostic technique and / or method to be used, such as immunohistochemical staining of tissue samples, flow cytometry, etc. Suitable labels for detection / analysis / diagnostic techniques and / or methods known in the art are well known to those skilled in the art.

[0185] Immunoconjugates

[0186] The present application also provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof of the present application, or a fusion protein of the present application. The immunoconjugate typically further comprises a drug linked (including but not limited to covalently linked, coupled, attached, adsorbed) to the antibody or antigen-binding fragment thereof or fusion protein.

[0187] As used herein, an antibody, or a fusion protein or fragment thereof of the present application can be linked to a drug. The linkage can be through one or more covalent bonds, or non-covalent interactions, and can include chelation. A variety of linkers (which can be known in the art) can be used to form the immunoconjugate. In some specific embodiments, the immunoconjugate can be an antibody-drug conjugate (ADC), having the formula Ab-(L-D)y, wherein: Ab is an anti-RORl antibody or antigen-binding fragment thereof, or a fusion protein thereof, of the present application; L is a linker; D is a drug moiety; and y is an arbitrary integer or decimal number (e.g., an integer or decimal number from 1 to 10, from 1 to 8, from 5 to 8, or from 7 to 8). In addition, immunoconjugates can be provided in the form of fusion proteins, which can be expressed from a polynucleotide encoding the immunoconjugate.

[0188] In immunoconjugates, the antibody and drug can be cross-linked via a linker, which can be a cleavable linker, such as a linker that can be enzymatically cleaved in vivo and / or in vitro, such as a peptide linker, a disulfide linker, or a hydrazone linker. Immunoconjugates can release the drug once metabolized in vivo (e.g., within a tumor cell) and / or in vitro. The linker is preferably a "cleavable linker" that can facilitate release of the drug in a cell (e.g., a tumor cell), such as an acid-labile linker, a peptidase-sensitive linker, a light-labile linker, a dimethyl linker, and a disulfide-containing linker. As a preferred aspect of the application, the linker is a tumor-specific linker, including a peptide linker that can be cleaved by intracellular proteases (e.g., lysosomal proteases or endosomal proteases) and an acid-cleavable linker.

[0189] In certain embodiments, the linker can include a dipeptide, such as a valine-citrulline (VC), valine-alanine (VA), or phenylalanine-lysine (FK) linker; a tripeptide, such as a glutamic acid 0 valine-citrulline (EVC) linker, a tetrapeptide, such as a glycine-glycine 0 phenylalanine-glycine (GGFG) linker, and a polypeptide having four or more amino acid residues, wherein one or more of the amino acid residues contain a lipophilic side chain. Other suitable linkers include linkers that are hydrolyzable at a pH less than 5.5, such as a hydrazone linker. Other suitable cleavable linkers include disulfide linkers, non-polymeric linkers, non-peptide linkers, or linkers that do not contain amino acid residues.

[0190] In certain embodiments, the linker can have a reactive functional group, which can include, for example, a nucleophilic group that is reactive toward an electrophilic group. Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, and acid anhydrides. Exemplary nucleophilic groups include hydrazides, oximes, amines, hydrazines, aminothioureas, carboxylic acid hydrazides, and arylhydrazines. In some embodiments, conjugation of the drug to the antibody can be formed by reaction with a maleimide group, which can also be referred to as a maleimide spacer. The maleimide group can be a maleimidocaproyl (MC), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC). In some embodiments, conjugation of the drug to the antibody can be formed by reaction with N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP) or N-succinimidyl 3(2-pyridyldithio) propionate (SPDP).

[0191] In certain embodiments, the linker can include a benzoic acid or benzyloxy group, or a derivative thereof. For example, the linker can comprise p-aminobenzoic acid (PABA), p-amino-benzyloxycarbonyl (PAB), or gamma-aminobutyric acid (GABA).

[0192] It is understood that the linker can comprise a peptide moiety, a reactive functional group, a benzoic acid (e.g., PABA) or benzyloxycarbonyl (PAB) group, or any combination thereof. Exemplary combinations include, but are not limited to: MC-VA, MC-VC, MC-VA-PAB, MC-VC-PAB, MC-GGFG, SC-VC-PAB. In some preferred embodiments, the linker can be, but is not limited to: SPDP, SMCC, SuO-VA-PAB, SuO-VC-PAB, MC-VA-PAB, MC-VC-PAB, MC-GGFG.

[0193] In some embodiments, the linker forms a covalent bond with a cysteine residue on the anti-ROR1 antibody or antigen-binding fragment thereof. In some preferred embodiments, the linker covalently binds to the anti-ROR1 antibody or antigen-binding fragment thereof at the succinimidyl, carbonyl, cyclooctene, quaternized vinylpyridine, or triazole group of the linker.

[0194] The linker can be conjugated to the anti-RORl antibody or antigen-binding fragment thereof in a variety of ways. Typically, the linker and drug moiety are synthesized and conjugated prior to attachment to the antibody, first forming a (L-D) structure, which is then conjugated to the Ab moiety. In some preferred embodiments,MC-GGFG-CPT2C, MC-VC-PAB-MMAE, MC-GGFG-Dxd, MC-VA-MMAE, MC-VC-MMAE, MC-VA-PAB-MMAE, MC-GGFG-MMAE, SC-VC-PAB-MMAE, MC-VA-MMAF, MC-VC-MMAF, MC-VA-PAB-MMAF, MC-GGFG-MMAF, SC-VC-PAB-MMAF, MC-VC-PAB-MMAF, MC-GGFG-camptothecin, MC-GGFG-20-deoxycamptothecin, MC-GGFG-10-methoxyamptothecin, MC-GGFG-9-methoxycamptothecin, MC-GGFG-10-hydroxycamptothecin, MC-GGFG-7-ethyl-10-hydroxycamptothecin, MC-GGFG-exatecan, MC-GGFG-DX-8951f, MC-GGFG-GG-211, MC-GGFG-topotecan, MC-GGFG-CPT11, MC-GGFG-simmitecan, MC-GGFG-sinotecan, MC-GGFG-TLC388, MC-GGFG-ST1481, MC-GGFG-BN-80915, MC-GGFG-CZ48, MC-GGFG-DLSFFZ93W4, MC-GGFG-NSC610457, MC-GGFG-HM910, MC-GGFG-SN38, MC-GGFG-GI-149893, MC-GGFG-cositecan, MC-GGFG-BN-80927, MC-GGFG-NSC609951, MC-GGFG-7-acetyl-Camptothecin, MC-GGFG-9-Glycineamidocamptothecin HCl, MC-GGFG-7-hydroxymethylcamptothecin, MC-GGFG-9-Glycineamidocamptothecin HCl, MC-GGFG-10-amino-11-hydroxy-camptothecin, MC-GGFG-diflomotecan, MC-GGFG-9-NC, MC-GGFG-9-AC, MC-GGFG-10-AC, MC-GGFG-BNP-1350, MC-GGFG-AR-67, CZY-8 (Please refer to CN117164601A,The relevant content of CN117164601A is hereby incorporated in its entirety.

[0195] In some embodiments, the immunoconjugate described herein has a structure according to Formula I:

[0196] wherein y in Formula I is any integer or decimal number (e.g., an integer or decimal number from 1 to 10, from 1 to 8, from 5 to 8, or from 7 to 8).

[0197] Methods for preparing the immunoconjugate should be known to those skilled in the art, for example, the antibody and / or fusion protein can be linked to a drug directly or through a spacer of suitable length, and the linkage can be chemical cross-linking or genetically engineered fusion expression, so as to obtain the immunoconjugate.

[0198] The immunoconjugate can also be a chimeric antigen receptor (CAR) that can be expressed in an immune cell. The "immune cell" and "immune effector cell" are used interchangeably herein and include T lymphocytes, NK cells, NKT cells, and the like, preferably NK cells and T lymphocytes. The chimeric antigen receptor generally comprises, in sequence: an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular binding domain comprises the antibody or fusion protein of the present application. The design of the transmembrane domain and the intracellular signaling domain based on CAR technology is well known in the art: for example, the transmembrane domain uses the transmembrane domain of CD8, CD28, and the like, and the intracellular signaling domain uses the intracellular signaling domain of the immune receptor tyrosine activation motif (ITAM) CD3 zeta chain or Fc epsilon RI gamma tyrosine activation, and the co-stimulatory signal molecules CD28, CD27, CD137, CD134, MyD88, CD40, and the like. More specifically, for T lymphocytes, the intracellular signaling domain only contains ITAM, which is the first generation of CAR T lymphocytes, wherein the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-ITAM, which can trigger an anti-tumor cytotoxic effect; the second generation of CAR T lymphocytes adds the intracellular signaling domain of CD28 or CD137 (also known as 4-1BB), wherein the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-ITAM or scFv-TM-CD137-ITAM; the B7 / CD28 or 4-1BBL / CD137 co-stimulation in the intracellular signaling domain causes the sustained proliferation of T lymphocytes, and can increase the level of cytokines such as IL-2 and IFN-gamma secreted by T lymphocytes, while increasing the survival period of CAR T in vivo and the anti-tumor effect; the third generation of CAR T lymphocytes, wherein the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further improves the survival period of CAR T in vivo and its anti-tumor effect.

[0199] According to the above, the chimeric antigen receptor prepared using the antibody or fusion protein of the present application can be an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain connected in sequence: the antibody or fusion protein of the present application, CD8, and CD3 zeta; the antibody or fusion protein of the present application, CD8, CD137, and CD3 zeta; the antibody or fusion protein of the present application, the transmembrane domain of the CD28 molecule (CD28a), the intracellular signaling domain of the CD28 molecule (CD28b), and CD3 zeta; or the antibody or fusion protein of the present application, the transmembrane domain of the CD28 molecule, the intracellular signaling domain of the CD28 molecule, CD137, and CD3 zeta.

[0200] The chimeric antigen receptor is expressed on the surface of immune effector cells, and the immune effector cells have highly specific cytotoxic effects on tumor cells expressing ROR1 while killing effects of the antibodies of the present application.

[0201] Pharmaceutical composition

[0202] The present application also provides a pharmaceutical composition comprising the anti-ROR1 antibody of the present application, or the fusion protein of the anti-ROR1 antibody of the present application, or the drug of the present application, or the immunoconjugate of the present application.

[0203] The pharmaceutical composition can further comprise various pharmaceutically acceptable carriers in the art. The pharmaceutically acceptable carriers are non-toxic to the recipient at the dosages and concentrations employed, and can specifically include but are not limited to: buffers such as acetate, Tris, phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; chlorhexidine; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum proteins, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicity-adjusting agents such as trehalose and sodium chloride; sugar alcohols such as sucrose, mannitol, trehalose or sorbitol; surfactants such as polysorbate; salt-forming counterions such as sodium; metal complexes (such as Zn-protein complexes); and / or non-ionic surfactants such as or polyethylene glycol (PEG). The pharmaceutical preparation for in vivo administration is generally sterile, and methods for achieving sterility of the pharmaceutical preparation should be known to those skilled in the art, for example, by filtration through a sterile filter membrane, etc. Those skilled in the art can also select appropriate pharmaceutically acceptable carriers according to the desired dosage form of the pharmaceutical composition, to prepare it into different dosage forms, for example, the pharmaceutical composition of the present application can be various dosage forms including but not limited to tablets, injections, lyophilized agents, etc.

[0204] The content of the fusion protein, drug, and immunoconjugate in the pharmaceutical composition is typically an effective amount, and the effective amount of the active ingredient can be determined according to the subject to be treated and the specific administration method. For example, the content of the fusion protein, drug, and immunoconjugate can range from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99%, based on the total mass of the pharmaceutical composition.

[0205] The fusion protein, drug, immunoconjugate, and pharmaceutical composition of the present application can be administered as a single active ingredient, or can be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy can be the fusion protein, drug, immunoconjugate, pharmaceutical composition in combination with at least one other anti-tumor drug. For another example, the combination therapy can be the fusion protein, drug, immunoconjugate, pharmaceutical composition in combination with an antibody targeting other tumor-specific antigens, such as, but not limited to, a combination of one or more of an anti-EGFR antibody, an anti-VEGF antibody, an anti-HER2 antibody, or an anti-Claudin18A2 antibody, and the like, and the inhibitor can preferably be a monoclonal antibody.

[0206] Use

[0207] The present application also provides the use of the antibody, fusion protein, drug, immunoconjugate, or pharmaceutical composition of the present application in the preparation of a medicament for the diagnosis, treatment, or prevention of a disease associated with cells expressing (or overexpressing) ROR1. In some specific embodiments, the disease is a tumor (including a primary tumor, a locally advanced tumor, a metastatic tumor); the tumor is preferably a tumor expressing ROR1; and more preferably, the tumor includes, but is not limited to, a lymphoma (including, but not limited to, B-cell leukemia, B-cell chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, Burkitt's lymphoma, mantle cell lymphoma), breast cancer (including, but not limited to, breast ductal carcinoma, triple-negative breast cancer), lung cancer (including, but not limited to, non-small cell lung cancer, such as lung adenocarcinoma, lung squamous cell carcinoma, lung large cell neuroendocrine carcinoma, lung lymphoepithelioma-like carcinoma, and the like, small cell lung cancer), ovarian cancer, neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, squamous cell carcinoma of the epithelium, melanoma, myeloma, gastric cancer, brain cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer, head and neck cancer, and combinations thereof.

[0208] A "therapeutically effective amount" of an antibody, fusion protein, drug, immunoconjugate, or pharmaceutical composition of the present application preferably results in a decrease in severity of a disease symptom, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of ROR1 -associated tumors (including, e.g., lymphoma, ovarian cancer, endometrial cancer, breast cancer, cervical cancer, etc.), a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80%, relative to a subject who has not received treatment. The ability to inhibit tumor growth can be assessed in an animal model system predictive of efficacy in human tumors. Alternatively, the ability to inhibit cell growth can also be assessed, which can be determined in vitro by assays known to those skilled in the art. A therapeutically effective amount of a fusion protein, drug, immunoconjugate, or pharmaceutical composition is generally capable of reducing tumor size, or otherwise ameliorating symptoms in a subject. Those skilled in the art can determine appropriate therapeutically effective amounts depending on the actual circumstances, e.g., the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen. Prescription of treatment (e.g., the decision to dose, etc.) can be made by a physician, and factors considered by the physician in making the determination can include, but are not limited to, the disease being treated, the individual patient status, the delivery site, the method of administration, and other factors. A prophylactically effective amount refers to an amount effective to prevent a desired effect, at a reasonable benefit / risk ratio applicable to any medical treatment. A prophylactically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. Ordinarily but not necessarily, a prophylactically effective amount is lower than a therapeutically effective amount. Typically, but not necessarily, a prophylactically effective amount is lower than a therapeutically effective amount because the prophylactic dose is used in subjects prior to or at the early stages of disease, and thus the disease has not progressed to the extent as would be the case in a therapeutic setting.

[0209] In particular embodiments of the present application, dosing regimens for animals such as mice are provided. Conversion of dosing amounts for animals such as mice to dosing amounts suitable for humans is readily made by those skilled in the art, for example, according to the Meeh-Rubner formula: A = k'(W2 / 3) / 10,000. In this formula, A is the body surface area in m2; W is the body weight in g; and k is a constant that varies with the species of animal, generally 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It is understood that conversion of dosing amounts can vary depending on the drug and the clinical situation, as assessed by an experienced clinician.

[0210] The present application also provides kits comprising the antibodies, fusion proteins, drugs, immunoconjugates, and / or pharmaceutical compositions described herein. In addition, the kits can include instructions for using the antibodies, fusion proteins, drugs, immunoconjugates, and / or pharmaceutical compositions in the kits.

[0211] The present application also provides a detection kit comprising the antibody, fusion protein or immunoconjugate of the present application. The kit can also comprise, as needed, containers, controls (negative or positive controls), buffers, adjuvants, etc., which can be selected by those skilled in the art according to the specific circumstances. The kit can also comprise instructions for use to facilitate the operation of those skilled in the art.

[0212] The present application further provides detection methods for detecting ROR1 protein using the antibody, including but not limited to qualitative detection, quantitative detection and localization detection. Specifically, the detection methods include but are not limited to immunofluorescence assay, immunohistochemistry and radioimmunoassay, etc.

[0213] A method for detecting the presence or absence of ROR1 protein in a sample can comprise contacting the sample with the antibody or antigen-binding fragment thereof of the present application; observing whether an antibody complex is formed, and if an antibody complex is formed, it indicates that ROR1 protein is present in the sample. The sample can be a cell and / or tissue sample; the sample can be fixed or dissolved in a medium; the level of ROR1 protein in the fixed or dissolved sample can be detected. In some embodiments, the detection object can be a cell-containing sample present in a cell preservation solution. In other embodiments, the antibody is also conjugated with a fluorescent dye, a chemical substance, a polypeptide, an enzyme, an isotope, a tag, etc. that can be used for detection or can be detected by other reagents.

[0214] The embodiments of the present application are described below by way of specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure in the specification. The present application can also be implemented or applied by other different specific embodiments, and the details in the specification can be modified or changed based on different views and applications without departing from the spirit of the present application.

[0215] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are used to describe the specific embodiments, but not to limit the scope of protection of the present application.

[0216] When the embodiments give numerical ranges, it should be understood that unless the application indicates otherwise, every numerical range's two endpoints, and any number between the two endpoints, can be selected. Unless otherwise defined, all technical and scientific terms used in the application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Except in the examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any method, device, material, or the like that is similar in principle to those described in the examples and that would be known or reasonably inferred by one of ordinary skill in the art in view of the present teachings and the general knowledge in the art can be used in the practice of the application.

[0217] Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the application all use conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the existing literature.

[0218] Example 1 Verification of immunization antigen activity

[0219] In this embodiment, the immunization antigen activity of hROR1-Fc is verified. hROR1-Fc (Sino Biological, Item No.: 13968-H02H1) contains the antigen active fragment Metl-Glu403, and the sequence of this fragment in human, cynomolgus monkey, and rhesus monkey ROR1 is homologous. The steps for verifying the immunization antigen activity of hROR1-Fc are as follows:

[0220] (1) Antigen coating: Prepare a concentration gradient dilution of human ROR1-Fc, denoted as hROR1-Fc (Sino Biological, Item No.: 13968-H02H1) solution, add 100 μl per well, and coat overnight at 4°C.

[0221] (2) Washing: Wash three times with PBST (containing 0.05% Tween 20) solution.

[0222] (3) Blocking: Block the antigen with DPBS (containing 3% BSA), add 200 μl per well, and incubate at room temperature for 1 h.

[0223] (4) Antibody binding: Dilute SPH-Cirm-01 (Biointron, Item No.: B266401) to 1 μg / mL with DPBS (containing 1% BSA) solution, add 100 μl per well, and incubate at 37°C for 1 h.

[0224] (5) Secondary antibody binding: Discard the reaction solution, wash three times with PBST solution, add the secondary antibody, add 100 μl per well, and incubate at 37°C for 1 h.

[0225] (6) ELISA detection: Discard the reaction solution, wash three times with PBST solution; add 100 μΐ TMB solution to each well, gently shake the reaction until blue appears; add 50 μΐ H2SO4 to each well, gently shake the reaction until yellow appears; detect OD value at 450 nm.

[0226] As shown in Figure 1, the results of the hROR1-Fc antigen activity experiment determined by ELISA showed that the hROR1-Fc antigen had good antigen activity and could be used as a mouse immunogen in the later hybridoma experiment.

[0227] Example 2 Preparation and screening of anti-ROR1 antibodies

[0228] Anti-ROR1 antibodies were obtained by immunizing 6-week-old female BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., 18 grams in weight). Certain biological properties of exemplary anti-ROR1 antibodies produced according to the method of this embodiment are described in detail in the examples set forth below.

[0229] 1. Preparation of hybridoma cells

[0230] Hybridoma technology is to fuse the spleen cells of mice immunized with antigens and mouse myeloma cells SP2 / 0, so as to maintain the main characteristics of the two cells at the same time. The main characteristic of the spleen cells of mice immunized with specific antigens (B lymphocytes) is its antibody secretion function, but it cannot be continuously cultured in vitro, while mouse myeloma cells have so-called immortality, which can divide and proliferate indefinitely under culture conditions. Under the action of selection medium, only hybrid cells formed by the fusion of B cells and myeloma cells can form cell clones with both antibody secretion function and cell immortality. In this experiment, the mice were immunized with human ROR1-Fc (Sino Biological, item number: 13968-H02H1) and RIBI adjuvant (Sigma, item number: 038M4131v) adjuvant, and the mice were immunized three times for protein cell mixed immunization. Then the obtained mouse spleen cells and SP2 / 0 cells were fused to obtain hybridoma cells capable of expressing positive antibodies.

[0231] 1.1 Animal immunization

[0232] The experimental animals and immunization information are shown in Table 1.

[0233] Table 1 Experimental animals and immunization information

[0234] 1.2 Detection of serum titer of immunized mice after immunization

[0235] Antigen coating: 1 μg / ml human ROR1-his (KACTUS, Cat: ROR-HM401) solution was prepared with DPBS solution, 100 μl was added to each well of 96-well plate, and coated at 4°C overnight.

[0236] Washing: PBST (containing 0.05% Tween 20) solution was used for washing three times.

[0237] Blocking: The antigen was blocked with DPBS (containing 3% BSA), 100 μl was added to each well, and incubated at room temperature for 1 h.

[0238] Antibody binding: The serum solution was diluted with DPBS (containing 1% BSA) solution, the first concentration was diluted 300 times, and then 2 times gradient dilution was performed, 8 concentrations were diluted, 100 μl was added to each well; the negative control was the serum of unimmunized mice. Incubate at room temperature for 1 h.

[0239] Secondary antibody binding: Discard the reaction solution, wash with PBST solution for three times; add secondary antibody at a dilution ratio of 1:5000, 100 μl was added to each well, and incubate at room temperature for 1 h.

[0240] ELISA detection: Discard the reaction solution, wash with PBST solution for three times; add 100 μl TMB solution to each well, gently shake until blue color appears; add 50 μl H2SO4 to each well, gently shake until yellow color appears; detect OD value at 450 nm.

[0241] The serum titers of multiple immunized mice are shown in Figure 2, and the values are shown in Table 2. The results show that 5 immunized mice all produce immune response, and the titer values are all above 218.7k, and subsequent boosting can be performed for fusion experiment.

[0242] Table 2 Serum titers of immunized mice

[0243] 2. Hybridoma fusion

[0244] 2.1 Mix the spleen cells and SP2 / 0 mouse myeloma cells at a ratio of 5:1 to 2:1, centrifuge, and co-precipitate. After removing the supernatant, resuspend the cells with an appropriate amount of fusion buffer, adjust the mixed cell density to 1×10 7 After the fusion is completed, the cells are placed in the electrode cartridge for 5 minutes, then the cells are added to 20% FBS 1×HAT selection culture medium, transferred to a 96-well cell culture plate, and incubated in a 37°C, 5% CO2 incubator. Change the fresh culture medium once after 7-8 days.

[0245] 2.2 After culturing for 10 days (or longer, depending on the cell growth state), the supernatant was taken and the amount of antibody expressed in the supernatant of the hybridoma was determined by FACS method, and hybridoma cells expressing specific anti-ROR1 antibodies were screened. The positive clones obtained by the preliminary screening were transferred to 24-well plates for culture, and then the supernatant was identified by re-screening, and positive clones were selected for 1-2 rounds of subcloning, and hybridoma monoclonal cells stably expressing specific anti-ROR1 antibodies were screened again, and the hybridoma cell line with the highest expression was selected, and the cell sample and Trizol sample were frozen.

[0246] The V region gene of the screened murine anti-ROR1 antibody was recombined with the C region gene of a human antibody to form a chimeric anti-ROR1 antibody, which was named Ab0. Then, the framework region was changed to form humanized anti-ROR1 antibodies Ab1-Ab8.

[0247] The CDRs, VH, VL, and heavy and light chain sequences of the chimeric anti-ROR1 antibody Ab0 and the humanized anti-ROR1 antibodies Ab1-Ab8 are given below.

[0248] CDRs of Ab0-Ab8:

[0249] HCDR1 (SEQ ID NO: 1): SYVMS

[0250] HCDR2 (SEQ ID NO: 2): SISSGGSTYYPDSVKG

[0251] HCDR3 (SEQ ID NO: 3): GPSTMINAMDY

[0252] LCDR1 (SEQ ID NO: 4): ITTTDIDDSMN

[0253] LCDR2 (SEQ ID NO: 5): EGNNLRP

[0254] LCDR3 (SEQ ID NO: 6): LQSDNLPYT

[0255] Ab0 VH (SEQ ID NO: 7, wherein the bold underlined part is the CDRs)

[0256] Ab0 VL (SEQ ID NO: 8, wherein the bold underlined part is the CDRs)

[0257] Ab1, Ab2, Ab3, Ab4 VH (SEQ ID NO: 9, wherein the bold underlined part is the CDRs)

[0258] Ab5, Ab6, Ab7, Ab8 VH (SEQ ID NO: 10, with CDRs bolded and underlined)

[0259] Ab1, Ab5 VL (SEQ ID NO: 11, with CDRs bolded and underlined)

[0260] Ab2, Ab6 VL (SEQ ID NO: 12, with CDRs bolded and underlined)

[0261] Ab3, Ab7 VL (SEQ ID NO: 13, with CDRs bolded and underlined)

[0262] Ab4, Ab8 VL (SEQ ID NO: 14, with CDRs bolded and underlined)

[0263] Positive control antibody VH (SEQ ID NO: 15, with CDRs bolded and underlined)

[0264] Positive control antibody VL (SEQ ID NO: 16, with CDRs bolded and underlined)

[0265] Negative control antibody (SEQ ID NO: 17, human IgGl)

[0266] Ab0 (human IgGl) heavy chain, with VH bolded and underlined (SEQ ID NO: 18)

[0267] Ab0 (human Kappa) light chain, with VL bolded and underlined (SEQ ID NO: 19)

[0268] Ab1 (human IgGl) heavy chain, with VH bolded and underlined (SEQ ID NO: 20)

[0269] Ab1 (human Kappa) light chain, with VL bolded and underlined (SEQ ID NO: 21)

[0270] Ab2 (human IgGl) heavy chain, where the bold underlined is VH (SEQ ID NO: 20)

[0271] Ab2 (human Kappa) light chain, where the bold underlined is VL (SEQ ID NO: 22)

[0272] Ab3 (human IgGl) heavy chain, where the bold underlined is VH (SEQ ID NO: 20)

[0273] Ab3 (human Kappa) light chain, where the bold underlined is VL (SEQ ID NO: 23)

[0274] Ab4 (human IgGl) heavy chain, where the bold underlined is VH (SEQ ID NO: 20)

[0275] Ab4 (human Kappa) light chain, where the bold underlined is VL (SEQ ID NO: 24)

[0276] Ab5 (human IgGl) heavy chain, where the bold underlined is VH (SEQ ID NO: 25)

[0277] Ab5 (human Kappa) light chain, where the bold underlined is VL (SEQ ID NO: 21)

[0278] Ab6 (human IgGl) heavy chain, where the bold underlined is VH (SEQ ID NO: 25)

[0279] Ab6 (human Kappa) light chain, where the bold underlined is VL (SEQ ID NO: 22)

[0280] Ab7 (human IgGl) heavy chain, where the bold underlined is VH (SEQ ID NO: 25)

[0281] Ab7 (human Kappa) light chain, where the bold underlined is VL (SEQ ID NO: 23)

[0282] Ab8 (human IgGl) heavy chain, where the bold underlined is VH (SEQ ID NO: 25)

[0283] Ab8 (human Kappa) light chain, where the bold underlined is VL (SEQ ID NO: 24)

[0284] Example 3 Binding of Anti-RORl Antibodies to MDA-MB-231 Cells

[0285] The binding ability of anti-RORl antibodies to human RORl expressed on the surface of triple negative breast cancer MDA-MB-231 cells was determined based on a flow cytometry assay. The binding ability of different anti-RORl antibodies of the present application was determined by comparing the binding curves of the different anti-RORl antibodies to human RORl expressed on the surface of MDA-MB-231 cells.

[0286] (1) Triple negative breast cancer MDA-MB-231 cells (Chinese Academy of Sciences Cell Bank, item number SCSP-5043) were plated in a 96-well plate.

[0287] (2) The positive control antibody (Uptake cross-linking, item number B013-DS-210602), the negative control antibody (human IgGl, novoprotein, item number NC002), and the antibody of the antigen binding protein of the present application were each prepared in PBS containing 2% FBS to a maximum concentration of 5 μg / ml, 5-fold dilution, 8 points, and the diluted samples were added to the 96-well plate, which was incubated at 4 degrees Celsius for 1 hour.

[0288] (3) The plate was washed 3 times with PBS containing 2% FBS.

[0289] (4) PE-labeled human IgG (Invitrogen, item number 12-4998-82) was diluted with 2% FBS in PBS according to the product instructions, and the diluted sample was added to the 96-well plate, which was incubated at 4 degrees Celsius for 0.5 hours.

[0290] (5) The plate was washed 2 times with PBS containing 2% FBS.

[0291] (6) The cells were resuspended with 2% FBS in PBS, and the median fluorescence value (MFI) in the PE channel was determined using a flow cytometer.

[0292] As shown in FIGS. 3 and 4, the binding curves of anti-RORl antibodies to human RORl expressed on the surface of MDA-MB-231 cells were determined based on a flow cytometry assay.

[0293] As shown in Table 3, the EC values and the highest MFI values of the binding of anti-RORl antibodies to human RORl expressed on the surface of MDA-MB-231 cells were determined based on a flow cytometry assay. 50

[0294] ​Table 3 EC of anti-RORl antibodies binding to RORl expressed on the surface of MDA-MB-231 cells 50 Values and highest MFI values

[0295] The above experiments show that the anti-RORl antibodies of the present application have binding activity to human RORl expressed on MDA-MB-231 cells, and 6 humanized anti-RORl antibodies have binding activity to RORl superior to the positive control antibody.

[0296] Example 4 Binding of anti-RORl antibodies to JeKo-1 cells

[0297] The binding ability of anti-RORl antibodies to JeKo-1 cells was determined based on a flow cytometry assay. The binding ability of different anti-RORl antibodies of the present application to JeKo-1 cells was determined by comparing their binding curves.

[0298] (1) JeKo-1 cells (SIBS, Cat. No. TCHu194) were plated in a 96-well plate.

[0299] (2) The positive control antibody (U-Clone, Cat. No. B013-DS-210602), the negative control antibody (human IgGl, novoprotein, Cat. No. NC002), and the anti-RORl antibodies of the present application were each prepared in PBS containing 2% FBS to a maximum concentration of 5 μg / ml, 5-fold dilution, 8 points, and the diluted samples were added to the 96-well plate, which was incubated at 4°C for 1 hour.

[0300] (3) The plate was washed 3 times with PBS containing 2% FBS.

[0301] (4) PE-labeled human IgG (Invitrogen, Cat. No. 12-4998-82) was diluted in PBS containing 2% FBS according to the product instructions, and the diluted sample was added to the 96-well plate, which was incubated at 4°C for 0.5 hour.

[0302] (5) The plate was washed 2 times with PBS containing 2% FBS.

[0303] (6) The cells were resuspended in PBS containing 2% FBS, and the median fluorescence value (MFI) in the PE channel was determined using a flow cytometer.

[0304] The binding curves of anti-RORl antibodies to JeKo-1 cells based on flow cytometry are shown in FIGS. 5 and 6.

[0305] EC of anti-RORl antibodies to JeKo-1 cells based on flow cytometry 50 The values and highest MFI values are shown in Table 4.

[0306] Table 4 EC of anti-RORl antibodies to JeKo-1 cells 50 Values and maximum MFI values

[0307] The above tests show that the anti-RORl antibodies of the present application have binding activity to JeKo-1 cells, and 6 anti-RORl antibodies are superior to the positive control antibody in activity.

[0308] Example 5 Binding of anti-RORl antibodies to A549 cells

[0309] The binding ability of anti-RORl antibodies to human lung adenocarcinoma A549 cells was determined based on a flow cytometry assay. The binding ability of different anti-RORl antibodies of the present application to A549 cells was determined by comparing their binding curves.

[0310] (1) Human lung adenocarcinoma A549 cells (Chinese Academy of Sciences Cell Bank, item number TCHu150) were plated in a 96-well plate.

[0311] (2) The positive control antibody (Uptake cross-linking, item number B013-DS-210602), the negative control antibody (human IgGl, novoprotein, item number NC002), and the antigen-binding protein antibodies of the present application were prepared in PBS containing 2% FBS at a maximum concentration of 5 μg / ml, 5-fold dilution, 8 points, and the diluted samples were added to the 96-well plate, which was incubated at 4°C for 1 hour.

[0312] (3) The plate was washed 3 times with PBS containing 2% FBS.

[0313] (4) PE-labeled human IgG (Invitrogen, item number 12-4998-82) was diluted with 2% FBS in PBS according to the product instructions, and the diluted sample was added to the 96-well plate, which was incubated at 4°C for 0.5 hours.

[0314] (5) The plate was washed 2 times with PBS containing 2% FBS.

[0315] (6) The cells were resuspended with 2% FBS in PBS, and the median fluorescence value (MFI) in the PE channel was determined using a flow cytometer.

[0316] As shown in FIGS. 7 and 8, the binding curves of anti-RORl antibodies to human RORl expressed on the surface of A549 cells were determined based on a flow cytometry assay.

[0317] As shown in Table 5, the EC of anti-RORl antibodies to human RORl expressed on the surface of A549 cells was determined based on a flow cytometry assay 50 Values and maximum MFI values.

[0318] Table 5 EC of anti-RORl antibodies to RORl binding expressed on the surface of A549 cells 50 Values and highest MFI values

[0319] The above experiments showed that the anti-RORl antibodies of the present application had binding activity to human RORl expressed on A549 cells, and 6 humanized anti-RORl antibodies had activity superior to the positive control antibody.

[0320] Example 6 Binding affinity of anti-RORl antibodies

[0321] The binding affinity of different anti-RORl antibodies to the antigen Human-cyno-RORl, His Tag (source: Acro Biosystem, item number: ROl-H522y) protein was detected by a molecular interaction instrument.

[0322] (1) Buffer preparation: 50 ml PBS added with 10 μl Tween 20, 220 μl per well added to the Buffer wells of a Bio-One 96-well black flat-bottom plate (source: Greiner, item number: 655209).

[0323] (2) The antibody was diluted to 5 μg / ml with PBST (0.02% Tween 20), and 220 μl per well was added to the antibody sample wells of a Bio-One 96-well black flat-bottom plate.

[0324] (3) Antigen preparation: Human-cyno-RORl, His Tag antigen was diluted to 100 nM with PBST (0.02% Tween 20), and 220 μl per well was added to the antigen sample wells of a Bio-One 96-well black flat-bottom plate.

[0325] (4) Regeneration solution: 220 μl per well of Glycine solution was added to the Regeneration wells of a Bio-One 96-well black flat-bottom plate.

[0326] (5) Put new Bio-One 96-well black flat-bottom plates on the left side of the MIA, put green tray on it, put ProteinA Dip and Read Biosensors (source: ForteBio, item number: 18-0015) in the first column of the tray. Put the Bio-One 96-well black flat-bottom plates containing prepared samples on the middle or right side of the MIA.

[0327] (6) Put the plates into the MIA for detection and analysis.

[0328] The binding affinities of different anti-ROR1 antibodies to Human-cyno-ROR1 antigenic proteins were detected by the MIA, as shown in Table 6.

[0329] Table 6 Binding affinities of anti-ROR1 antibodies to Human-cyno-ROR1 antigenic proteins

[0330] The results show that the anti-ROR1 antibodies of the application have strong binding affinities to Human-cyno-ROR1 antigens, and are comparable to the affinities of the positive control antibodies.

[0331] Example 7 Cross-reaction test of anti-ROR1 antibodies

[0332] In this example, the binding of anti-ROR1 antibodies to human ROR2-His (source: Acro, item number: RO2-H52E5) protein was detected.

[0333] (1) Prepare ROR2 His antigen 0.5 μg / ml with PBS, add to 96-well plates (Corning, item number: 9018), and incubate at 4°C overnight.

[0334] (2) The next day, discard the coating solution, and wash the plates 3 times with 0.05% Tween 20 in PBS.

[0335] (3) Incubate with 3% BSA in PBS at 37°C for 2 hours, and wash the plates 3 times with 0.05% Tween 20 in DPBS.

[0336] (4) Prepare the test antibodies with 1% BSA in PBS at a maximum concentration of 15 μg / ml, 5-fold dilution, and 8 concentration points. Add to the 96-well plates, and incubate at room temperature for 1 hour. Wash the plates 3 times with 0.05% Tween 20 in PBS.

[0337] (5) The secondary antibody HRP-Goat anti-Human IgG Fc (Jackson, item number 109-035-098) was diluted with PBS containing 1% BSA according to the product manual, and the diluted sample was added to the 96-well plate, and incubated at room temperature for 0.5 hours. The plate was washed 3 times with PBS containing 0.05% Tween 20.

[0338] (6) 100 μl of TMB substrate color developing liquid was added to each well, and incubated at room temperature for 15 minutes.

[0339] (7) 100 μl of 2N H2SO4 termination liquid was added to each well, and the value was read at 450 nm wavelength with an enzyme-labeled instrument and recorded.

[0340] The binding curve of the anti-RORl antibody to the human ROR2 antigen is shown in Figure 9.

[0341] The binding ability of the anti-RORl antibody to the human ROR2 antigen is shown in Table 7.

[0342] Table 7 Binding ability of the humanized anti-RORl antibody to the human ROR2 antigen

[0343] The above experiment shows that the anti-RORl antibody of the present application has no binding activity to the human ROR2-His antigen, indicating that the anti-RORl antibody of the present application has binding specificity to the RORl antigen.

[0344] Example 8 Endocytosis biological activity of the anti-RORl antibody

[0345] After gradient dilution of the antibody to be tested, the antibody was incubated with the DT3C diluent at room temperature to couple the antibody to the toxin, and then added to the 96-well plate to co-incubate with the CHOKl / hRORl cells for 3 days, and then MTS was added to detect the toxicity of the toxin-coupled antibody to the cells. DT3C has little toxicity to the cells outside the cells, and only after the antibody is endocytosed into the cells will it cause growth inhibition, so the amount of endocytosis of the antibody into the cells is reflected by the degree of growth inhibition of the cells.

[0346] (1) On the first day, the CHOKl / hRORl cells were digested, the cells were resuspended and counted, the supernatant was removed by centrifugation, and the cell density was adjusted to 4E4.

[0347] (2) The cells were plated in the 96-well cell culture plate, 50 μL was added to each well, i.e. 2E3 cells were seeded in each well, and the cell culture plate was gently shaken to mix, and then incubated in a 37°C cell culture incubator for 16 hours.

[0348] (3) The next day, dilute DT3C to 2X (2 μg / mL) working concentration with F12K / 10% FBS. Dilute the antibody from the maximum concentration of 5 μg / ml (2X) with 2-fold dilution, 9 points. Add 50 μL per well to make the sample concentration as shown in the experimental design. Set two wells with 50 μl of complete medium consistent with the DT3C concentration of the experimental group, and the rest of the wells are supplemented with F12K complete medium to 100 μL.

[0349] (4) Gently mix and place the cell culture plate in a 37°C cell incubator for 72 hours.

[0350] (5) After 3 days of culture, add 7 μL of Triton-X 100 to the corresponding wells of each plate, and incubate at 37°C for 15 min (this step is to lyse the cells, which is the positive control well for the cytotoxicity experiment), and at the same time, pre-melt the MTS at room temperature in the dark.

[0351] (6) Take the cell plate out of the incubator, and add 20 μL of MTS to each well. After gently mixing, incubate in a 37°C constant temperature incubator for 2.5 h, and observe the color change and gently shake during the incubation.

[0352] (7) After the color has a clear distinction, use an enzyme-labeled instrument to detect: OD490 reading.

[0353] Calculation formula: Background value = cell only-Lysis; Inhibition rate = 1- (experimental group-Lysis) / (background value) * 100%.

[0354] As shown in Figure 10, the anti-ROR1 antibody has good endocytosis activity, and the endocytosis activity of the anti-ROR1 antibody Ab2 is better than that of the positive control antibody.

[0355] As shown in Table 8, the anti-ROR1 antibody has a concentration-dependent effect on cell growth inhibition.

[0356] Table 8 Anti-ROR1 antibody endocytosis activity detection in CHOK1 / hROR1 cells

[0357] The above experiments show that the anti-ROR1 antibody of the present application has a concentration-dependent effect on cell growth inhibition and has good endocytosis activity.

[0358] Example 9 Preparation of Ab2-ADC7

[0359] Synthesis of compound 10:

[0360] Compound 10 was synthesized by dissolving (2-bromoethoxy)(tert-butyl)diphenylsilane (22.5 g, refer to Synlett 2014; 25(19): 2802-2805) and 1H-pyrazole-4-carbaldehyde (5.0 g, CAS: 35344-95-7, purchased from Shaoyuan Technology (Shanghai) Co., Ltd.) in 100 mL of anhydrous N,N-dimethylformamide, adding potassium carbonate (8.6 g) at room temperature, heating to 85 °C for 4 hours. TLC monitoring of the complete reaction of raw materials, cooling to room temperature. Add 200 mL of water to quench the reaction, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter, concentrate under reduced pressure, and then separate by column chromatography to obtain compound 10 as a colorless oil 12.8 g in 65% yield. 1 H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 8.02 (s, 1H), 7.98 (s, 1H), 7.54-7.48 (m, 4H), 7.45-7.33 (m, 6H), 4.27 (t, J = 5.1 Hz, 2H), 4.00 (t, J = 5.1 Hz, 2H), 1.00 (s, 9H).

[0361] Synthesis of compound K4:

[0362] Step 1: Synthesis of compound 11

[0363] Compound 9 (1.0 g, synthesis method refer to patent CN111470998A, example 1) was dissolved in 15 mL of anhydrous tetrahydrofuran under nitrogen protection and cooled at -78 °C for half an hour. A solution of n-butyllithium in hexane (2.5 M, 3.5 mL) was added dropwise at low temperature, and a solution of compound 10 (1.5 g) in anhydrous tetrahydrofuran (5 mL) was added quickly after the dropwise addition was completed. The reaction was continued at -78 °C for one hour. After TLC detection of the complete reaction of the raw materials, the reaction was quenched with saturated ammonium chloride solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by column chromatography to obtain compound 11 as a light yellow oil 0.6 g in 25% yield. 1 H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.95 (d, J = 11.6 Hz, 1H), 7.52-7.28 (m, 14H), 6.91 (d, J = 8.2 Hz, 1H), 5.86 (s, 1H), 4.24-4.13 (m, 2H), 3.99-3.87 (m, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 0.96 (s, 9H). MS (ESI) m / z = 546.3 (M+H + ).

[0364] Step 2: Synthesis of compound 12

[0365] Compound 11 (0.6 g) was dissolved in 15 mL acetonitrile under nitrogen protection, and manganese dioxide (0.86 g) was added. The reaction was heated to reflux for 3 hours. TLC was used to monitor the reaction. After the reaction was completed, the reaction mixture was filtered through celite and the filtrate was evaporated to dryness. Compound 12 was obtained as colorless oil by column chromatography. Yield: 0.55 g, 92%. 1 H NMR (400 MHz, CDC13) δ 10.82 (s, 1H), 8.39 (d, J = 12.4 Hz, 1H), 7.99 (s, 1H), 7.91 (s, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.55 - 7.33 (m, 10H), 4.30 (t, J = 5.0 Hz, 2H), 4.03 (t, J = 5.0 Hz, 2H), 2.27 - 2.15 (m, 6H), 1.00 (s, 9H). MS (ESI) m / z = 544.2 (M+H + ).

[0366] Step 3: Synthesis of compound 13

[0367] Compound 12 (0.55 g) was dissolved in 10 mL ethanol under nitrogen protection, and 3 mL concentrated hydrochloric acid was added. The reaction was heated to reflux for 3 hours. White solid was precipitated. TLC was used to monitor the reaction. After the reaction was completed, the reaction mixture was filtered and dried. Compound 13 was obtained as 0.19 g white solid. Yield: 80%. No further purification was performed, and the product was used directly in the next step.

[0368] Step 4: Synthesis of compound K4

[0369] Compound 13 (0.19 g) and compound B (0.22 g, CAS: 110351-94-5, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) were dissolved in 10 mL toluene and 5 mL acetic acid under nitrogen protection. The reaction was heated to 100°C overnight. TLC was used to monitor the reaction. After the reaction was completed, the reaction mixture was evaporated to dryness. Compound K4 was obtained as 0.28 g white solid by column chromatography. Yield: 80%. 1 H NMR (400 MHz, DMSO) δ 8.43 (s, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.08 (s, 1H), 7.91 (d, J = 10.8 Hz, 1H), 7.33 (s, 1H), 5.42 (s, 2H), 5.38 - 5.12 (m, 2H), 4.34 (t, J = 5.5 Hz, 2H), 3.88 (t, J = 5.6 Hz, 2H), 2.47 (s, 3H), 2.00 - 1.73 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). MS (ESI) m / z = 491.2 (M+H+).

[0370] Synthesis of compound mc-GGFG-CPT2C:

[0371] Step 1: Preparation of compound 15

[0372] Compound K4 (100 mg) and compound 14 (150 mg, CAS: 1599440-06-8, purchased from MCE company) were dissolved in 10 mL of anhydrous tetrahydrofuran under nitrogen protection, and pyridine p-toluenesulfonate (25 mg) was added at room temperature, and heated to 55 °C overnight. The reaction was quenched with saturated sodium bicarbonate solution, extracted with ethyl acetate, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by column chromatography to obtain compound 15, white solid 50 mg, yield 30%.

[0373] 1 H NMR (400 MHz, DMSO) δ 8.76 (t, J = 6.6 Hz, 1H), 8.40 (s, 1H), 8.11-8.01 (m, 2H), 7.85-7.77 (m, 3H), 7.65-7.55 (m, 3H), 7.39-7.22 (m, 5H), 6.52 (s, 1H), 5.40 (s, 2H), 5.22-5.05 (m, 2H), 4.63 (d, J = 6.6 Hz, 2H), 4.46 (t, J = 5.2 Hz, 2H), 4.25-4.18 (m, 2H), 4.17-4.10 (m, 1H), 3.90 (t, J = 5.4 Hz, 2H), 3.65 (d, J = 6.0 Hz, 2H), 2.42 (s, 3H), 1.90-1.81 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). MS (ESI) m / z = 799.3 (M+H + ).

[0374] Step 2: Preparation of compound 16

[0375] Compound 15 (50 mg) was dissolved in 5 mL of anhydrous tetrahydrofuran under nitrogen protection, and diethylamine (0.5 mL) was added at room temperature, and reacted at room temperature for 2 hours, and a solid was precipitated. TLC monitoring showed that the raw material was completely reacted, filtered and dried under vacuum to obtain compound 16, yellowish solid 30 mg, yield 83%. Without further purification, it was directly used in the next step reaction. MS (ESI) m / z = 577.2 (M+H + ).

[0376] Step 3: Preparation of compound mc-GGFG-CPT2C

[0377] Compound 17 (30 mg, CAS: 1599440-15-9, purchased from MCE company) was dissolved in 2 mL of anhydrous N,N-dimethylformamide under nitrogen protection, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (18.5 mg) and N,N-diisopropylethylamine (10 μL) were added at room temperature, and the reaction was carried out at room temperature for 2 hours. After the reaction solution became clear, compound 16 (30 mg) was added at room temperature and the reaction was carried out at room temperature overnight. LC-MS monitoring showed that the raw material 16 was completely reacted, and after direct spin drying, compound mc-GGFG-CPT2C was obtained by C18 reverse phase column purification, 27 mg of light yellow solid, yield 50%. 1 H NMR (400 MHz, DMSO) δ 8.57 (t, J = 6.6 Hz, 1H), 8.45 (s, 1H), 8.30 (t, J = 5.8 Hz, 1H), 8.17-8.07 (m, 3H), 8.04 (t, J = 5.7 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.90 (d, J = 10.7 Hz, 1H), 7.33 (s, 1H), 7.26-7.10 (m, 5H), 6.98 (s, 2H), 6.51 (s, 1H), 5.42 (s, 2H), 5.35-5.18 (m, 2H), 4.62 (d, J = 6.6 Hz, 2H), 4.51-4.41 (m, 3H), 3.89 (t, J = 5.4 Hz, 2H), 3.79-3.69 (m, 3H), 3.67-3.63 (m, 2H), 3.58 (dd, J = 16.7, 5.4 Hz, 1H), 3.35 (t, J = 7.1 Hz, 2H), 3.01 (dd, J = 13.8, 4.5 Hz, 1H), 2.76 (dd, J = 13.7, 9.7 Hz, 1H), 2.47 (s, 3H), 2.09 (t, J = 7.4 Hz, 2H), 1.94-1.79 (m, 2H), 1.52-1.39 (m, 4H), 1.23-1.12 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). MS (ESI) m / z = 1031.4 (M+H + )。

[0378] Ab2-ADC7 was prepared in the following formula, wherein Ab is Ab2, ADC7 is mc-GGFG-CPT2C:

[0379] To the antibody Ab2-containing PBS buffer (10 mM PBS buffer, pH = 7.4; 5.0 mg / mL, 4.0 mL, 133 nmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 112.7 uL, 1127 nmol) at 37 °C, and the reaction was stopped after 1.5 hours of oscillation at 37 °C in a water bath shaker.

[0380] The reaction solution was then cooled to 25 °C. Compound mc-GGFG-CPT2C (2.1 mg, 2036 nmol) was dissolved in 210 uL of DMSO and added to the above reaction solution, which was then placed in a water bath shaker and reacted for 0.5 hours at 25 °C. The reaction solution was purified using a MabSelect SuRe affinity chromatography column (mobile phase A: 10 mM PBS buffer, pH = 7.4; mobile phase B: 0.1 M acetic acid solution), and the sample was then exchanged into 10 mM PBS buffer, pH = 7.4, by passing through a Sephadex G25 desalting column to obtain Ab2-ADC7 PBS buffer (4.0 mg / mL, 4.5 mL), which was then stored at 4 °C.

[0381] The average drug loading calculated by RP-HPLC was y = 7.9.

[0382] Example 10 Preparation of Ab7-ADC7

[0383] Ab7-ADC7 of the following formula was prepared, where Ab is Ab7 and ADC7 is mc-GGFG-CPT2C:

[0384] To the antibody Ab7-containing PBS buffer (10 mM PBS buffer, pH = 7.4; 5.0 mg / mL, 2.0 mL, 67 nmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 66.7 uL, 667 nmol) at 37 °C, and the reaction was stopped after 1.5 hours of oscillation at 37 °C in a water bath shaker.

[0385] The reaction solution was then cooled to 25°C. Compound mc-GGFG-CPT2C (0.82 mg, 795 nmol) was dissolved in 82 uL DMSO and added to the above reaction solution, which was placed in a water bath shaker and shaken at 25°C for 0.5 hours. The reaction solution was purified by MabSelect SuRe affinity chromatography column (mobile phase A: 10 mM PBS buffer, pH = 7.4, mobile phase B: 0.1 M acetic acid solution), and then the sample was exchanged into 10 mM PBS buffer, pH = 7.4, by a Sephadex G25 desalting column to obtain the PBS buffer of Ab7-ADC7 (3.6 mg / mL, 2.1 mL), which was then stored at 4°C.

[0386] The average drug loading calculated by RP-HPLC: y = 7.9.

[0387] Example 11 Positive Control Antibody-ADC1 Preparation

[0388] A positive control antibody-ADC1 of the following formula was prepared, wherein Ab is a positive control antibody and ADC1 is mc-vc-PAB-MMAE:

[0389] A prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 50 uL, 500 nmol) was added to the PBS buffer (10 mM PBS buffer, pH = 7.4; 5.0 mg / mL, 6.0 mL, 200 nmol) containing the antibody positive control antibody, which was placed in a water bath shaker and shaken at 37°C for 1.5 hours, and then the reaction was stopped.

[0390] The reaction solution was then cooled to 25°C. Compound mc-vc-PAB-MMAE (2.1 mg, 1595 nmol) was dissolved in 210 uL DMSO and added to the above reaction solution, which was placed in a water bath shaker and shaken at 25°C for 0.5 hours. The reaction solution was purified by MabSelect SuRe affinity chromatography column (mobile phase A: 10 mM PBS buffer, pH = 7.4, mobile phase B: 0.1 M acetic acid solution), and then the sample was exchanged into 10 mM PBS buffer, pH = 7.4, by a Sephadex G25 desalting column to obtain the PBS buffer of the positive control antibody-ADC1 (8.4 mg / mL, 4.0 mL), which was then stored at 4°C.

[0391] The average drug loading calculated by RP-HPLC: y = 3.9.

[0392] Example 12 Ab7-ADC6 Preparation A prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 50 uL, 500 nmol) was added to the PBS buffer (10 mM PBS buffer, pH = 7.4; 5.0 mg / mL, 6.0 mL, 200 nmol) containing the antibody positive control antibody, which was placed in a water bath shaker and shaken at 37°C for 1.5 hours, and then the reaction was stopped.

[0393] Structure of mc-GGFG-Dxd:

[0394] To the PBS aqueous solution of antibody Ab7 (0.05 M PBS aqueous solution, pH = 6.5; 10 mg / mL, 3 mL, 200 nmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 160 μL, 1600 nmol) at 37 °C, and the reaction was stopped after 3 hours of oscillation in a water bath shaker at 37 °C.

[0395] The reaction solution was then cooled to 25 °C with a water bath. Compound mc-GGFG-Dxd (3.1 mg, 2998 nmol, CAS: 1599440-13-7, purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd.) was dissolved in 500 μL of DMSO and added to the above reaction solution, which was placed in a water bath shaker and reacted at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified by Sephadex G25 gel column (mobile phase: 0.05 M PBS aqueous solution, pH 6.5, containing 0.001 M EDTA), to obtain Ab7-ADC6 PBS buffer (4.5 mg / mL, 4.9 mL), which was then stored at 4 °C.

[0396] Average drug loading calculated by RP-HPLC: y = 7.8.

[0397] Example 13 Anti-tumor efficacy test of anti-ROR1 antibody-ADC7 in NOG mice subcutaneously transplanted with JeKo-1 cells

[0398] This test used a NOG mouse subcutaneously inoculated with JeKo-1 cell animal model to determine the anti-tumor effect of humanized anti-ROR1 antibody-ADC7.

[0399] NOG mice: Female NOG mice (6 weeks) were purchased from Beijing Vantoll Life Science and Technology Co., Ltd. The mice were adaptively fed for 7 days after arrival, and then the study began.

[0400] Cells: Human T cell lymphoma JeKo-1 cells (from the Shanghai Institute of Cell Biology) were routinely subcultured according to the instructions; the cells were resuspended in serum-free medium and the cell density was adjusted, and the cell suspension was subcutaneously inoculated into the right axillary fossa of female NOG mice on day 0 to establish a JeKo-1 tumor-bearing mouse model.

[0401] Dosing:

[0402] On the 6th day after tumor cell inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes of 107.45 mm 3 ~ 133.77 mm3 The mice in the range were divided into groups (5 mice per group) according to the average tumor volume, and were administered on the 6th and 20th day after inoculation, respectively, with a negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., item number: HSP067-F1), a positive control antibody-ADC1 (coupled with toxin MMAE) (SPH022-PC-2, upper drug crosslinking, item number: B013-DS-210602), and the antibody Ab2-ADC7 (coupled with toxin CPT2C) of the present application. The changes in tumor volume and body weight of the mice in each group were monitored during the administration period, with a monitoring frequency of 2 times / week for 3 consecutive weeks. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 27th day after inoculation, according to the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100; where Ti: the average tumor volume of the administration group, T0: the average tumor volume of the administration group on D0, Vi: the average tumor volume of the isotype control group, and V0: the average tumor volume of the isotype control group on D0. Tumor volume determination: the long diameter (a) and the wide diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2 x a x b 2 The body weight was measured using an electronic balance.

[0403] The administration dose and method are shown in Table 9.

[0404] Table 9 Administration test design

[0405] * Administration once every two weeks, for a total of 2 times.

[0406] As shown in Figure 11, the anti-tumor efficacy of Ab2-ADC7 was detected in the NOG mouse animal model of subcutaneously transplanted JeKo-1 cells.

[0407] As shown in Figure 12, the body weight changes of Ab2-ADC7 in the NOG mouse animal model of subcutaneously transplanted JeKo-1 cells.

[0408] As shown in Table 10, on the 27th day after inoculation, the tumor volume inhibition rate of the positive control antibody-ADC1 was 98.31%; Ab2-ADC7 significantly inhibited the growth of tumors at a dose of 3 mg / kg, with a tumor volume inhibition rate of 100.84%.

[0409] Table 10 Inhibition of tumor growth by Ab2-ADC7 in vivo on the 27th day

[0410] Note: **: P<0.01 compared with the tumor volume of the negative control antibody.

[0411] The above experiment showed that Ab2-ADC7 significantly inhibited the growth of JeKo-1 cell tumors and had no significant effect on the body weight of NOG mice.

[0412] Example 14 Anti-tumor efficacy test of anti-ROR1 antibody-ADC7 on NOG mice subcutaneously transplanted with HCC70 cell animal models

[0413] In this experiment, the anti-tumor effect of humanized anti-ROR1 antibody-ADC7 was determined using a NOG mouse subcutaneously inoculated with HCC70 cell animal model.

[0414] NOG mice: Female NOG mice (6 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were adaptively fed for 7 days after arrival, and then the study began.

[0415] Cells: Human breast ductal carcinoma HCC70 cells (ATCC source) were routinely subcultured according to the instructions; the cells were resuspended in serum-free medium and the cell density was adjusted, and the cell suspension was subcutaneously inoculated into the right front limb axillary fossa of female NOG mice on day 0 to establish a HCC70 tumor-bearing mouse model.

[0416] Dosing:

[0417] On day 9 after tumor cell inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes in the range of 137.50mm 3 ~ 229.45mm 3 were selected and evenly divided into groups according to tumor volume (5 mice per group). The negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), the positive control antibody-ADC1 (coupled with toxin MMAE) (SPH022-PC-2, source: Shangyi Crosslinking, catalog number: B013-DS-210602), and the antibody Ab2-ADC7 (coupled with toxin CPT2C) of the present application were administered on days 9, 16, 23, and 30 after inoculation, respectively. The changes in tumor volume and body weight of mice in each group were monitored during the administration period, with a monitoring frequency of 2 times / week for a continuous period of 10 weeks. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on day 82 after inoculation, using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100; where Ti: mean tumor volume of the administration group, T0: mean tumor volume of the administration group on day 0, Vi: mean tumor volume of the isotype control group, V0: mean tumor volume of the isotype control group on day 0. Tumor volume determination: The long diameter (a) and the wide diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2 x a x b 2 . The body weight was measured using an electronic balance.

[0418] The administration dose and mode are shown in Table 11.

[0419] Table 11 Administration test design

[0420] * Administration once a week, for a total of 4 times.

[0421] As shown in Figure 13, the anti-tumor efficacy of Ab2-ADC7 was tested in a NOG mouse animal model subcutaneously transplanted with HCC70 cells.

[0422] As shown in Figure 14, the body weight change of Ab2-ADC7 was tested in a NOG mouse animal model subcutaneously transplanted with HCC70 cells.

[0423] As shown in Table 12, on the 82nd day after inoculation, the tumor volume inhibition rate of the positive control antibody-ADC1 was 56%; Ab2-ADC7 significantly inhibited the growth of tumors at a dose of 5 mg / kg, and the tumor volume inhibition rate was 96%.

[0424] Table 12 Inhibition of tumor growth by Ab2-ADC7 on the 82nd day

[0425] Note: **: P < 0.01 compared with the tumor volume of the negative control antibody.

[0426] The above test shows that Ab2-ADC7 significantly inhibits the growth of HCC70 cell tumors and has no significant effect on the body weight of NOG mice.

[0427] Example 15 Anti-tumor efficacy test of anti-ROR1 antibody-ADC7 in a BALB / c nude mouse animal model subcutaneously transplanted with MDA-MB-231 cells

[0428] This test uses a BALB / c nude mouse animal model subcutaneously inoculated with MDA-MB-231 cells to determine the anti-tumor effect of humanized anti-ROR1 antibody-ADC7.

[0429] BALB / c nude mice: Female BALB / c nude mice (6 weeks) were purchased from Shanghai Sino-British Biotech Co., Ltd. The mice were adaptively fed for 7 days after arrival, and then the study began.

[0430] Cells: Human triple-negative breast cancer MDA-MB-231 cells (from the Shanghai Academy of Sciences Cell Bank) were routinely subcultured according to the instructions; the cells were resuspended in serum-free medium and the cell density was adjusted, and on day 0, the cell suspension was subcutaneously inoculated into the right axillary fossa of female BALB / c nude mice to establish a MDA-MB-231 tumor-bearing mouse model.

[0431] Administration:

[0432] On the 10th day after tumor cell inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes in the range of 139.65 mm 3 ~ 288.26 mm 3 were selected and evenly divided into groups according to tumor volume (5 mice per group). The negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number: HSP067-F1), the antibody Ab7-ADC6 (coupled with toxin Dxd), and Ab7-ADC7 (coupled with toxin CPT2C) of the present application were administered on the 10th, 17th, 24th, and 31st days after inoculation. During the administration period, the tumor volume and body weight changes of the mice in each group were monitored, with a frequency of 2 times / week for a continuous period of 6 weeks. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 52nd day after inoculation, using the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100; where Ti: the mean tumor volume of the administration group, T0: the mean tumor volume of the administration group on D0, Vi: the mean tumor volume of the isotype control group, and V0: the mean tumor volume of the isotype control group on D0. Tumor volume determination: the long diameter (a) and the wide diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2 x a x b 2 The body weight was measured using an electronic balance.

[0433] The administration dose and method are shown in Table 13.

[0434] Table 13 Administration test design

[0435] * Administration once a week for a total of 4 times.

[0436] As shown in Figure 15, the anti-tumor efficacy of Ab7-ADC6 and Ab7-ADC7 was detected in the animal model of BALB / c nude mice subcutaneously transplanted with MDA-MB-231 cells.

[0437] As shown in Figure 16, the body weight changes of Ab7-ADC6 and Ab7-ADC7 were detected in the animal model of BALB / c nude mice subcutaneously transplanted with MDA-MB-231 cells.

[0438] As shown in Table 14, on the 52nd day after inoculation, Ab7-ADC6 and Ab7-ADC7 significantly inhibited tumor growth at a dose of 3 mg / kg, with tumor volume inhibition rates of 109.25% and 111.51%, respectively.

[0439] Table 14 Inhibition of tumor growth by Ab7-ADC7 in vivo on the 52nd day

[0440] Note: **: P<0.01 compared with the tumor volume of the negative control antibody.

[0441] The above experiments showed that Ab7-ADC6 and Ab7-ADC7 both significantly inhibited the growth of MDA-MB-231 cell tumors, and had no significant effect on the body weight of BALB / c nude mice.

[0442] Example 16 Anti-tumor efficacy test of anti-ROR1 antibody-ADC7 in BALB / c nude mice subcutaneously transplanted with MDA-MB-231 cells

[0443] This experiment used a BALB / c nude mouse subcutaneously inoculated with MDA-MB-231 cell animal model to determine the anti-tumor effect of humanized anti-ROR1 antibody-ADC7.

[0444] BALB / c nude mice: Female BALB / c nude mice (6 weeks) were purchased from Shanghai Eprui Biotechnology Co., Ltd. The mice were adaptively fed for 7 days after arrival, and then the study began.

[0445] Cells: Human triple-negative breast cancer MDA-MB-231 cells (from Shanghai Institute of Cell Biology) were routinely subcultured according to the instructions; the cells were resuspended in serum-free medium and the cell density was adjusted, and on day 0, the cell suspension was subcutaneously inoculated into the right axillary fossa of female BALB / c nude mice to establish a MDA-MB-231 tumor-bearing mouse model.

[0446] Dosing:

[0447] On day 9 after tumor cell inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes of 139.65 mm 3 ~ 288.26 mm 3The mice in the range were divided into groups (5 mice per group) according to the average tumor volume, and were administered on the 9th, 16th, 23rd, and 30th days after inoculation, respectively, with a negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., item number: HSP067-F1), a positive control antibody-ADC1 (coupled with toxin MMAE) (SPH022-PC-2, source: Shangyi Crosslinking, item number: B013-DS-210602), and the antibody Ab2-ADC7 (coupled with toxin CPT2C) of the present application. The changes in tumor volume and body weight of the mice in each group were monitored during the administration period, with a monitoring frequency of 2 times / week, and the monitoring was continued for 8 weeks. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 65th day after inoculation, according to the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100; wherein Ti: the average tumor volume of the administration group, T0: the average tumor volume of the administration group on D0, Vi: the average tumor volume of the isotype control group, and V0: the average tumor volume of the isotype control group on D0. Tumor volume determination: the long diameter (a) and the wide diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2x a x b 2 The body weight was measured using an electronic balance.

[0448] The administration dose and mode are shown in Table 15.

[0449] Table 15 Administration test design

[0450] * Administration once a week for a total of 4 times.

[0451] As shown in FIG. 17, the anti-tumor efficacy of Ab2-ADC7 was detected in a BALB / c nude mouse animal model of subcutaneously transplanted MDA-MB-231 cells.

[0452] As shown in FIG. 18, the body weight changes of Ab2-ADC7 were detected in a BALB / c nude mouse animal model of subcutaneously transplanted MDA-MB-231 cells.

[0453] As shown in Table 16, on the 65th day after inoculation, the tumor volume inhibition rate of the positive control antibody-ADC1 was 88.56%; Ab2-ADC7 significantly inhibited the growth of tumors at a dose of 3 mg / kg, and the tumor volume inhibition rate was 101.02%.

[0454] Table 16 Inhibition of tumor growth by Ab2-ADC7 in vivo on the 65th day

[0455] Note: **: P<0.01 compared with the tumor volume of the negative control antibody.

[0456] The above experiment showed that Ab2-ADC7 could significantly inhibit the growth of MDA-MB-231 cell tumors, and had no significant effect on the body weight of BALB / c nude mice.

[0457] Example 17 Anti-tumor efficacy test of anti-ROR1 antibody-ADC on breast cancer PDX animal model LD1-2009-362153

[0458] In this experiment, the anti-tumor effect of anti-ROR1 antibody-ADC was determined by subcutaneously inoculating NCG mice with human breast cancer LD1-2009-362153 tumor tissue.

[0459] NCG mice: Female NCG mice (6 weeks) were purchased from Chengdu Yakang Biotechnology Co., Ltd. The mice were adaptively fed for 3 days after arrival, and then the study began.

[0460] Tumor tissue: Human breast cancer LD1-2009-362153 tumor tissue (from Xi'an Lidi Biotechnology Co., Ltd.) was passed to FP3+5 generation for this efficacy experiment. On day 0, the tumor mass of LD1-2009-LD1-2009-362153 human breast cancer xenograft was cut into tumor tissue with a size of about 3 mm x 3 mm x 3 mm (about 45-60 mg), and it was inoculated into NCG mice skin to establish a tumor-bearing mouse model.

[0461] Dosing:

[0462] On day 32 of tumor tissue inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes of 100 mm 3 ~200 mm 3The mice in the range were divided into groups (5 mice per group) according to the average tumor volume, and were administered on the 32nd, 39th, 46th, and 53rd days after inoculation, respectively, a negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., item number: HSP067-F1), a positive control antibody-ADC1 (coupled with toxin MMAE) (SPH022-PC-2, upper drug crosslinking, item number: B013-DS-210602), and the antibody Ab2-ADC7 of the present application (coupled with toxin CPT2C). The changes in tumor volume and body weight of the mice in each group were monitored during the administration period, and the monitoring frequency was 2 times / week, and the monitoring was continuously performed for 6 weeks. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 38th day after administration, according to the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100; wherein Ti: the average tumor volume of the administration group, T0: the average tumor volume of the administration group on D0, Vi: the average tumor volume of the isotype control group, and V0: the average tumor volume of the isotype control group on D0. Tumor volume determination: the long diameter (a) and the wide diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2x a x b 2 The body weight was measured using an electronic balance.

[0463] The administration dose and mode are shown in Table 17.

[0464] Table 17 Administration test design

[0465] * 1 administration per week, a total of 4 times.

[0466] Figures 19 and 20 show the anti-tumor efficacy of Ab2-ADC7 on the NCG mouse subcutaneously transplanted breast cancer PDX animal model LD1-2009-362153 and the change in body weight.

[0467] As shown in Table 18, on the 38th day after administration, the tumor volume inhibition rate of the positive control antibody-ADC1 was 18.74%; Ab2-ADC7 significantly inhibited the growth of tumors at a dose of 3 mg / kg, and the tumor volume inhibition rate was 103.40%.

[0468] Table 18 Inhibition of tumor growth by Ab2-ADC7 in vivo on the 38th day

[0469] Note: ****: P<0.0001 compared with the tumor volume of the negative control antibody.

[0470] The above test results show that Ab2-ADC7 significantly inhibits the growth of the breast cancer PDX animal model LD1-2009-362153 and does not have a significant effect on the body weight of the NCG mice.

[0471] Example 18 Anti-tumor efficacy test of anti-ROR1 antibody-ADC on breast cancer PDX animal model LD1-2009-361973

[0472] This test was used to determine the anti-tumor effect of anti-ROR1 antibody-ADC on human breast cancer LD1-2009-361973 tumor tissue subcutaneously inoculated in Nu / Nu mice.

[0473] Nu / Nu mice: Female Nu / Nu mice (6 weeks) were purchased from Chengdu Yakang Biotechnology Co., Ltd. The mice were adaptively fed for 3 days after arrival, and then the study began.

[0474] Tumor tissue: Human breast cancer LD1-2009-361973 tumor tissue (from Xi'an Lidi Biotechnology Co., Ltd.) was passed to FP2+5 generations for this efficacy test. On day 0, the tumor mass of LD1-2009-361973 human breast cancer xenograft was cut into tumor tissue with a size of about 3mm x 3mm x 3mm (about 45-60mg), and it was inoculated in Nu / Nu mice to establish a tumor-bearing mouse model.

[0475] Dosing: On the 34th day after tumor tissue inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes in the range of 100mm 3 ~200mm 3 were selected and evenly divided into groups according to tumor volume (5 mice per group). The negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), the positive control antibody-ADC1 (conjugated with toxin MMAE) (SPH022-PC-2, upper drug crosslinking, catalog number: B013-DS-210602), and the antibody Ab2-ADC7 (conjugated with toxin CPT2C) of the present application were administered on the 34th day after inoculation, respectively. The tumor volume and body weight changes of the mice in each group were monitored during the administration period, with a monitoring frequency of 2 times / week, and continuous monitoring for 8 weeks. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 35th day after administration, according to the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)] x 100; where Ti: mean tumor volume of the administration group, T0: mean tumor volume of the administration group on D0, Vi: mean tumor volume of the isotype control group, V0: mean tumor volume of the isotype control group on D0. Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2 x a x b2. The body weight was measured using an electronic balance.

[0476] The dosing dose and method are shown in Table 19.

[0477] Table 19 Dosing test design

[0478] Single dose.

[0479] Fig. 21 and Fig. 22 show the anti-tumor efficacy test of Ab2-ADC7 on the breast cancer PDX animal model LD1-2009-361973 subcutaneously transplanted in Nu / Nu mice and the body weight change.

[0480] As shown in Table 20, on the 35th day after administration, the tumor volume inhibition rate of the positive control antibody-ADC1 was 23.37%; Ab2-ADC7 significantly inhibited the growth of tumors at a dose of 3 mg / kg, and the tumor volume inhibition rate was 108.23%.

[0481] Table 20 Inhibition of tumor growth by Ab2-ADC7 in vivo on the 35th day

[0482] Note:***: P < 0.001 compared with the tumor volume of the negative control antibody.

[0483] The above test results show that Ab2-ADC7 significantly inhibits the growth of breast cancer PDX animal model LD1-2009-361973 tumors, and has no obvious effect on the body weight of Nu / Nu mice.

[0484] Example 19 Anti-tumor efficacy test of anti-ROR1 antibody-ADC on lymphoma PDX animal model LD1-0026-410827

[0485] This test uses human lymphoma LD1-0026-410827 tumor tissue to subcutaneously inoculate NU / NU mice to determine the anti-tumor effect of anti-ROR1 antibody-ADC.

[0486] NU / NU mice: Female NU / NU mice (6 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were adaptively fed for 3 days after arrival, and then the study began.

[0487] Tumor tissue: Human lymphoma LD1-0026-410827 tumor tissue (from Xi'an Lide Biotechnology Co., Ltd.) was passed to FP1+2 generations for this efficacy test. On the 0th day, the LD1-0026-410827 human lymphoma in vivo transplanted tumor was cut into tumor tissue with a size of about 3 mm x 3 mm x 3 mm (about 45-60 mg), and was inoculated subcutaneously in NU / NU mice to establish a tumor-bearing mouse model.

[0488] Administration: On the 29th day after tumor tissue inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes of 100 mm 3 ~ 200 mm 3The mice in the range were divided into groups (5 mice per group) according to the average tumor volume, and were administered on days 29, 36, 43, and 50 after inoculation, respectively, with a negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., item number: HSP067-F1), a positive control antibody-ADC1 (coupled with toxin MMAE) (SPH022-PC-2, upper drug crosslinking, item number: B013-DS-210602), and the antibody Ab2-ADC7 of the present application (coupled with toxin CPT2C). During the administration period, the changes in tumor volume and body weight of the mice in each group were monitored, with a monitoring frequency of 2 times / week, and continuous monitoring for 4 weeks. Tumor volume determination: the long diameter (a) and the wide diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2 x a x b 2 The body weight was measured using an electronic balance.

[0489] The administration dose and mode are shown in Table 21.

[0490] Table 21 Administration test design

[0491] * Administered once a week for a total of 4 times.

[0492] Figures 23 and 24 show the anti-tumor efficacy of Ab2-ADC7 on the PDX animal model LD1-0026-410827 of subcutaneously transplanted lymphoma in NU / NU mice and the changes in body weight.

[0493] As shown in Table 22, on day 25 after inoculation, the tumor volume inhibition rate of the positive control antibody-ADC1 was 34.99%; Ab2-ADC7 significantly inhibited the growth of tumors at a dose of 3 mg / kg, and the tumor volume inhibition rate was 98.91%.

[0494] Table 22 Inhibition of tumor growth by Ab2-ADC7 in vivo on day 25

[0495] Note: ***: P < 0.001 compared with the tumor volume of the negative control antibody.

[0496] The above test results show that Ab2-ADC7 significantly inhibits the growth of the PDX animal model LD1-0026-410827 of lymphoma and has no obvious effect on the body weight of the NU / NU mice.

[0497] Example 20 Anti-tumor efficacy test of anti-ROR1 antibody-ADC on the PDX animal model LU-01-1621 of lung cancer

[0498] The experiment adopts human breast cancer LU-01-1621 tumor tissue to inoculate BALB / c nude mice subcutaneously to determine the anti-tumor effect of the anti-ROR1 antibody-ADC.

[0499] BALB / c nude mice: female BALB / c nude mice (7-9 weeks) were purchased from Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd. After arrival, the mice were adaptively fed for 3 days, and then the study began.

[0500] Tumor tissue: human lung cancer LU-01-1621 tumor tissue (from Shanghai Pharming Kangde New Drug Development Co., Ltd.) was transmitted to FP5 generation for this efficacy experiment. On day 0, the tumor mass of the LU-01-1621 human lung cancer xenograft was cut into tumor tissue with a size of about 30mm 3 , and was inoculated subcutaneously in BALB / c nude mice to establish a tumor-bearing mouse model.

[0501] Dosing: on the 33rd day after tumor tissue inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes in the range of 100mm 3 -200mm 3 were selected and evenly grouped according to tumor volume (5 mice per group). The negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., product number: HSP067-F1), the positive control antibody-ADC1 (coupled with toxin MMAE) (SPH022-PC-2, upper drug crosslinking, product number: B013-DS-210602), and the antibody Ab2-ADC7 (coupled with toxin CPT2C) of the application were administered on the 33rd, 40th, 47th, and 54th days after inoculation, respectively. During the administration period, the tumor volume and body weight changes of the mice in each group were monitored, with a monitoring frequency of 2 times / week, and the monitoring was continued for 4 weeks. The body weight and tumor volume were measured before each administration, and the tumor volume inhibition rate (TGI%) was calculated on the 28th day after administration, according to the following formula: TGI (%) = [1-(Ti-T0) / (Vi-V0)]x100; where Ti: the mean tumor volume of the administration group, T0: the mean tumor volume of the administration group on day 0, Vi: the mean tumor volume of the isotype control group, and V0: the mean tumor volume of the isotype control group on day 0. Tumor volume determination: the long diameter (a) and the wide diameter (b) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: TV = 1 / 2x a x b2. The body weight was measured using an electronic balance.

[0502] The administration dose and method are shown in Table 23.

[0503] Table 23 Administration test design

[0504] * Single administration.

[0505] Figures 25 and 26 show the anti-tumor efficacy of Ab2-ADC7 in the lung cancer PDX animal model LU-01-1621 subcutaneously transplanted in BALB / c nude mice and the body weight change.

[0506] As shown in Table 24, on day 28 after administration, the tumor volume inhibition rate of the positive control antibody-ADC1 was 43.81%; Ab2-ADC7 significantly inhibited the growth of tumors at a dose of 5 mg / kg, and the tumor volume inhibition rate was 59.39%.

[0507] Table 24: Inhibition of tumor growth by Ab2-ADC7 in vivo on day 35

[0508] Note: *: P < 0.05 compared with the tumor volume of the negative control antibody.

[0509] The above test results show that Ab2-ADC7 significantly inhibits the growth of tumors in the lung cancer PDX animal model LU-01-1621, and does not have a significant effect on the body weight of BALB / c nude mice.

[0510] The above-described examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. At the same time, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference.

Claims

1. An anti-RORl antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: an immunoglobulin heavy chain variable region (VH) comprising a heavy chain HCDR1, HCDR2, and HCDR3, wherein the HCDR1 is or comprises an amino acid sequence as set forth in SEQ ID NO: 1, the HCDR2 is or comprises an amino acid sequence as set forth in SEQ ID NO: 2, and the HCDR3 is or comprises an amino acid sequence as set forth in SEQ ID NO: 3; and an immunoglobulin light chain variable region (VL) comprising a light chain LCDR1, LCDR2, and LCDR3, wherein the LCDR1 is or comprises an amino acid sequence as set forth in SEQ ID NO: 4, the LCDR2 is or comprises an amino acid sequence as set forth in SEQ ID NO: 5, and the LCDR3 is or comprises an amino acid sequence as set forth in SEQ ID NO:

6.

2. The anti-RORl antibody or antigen-binding fragment thereof of claim 1, wherein, the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO: 7, 9, or 10, or at least 85% identical to the amino acid sequence as set forth in SEQ ID NO: 7, 9, or 10, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 8, 11, 12, 13, or 14, or at least 85% identical to the amino acid sequence as set forth in SEQ ID NO: 8, 11, 12, 13, or 14.

3. The anti-RORl antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 7, and a light chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 8; or the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 11; or the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 12; or the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 13; or the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 14; or the anti-RORl antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region of an amino acid sequence identical to or at least about 85% identical to SEQ ID NO: 14; or the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to, or having at least about 85% identity with, SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to, or having at least about 85% identity with, SEQ ID NO: 11; or the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to, or having at least about 85% identity with, SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to, or having at least about 85% identity with, SEQ ID NO: 12; or the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to, or having at least about 85% identity with, SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to, or having at least about 85% identity with, SEQ ID NO: 13; or the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of an amino acid sequence identical to, or having at least about 85% identity with, SEQ ID NO: 10, and a light chain variable region of an amino acid sequence identical to, or having at least about 85% identity with, SEQ ID NO:

14.

4. The anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein, it comprises: a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 9 and SEQ ID NO: 11, respectively; or the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 9 and SEQ ID NO: 12, respectively; or the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 9 and SEQ ID NO: 13, respectively; or the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 9 and SEQ ID NO: 14, respectively; or the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 11, respectively; or the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 12, respectively; or the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 13, respectively; or the VH region and the VL region are or comprise amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 14, respectively.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein, wherein the anti-RORl antibody or antigen-binding fragment thereof is or comprises a heavy chain that is or comprises an amino acid sequence as set forth in SEQ ID NO: 18, 20, or 25, and a light chain that is or comprises an amino acid sequence as set forth in SEQ ID NO: 19, 21, 22, 23, or 24, or that is at least about 85% identical thereto; Preferably, the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 18; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 19; or the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 21; or the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 22; or the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 23; or the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 20; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 24; or the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 21; or the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 22; or the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 23; or the heavy chain is or comprises an amino acid sequence as set forth in SEQ ID NO: 25; and the light chain is or comprises an amino acid sequence as set forth in SEQ ID NO:

24.

6. The anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein, the anti-RORl antibody or antigen-binding fragment thereof is a murine, humanized, chimeric, or fully human antibody.

7. A polynucleotide encoding the anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. An immunoconjugate comprising: the anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-6; and a drug attached thereto; Preferably, the immunoconjugate has the formula Ab-(L-D)y, wherein: Ab is the anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-6; L is a linker; D is a drug; and y is a number from 1 to 10, either an integer or a decimal; more preferably, y is an integer or a decimal from 1 to 8, more preferably an integer or a decimal from 7 to 8. More preferably, the immunoconjugate has a structure according to Formula I: wherein Ab is the anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-6, and y is an integer or a decimal from 1 to 8, more preferably an integer or a decimal from 7 to 8.

9. The immunoconjugate of claim 8, wherein, The drug comprises a molecule that inhibits a tumor, a molecule that targets a tumor surface marker, a molecule that targets a surface marker of an immune cell, a radioactive moiety, a detectable label, or a combination thereof; Preferably, the molecule that targets a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker; or the molecule that inhibits a tumor is an anti-tumor cytokine or an anti-tumor toxin; More preferably, The antibody that binds to a tumor surface marker is an antibody that recognizes an antigen other than ROR1, the antigen other than ROR1 comprising: EGFR, EGFRvIII, mesothelin, HER2, EphA2, Her3, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, ASGPR1, or CDH16; and / or The anti-tumor cytokine comprises: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, or a variant thereof; and / or The anti-tumor toxin includes: toxins acting on tubulin; toxins acting on DNA or derivatives thereof; compounds acting on intracellular metabolism, transcription, translation or signal transduction or derivatives thereof; more preferably including CPT2C, camptothecin, 20-deoxycamptothecin, 10-methoxyamptothecin, 9-methoxycamptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, exatecan, deruxtecan (Dxd), irinotecan (DX-8951f), lurtotecan (GG-211), topotecan, irinotecan (CPT11), simmitecan, sinotecan, lipotecan (TLC388), gimmitecan (ST1481), diflomotecan (BN-80915), CZ48, Camptothecin chloroacetate (DLSFFZ93W4), Camptothecin lysinate (NSC610457), HM910, SN38, GI-149893, cositecan (karenitecin), Elomotecan (BN-80927), 11-cyanocamptothecin (NSC609951), 7-acetyl-Camptothecin (SCHEMBL6851483), 9-Glycineamidocamptothecin HCl (MLS000756803), 7-hydroxymethylcamptothecin, 9-Glycineamidocamptothecin HCl (MLS000756803), 10-amino-11-hydroxy-camptothecin (BDBM50285230), diflomotecan, 9-nitrocamptothecin (9-NC), 9-aminocamptothecin (9-AC), 10-aminocamptothecin (10-AC), kanitecan (BNP-1350), or 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (AR-67); maytansinoid; monomethyl auristatin DE and DF (MMAE and MMAF); dolastatin;calicheamicin or a derivative thereof; an anthracycline, doxorubicin or daunorubicin; methotrexate; vindesine; a taxane, docetaxel, paclitaxel, larotaxel, tesetaxel or ortataxel; a trichothecene; a duocarmycin; a pyrrolobenzodiazepine (PBD); SN-38; GX-2; CC1065; The CPT2C has a structure as shown in the following formula II:

10. The immunoconjugate of claim 8 or 9, wherein The antibody or antigen-binding fragment thereof comprises: a heavy chain variable (VH) region and a light chain variable (VL) region, wherein: the VH region and the VL region are or comprise the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 12, respectively; or the VH region and the VL region are or comprise the amino acid sequences set forth in SEQ ID NO: 10 and SEQ ID NO: 13, respectively; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein: the heavy chain is or comprises the amino acid sequence set forth in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence set forth in SEQ ID NO: 22; or the heavy chain is or comprises the amino acid sequence set forth in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence set forth in SEQ ID NO:

23.

11. The immunoconjugate of any one of claims 8 to 10, wherein, The linker is a cleavable linker; Preferably, the linker is a tumor-specific cleavable linker; More preferably, the linker is an acid-labile linker, a peptidase-sensitive linker, a photo-labile linker, a dimethyl linker, or a disulfide-containing linker; More preferably, the linker comprises a peptide moiety, a reactive functional group, a benzoic acid or a benzyloxy carbonyl group, or any combination thereof; wherein the peptide moiety comprises SC, VC, VA, FK, EVC, GGFG; the reactive functional group comprises MC, SMCC, sulfo-SMCC, SPP, SPDP; the benzoic acid or the benzyloxy carbonyl group comprises PABA, PAB, GABA; More preferably, the linker comprises MC-VA, MC-VC, SC-VC-PAB, SPDP, SMCC, SuO-VA-PAB, SuO-VC-PAB, MC-VA-PAB, MC-VC-PAB, MC-GGFG.

12. The immunoconjugate of any one of claims 8 to 11, wherein, MC-GGFG-CPT2C, MC-VC-PAB-MMAE, MC-GGFG-Dxd, MC-VA-MMAE, MC-VC-MMAE, MC-VA-PAB-MMAE, MC-GGFG-MMAE, SC-VC-PAB-MMAE, MC-VA-MMAF, MC-VC-MMAF, MC-VA-PAB-MMAF, MC-GGFG-MMAF, SC-VC-PAB-MMAF, MC-VC-PAB-MMAF, MC-GGFG-camptothecin, MC-GGFG-20-deoxycamptothecin, MC-GGFG 010-methoxyamptothecin, MC-GGFG-9-methoxycamptothecin, MC-GGFG-10-hydroxycamptothecin, MC-GGFG-7-ethyl-10-hydroxycamptothecin, MC-GGFG-exatecan, MC-GGFG-DX-8951f, MC-GGFG-GG-211, MC-GGFG-topotecan, MC-GGFG-CPT11, MC-GGFG-simmitecan, MC-GGFG-sinotecan, MC-GGFG-TLC388, MC-GGFG-ST1481, MC-GGFG-BN-80915, MC-GGFG-CZ48, MC-GGFG-DLSFFZ93W4, MC-GGFG-NSC610457, MC-GGFG-HM910, MC-GGFG-SN38, MC-GGFG-GI-149893, MC-GGFG-cositecan, MC-GGFG-BN-80927, MC-GGFG-NSC609951, MC-GGFG-7-acetyl-Camptothecin, MC-GGFG-9-Glycineamidocamptothecin HCl, MC-GGFG-7-hydroxymethylcamptothecin, MC-GGFG-9-Glycineamidocamptothecin HCl, MC-GGFG-10-amino-11-hydroxy-camptothecin, MC-GGFG-diflomotecan, MC-GGFG-9-NC, MC-GGFG-9-AC, MC-GGFG-10-AC, MC-GGFG-BNP-1350, MC-GGFG-AR-67, CZY-8.

13. A pharmaceutical composition comprising: The anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-6, or the immunoconjugate of any one of claims 8-12.

14. Use of the anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-6, the immunoconjugate of any one of claims 8-12, or the pharmaceutical composition of claim 13 in the manufacture of a medicament, kit or pharmaceutical kit for the diagnosis or treatment of a tumor, preferably a tumor expressing RORl. More preferably, the tumor comprises a primary tumor, a locally advanced tumor, a metastatic tumor; and / or, the tumor comprises: lymphoma, breast cancer, lung cancer, ovarian cancer, neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, squamous cell carcinoma of the epithelium, melanoma, myeloma, gastric cancer, brain cancer, pancreatic cancer, cervical cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer, head and neck cancer, and combinations thereof; More preferably, the lymphoma comprises B-cell leukemia, B-cell chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, Burkitt's lymphoma, mantle cell lymphoma; the breast cancer comprises breast ductal carcinoma, triple negative breast cancer; the lung cancer comprises non-small cell lung cancer, small cell lung cancer; more preferably, the non-small cell lung cancer comprises lung adenocarcinoma, lung squamous cell carcinoma, lung large cell neuroendocrine carcinoma, lung lymphoepithelioma-like carcinoma.

15. A kit or pharmaceutical kit comprising the anti-RORl antibody or antigen-binding fragment thereof of any one of claims 1-6, or the polynucleotide of claim 7, or the immunoconjugate of any one of claims 8-12, or the pharmaceutical composition of claim 13.

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