WO2025218776A1 - Anti-ror1 antibody, immunoconjugate thereof and use thereof - Google Patents

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

This application claims priority to patent application number CN 202410470842.7, filed on April 18, 2024, entitled “Anti-ROR1 antibodies, immunoconjugates thereof and their applications”, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention belongs to the field of biomedicine, and more specifically, relates to anti-ROR1 antibodies, immunoconjugates and applications thereof.

Background Art

Triple-negative breast cancer (TNBC) refers to breast cancer that is negative for estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2, and accounts for approximately 20% of all breast cancer types. Currently, TNBC is treated primarily with surgery and chemotherapy, with a lack of targeted therapies and high-response therapies. Compared with other types of breast cancer, TNBC is more likely to spread beyond the breast and recur after treatment. It is also more aggressive and has a poorer prognosis. TNBC is one of the most deadly diseases in women, presenting urgent clinical needs and significant market potential.

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 a member of the receptor RTKs family with a molecular weight of approximately 105 kDa. Human ROR1 is composed of an extracellular immunoglobulin-like domain (Ig), two cysteine-rich domains (FZD), a membrane-proximal kringle domain (KRD), a single-pass transmembrane structure, an intracellular tyrosine kinase domain (TKD), two serine/threonine-rich domains (S/TRD), and a proline-rich domain (PRD).

During early embryonic development, ROR1 regulates cell division, proliferation, migration, and chemotaxis by mediating signaling through the Wnt signaling pathway, playing a physiological role in the development of embryonic neural, auditory, skeletal, and vascular organs. ROR1 expression gradually decreases during embryonic development, reaching very low levels in most normal tissues of children and adults, but is highly upregulated in cancerous tissues. ROR1 is highly expressed in a variety of hematologic malignancies, including B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), and myeloid hematologic cancers; and in solid tumors, including TNBC, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, and melanoma. ROR1 in tumor cells binds to Wnt5a and activates the non-canonical Wnt signaling pathway, promoting cell migration, invasion, EMT, and metastasis. Furthermore, MET can phosphorylate ROR1, regulating tumor cell growth. Reports indicate that ROR1 expression is associated with activation of YAP/TAZ transcription, thereby enhancing tumorigenesis and chemoresistance. ROR1 expression is upregulated in HER2+ breast cancer patients treated with T-DM1, leading to drug resistance. Furthermore, ROR1-high expressing tumor cells exhibit increased stemness and self-renewal capacity. ROR1 expression is also closely associated with disease progression and treatment efficacy. High ROR1 expression is associated with a poor prognosis in TNBC. Compared with patients with lower ROR1 expression, TNBC patients with higher ROR1 expression have significantly decreased disease-free survival and overall survival. Therefore, ROR1 is an ideal anti-tumor target and tumor diagnostic marker.

Therefore, there is a need in the art to develop antibodies and immunoconjugates targeting ROR1 and apply them to treat tumors.

Summary of the Invention

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

In one or more embodiments, the anti-ROR1 antibody or its antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7, 9 or 10 or at least 85% identity with the amino acid sequence as shown in SEQ ID NO: 7, 9 or 10, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 8, 11, 12, 13 or 14 or at least 85% identity with the amino acid sequence as shown in SEQ ID NO: 8, 11, 12, 13 or 14.

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

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

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

The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 13; or

The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is 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 having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 11; or

The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 12; or

The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 13; or

The anti-ROR1 antibody or its antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence that is identical to or has at least about 85% identity with SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to or has at least about 85% identity with SEQ ID NO: 14.

In one or more embodiments, 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 shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown 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 shown in SEQ ID NO: 9 and SEQ ID NO: 13, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 14, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 12, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 13, respectively; or

The VH region and the VL region are or contain the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, respectively.

In one or more embodiments, the anti-ROR1 antibody or its antigen-binding fragment is or comprises an amino acid sequence as shown in SEQ ID NO: 18, 20 or 25, or a heavy chain having at least about 85% identity thereto, and an amino acid sequence as shown in SEQ ID NO: 19, 21, 22, 23 or 24, or a light chain having at least about 85% identity thereto.

In one or more embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain (HC) and a light chain (LC), wherein: the heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 18; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 19; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 21; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 22; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 23; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 24; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 21; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 22; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 23; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 24.

In one or more embodiments, the anti-ROR1 antibody or antigen-binding fragment thereof is a murine antibody, a humanized antibody, a chimeric antibody, or a fully human antibody.

In a second aspect of the present invention, a polynucleotide is provided, which encodes the anti-ROR1 antibody or antigen-binding fragment thereof according to any embodiment of the present invention.

In a third aspect of the present invention, an immunoconjugate is provided, comprising: the antibody according to any embodiment of the present invention; and a drug linked thereto.

In one or more embodiments, the immunoconjugate has the chemical formula Ab-(L-D)y, wherein: Ab is the antibody or antigen-binding fragment thereof according to any embodiment of the present invention; L is a linker; D is a drug; y is a decimal or integer from 1 to 10; more preferably, y is an integer or decimal from 1 to 8, more preferably an integer or decimal from 7 to 8.

In one or more embodiments, the immunoconjugate has a structure as shown in Chemical Formula I:

Wherein, Ab is the antibody or antigen-binding fragment thereof according to any embodiment of the present invention, and y is an integer or decimal of 1 to 8, more preferably an integer or decimal of 7 to 8.

In one or more embodiments, the drug comprises: a molecule that inhibits tumors, a molecule that targets a tumor surface marker, a molecule that targets a surface marker of an immune cell, a radioactive group, a detectable label, or a combination thereof.

In one or more embodiments, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker; or the tumor-suppressing molecule is an anti-tumor cytokine or an anti-tumor toxin.

In one or more embodiments, the antibody that binds to a tumor surface marker is an antibody that recognizes antigens other than ROR1, and the other antigens include: 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.

In one or more embodiments, the anti-tumor cytokine comprises IL-2, IL-12, IL-15, IFN-beta, TNF-alpha or variants thereof.

In one or more embodiments, the anti-tumor toxins include: toxins that act on microtubules; toxins or derivatives thereof that act on DNA; compounds or derivatives thereof that act on intracellular metabolism, transcription, translation or signal transduction; more preferably include CPT2C, camptothecin, 20-deoxycamptothecin, 10-methoxycamptothecin, 9-methoxycamptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, exatecan, deruxtecan (Dxd ), Exatecan (DX-8951f), Rutotecan (GG-211), Topotecan, Irinotecan (CPT11), Simmitecan, Sinotecan, Ripotecan (TLC388), Gimatidecan (ST1481), Diflutecan (BN-80915), CZ48, Camptothecin chloroacetate (DLSFFZ93W4), Camptothecin lysinate (NSC610457), HM910, SN38, GI-149893, Cositecan (karenitecin), Elomotecan (BN-80927), 11-cyanocamptothecin (NSC609 951), 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), carninotecan (BNP-1350), or 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (AR-67 ); maytansinoids; monomethyl auristatins DE and DF (MMAE and MMAF); dolastatin; calicheamicin or its derivatives; anthracyclines, daunomycin, or doxorubicin; methotrexate; vindesine; taxanes, docetaxel, paclitaxel, larotaxel, tesetaxel, or ortataxel; trichothecenes; duocarmycins; pyrrolobenzodiazepines (PBDs); SN-38; GX-2; CC1065.

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

In one or more embodiments, in the immunoconjugate, the anti-ROR1 antibody or its antigen-binding fragment 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 shown 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 shown in SEQ ID NO: 10 and SEQ ID NO: 13, respectively.

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

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

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

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

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.

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-deox ycamptothecin, 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-HM9 10. MC-GGFG-SN38, MC-GGFG-GI-149893, MC-GGFG-cositecan, MC-GGFG-BN-80927, MC-GGFG-NSC609951, MC-GGFG-7-acetyl-Camptothecin, MC-GGF G-9-Glycineamidocamptothecin HCl, MC-GGFG-7-hydroxymethylcamptothecin, MC-GGFG-9-Glycineamidocamptothecin HCl, MC-GGFG-10-amin o-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.

In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising: the antibody or antigen-binding fragment thereof according to any embodiment of the present invention, or the immunoconjugate according to any embodiment of the present invention.

In the fifth aspect of the present invention, provided is the use of the antibody or antigen-binding fragment thereof described in any embodiment of the present invention, the immunoconjugate described in any embodiment of the present invention, or the pharmaceutical composition described in any embodiment of the present invention in the preparation of a preparation, kit or medicine box for diagnosing or treating tumors.

In one or more embodiments, the tumor is a ROR1-expressing tumor.

In one or more embodiments, 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, epithelial squamous cell carcinoma, 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.

In one or more embodiments, the lymphoma includes B-cell leukemia, B-cell chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, Burkitt's lymphoma, and mantle cell lymphoma; the breast cancer includes mammary ductal carcinoma and triple-negative breast cancer; the lung cancer includes non-small cell lung cancer and small cell lung cancer; more preferably, the non-small cell lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, lung large cell neuroendocrine carcinoma, and lung lymphoepithelioma-like carcinoma.

In the sixth aspect of the present invention, a kit or a medicine box is provided, comprising the antibody or antigen-binding fragment thereof described in any embodiment of the present invention, or the polynucleotide described in any embodiment of the present invention, or the immunoconjugate described in any embodiment of the present invention, or the pharmaceutical composition described in any embodiment of the present invention.

Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. ELISA detection of hROR1-Fc antigen activity.

Figure 2. Serum titers of immunized mice.

Figure 3. Binding curves of humanized anti-ROR1 antibodies (Ab0-4, positive control antibody, negative control antibody) and MDA-MB-231 cells.

Figure 4. Binding curves of humanized anti-ROR1 antibodies (Ab0, Ab5-8, positive control antibody, negative control antibody) and MDA-MB-231 cells.

Figure 5. Binding curves of humanized anti-ROR1 antibodies (Ab0-4, positive control antibody, negative control antibody) and JeKo-1 cells.

Figure 6. Binding curves of humanized anti-ROR1 antibodies (Ab0, Ab5-8, positive control antibody, negative control antibody) and JeKo-1 cells.

Figure 7. Binding curves of humanized anti-ROR1 antibodies (Ab0-4, positive control antibody, negative control antibody) and A549 cells.

Figure 8. Binding curves of humanized anti-ROR1 antibodies (Ab0, Ab5-8, positive control antibody, negative control antibody) and A549 cells.

Figure 9. Binding curves of humanized anti-ROR1 antibodies (Ab0, Ab1-8, positive control antibodies, negative control antibodies) and human ROR2 antigen.

Figure 10. Detection of endocytic activity of humanized anti-ROR1 antibodies (Ab0, Ab2, Ab7, positive control antibody, negative control antibody) in CHOK1/hROR1 cells.

Figure 11. Anti-tumor efficacy test of Ab2-ADC7 in the NOG mouse subcutaneously transplanted JeKo-1 cell animal model.

FIG12 . Ab2-ADC7 affects the body weight changes in the NOG mouse subcutaneously transplanted JeKo-1 cell animal model.

Figure 13. Anti-tumor efficacy test of Ab2-ADC7 on the NOG mouse subcutaneously transplanted HCC70 cell animal model.

FIG14 . Ab2-ADC7 affects the body weight changes of NOG mice subcutaneously transplanted with HCC70 cells.

Figure 15. Anti-tumor efficacy test of Ab7-ADC6 and Ab7-ADC7 in the animal model of subcutaneously transplanted MDA-MB-231 cells in BALB/c nude mice.

Figure 16. Effects of Ab7-ADC6 and Ab7-ADC7 on body weight changes in the BALB/c nude mouse subcutaneously transplanted MDA-MB-231 cell animal model.

Figure 17. Antitumor efficacy test of Ab2-ADC7 in the animal model of subcutaneously transplanted MDA-MB-231 cells in BALB/c nude mice.

Figure 18. Effect of Ab2-ADC7 on body weight changes in the BALB/c nude mouse subcutaneously transplanted MDA-MB-231 cell animal model.

Figure 19. Antitumor efficacy test of Ab2-ADC7 on the subcutaneously transplanted breast cancer PDX animal model LD1-2009-362153 in NCG mice.

FIG20 . Ab2-ADC7 affects the body weight changes of NCG mice subcutaneously transplanted with breast cancer PDX animal model LD1-2009-362153.

Figure 21. Antitumor efficacy test of Ab2-ADC7 on the subcutaneously transplanted breast cancer PDX animal model LD1-2009-361973 in Nu/Nu mice.

FIG22 . Ab2-ADC7 affects the body weight changes of Nu/Nu mice subcutaneously transplanted with breast cancer PDX animal model LD1-2009-361973.

Figure 23. Antitumor efficacy test of Ab2-ADC7 on the subcutaneously transplanted lymphoma PDX animal model LD1-0026-410827 in NU/NU mice.

FIG24 : Ab2-ADC7 affects the body weight changes of NU/NU mice subcutaneously transplanted with lymphoma PDX animal model LD1-0026-410827.

Figure 25. Antitumor efficacy test of Ab2-ADC7 on the subcutaneously transplanted lung cancer PDX animal model LU-01-1621 in BALB/c nude mice.

FIG26 : Effects of Ab2-ADC7 on body weight changes of BALB/c nude mice subcutaneously transplanted with lung cancer PDX model LU-01-1621.

DETAILED DESCRIPTION

After in-depth research, the present inventors provide an anti-ROR1 antibody, an immunoconjugate comprising the anti-ROR1 antibody, and use of the immunoconjugate in the preparation of a medicament for treating and/or preventing diseases, diseases associated with overexpression of ROR1, such as tumors.

the term

The term "ROR1" or "receptor tyrosine kinase-like orphan receptor 1" refers to the 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).

The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-domain antibodies (VHH), single-chain Fv (scFv), multispecific antibodies (e.g., bispecific antibodies generated from at least two intact antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antigenic determinants of antibodies, and any other modified immunoglobulin molecules containing an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (subtypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively, based on the identity of their heavy chain constant regions. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, and the like.

The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to the light chain complementary determining region 1 (LCDR1), the light chain complementary determining region 2 (LCDR2), and the light chain complementary determining region 3 (LCDR3); the three CDR regions of the heavy chain refer to the heavy chain complementary determining region 1 (HCDR1), the heavy chain complementary determining region 2 (HCDR2), and the heavy chain complementary determining region 3 (HCDR3).

Antibody CDRs can be identified by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat/Chothia. These coding 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 for antibodies, the CDRs of the antibodies can be identified according to the Kabat numbering system (see, for example, 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).

As used in the present invention, the "antigen binding fragment" of an antibody or immunoglobulin chain (heavy chain or light chain) includes a part of an antibody that lacks some amino acids compared to the full-length chain, but can specifically bind to an antigen. This fragment can be considered to have biological activity because it can specifically bind to the target antigen or can compete with other antibodies or antigen binding fragments for binding to a specific antigenic determinant. In some embodiments, this fragment comprises at least one CDR present in the full-length light chain or heavy chain, and in some instances, it includes a short chain heavy chain and/or light chain or a portion thereof. This biologically active fragment can be produced by recombinant DNA technology or can be produced, for example, by enzymatically or chemically cutting a complete antibody. Immune function immunoglobulin fragments include Fab, Fab, F(ab)2, scFab, dsFv, Fv, scFv, scFv-Fc, bifunctional antibodies, miniantibodies, scAb and dAb, but are not limited thereto, and can be derived from any mammal, including but not limited to humans, mice, rats, camels or rabbits. The functional portion of the antibody (such as one or more CDRs disclosed in the present invention) can be linked to a secondary protein or a small molecule compound through a covalent bond and thereby used as a targeted therapeutic agent for a specific target.

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, the Cold Spring Harbor Manual of Antibody Laboratory Techniques, Chapters 5-8 and 15. For example, mice can be immunized with human ROR1 or a fragment thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described herein are engineered to have one or more human FR regions added to the non-human CDR regions using genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, or from the Journal of Immunoglobulins, 2001 ISBN 012441351.

The term "anti-ROR1 antibody" or "antibody that binds to ROR1" means an antibody that is capable of binding to ROR1 with sufficient affinity such that the antibody targeting ROR1 is effective as a diagnostic and/or therapeutic agent. The extent of binding of the anti-ROR1 antibody to an unrelated, non-ROR1 protein (e.g., ROR2) is about 10% less than the extent of binding of the antibody to ROR1 as measured, for example, by radioimmunoassay (RIA) or surface plasmon resonance (SPR) techniques.

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

The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibody framework sequences. This can overcome the heterologous reactions induced by chimeric antibodies due to the large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In order to avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

" humanized antibody " can retain the antigen specificity that is similar to the original antibody. " humanized " form of non-human (for example mouse) antibody can comprise the chimeric antibody of the sequence derived from non-human immunoglobulin at least.In some cases, the CDR district residues in human immunoglobulin (receptor antibody) can be replaced with the CDR district residues of the non-human species (donor antibody) (such as mouse, rat, rabbit or non-human primate) with desired property, affinity and/or ability.In some cases, the FR district residues of human immunoglobulin can be replaced with corresponding non-human residues.In addition, humanized antibody can be included in the amino acid modification that does not have in the receptor antibody or in the donor antibody.Carrying out these modifications can be in order to further improve the performance of antibody, such as binding affinity.

The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region 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 multispecific antibodies formed from antibody fragments.

"Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating sequence identity or the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program from the NCBI database can be used to determine identity.

An "effective amount" of an agent is that amount necessary to effect a physiological change in the cell or tissue to which it is administered.

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 preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

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.

The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredient contained therein is effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

The term "pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The term "treatment/prevention" (and grammatical variations thereof) refers to an attempt to alter the natural course of a disease in a treated individual and can be a clinical intervention performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of the disease or slow the progression of the condition.

The term "detectable label" refers to a marker that can be attached to an antibody and used to determine the presence and amount of a specific target in a subject to be detected. The "detectable label" may include, but is not limited to, enzymes, fluorescent markers, radionuclides, quantum dots, colloidal gold, and the like. More specifically, it may be selected from, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

In the present invention, the tumor is a tumor expressing ROR1; preferably, the tumor expressing ROR1 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, such as 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, epithelial squamous cell carcinoma, 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 estrogen receptor (ER), progesterone receptor (PR) or Her2/neu genes.

anti-ROR1 antibody

The present invention 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), which comprises a heavy chain HCDR1, HCDR2 and HCDR3, wherein the HCDR1 is or comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 is or comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 is or comprises the amino acid sequence shown in SEQ ID NO: 3; and an immunoglobulin light chain variable region (VL), which comprises a heavy chain LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is or comprises the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 is or comprises the amino acid sequence shown in SEQ ID NO: 5, and the LCDR3 is or comprises the amino acid sequence shown in SEQ ID NO: 6.

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

The present invention provides an anti-ROR1 antibody, comprising HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 8; or

The anti-ROR1 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 9, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 11; or

The anti-ROR1 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 9, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 12; or

The anti-ROR1 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 9, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 13; or

The anti-ROR1 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 9, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 14; or

The anti-ROR1 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 10, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 11; or

The anti-ROR1 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 10, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 12; or

The anti-ROR1 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 10, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 13; or

The anti-ROR1 antibody comprises HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 10, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 14.

The present invention provides an anti-ROR1 antibody, comprising HCDR1, HCDR2 and HCDR3 contained in the amino acid sequence shown in SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 contained in the amino acid sequence shown in SEQ ID NO: 8; and

The anti-ROR1 antibody comprises an amino acid mutation at position 44 and/or position 49 of the amino acid sequence shown in SEQ ID NO: 7, preferably the mutation is G44R or S49A; and/or

The anti-ROR1 antibody comprises an amino acid mutation at one, two, three or all of positions 2, 49, 64 and 67 of the amino acid sequence shown in SEQ ID NO: 8, preferably the mutation is I2T, Y49D, G64S, S67Y; and/or

The anti-ROR1 antibody comprises an amino acid mutation at one, two or all of positions 4, 22 and 73 of the amino acid sequence shown in SEQ ID NO: 8, preferably the mutation is M4V, T22R, L73F; and/or

The anti-ROR1 antibody comprises an amino acid mutation at position 13 of the amino acid sequence shown in SEQ ID NO: 8, and preferably the mutation is A13M.

The present invention provides an anti-ROR1 antibody, comprising a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 7, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 8; or

The anti-ROR1 antibody comprises a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 11; or

The anti-ROR1 antibody comprises a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 12; or

The anti-ROR1 antibody comprises a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 13; or

The anti-ROR1 antibody comprises a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 14; or

The anti-ROR1 antibody comprises a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 11; or

The anti-ROR1 antibody comprises a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 12; or

The anti-ROR1 antibody comprises a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 13; or

The anti-ROR1 antibody comprises a heavy chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 14.

The present invention provides an anti-ROR1 antibody comprising: 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 shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown 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 shown in SEQ ID NO: 9 and SEQ ID NO: 13, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 14, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 12, respectively; or

The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 13, respectively; or

The VH region and the VL region are or contain the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, respectively.

The present invention provides an anti-ROR1 antibody, comprising a heavy chain (HC) having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 18, 20 or 25, and a light chain (LC) having an amino acid sequence that is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, SEQ ID NO: 19, 21, 22, 23 or 24. The present invention provides an anti-ROR1 antibody comprising: a heavy chain (HC) and a light chain (LC), wherein:

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 18, 20 or 25;

The light chain is or comprises an amino acid sequence as shown in SEQ ID NO: 19, 21, 22, 23 or 24.

The present invention provides an anti-ROR1 antibody comprising: a heavy chain (HC) and a light chain (LC), wherein:

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 18; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 19; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 21; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 22; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 23; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 24; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 21; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 22; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 23; or

The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 24.

The anti-ROR1 antibodies provided by the present invention can efficiently bind to human ROR1 specifically, can efficiently block the action of ROR1, and can efficiently carry another substance coupled thereto to the target cell. The anti-ROR1 antibodies provided by the present invention may include but are not limited to monoclonal antibodies, bispecific antibodies, double antibodies, multispecific antibodies, multiple antibodies, miniantibodies, domain antibodies, antibody mimetics (or synthetic antibodies), chimeric antibodies or antibody fusions (or antibody conjugates) and fragments thereof, and include various forms of antibodies disclosed herein. The antibody fragments of the antibodies disclosed in the present invention may include Fab, Fab', F(ab')2, scFab, Fv, dsFv, scFv, scFv-Fc, miniantibodies, bifunctional antibodies, scAb or dAb.

The antibodies disclosed in the present invention may be composed of only heavy chains comprising a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, 9 or 10, or only light chains comprising a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8, 11, 12, 13 or 14.

An antibody disclosed herein may share specific regions or sequences with another antibody disclosed herein. In some embodiments, they may share the constant region of an antibody or antigen-binding fragment. In some embodiments, they may share the Fc region. In some embodiments, they may share the framework of a variable region.

In some embodiments, the antibody has a typical structure of an antibody found in nature. Camelids produce antibodies composed of a single heavy chain, but the structural unit of such an antibody typically comprises a tetrameric polypeptide, wherein the tetramer comprises a pair of two polypeptide chain bodies composed of two different polypeptide chains. In a typical antibody, a pair of polypeptide chain bodies comprises a full-length light chain (about 25 kDa) and a full-length heavy chain (about 50 to 70 kDa). Each chain displays a characteristic folding pattern and is composed of several immunoglobulin domains, each of which consists 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 includes a portion called a variable region or V region that recognizes the antigen. The carboxyl-terminal portion is more evolutionarily conserved than the amino-terminus and includes a portion called a constant region or C region. Human light chains are typically classified as kappa (κ) or lambda (λ) light chains, and these respectively include a variable region and a constant region.

Heavy chains are typically classified as mu (μ), delta (δ), gamma (γ), alpha (αα), or epsilon (ε) chains, and these are defined as IgM, IgD, IgG, IgA, and IgE isotypes, respectively. IgG has multiple 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 contain four heavy chains and four light chains.

The IgG and IgE isotypes contain two heavy chains and two light chains, and the IgM isotype contains five heavy chains and five light chains. The heavy chain constant region generally includes at least one domain that exhibits effector functions. The number of heavy chain constant regions varies depending on the isotype. For example, the IgG heavy chain contains three C regions, referred to as CH1, CH2, and CH3, respectively. The antibodies disclosed herein can be any of these isotypes and subtypes. In some embodiments, the antibodies are of the IgG1, IgG2a, IgG2b, IgG3, or IgG4 subtypes. In some embodiments, the antibodies of the present invention are of the IgG1 or IgG2 type. In another embodiment, the antibodies of the present invention are of the IgG1 type.

The heavy chain variable region and light chain variable region according to the present invention 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, anti-body-dependent cellular phagocytosis, and/or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. In addition, human IgG1 and IgG3 induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be either λ or κ.

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

Each of the heavy and light chain variable regions disclosed in the present invention can be combined with various target heavy and light chain constant regions to form the heavy and light chains of complete antibodies, respectively. In addition, the individual heavy and light chain sequences combined with the constant regions can be further combined to form complete antibody structures.

Any variable region of the heavy chain or light chain of the antibody according to the present invention can be connected to at least a portion of the constant region. The constant region can be selected according to whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and/or complement-dependent cytotoxicity are needed. Those skilled in the art will be able to know that other variable regions, including IgG1, 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 in the present invention to obtain target characteristics (such as stability, expression, manufacturability or other).

A portion of the antibodies of the present invention may comprise conservative amino acid substitutions in one or more of the residues in the heavy chain, light chain, variable region, or CDR sequences disclosed above. Conservative amino acid substitutions are substitutions that do not substantially affect the activity or antigenicity of the polypeptide. In some embodiments, conservative amino acid substitutions are substitutions with another residue belonging to the same class of amino acids as those described below. Naturally occurring amino acids can be categorized by common side chain properties as follows: 1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; 2) Neutral Hydrophilic: Cys, Ser, Thr, Gln; 3) Acidic: Asp, Glu; 4) Basic: His, Lys, Arg; 5) Residues that affect chain direction: Gly, Pro; and 6) Aromatic: Trp, Tyr, Phe. Conservative amino acid substitutions may also include non-naturally occurring amino acid residues, such as peptidomimetics, and such residues are typically introduced through chemical synthesis rather than cell-mediated introduction. Non-conservative substitutions include substitutions with residues belonging to other classes within the above categories. Such substitutions may be made in regions of the antibody that are homologous to the antibodies of the invention or in non-homologous regions thereof.

The present invention also includes a fusion protein of an anti-ROR1 antibody, comprising a first domain of an antibody or antibody fragment as described in the present invention and a second domain for extending the half-life in vivo and/or having a binding effect on effector cells. The fusion protein may be a binding molecule that is capable of specifically binding to cells expressing ROR1. In the second domain, the fragment for extending the half-life in vivo may include serum albumin or a fragment thereof, polyethylene glycol, a domain that binds serum albumin (such as an antibody against serum albumin), a polyethylene glycol-liposome complex, and the like. In the second domain, the fragment that has a binding effect on effector cells may include an immunoglobulin Fc region, etc., preferably selected from the human immunoglobulin Fc region. The human immunoglobulin Fc region includes mutations for changing Fc-mediated effector functions, and the effector functions include one or more combinations of CDC activity, ADCC activity, and ADCP activity. The immunoglobulin can be selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, IgM, etc., and the IgG can specifically be selected from a combination of one or more of IgG1, IgG2, IgG3 or IgG4 subtypes, etc. The immunoglobulin Fc region contained in the antibody fusion protein can cause 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 invention, the immunoglobulin Fc region can be the Fc region of human IgG1, more specifically, it can be a wild-type IgG1 Fc sequence, and the sequence can be introduced with mutations for changing Fc-mediated effector functions, for example, a) mutations that change Fc-mediated CDC activity; b) mutations that change Fc-mediated ADCC activity; or c) mutations that change Fc-mediated ADCP activity.

In the fusion protein of the anti-ROR1 antibody provided by the present invention, a connecting peptide may be provided between the first domain and the second domain. The connecting peptide may be a flexible polypeptide chain composed of alanine (A) and/or serine (S) and/or glycine (G), and the length of the connecting peptide may be 3 to 30 amino acids, preferably 3-9, 9-12, 12-16, or 16 to 20 amino acids. For example, the length of the connecting peptide may be 8 or 15.

In a specific embodiment of the present invention, the inventors conjugated the humanized anti-ROR1 antibody with a drug to prepare an antibody-drug conjugate, and the results showed that it had a significant killing effect on tumor cells.

The present invention also provides an isolated polynucleotide encoding the antibody of the present invention or the fusion protein. The polynucleotide may be RNA, DNA, or cDNA. Methods for providing the isolated polynucleotide should be known to those skilled in the art. For example, the isolated polynucleotide may be prepared by automated DNA synthesis and/or recombinant DNA technology, or may be isolated from a suitable natural source.

drug

The present invention also provides a drug that can be conjugated with the antibody of the present invention to form an immunoconjugate (e.g., an antibody-drug conjugate). The drug includes (but is not limited to): a tumor-suppressing molecule, a molecule targeting a tumor surface marker, a molecule targeting a surface marker of an immune cell, a radioactive group, a detectable marker, or a combination thereof.

In some embodiments, the drug of the present invention can be a tumor-suppressing molecule. Specifically, the tumor-suppressing molecule includes (but is not limited to): camptothecin (CPT), such as but not limited to exatecan and its derivatives deruxtecan (Dxd), exatecan (DX-8951f), rutotecan (GG-211), CZ48, HM910, cilotecan, ripotecan (TLC388), 9-nitrocamptothecin (9-NC), 9-aminocamptothecin (9-AC), carninotecan (BNP-1350), gimatidecan (ST1481), diflutecan (BN-80915), 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (AR-67), CPT2C; maytansinoid; auristatin, such as monomethyl auristatin. Restatin drugs include DE and DF (MMAE and MMAF); dolastatins; dolastatin; calicheamicin or its derivatives; anthracyclines, such as daunomycin or doxorubicin; methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; CC1065; and cytokines, such as IL-2, IL-12, IL-15, IFN-beta, and TNF-alpha. In terms of mechanism of action, the tumor-suppressing molecules can be anti-tumor toxins, including toxins that act on microtubules, such as monomethyl auristatin (MMAE)-related compounds and their derivatives, maytansinoid-related compounds and their derivatives; toxins that act on DNA, such as duocarmycin, calicheamicin, pyrrolobenzodiazepines (PBDs), SN-38, Dxd, CPT2C and other related compounds and their derivatives; and related compounds and their derivatives that act on other functions in cells, such as metabolism, transcription, translation, signal transduction, etc. The present invention also includes analogs, isomers, precursors, etc. of these small molecule compounds that act as toxins.

As a preferred embodiment of the present invention, the drug of the present invention is a camptothecin (CPT) molecule, such as but not limited to camptothecin, 20-deoxycamptothecin, 10-methoxycamptothecin, 9-methoxycamptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, ... cin), exatecan, deruxtecan (Dxd), exatecan (DX-8951f), rutotecan (GG-211), topotecan, irinotecan (CPT11), simmitecan, cilnotecan, ripotecan (TLC388), gimatidecan (ST1481), diflutecan (BN-80915), CZ48, Camptothecin chloroacetate (DLSFFZ93W4), Camptothecin n 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-Glycine amidocamptothecin HCl (MLS000756803), 10-amino-11-hydroxy-camptothecin (BDBM50285230), diflomotecan, 9-nitrocamptothecin (9-NC), 9-aminocamptothecin (9-AC), 10-aminocamptothecin (10-AC), caninotecan (BNP-1350), 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (AR-67), GX-2 (refer to CN117164601A, the relevant content of CN117164601A is incorporated herein in its entirety), and CPT2C.

As a preferred embodiment of the present invention, the CPT2C of the present invention has a structure as shown in Chemical Formula II.

In some embodiments, the drug of the present invention may also be an enzymatically active toxin or a fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii toxin, ricin A chain, abrin A chain, alpha-sarcin ... rdii) proteins, carnation (dianthin) proteins, Phytolaca americana (Phytolaca americana) proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecenes.

In some embodiments, the drug can be at least one molecule that targets a tumor surface marker. The molecule that targets a tumor surface marker can, for example, synergize with the antibody of the present invention to more accurately target tumor cells. In other embodiments, the drug can also be a molecule that targets a surface marker of an immune cell. The molecule that targets a surface marker of an immune cell can recognize an immune cell, which carries the antibody of the present invention to the immune cell. At the same time, the antibody of the present invention can target the immune cell to the tumor cell, thereby utilizing the killing effect of the antibody of the present invention while also inducing the immune cell to specifically kill the tumor.

For therapeutic purposes, the drug may be a radioactive group, i.e. a group consisting of or comprising a radioisotope or radionuclide (e.g. 3H, 14C, 15N, 33P, 35S, 90Y, 99Tc, 111In, 123I, 125I, 131I, 201TI, 213Bi), a toxin or a cytotoxic group, e.g. a cytostatic group.

For detection purposes, the drug can be a detectable marker. Detectable markers include, but are not limited to, fluorescent markers, chromogenic markers, and protein tags, such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-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 groups, biotin groups, metal particles (e.g., gold particles), magnetic particles (e.g., having a core containing magnetite (Fe3O4) and/or maghemite (Fe2O3)), predetermined polypeptide groups, and the like. More than one marker may be included. The label used to label the antibody for detection and/or analysis and/or diagnosis depends on the specific detection/analysis/diagnosis technique and/or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels for detection/analysis/diagnosis techniques and/or methods known in the art are well known to those skilled in the art.

Immunoconjugates

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

As used in the present invention, the antibody of the present invention, or the fusion protein of the present invention or its fragment can be connected to a drug. The connection can be through one or more covalent bonds, or non-covalent interactions, and can include chelation. A variety of linkers (the linkers can be known in the art) can be used to form an immunoconjugate. In some specific embodiments, the immunoconjugate can be an antibody-drug conjugate (ADC) having the chemical formula Ab-(L-D)y, wherein: Ab is an anti-ROR1 antibody or antigen-binding fragment thereof, or a fusion protein thereof, according to the present invention; L is a linker; D is a drug moiety; y is any integer or decimal (e.g., an integer or decimal of 1 to 10, 1 to 8, 5 to 8, or 7 to 8). In addition, the immunoconjugate can be provided in the form of a fusion protein, which can be expressed by a polynucleotide encoding the immunoconjugate.

In the immunoconjugate, the antibody and the drug can be cross-linked through a linker, which can be a cleavable linker, for example, a linker that can be enzymatically cleaved in vivo and/or in vitro, for example, a peptide linker, a disulfide linker, or a hydrazone linker. Once metabolized in vivo (e.g., in tumor cells) and/or in vitro, the immunoconjugate can release the drug. The linker is preferably a "cleavable linker" that can promote the release of the drug in cells (e.g., tumor cells), such as an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, and a disulfide-containing linker. As a preferred embodiment of the present invention, the linker is a tumor-specific linker, including a peptide linker and an acid-cleavable linker that can be cleaved by intracellular proteases (e.g., lysosomal proteases or endosomal proteases).

In certain embodiments, the linker may include a dipeptide, such as a valine-citrulline (VC), a valine-alanine (VA), or a 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 these amino acid residues contains a lipophilic side chain. Other suitable linkers include linkers that are hydrolyzable at a pH of 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.

In certain embodiments, the linker can have a reactive functional group, which can include, for example, a nucleophilic group reactive to an electrophilic group. Exemplary electrophilic groups include carbonyls, such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, and anhydrides. Exemplary nucleophilic groups include hydrazides, oximes, amino groups, hydrazines, thiosemicarbazides, carboxylic acid hydrazides, and arylhydrazines. In some embodiments, the conjugation of a drug to an antibody can be formed by reacting with a maleimide group (which can also be referred to as a maleimide spacer). The maleimide group can be maleimidocaproyl (MC), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylates (SMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylates (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 4-(2-pyridyldithio)-pentanoate (SPDP).

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

It should be understood that the linker can comprise a peptide portion, 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, and 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, and MC-GGFG.

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 is covalently bound to the anti-ROR1 antibody or antigen-binding fragment thereof at a succinimide, carbonyl, cyclooctene, quaternized vinylpyridine, or triazole group of the linker.

The linker can be conjugated to the anti-ROR1 antibody or antigen-binding fragment thereof in a variety of ways. Typically, the linker and drug moiety are synthesized and conjugated before being attached to the antibody, first forming a (L-D) structure, which is then conjugated to the Ab moiety. In some preferred embodiments, the (L-D) structure includes but is not limited to: 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 thecin, MC-GGFG-10-methoxyamptothecin, MC-GGFG-9-methoxycamptothecin, MC-GGFG-10-hydroxycamptothecin, MC-GGFG-7-ethyl-10-hydroxycampt othecin, 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 H Cl, 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 (refer to CN117164601A, the relevant content of CN117164601A is incorporated herein in its entirety).

In some embodiments, the immunoconjugates described herein have a structure shown in Chemical Formula I:

Wherein, y in Chemical Formula I is any integer or decimal (for example, an integer or decimal of 1 to 10, 1 to 8, 5 to 8, or 7 to 8).

The method 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 the drug directly or through a spacer of appropriate length. The connection method can be chemical cross-linking or genetic engineering fusion expression to obtain the immunoconjugate.

The immunoconjugate can also be a chimeric antigen receptor (CAR), which can be expressed in immune cells. The "immune cells" and "immune effector cells" can be used interchangeably, and include: T lymphocytes, NK cells or NKT cells, etc., preferably, NK cells and T lymphocytes. The chimeric antigen receptor generally comprises: an extracellular binding region, a transmembrane region and an intracellular signaling region connected in sequence, wherein the extracellular binding region comprises the antibody or fusion protein of the present invention. The design of the transmembrane region and the intracellular signaling region based on CAR technology is well known in the art: for example, the transmembrane region adopts the transmembrane region of molecules such as CD8 and CD28, and the intracellular signaling region adopts the intracellular signaling region of the immunoreceptor tyrosine activation motif (ITAM) CD3ζ chain or FcεRIγ tyrosine activation continuation and co-stimulatory signal molecules CD28, CD27, CD137, CD134, MyD88, CD40, etc. More specifically, for T lymphocytes, the first generation of CAR T lymphocytes contains only ITAM in the intracellular signaling region, in which the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-ITAM, which can stimulate anti-tumor cytotoxic effects; the second generation of CAR T lymphocytes adds the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), in which the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-ITAM or scFv-TM-/CD137-ITAM; the intracellular signaling region contains B7/ The co-stimulation of CD28 or 4-1BBL/CD137 causes the continuous proliferation of T lymphocytes and can increase the level of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, while also improving the survival cycle and anti-tumor effect of CAR T in the body; the third-generation CAR T lymphocytes, in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further improve the survival cycle of CAR T in the body and its anti-tumor effect.

According to the above, the chimeric antigen receptor prepared using the antibody or fusion protein of the present invention can be a chimeric antigen receptor comprising the following sequentially connected extracellular binding regions, transmembrane regions and intracellular signaling regions: the antibody or fusion protein of the present invention, CD8 and CD3ζ; the antibody or fusion protein of the present invention, CD8, CD137 and CD3ζ; the antibody or fusion protein of the present invention, the transmembrane region (CD28a) of the CD28 molecule, the intracellular signaling region (CD28b) of the CD28 molecule and CD3ζ; or the antibody or fusion protein of the present invention, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, CD137 and CD3ζ.

The chimeric antigen receptor is expressed on the surface of immune effector cells, and while utilizing the killing effect of the antibody of the present invention, the immune effector cells can also have a highly specific cytotoxic effect on tumor cells expressing ROR1.

Pharmaceutical composition

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

The pharmaceutical composition may also include various pharmaceutically acceptable carriers in the art. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used, and may include, but are not limited to: buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parahydroxybenzoates such as methyl parahydroxybenzoate or propyl parahydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as blood albumin, gelatin or immunoglobulin; 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 dextrin; chelating agents such as EDTA; tonicity modifiers such as trehalose and sodium chloride; sugars 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 nonionic surfactants such as Or polyethylene glycol (PEG). Pharmaceutical preparations for in vivo administration are generally sterile. Methods for achieving sterility of pharmaceutical preparations should be known to those skilled in the art. For example, it can be achieved by filtration through a sterile filtration membrane. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier 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 invention can be in various dosage forms including but not limited to tablets, injections, lyophilized agents, etc.

In the pharmaceutical composition, the content of the fusion protein, drug and immunoconjugate is generally an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined according to the subject to be treated and the specific administration method. For example, based on the total mass of the pharmaceutical composition, 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%.

The fusion proteins, drugs, immunoconjugates, and pharmaceutical compositions of the present invention can be administered as a single active ingredient or in combination therapy, i.e., in combination with other agents. For example, the combination therapy can include the fusion protein, drug, immunoconjugate, or pharmaceutical composition in combination with at least one other anti-tumor drug. As another example, the combination therapy can include the fusion protein, drug, immunoconjugate, or pharmaceutical composition in combination with antibodies targeting other tumor-specific antigens, such as, but not limited to, anti-EGFR antibodies, anti-VEGF antibodies, anti-HER2 antibodies, or anti-Claudin18A2 antibodies. The inhibitor can preferably be a monoclonal antibody.

use

The present invention also provides use of the antibody, fusion protein, drug, immunoconjugate or pharmaceutical composition of the present invention in the preparation of a drug for diagnosing, treating or preventing a disease associated with cells expressing (or overexpressing) ROR1. In some specific embodiments, the disease is a tumor (including primary tumors, locally advanced tumors, metastatic tumors); the tumor is preferably a tumor expressing ROR1; preferably, 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, such as 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, epithelial squamous cell carcinoma, 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 "therapeutically effective amount" of the antibodies, fusion proteins, drugs, immunoconjugates, and pharmaceutical compositions provided by the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevents damage or disability caused by the pain of the disease. For example, for the treatment of ROR1-related tumors (including lymphoma, ovarian cancer, endometrial cancer, breast cancer, cervical cancer, etc.), relative to untreated subjects, a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts the efficacy of the drug in human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which inhibition can be determined in vitro by tests 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 alleviating the symptoms of the subject. Those skilled in the art can select a suitable therapeutically effective amount according to actual conditions, for example, the size of the subject, the severity of the subject's symptoms and the specific composition or route of administration selected. The prescription for treatment (for example, the decision on dosage, etc.) can be determined by a doctor, and the factors usually considered include but are not limited to the disease being treated, the individual patient's condition, the delivery site, the method of administration and other factors. A prophylactic effective amount refers to an amount that effectively achieves the desired prophylactic effect at the necessary dosage and time. Usually, but not necessarily, since a prophylactic dose is used for a subject before the onset of the disease or in the early stages of the disease, a "prophylactic effective amount" is usually lower than a "therapeutically effective amount."

In the specific embodiments of the present invention, some dosing regimens for animals such as mice are provided. It is easy for those skilled in the art to convert the dosage for animals such as mice into a dosage suitable for humans. For example, the calculation can be performed according to the Meeh-Rubner formula: Meeh-Rubner formula: A = k'(W2/3)/10,000. In the formula, A is the body surface area, calculated in m2; W is the body weight, calculated in g; K is a constant that varies with the animal species. Generally speaking, it is 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 should be understood that the dosage conversion can vary depending on the drug and clinical situation, based on the evaluation of an experienced pharmacist.

The present invention also provides a kit containing the antibody, fusion protein, drug, immunoconjugate and/or pharmaceutical composition. In addition, the kit may also include instructions for using the antibody, fusion protein, drug, immunoconjugate and/or pharmaceutical composition in the kit.

The present invention also provides a detection kit comprising the antibody, fusion protein, or immunoconjugate described herein. The kit may further include, as needed, a container, controls (negative or positive controls), a buffer, adjuvants, etc., which can be selected by those skilled in the art based on specific circumstances. The kit may also include instructions for use to facilitate operation by those skilled in the art.

The present invention further provides a method for detecting ROR1 protein using the antibody, including but not limited to qualitative detection, quantitative detection and localization detection. Specifically, the detection method includes but is not limited to immunofluorescence assay, immunohistochemistry and radioimmunoassay.

A method for detecting the presence of ROR1 protein in a sample may include: contacting the sample with the antibody of the present invention or its antigen-binding fragment; observing whether an antibody complex is formed, the formation of which indicates the presence of ROR1 protein in the sample. The sample may be a cell and/or tissue sample; the sample may be fixed or dissolved in a medium; and the level of ROR1 protein in the fixed or dissolved sample is detected. In some embodiments, the subject of detection may be a cell-containing sample present in a cell preservation solution. In other embodiments, the antibody is further conjugated with a fluorescent dye, chemical substance, peptide, enzyme, isotope, label, etc. that can be used for detection or can be detected by other reagents.

The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, which are well described in the existing literature.

Example 1 Verification of immune antigen activity

In this example, the immunogenic activity of hROR1-Fc was verified. hROR1-Fc (Sino Biological, Catalog No. 13968-H02H1) contains the antigenically active fragment Metl-Glu403, which is homologous to the sequence of human, cynomolgus monkey, and rhesus monkey ROR1. The steps for verifying the immunogenic activity of hROR1-Fc are as follows:

(1) Antigen coating: Prepare a gradient dilution of human ROR1-Fc (hROR1-Fc (Sino Biological, catalog number: 13968-H02H1) solution using DPBS solution, add 100 μl to each well, and coat overnight at 4°C.

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

(3) Blocking: Use DPBS (containing 3% BSA) to block the antigen, add 200 μl to each well, place at room temperature, and block for 1 hour.

(4) Antibody binding: Dilute SPH-Cirm-01 (Biointron, catalog number: B266401) to 1 μg/mL using DPBS (containing 1% BSA) solution, add 100 μl to each well, and incubate at 37°C for 1 hour.

(5) Secondary antibody binding: discard the reaction solution, wash three times with PBST solution, add secondary antibody, add 100 μl to each well, and incubate at 37°C for 1 h.

(6) ELISA test: discard the reaction solution and wash three times with PBST solution; add 100μl TMB solution to each well and shake gently until the color turns blue; add 50μl H2SO4 to each well and shake gently until the color turns yellow; detect the OD value at 450nm.

As shown in FIG1 , the results of the hROR1-Fc antigen activity assay determined by ELISA showed that the hROR1-Fc antigen had good antigenic activity and could be used as a mouse immunogen in subsequent hybridoma experiments.

Example 2 Preparation and Screening of Anti-ROR1 Antibodies

Anti-ROR1 antibodies were obtained by immunizing 6-week-old female BALB/c mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., weighing 18 g). Certain biological properties of exemplary anti-ROR1 antibodies produced according to the methods of this example are described in detail in the examples set forth below.

1. Preparation of Hybridoma Cells

Hybridoma technology is achieved by fusing mouse spleen cells immunized with antigens and mouse myeloma cells SP2/0, thereby maintaining the main characteristics of both cells at the same time. The main characteristic of mouse spleen cells (B lymphocytes) immunized with specific antigens is their antibody secretion function, but they cannot be continuously cultured in vitro, while mouse myeloma cells have the so-called immortality and can divide and proliferate indefinitely under culture conditions. Under the action of selective culture medium, only hybrid cells fused with B cells and myeloma cells can form cell clones with both antibody secretion function and cell immortality. In this experiment, mice were immunized with human ROR1-Fc (Sino Biological, Catalog No.: 13968-H02H1) and RIBI adjuvant (Sigma, Catalog No.: 038M4131v). The three immunizations were mixed immunizations of protein cells. The obtained mouse spleen cells were then fused with SP2/0 cells to obtain hybridoma cells that can express positive antibodies.

1.1 Animal immunization

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

Table 1 Experimental animals and immunization information

1.2 Detection of serum titer of immunized mice after immunization

Antigen coating: Prepare 1 μg/ml human ROR1-his (KACTUS, Catalog No.: ROR-HM401) solution in DPBS solution, add 100 μl to each well of a 96-well plate, and coat overnight at 4°C.

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

Blocking: Use DPBS (containing 3% BSA) to block the antigen, add 100 μl to each well, place at room temperature, and block for 1 hour.

Antibody Binding: Dilute the serum solution with DPBS (containing 1% BSA) at a 300-fold initial concentration. Perform a 2-fold serial dilution through eight concentrations, adding 100 μl to each well. Serum from unimmunized mice was used as a negative control. Incubate at room temperature for 1 hour.

Secondary antibody binding: discard the reaction solution, wash three times with PBST solution; add secondary antibody at a dilution ratio of 1:5000, add 100 μl to each well, and react at room temperature for 1 hour.

ELISA test: discard the reaction solution and wash three times with PBST solution; add 100μl TMB solution to each well and shake gently until blue appears; add 50μl H2SO4 to each well and shake gently until yellow appears; detect OD value at 450nm.

The serum titers of multiple immunized mice are shown in Figure 2, and the values are shown in Table 2. The results showed that all five immunized mice produced immune responses, with titers reaching 218.7k or above, and fusion experiments were performed after subsequent flushing.

Table 2 Serum titers of immunized mice

2. Hybridoma Fusion

2.1 Mix spleen cells with SP2/0 mouse myeloma cells at a ratio of 5:1 to 2:1, centrifuge, and co-precipitate. Remove the supernatant and resuspend the cells in an appropriate amount of fusion buffer. Adjust the cell density to 1× ¹⁰⁷ cells/ml for electrofusion. After fusion, allow the cells to rest in the electrode holder for 5 minutes. Then, add the cells to 1×HAT selection medium containing 20% FBS, transfer them to a 96-well cell culture plate, and culture in a 37°C, 5% CO2 incubator. Replace the culture medium with fresh medium every 7-8 days.

2.2 After culturing for 10 days (or longer, depending on cell growth status), collect the supernatant and measure the antibody expression in the hybridoma supernatant by FACS to screen for hybridoma cells expressing specific anti-ROR1 antibodies. Positive clones obtained from the initial screening are transferred to 24-well plates for culture. The supernatant is then screened again for identification. Positive clones are selected for 1-2 rounds of subcloning. Hybridoma monoclonal cells that stably express specific anti-ROR1 antibodies are screened again. Based on this, the hybridoma cell line with the highest expression level is selected and the cell samples and Trizol samples are frozen.

The V region gene of the screened mouse anti-ROR1 antibody was recombined with the C region gene of the human antibody to create a chimeric anti-ROR1 antibody named Ab0. Then, the framework region was modified to transform Ab0 into humanized anti-ROR1 antibodies Ab1 to Ab8.

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

CDRs of Ab0 to Ab8:

HCDR1(SEQ ID NO：1)：SYVMS

HCDR2(SEQ ID NO：2)：SISSGGSTYYPDSVKG

HCDR3 (SEQ ID NO: 3): GPSTMINAMDY

LCDR1(SEQ ID NO：4)：ITTTDDDSMN

LCDR2(SEQ ID NO：5)：EGNNLRP

LCDR3 (SEQ ID NO: 6): LQSDNLPYT

Ab0 VH (SEQ ID NO: 7, where the bold underlined parts are CDRs)

Ab0 VL (SEQ ID NO: 8, where the bold underlined parts are CDRs)

Ab1, Ab2, Ab3, Ab4 VH (SEQ ID NO: 9, where the bold underlined parts are CDRs)

Ab5, Ab6, Ab7, Ab8 VH (SEQ ID NO: 10, where the bold underlined parts are CDRs)

Ab1, Ab5 VL (SEQ ID NO: 11, where the bold underlined parts are CDRs)

Ab2, Ab6 VL (SEQ ID NO: 12, where the bold underlined parts are CDRs)

Ab3, Ab7 VL (SEQ ID NO: 13, where the bold underlined parts are CDRs)

Ab4, Ab8 VL (SEQ ID NO: 14, where the bold underlined parts are CDRs)

Positive control antibody VH (SEQ ID NO: 15, where the bold underlined parts are CDRs)

Positive control antibody VL (SEQ ID NO: 16, where the bold underlined parts are CDRs)

Negative control antibody (SEQ ID NO: 17, human IgG1)

Ab0 (human IgG1) heavy chain, where the bold underline is VH (SEQ ID NO: 18)

Ab0 (human Kappa) light chain, where the bold underline is VL (SEQ ID NO: 19)

Ab1 (human IgG1) heavy chain, where the bold underline is VH (SEQ ID NO: 20)

Ab1 (human Kappa) light chain, where the bold underline is VL (SEQ ID NO: 21)

Ab2 (human IgG1) heavy chain, in which the bold underline is VH (SEQ ID NO: 20)

Ab2 (human Kappa) light chain, in which the bold underline is VL (SEQ ID NO: 22)

Ab3 (human IgG1) heavy chain, where the bold underline is VH (SEQ ID NO: 20)

Ab3 (human Kappa) light chain, where the bold underline is VL (SEQ ID NO: 23)

Ab4 (human IgG1) heavy chain, where the bold underline is VH (SEQ ID NO: 20)

Ab4 (human Kappa) light chain, where the bold underline is VL (SEQ ID NO: 24)

Ab5 (human IgG1) heavy chain, where the bold underline is VH (SEQ ID NO: 25)

Ab5 (human Kappa) light chain, where the bold underline is VL (SEQ ID NO: 21)

Ab6 (human IgG1) heavy chain, where the bold underline is VH (SEQ ID NO: 25)

Ab6 (human Kappa) light chain, where the bold underline is VL (SEQ ID NO: 22)

Ab7 (human IgG1) heavy chain, where the bold underline is VH (SEQ ID NO: 25)

Ab7 (human Kappa) light chain, where the bold underline is VL (SEQ ID NO: 23)

Ab8 (human IgG1) heavy chain, where the bold underline is VH (SEQ ID NO: 25)

Ab8 (human Kappa) light chain, where the bold underline is VL (SEQ ID NO: 24)

Example 3 Binding of anti-ROR1 antibodies to MDA-MB-231 cells

The binding ability of the anti-ROR1 antibodies to human ROR1 expressed on the surface of triple-negative breast cancer MDA-MB-231 cells was determined by flow cytometry. The binding ability of different anti-ROR1 antibodies of the present invention to human ROR1 expressed on the surface of MDA-MB-231 cells was determined by comparing the binding curves.

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

(2) The positive control antibody (cross-linked with the upper drug, catalog number B013-DS-210602), the negative control antibody (human IgG1, novoprotein, catalog number NC002) and the antigen-binding protein antibody of the present invention 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 a 96-well plate and incubated at 4 degrees Celsius for 1 hour.

(3) Wash the plate three times with PBS containing 2% FBS.

(4) PE-labeled human IgG (Invitrogen, catalog number 12-4998-82) was diluted with 2% FBS-PBS according to the product instructions. The diluted samples were added to a 96-well plate and incubated at 4°C for 0.5 h.

(5) Wash the plate twice with PBS containing 2% FBS.

(6) Resuspend the cells in PBS containing 2% FBS and measure the median fluorescence intensity (MFI) of the PE channel using a flow cytometer.

As shown in FIG3 and FIG4 , the binding curves of the anti-ROR1 antibody and human ROR1 expressed on the surface of MDA-MB-231 cells were determined based on flow cytometry.

As shown in Table 3, the EC ₅₀ values and the highest MFI values of the anti-ROR1 antibodies binding to human ROR1 expressed on the surface of MDA-MB-231 cells were determined based on flow cytometry.

Table 3 _EC50 values and maximum MFI values of anti-ROR1 antibodies binding to ROR1 expressed on the surface of MDA-MB-231 cells

The above experiments show that the anti-ROR1 antibodies of the present invention have binding activity to human ROR1 expressed on MDA-MB-231 cells, and the binding activity of 6 humanized anti-ROR1 antibodies to ROR1 is better than that of the positive control antibody.

Example 4 Binding of anti-ROR1 antibodies to JeKo-1 cells

The binding ability of the anti-ROR1 antibodies to JeKo-1 cells was determined based on flow cytometry. The binding ability of the different anti-ROR1 antibodies of the present invention to JeKo-1 cells was determined by comparing their binding curves.

(1) JeKo-1 cells (SIBS, catalog number TCHu194) were plated in a 96-well plate.

(2) The positive control antibody (cross-linked with the upper drug, product number B013-DS-210602), the negative control antibody (human IgG1, novoprotein, product number NC002) and the antigen-binding protein antibody of the present invention 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 a 96-well plate and incubated at 4 degrees Celsius for 1 hour.

(3) Wash the plate three times with PBS containing 2% FBS.

(4) PE-labeled human IgG (Invitrogen, catalog number 12-4998-82) was diluted with 2% FBS-PBS according to the product instructions. The diluted samples were added to a 96-well plate and incubated at 4°C for 0.5 h.

(5) Wash the plate twice with PBS containing 2% FBS.

(6) Resuspend the cells in PBS containing 2% FBS and measure the median fluorescence intensity (MFI) of the PE channel using a flow cytometer.

The binding curves of anti-ROR1 antibodies to JeKo-1 cells determined by flow cytometry are shown in Figures 5 and 6 .

The EC ₅₀ values and maximum MFI values of anti-ROR1 antibodies against JeKo-1 cells were determined by flow cytometry and are shown in Table 4 .

Table 4 _EC50 values and maximum MFI values of anti-ROR1 antibodies on JeKo-1 cells

The above experiments show that the anti-ROR1 antibodies of the present invention have binding activity to JeKo-1 cells, and the activities of 6 anti-ROR1 antibodies are better than those of the positive control antibody.

Example 5 Binding of anti-ROR1 antibodies to A549 cells

The binding ability of the anti-ROR1 antibodies to human lung adenocarcinoma A549 cells was determined based on flow cytometry. The binding ability of different anti-ROR1 antibodies of the present invention to A549 cells was determined by comparing their binding curves.

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

(2) The positive control antibody (cross-linked with the upper drug, product number B013-DS-210602), the negative control antibody (human IgG1, novoprotein, product number NC002) and the antigen-binding protein antibody of the present invention 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 a 96-well plate and incubated at 4 degrees Celsius for 1 hour.

(3) Wash the plate three times with PBS containing 2% FBS.

(4) PE-labeled human IgG (Invitrogen, catalog number 12-4998-82) was diluted with 2% FBS-PBS according to the product instructions. The diluted samples were added to a 96-well plate and incubated at 4°C for 0.5 h.

(5) Wash the plate twice with PBS containing 2% FBS.

(6) Resuspend the cells in PBS containing 2% FBS and measure the median fluorescence intensity (MFI) of the PE channel using a flow cytometer.

As shown in FIG7 and FIG8 , the binding curves of the anti-ROR1 antibody and human ROR1 expressed on the surface of A549 cells were determined based on flow cytometry.

As shown in Table 5, the EC ₅₀ values and the highest MFI values of the anti-ROR1 antibodies binding to human ROR1 expressed on the surface of A549 cells were determined based on flow cytometry.

Table 5 _EC50 values and maximum MFI values of anti-ROR1 antibodies binding to ROR1 expressed on the surface of A549 cells

The above experiments show that the anti-ROR1 antibodies of the present invention have binding activity to human ROR1 expressed on A549 cells, and the activities of the six humanized anti-ROR1 antibodies are better than those of the positive control antibodies.

Example 6 Binding affinity of anti-ROR1 antibodies

A molecular interaction instrument was used to detect the binding affinity of different anti-ROR1 antibodies to the antigen Human-cyno-ROR1, His Tag (source: Acro Biosystem, product number: RO1-H522y) protein.

(1) Buffer preparation: Add 10 μl Tween 20 to 50 ml PBS and add 220 μl per well to the buffer well of a Bio-One 96-well black flat-bottom plate (source: Greiner, catalog number: 655209).

(2) Dilute the antibody to 5 μg/ml with PBST (0.02% Tween 20) and add 220 μl per well to the antibody sample wells of the Bio-One 96-well black flat-bottom plate.

(3) Antigen preparation: Dilute the Human-cyno-ROR1, His Tag antigen to 100 nM with PBST (0.02% Tween 20) and add 220 μl per well to the antigen sample wells of the Bio-One 96-well black flat-bottom plate.

(4) Regeneration solution: Add 220 μl of Glycine solution per well to the Regeneration well of the Bio-One 96-well black flat-bottom plate.

(5) Place a new Bio-One 96-well black flat-bottom plate on the left side of the Molecular Interaction Analyzer. Place the green tray on top of it. Place the Protein A Dip and Read Biosensors (source: ForteBio, catalog number: 18-0015) in the first column of the tray. Place the Bio-One 96-well black flat-bottom plate with the sample preparation on the middle or right side of the Molecular Interaction Analyzer.

(6) On-machine testing and analysis.

The binding affinity of different anti-ROR1 antibodies to Human-cyno-ROR1 antigen protein was detected using a molecular interaction analyzer, as shown in Table 6.

Table 6 Binding affinity of anti-ROR1 antibodies to Human-cyno-ROR1 antigen protein

The results showed that the anti-ROR1 antibody of the present invention had a strong binding affinity to the Human-cyno-ROR1 antigen, and the affinity was comparable to that of the positive control antibody.

Example 7 Cross-reaction test of anti-ROR1 antibodies

This example detects the binding of anti-ROR1 antibodies to human ROR2-His (source: Acro, catalog number: RO2-H52E5) protein.

(1) Prepare ROR2 His antigen at 0.5 μg/ml in PBS, add it to a 96-well plate (Corning, Cat. No. 9018), and incubate at 4°C overnight.

(2) The next day, discard the coating solution and wash the plate three times with 0.05% Tween 20 in PBS.

(3) Incubate with PBS containing 3% BSA at 37°C for 2 hours, and wash the plate three times with DPBS containing 0.05% Tween20.

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

(5) Dilute the secondary antibody HRP-Goat anti-Human IgG Fc (Jackson, Cat. No. 109-035-098) in PBS containing 1% BSA according to the product instructions. Add the diluted sample to a 96-well plate and incubate at room temperature for 0.5 h. Wash the plate three times with PBS containing 0.05% Tween 20.

(6) Add 100 μl TMB substrate colorimetric solution to each well and incubate at room temperature for 15 minutes.

(7) Add 100 μl of 2N H2SO4 stop solution to each well, read the value at a wavelength of 450 nm using an enzyme-labeled reader and record it.

The binding curve of anti-ROR1 antibody to human ROR2 antigen is shown in FIG9 .

The binding ability of anti-ROR1 antibodies to human ROR2 antigen is shown in Table 7.

Table 7 Binding ability of humanized anti-ROR1 antibodies to human ROR2 antigen

The above experiments show that the anti-ROR1 antibody of the present invention has no binding activity with the human ROR2-His antigen, indicating that the anti-ROR1 antibody of the present invention has binding specificity with the ROR1 antigen.

Example 8 Endocytic biological activity of anti-ROR1 antibodies

The test antibody is serially diluted and incubated with DT3C diluent at room temperature to conjugate the antibody toxin. The antibody is then incubated with CHOK1/hROR1 cells in a 96-well plate for three days. MTS is then added to measure the cytotoxicity of the toxin-conjugated antibody. DT3C is virtually non-toxic to cells outside the body and only causes growth inhibition after the antibody is internalized. Therefore, the degree of cell growth inhibition reflects the amount of antibody internalized by the cells.

(1) On the first day, CHOK1/hROR1 cells were digested separately, resuspended and counted, centrifuged and the supernatant removed, and the cell density was adjusted to 4E4.

(2) Cells were plated in 96-well cell culture plates, 50 μL was added to each well, i.e., 2E3 cells were seeded per well. The plates were gently tapped to mix, and the plates were placed in a 37°C cell culture incubator and incubated for 16 hours.

(3) The next day, dilute DT3C to a 2X (2 μg/mL) working concentration using F12K/10% FBS. Dilute the antibody from its maximum concentration of 5 μg/ml (2X) in 2-fold dilutions for 9 points. Add 50 μL to each well to achieve the sample concentration as indicated in the experimental design. Add 50 μL of complete medium to two wells containing the same DT3C concentration as the experimental group, and fill the remaining wells to 100 μL with F12K complete medium.

(4) Gently tap to mix, and place the cell culture plate in a 37°C cell culture incubator for 72 hours.

(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 and serves as the positive control well for the cytotoxicity experiment). At the same time, MTS is thawed in advance at room temperature in the dark.

(6) Remove the cell plate from the incubator and add 20 μL of MTS to each well. Gently tap the plate and incubate in a 37°C incubator for 2.5 h. During this time, observe the color change and gently shake to mix.

(7) After the colors are clearly differentiated, use a microplate reader to detect: OD490 reading.

Calculation formula: background value = cell only - Lysis; inhibition rate = 1 - (experimental group - Lysis) / (background value) * 100%.

As shown in FIG10 , the anti-ROR1 antibody has good endocytic activity, and the endocytic activity of the anti-ROR1 antibody Ab2 is better than that of the positive control antibody.

As shown in Table 8, the anti-ROR1 antibody inhibited cell growth in a concentration-dependent manner.

Table 8 Detection of endocytic activity of anti-ROR1 antibodies in CHOK1/hROR1 cells

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

Example 9 Preparation of Ab2-ADC7

Synthesis of compound 10:

Under nitrogen protection, (2-bromoethoxy)(tert-butyl)diphenylsilane (22.5 g, synthesized according to Synlett 2014; 25(19): 2802-2805) and 1H-pyrazole-4-carboxaldehyde (5.0 g, CAS: 35344-95-7, purchased from Shaoyuan Technology (Shanghai) Co., Ltd.) were dissolved in 100 mL of anhydrous N,N-dimethylformamide. Potassium carbonate (8.6 g) was added at room temperature, and the mixture was heated to 85°C for 4 hours. TLC monitoring showed that the reaction of the raw materials was complete, and the mixture was cooled to room temperature. 200 mL of water was added to quench the reaction, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain compound 10 as a colorless oil (12.8 g, yield 65%). ¹ H NMR (400MHz, CDCl ₃ )δ9.86 (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=5.1Hz, 2H), 4.00 (t, J=5.1Hz, 2H), 1.00 (s, 9H).

Synthesis of compound K4:

Step 1: Synthesis of compound 11

Under nitrogen protection, compound 9 (1.0 g, synthesis method refers to Example 1 of patent CN111470998A) was dissolved in 15 mL of anhydrous tetrahydrofuran and cooled at -78°C for half an hour. At low temperature, a hexane solution of n-butyllithium (2.5 M, 3.5 mL) was added dropwise. After the dropwise addition, a solution of compound 10 (1.5 g) in anhydrous tetrahydrofuran (5 mL) was quickly added and 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, and concentrated under reduced pressure. Compound 11 was isolated by column chromatography to obtain 0.6 g of a light yellow oil in a yield of 25%. ¹ H NMR (400MHz, CDCl ₃ )δ8.69 (s, 1H), 7.95 (d, J=11.6Hz, 1H), 7.52-7.28 (m, 14H), 6.91 (d, J=8.2Hz, 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 ⁺ ).

Step 2: Synthesis of compound 12

Under nitrogen, compound 11 (0.6 g) was dissolved in 15 mL of acetonitrile, and manganese dioxide (0.86 g) was added. The mixture was heated under reflux for three hours. After TLC analysis of the reaction, the mixture was filtered through celite, and the filtrate was dried and separated by column chromatography to afford compound 12 as a colorless oil (0.55 g, 92% yield). ¹ H NMR (400MHz, CDCl ₃ )δ10.82 (s, 1H), 8.39 (d, J = 12.4Hz, 1H), 7.99 (s, 1H), 7.91 (s, 1H), 7.62 (d, J = 8.3Hz, 1H), 7.55-7.33 (m, 1 0H), 4.30 (t, J=5.0Hz, 2H), 4.03 (t, J=5.0Hz, 2H), 2.27-2.15 (m, 6H), 1.00 (s, 9H).MS (ESI) m/z=544.2 (M+H ⁺ ).

Step 3: Synthesis of compound 13

Under nitrogen, compound 12 (0.55 g) was dissolved in 10 mL of ethanol, and 3 mL of concentrated hydrochloric acid was added. The mixture was heated under reflux for 3 hours, resulting in the precipitation of a white solid. TLC confirmed the complete reaction of the starting material. The product was filtered and dried to afford 0.19 g of compound 13, in an 80% yield. The product was used directly in the next reaction without further purification.

Step 4: Synthesis of compound K4

Under nitrogen, 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 of toluene and 5 mL of acetic acid. The mixture was heated to 100°C and reacted overnight. TLC confirmed the complete reaction. The product was then dried and separated by column chromatography to yield 0.28 g of K4 as a white solid, with a yield of 80%. ¹ H NMR (400MHz, DMSO) δ8.43 (s, 1H), 8.16 (d, J = 8.2Hz, 1H), 8.08 (s, 1H), 7.91 (d, J = 10.8Hz, 1H), 7.33 (s, 1H), 5.42 (s, 2H), 5.38-5.12 (m , 2H), 4.34 (t, J=5.5Hz, 2H), 3.88 (t, J=5.6Hz, 2H), 2.47 (s, 3H), 2.00-1.73 (m, 2H), 0.89 (t, J=7.3Hz, 3H). MS (ESI) m/z=491.2 (M+H+).

Synthesis of compound mc-GGFG-CPT2C:

Step 1: Preparation of compound 15

Under nitrogen, compound K4 (100 mg) and compound 14 (150 mg, CAS: 1599440-06-8, purchased from MCE) were dissolved in 10 mL of anhydrous tetrahydrofuran. Pyridinium 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 over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 15 was isolated by column chromatography to yield 50 mg of a white solid in a 30% yield.

¹ H NMR (400MHz, DMSO) δ8.76 (t, J=6.6Hz, 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=6.6Hz, 2H), 4.46 (t, J=5.2Hz, 2H), 4.25-4.18 (m, 2H), 4.17-4.10 (m, 1H), 3.90 (t, J =5.4Hz, 2H), 3.65 (d, J=6.0Hz, 2H), 2.42 (s, 3H), 1.90-1.81 (m, 2H), 0.89 (t, J=7.3Hz, 3H). MS (ESI) m/z = 799.3 (M+H ⁺ ).

Step 2: Preparation of compound 16

Under nitrogen, compound 15 (50 mg) was dissolved in 5 mL of anhydrous tetrahydrofuran. Diethylamine (0.5 mL) was added at room temperature and allowed to react for 2 hours, resulting in the precipitation of a solid. TLC confirmed the complete reaction of the starting material. The product was then filtered and dried under vacuum to afford compound 16 as a pale yellow solid (30 mg, 83% yield). The product was used directly in the next step without further purification. MS (ESI) m/z = 577.2 (M+H ⁺ ).

Step 3: Preparation of compound mc-GGFG-CPT2C

Under nitrogen, compound 17 (30 mg, CAS: 1599440-15-9, purchased from MCE) was dissolved in 2 mL of anhydrous N,N-dimethylformamide. 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (18.5 mg) and N,N-diisopropylethylamine (10 μL) were added at room temperature and allowed to react for 2 hours. After the reaction solution became clear, compound 16 (30 mg) was added at room temperature and allowed to react overnight. LC-MS confirmed the complete reaction of raw material ^16. The product was then directly spin-dried and purified on a C18 reverse-phase column to obtain compound mc-GGFG-CPT2C as a light yellow solid (27 mg, 50% yield). NMR (400MHz, DMSO) δ8.57 (t, J=6.6Hz, 1H), 8.45 (s, 1H), 8.30 (t, J=5.8Hz, 1H), 8.17-8.07 (m, 3H), 8.04 (t, J=5.7Hz, 1H), 7.99 (t, J=5.6Hz, 1H), 7.9 0(d, J=10.7Hz, 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=6.6Hz, 2H), 4.51-4.41 (m , 3H), 3.89 (t, J=5.4Hz, 2H), 3.79-3.69 (m, 3H), 3.67-3.63 (m, 2H), 3.58 ( dd, J＝16.7, 5.4Hz, 1H), 3.35 (t, J＝7.1Hz, 2H), 3.01 (dd, J＝13.8, 4.5Hz, 1H ), 2.76 (dd, J=13.7, 9.7Hz, 1H), 2.47 (s, 3H), 2.09 (t, J=7.4Hz, 2H), 1.94- 1.79 (m, 2H), 1.52-1.39 (m, 4H), 1.23-1.12 (m, 2H), 0.88 (t, J=7.3Hz, 3H). MS (ESI) m/z=1031.4(M+H ⁺ ).

Prepare Ab2-ADC7 of the following formula, wherein Ab is Ab2 and ADC7 is mc-GGFG-CPT2C:

At 37°C, add the prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 112.7 uL, 1127 nmol) to the PBS buffer (10 mM PBS buffer at pH = 7.4; 5.0 mg/mL, 4.0 mL, 133 nmol) containing the antibody Ab2, place in a water bath shaker, and shake at 37°C for 1.5 hours before stopping the reaction.

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

The average drug loading calculated by RP-HPLC was: y=7.9.

Example 10 Preparation of Ab7-ADC7

Prepare Ab7-ADC7 of the following formula, wherein Ab is Ab7 and ADC7 is mc-GGFG-CPT2C:

At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 66.7 uL, 667 nmol) to the PBS buffer (10 mM PBS buffer at pH = 7.4; 5.0 mg/mL, 2.0 mL, 67 nmol) containing the antibody Ab7, place in a water bath shaker, and shake at 37°C for 1.5 hours before stopping the reaction.

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

The average drug loading calculated by RP-HPLC was: y=7.9.

Example 11 Preparation of positive control antibody-ADC1

Prepare a positive control antibody-ADC1 of the following formula, wherein Ab is a positive control antibody and ADC1 is mc-vc-PAB-MMAE:

At 37°C, add the prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 50 uL, 500 nmol) to the PBS buffer (10 mM PBS buffer at pH = 7.4; 5.0 mg/mL, 6.0 mL, 200 nmol) containing the antibody positive control antibody, place in a water bath shaker, and shake at 37°C for 1.5 hours before stopping the reaction.

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

The average drug loading value calculated by RP-HPLC was: y=3.9.

Example 12 Preparation of Ab7-ADC6

The structure of mc-GGFG-Dxd:

At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 160 μL, 1600 nmol) was added to a PBS buffer solution of antibody Ab7 (0.05 M PBS buffer solution at pH 6.5; 10 mg/mL, 3 mL, 200 nmol), and the mixture was placed in a water bath shaker. After shaking at 37°C for 3 hours, the reaction was stopped.

The reaction solution was then cooled to 25°C in 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 DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and oscillated at 25°C for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (mobile phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain Ab7-ADC6 in PBS buffer (4.5 mg/mL, 4.9 mL), which was then stored at 4°C.

The average drug loading value calculated by RP-HPLC was: y=7.8.

Example 13 Anti-tumor efficacy test of anti-ROR1 antibody-ADC7 on the NOG mouse subcutaneously transplanted JeKo-1 cell animal model

This study used the NOG mouse subcutaneous inoculation of JeKo-1 cells as an animal model to determine the anti-tumor effect of humanized anti-ROR1 antibody-ADC7.

NOG mice: Female NOG mice (6 weeks old) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The mice were acclimated for 7 days after arrival before the study began.

Human mantle cell lymphoma JeKo-1 cells (from the Shanghai Chinese Academy of Sciences Cell Bank) were routinely subcultured according to the manufacturer's instructions. Cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of female NOG mice to establish a JeKo-1 tumor-bearing mouse model.

Dosage:

On the 6th day after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes ranging from 107.45 mm ³ to 133.77 mm ³ were selected and evenly grouped according to tumor volume (5 mice per group). They were administered with negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), positive control antibody-ADC1 (conjugated toxin MMAE) (SPH022-PC-2, cross-linked with the drug, catalog number: B013-DS-210602), and antibody Ab2-ADC7 of the present invention (conjugated toxin CPT2C) on the 6th and 20th days after inoculation, respectively. During the administration period, the changes in tumor volume and body weight of each group of mice were monitored twice a week for 3 consecutive weeks. Body weight and tumor volume were measured before each administration. Tumor volume inhibition rate (TGI%) was calculated on day 27 after inoculation using the following formula: TGI (%) = [1-(Ti - T0) / (Vi - V0)] × 100; where Ti: mean tumor volume of the treatment group, T0: mean tumor volume of the treatment group on day D0, Vi: mean tumor volume of the isotype control group, and V0: mean tumor volume of the isotype control group on day D0. Tumor volume measurement: The major diameter (a) and the major diameter (b) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: TV = 1/2 × a × ^b² . Body weight was measured using an electronic balance.

The dosage and method of administration are shown in Table 9.

Table 9 Dosage trial design

* Administer once every two weeks for a total of 2 times.

As shown in FIG11 , the anti-tumor efficacy of Ab2-ADC7 was tested in an animal model of subcutaneously transplanted JeKo-1 cells in NOG mice.

As shown in FIG12 , Ab2-ADC7 affects the body weight changes of NOG mice subcutaneously transplanted with JeKo-1 cells.

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

Table 10 Inhibitory effect of Ab2-ADC7 on tumor growth in vivo on day 27

Note: **: Compared with the tumor volume of negative control antibody, P < 0.01.

The above experiments 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.

Example 14 Anti-tumor efficacy test of anti-ROR1 antibody-ADC7 on the NOG mouse subcutaneously transplanted HCC70 cell animal model

This study used the NOG mouse subcutaneous inoculation of HCC70 cells as an animal model to determine the anti-tumor effect of humanized anti-ROR1 antibody-ADC7.

NOG mice: Female NOG mice (6 weeks old) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The mice were acclimated for 7 days after arrival before the study began.

Human breast ductal carcinoma HCC70 cells (ATCC source) were routinely subcultured according to the manufacturer's instructions. Cells were resuspended in serum-free medium and the cell density was adjusted. On day 0, the cell suspension was subcutaneously inoculated into the right axilla of female NOG mice to establish an HCC70 tumor-bearing mouse model.

Dosage:

On the 9th day after tumor cell inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes ranging from 137.50 mm ³ to 229.45 mm ³ were selected and evenly grouped according to tumor volume (5 mice per group). They were administered with negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), positive control antibody-ADC1 (conjugated toxin MMAE) (SPH022-PC-2, source: Shanghai Pharmaceutical Cross-linking, catalog number: B013-DS-210602), and antibody Ab2-ADC7 of the present invention (conjugated toxin CPT2C) on days 9, 16, 23, and 30 after inoculation. During the administration period, the changes in tumor volume and body weight of each group of mice were monitored twice a week for 10 consecutive weeks. Body weight and tumor volume were measured before each administration. Tumor volume inhibition rate (TGI%) was calculated on day 82 after inoculation using the following formula: TGI (%) = [1-(Ti - T0) / (Vi - V0)] × 100; where Ti: mean tumor volume of the treatment group, T0: mean tumor volume of the treatment group on day D0, Vi: mean tumor volume of the isotype control group, and V0: mean tumor volume of the isotype control group on day D0. Tumor volume measurement: The major diameter (a) and the major diameter (b) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: TV = 1/2 × a × ^b² . Body weight was measured using an electronic balance.

The dosage and method of administration are shown in Table 11.

Table 11 Dosage trial design

* Administer once a week for a total of 4 times.

As shown in FIG13 , the anti-tumor efficacy of Ab2-ADC7 was tested on the HCC70 cell subcutaneously transplanted animal model in NOG mice.

As shown in FIG14 , Ab2-ADC7 affects the body weight changes in the NOG mouse subcutaneously transplanted HCC70 cell animal model.

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

Table 12 Ability of Ab2-ADC7 to inhibit tumor growth in vivo on day 82

Note: **: Compared with the tumor volume of negative control antibody, P < 0.01.

The above experiments showed that Ab2-ADC7 significantly inhibited the growth of HCC70 cell tumors and had no significant effect on the body weight of NOG mice.

Example 15 Anti-tumor efficacy test of anti-ROR1 antibody-ADC7 on the BALB/c nude mouse subcutaneous transplantation of MDA-MB-231 cells animal model

In this study, the anti-tumor effect of humanized anti-ROR1 antibody ADC7 was determined using an animal model in which BALB/c nude mice were subcutaneously inoculated with MDA-MB-231 cells.

BALB/c nude mice: Female BALB/c nude mice (6 weeks old) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were acclimated for 7 days after arrival before the study began.

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

Dosage:

Ten days after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes ranging from 139.65 mm ³ to 288.26 mm ³ were selected and evenly grouped according to tumor volume (5 mice per group). They were administered with negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), antibody Ab7-ADC6 (conjugated toxin Dxd) of the present invention, and Ab7-ADC7 (conjugated toxin CPT2C) on days 10, 17, 24, and 31 after inoculation. During the administration period, the tumor volume and body weight of each group of mice were monitored twice a week for 6 consecutive weeks. Body weight and tumor volume were measured before each administration. Tumor volume inhibition rate (TGI%) was calculated on day 52 after inoculation using the following formula: TGI (%) = [1-(Ti - T0) / (Vi - V0)] × 100; where Ti: mean tumor volume of the treatment group, T0: mean tumor volume of the treatment group on day D0, Vi: mean tumor volume of the isotype control group, and V0: mean tumor volume of the isotype control group on day D0. Tumor volume measurement: The major diameter (a) and the major diameter (b) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: TV = 1/2 × a × ^b² . Body weight was measured using an electronic balance.

The dosage and administration method are shown in Table 13.

Table 13 Dosage trial design

* Administer once a week for a total of 4 times.

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

As shown in Figure 16, Ab7-ADC6 and Ab7-ADC7 affect the body weight changes of the BALB/c nude mouse subcutaneously transplanted MDA-MB-231 cell animal model.

As shown in Table 14, on day 52 after inoculation, both 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.

Table 14 Inhibitory effect of Ab7-ADC7 on tumor growth in vivo on day 52

Note: **: Compared with the tumor volume of negative control antibody, P < 0.01.

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

Example 16 Anti-tumor efficacy test of anti-ROR1 antibody-ADC7 on the BALB/c nude mouse subcutaneously transplanted MDA-MB-231 cell animal model

In this study, the anti-tumor effect of humanized anti-ROR1 antibody ADC7 was determined using an animal model in which BALB/c nude mice were subcutaneously inoculated with MDA-MB-231 cells.

BALB/c nude mice: Female BALB/c nude mice (6 weeks old) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The mice were acclimated for 7 days after arrival before the study began.

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

Dosage:

On the 9th day after tumor cell inoculation, the tumor volume of each mouse was detected, and mice with tumor volumes ranging from 139.65 mm ³ to 288.26 mm ³ were selected and evenly grouped according to tumor volume (5 mice per group). They were administered with negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), positive control antibody-ADC1 (conjugated toxin MMAE) (SPH022-PC-2, source: Shanghai Pharmaceutical Cross-linking, catalog number: B013-DS-210602), and antibody Ab2-ADC7 of the present invention (conjugated toxin CPT2C) on days 9, 16, 23, and 30 after inoculation. During the administration period, the changes in tumor volume and body weight of each group of mice were monitored twice a week for 8 consecutive weeks. Body weight and tumor volume were measured before each administration. Tumor volume inhibition rate (TGI%) was calculated on day 65 after inoculation using the following formula: TGI (%) = [1-(Ti - T0) / (Vi - V0)] × 100; where Ti: mean tumor volume of the treatment group, T0: mean tumor volume of the treatment group on day D0, Vi: mean tumor volume of the isotype control group, and V0: mean tumor volume of the isotype control group on day D0. Tumor volume measurement: The major diameter (a) and the major diameter (b) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: TV = 1/2 × a × ^b² . Body weight was measured using an electronic balance.

The dosage and method of administration are shown in Table 15.

Table 15 Dosage study design

* Administer once a week for a total of 4 times.

As shown in Figure 17, the anti-tumor efficacy of Ab2-ADC7 was tested in an animal model of subcutaneously transplanted MDA-MB-231 cells in BALB/c nude mice.

As shown in Figure 18, Ab2-ADC7 affects the body weight changes of the BALB/c nude mouse subcutaneously transplanted MDA-MB-231 cell animal model.

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

Table 16 Inhibitory effect of Ab2-ADC7 on tumor growth in vivo on day 65

Note: **: Compared with the tumor volume of negative control antibody, P < 0.01.

The above experiments 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/cnude mice.

Example 17 Antitumor efficacy test of anti-ROR1 antibody-ADC on breast cancer PDX animal model LD1-2009-362153

In this study, human breast cancer LD1-2009-362153 tumor tissue was subcutaneously inoculated into NCG mice to determine the anti-tumor effect of anti-ROR1 antibody-ADC.

NCG mice: Female NCG mice (6 weeks old) were purchased from Chengdu Yaokang Biotechnology Co., Ltd. The mice were acclimated for 3 days after arrival before the study began.

Tumor tissue: Human breast cancer LD1-2009-362153 tumor tissue (provided by Xi'an Lidi Biotechnology Co., Ltd.), passaged FP3+5, was used for this efficacy study. On day 0, LD1-2009-LD1-2009-362153 human breast cancer xenografts were cut into approximately 3 mm × 3 mm × 3 mm (approximately 45-60 mg) pieces and inoculated subcutaneously into NCG mice to establish a tumor-bearing mouse model.

Dosage:

On the 32nd day after tumor tissue inoculation, the tumor volume of each mouse was detected. Mice with tumor volumes ranging from 100 mm ³ to 200 mm ³ were selected and evenly grouped according to tumor volume (5 mice per group). They were administered with negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), positive control antibody-ADC1 (conjugated toxin MMAE) (SPH022-PC-2, cross-linked with the drug, catalog number: B013-DS-210602), and antibody Ab2-ADC7 of the present invention (conjugated toxin CPT2C) on days 32, 39, 46, and 53 after inoculation, respectively. During the administration period, the changes in tumor volume and body weight of each group of mice were monitored twice a week for 6 consecutive weeks. Body weight and tumor volume were measured before each dosing. Tumor volume inhibition rate (TGI%) was calculated 38 days after dosing using the following formula: TGI (%) = [1-(Ti - T0) / (Vi - V0)] × 100; where Ti: mean tumor volume of the dosing group, T0: mean tumor volume of the dosing group on day D0, Vi: mean tumor volume of the isotype control group on day D0, and V0: mean tumor volume of the isotype control group on day D0. Tumor volume measurement: The major diameter (a) and the major diameter (b) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: TV = 1/2 × a × ^b² . Body weight was measured using an electronic balance.

The dosage and administration method are shown in Table 17.

Table 17 Dosage trial design

* Administer once a week for a total of 4 times.

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

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

Table 18 Inhibitory effect of Ab2-ADC7 on tumor growth in vivo on day 38

Note: ****: Compared with the tumor volume of negative control antibody, P<0.0001.

The above test results showed that Ab2-ADC7 significantly inhibited the growth of tumors in the breast cancer PDX animal model LD1-2009-362153, and had no significant effect on the body weight of NCG mice.

Example 18 Antitumor efficacy test of anti-ROR1 antibody-ADC on breast cancer PDX animal model LD1-2009-361973

In this study, human breast cancer LD1-2009-361973 tumor tissue was subcutaneously inoculated into Nu/Nu mice to determine the anti-tumor effect of anti-ROR1 antibody-ADC.

Nu/Nu mice: Female Nu/Nu mice (6 weeks old) were purchased from Chengdu Yaokang Biotechnology Co., Ltd. The mice were acclimated for 3 days after arrival before the study began.

Tumor tissue: Human breast cancer LD1-2009-361973 tumor tissue (provided by Xi'an Lidi Biotechnology Co., Ltd.), passaged FP2+5, was used for this efficacy study. On day 0, LD1-2009-361973 human breast cancer xenografts were cut into approximately 3 mm × 3 mm × 3 mm (approximately 45-60 mg) pieces and inoculated subcutaneously into Nu/Nu mice to establish a tumor-bearing mouse model.

Administration: On the 34th day after tumor tissue inoculation, the tumor volume of each mouse was detected. Mice with tumor volumes ranging from 100 ^mm3 to 200 ^mm3 were selected and evenly grouped according to tumor volume (5 mice per group). They were administered with negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), positive control antibody-ADC1 (conjugated toxin MMAE) (SPH022-PC-2, cross-linked with the above drug, catalog number: B013-DS-210602), and antibody Ab2-ADC7 of the present invention (conjugated toxin CPT2C) on the 34th day after inoculation. During the administration period, the changes in tumor volume and body weight of each group of mice were monitored twice a week for 8 consecutive weeks. Body weight and tumor volume were measured before each dosing. Tumor volume inhibition (TGI%) was calculated 35 days after dosing using the following formula: TGI (%) = [1-(Ti - T0) / (Vi - V0)] × 100; where Ti: mean tumor volume of the dosing group, T0: mean tumor volume of the dosing group on D0, Vi: mean tumor volume of the isotype control group on D0. Tumor volume was measured using a vernier caliper to measure the major diameter (a) and the major diameter (b). Tumor volume was calculated using the following formula: TV = 1/2 × a × b². Body weight was measured using an electronic balance.

The dosage and administration method are shown in Table 19.

Table 19 Dosage trial design

*Single dose.

Figures 21 and 22 show the anti-tumor efficacy of Ab2-ADC7 on the subcutaneously transplanted breast cancer PDX animal model LD1-2009-361973 in Nu/Nu mice and the changes in body weight.

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

Table 20 Inhibitory effect of Ab2-ADC7 on tumor growth in vivo on day 35

Note: ***: Compared with the tumor volume of negative control antibody, P < 0.001.

The above experimental results showed that Ab2-ADC7 significantly inhibited the growth of tumors in the breast cancer PDX animal model LD1-2009-361973, and had no significant effect on the body weight of Nu/Nu mice.

Example 19 Antitumor efficacy test of anti-ROR1 antibody-ADC on lymphoma PDX animal model LD1-0026-410827

In this study, human lymphoma LD1-0026-410827 tumor tissue was subcutaneously inoculated into NU/NU mice to determine the anti-tumor effect of anti-ROR1 antibody-ADC.

NU/NU mice: Female NU/NU mice (6 weeks old) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The mice were acclimated for 3 days after arrival before the study began.

Tumor tissue: Human lymphoma LD1-0026-410827 tumor tissue (provided by Xi'an Lidi Biotechnology Co., Ltd.), passaged FP1+2, was used for this efficacy study. On day 0, the LD1-0026-410827 human lymphoma xenograft was cut into approximately 3 mm × 3 mm × 3 mm (approximately 45-60 mg) pieces and inoculated subcutaneously into NU/NU mice to establish a tumor-bearing mouse model.

Dosing: 29 days after tumor tissue inoculation, tumor volume was measured in each mouse. Mice with tumor volumes between 100 ^mm³ and 200 ^mm³ were selected and divided into groups (5 mice per group) based on tumor volume. Dosing was performed on days 29, 36, 43, and 50 after inoculation, respectively. A negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), a positive control antibody - ADC1 (conjugated toxin MMAE) (SPH022-PC-2, cross-linked with the drug, catalog number: B013-DS-210602), and the antibody Ab2-ADC7 of the present invention (conjugated toxin CPT2C) were administered. Tumor volume and body weight were monitored twice weekly for 4 weeks. Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: TV = 1/2 × a × ^b² . Body weight was measured using an electronic balance.

The dosage and method of administration are shown in Table 21.

Table 21 Dosage trial design

* Administer once a week for a total of 4 times.

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

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

Table 22 Ability of Ab2-ADC7 to inhibit tumor growth in vivo on day 25

Note: ***: Compared with the tumor volume of negative control antibody, P < 0.001.

The above experimental results showed that Ab2-ADC7 significantly inhibited the growth of tumors in the lymphoma PDX animal model LD1-0026-410827, and had no significant effect on the body weight of NU/NU mice.

Example 20 Antitumor efficacy test of anti-ROR1 antibody-ADC on lung cancer PDX animal model LU-01-1621

In this study, human breast cancer LU-01-1621 tumor tissue was subcutaneously inoculated into BALB/c nude mice to determine the anti-tumor effect of anti-ROR1 antibody-ADC.

BALB/c nude mice: Female BALB/c nude mice (7–9 weeks) were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd. Mice were acclimated for 3 days after arrival before the study began.

Tumor tissue: Human lung cancer LU-01-1621 tumor tissue (Shanghai WuXi AppTec Pharmaceutical Development Co., Ltd.), passaged at FP5, was used for this efficacy study. On day 0, LU-01-1621 human lung cancer xenografts were excised into approximately 30 ^mm³ sections and inoculated subcutaneously into BALB/c nude mice to establish a tumor-bearing mouse model.

Administration: On the 33rd day after tumor tissue inoculation, the tumor volume of each mouse was detected. Mice with tumor volumes ranging from 100 mm ³ to 200 mm ³ were selected and evenly grouped according to tumor volume (5 mice per group). Drugs were administered on days 33, 40, 47, and 54 after inoculation, respectively. The negative control antibody (human IgG Control, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP067-F1), the positive control antibody - ADC1 (conjugated toxin MMAE) (SPH022-PC-2, cross-linked with the above drug, catalog number: B013-DS-210602), and the antibody Ab2-ADC7 of the present invention (conjugated toxin CPT2C) were administered. During the administration period, the changes in tumor volume and body weight of each group of mice were monitored twice a week for 4 consecutive weeks. Body weight and tumor volume were measured before each dosing. Tumor volume inhibition (TGI%) was calculated 28 days after dosing using the following formula: TGI (%) = [1-(Ti - T0) / (Vi - V0)] × 100; where Ti: mean tumor volume of the dosing group, T0: mean tumor volume of the dosing group on day D0, Vi: mean tumor volume of the isotype control group on day D0, and V0: mean tumor volume of the isotype control group on day D0. Tumor volume measurement: The major diameter (a) and the major diameter (b) of the tumor were measured using a vernier caliper. Tumor volume was calculated using the following formula: TV = 1/2 × a × b². Body weight was measured using an electronic balance.

The dosage and method of administration are shown in Table 23.

Table 23 Dosage trial design

*Single dose.

Figures 25 and 26 show the antitumor efficacy of Ab2-ADC7 on the subcutaneously transplanted lung cancer PDX animal model LU-01-1621 in BALB/c nude mice and the changes in body weight.

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 tumor growth at a dose of 5 mg/kg, with a tumor volume inhibition rate of 59.39%.

Table 24 Inhibitory effect of Ab2-ADC7 on tumor growth in vivo on day 35

Note: *: Compared with the tumor volume of negative control antibody, P < 0.05.

The above experimental results show that Ab2-ADC7 significantly inhibits the growth of lung cancer PDX animal model LU-01-1621 tumor, and has no obvious effect on the body weight of BALB/c nude mice.

The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art can make a number of variations and improvements without departing from the concept of the present invention, and these are all within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the appended claims. At the same time, all documents mentioned in the present invention are cited as references in the present invention, just as if each document was cited as a reference individually.

Claims (15)

An anti-ROR1 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: an immunoglobulin heavy chain variable region (VH), which comprises heavy chain HCDR1, HCDR2 and HCDR3, wherein the HCDR1 is or comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 is or comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 is or comprises the amino acid sequence shown in SEQ ID NO: 3; and an immunoglobulin light chain variable region (VL), which comprises light chain LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is or comprises the amino acid sequence shown in SEQ ID NO: 4, the LCDR2 is or comprises the amino acid sequence shown in SEQ ID NO: 5, and the LCDR3 is or comprises the amino acid sequence shown in SEQ ID NO: 6. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1, characterized in that the anti-ROR1 antibody or antigen-binding fragment thereof comprises: an amino acid sequence as shown in SEQ ID NO: 7, 9 or 10, or a heavy chain variable region having at least 85% identity with the amino acid sequence as shown in SEQ ID NO: 7, 9 or 10, and a light chain variable region having at least 85% identity with the amino acid sequence as shown in SEQ ID NO: 8, 11, 12, 13 or 14. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or has at least about 85% identity with SEQ ID NO: 7, and a light chain variable region having an amino acid sequence that is identical to or has at least about 85% identity with SEQ ID NO: 8; or The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 11; or The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 12; or The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 13; or The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 9, and a light chain variable region having an amino acid sequence that is 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 having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 11; or The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 12; or The anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to or at least about 85% identical to SEQ ID NO: 13; or The anti-ROR1 antibody or its antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence that is identical to or has at least about 85% identity with SEQ ID NO: 10, and a light chain variable region having an amino acid sequence that is identical to or has at least about 85% identity with SEQ ID NO: 14. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that 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 the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 11, respectively; or The VH region and the VL region are or comprise the amino acid sequences shown 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 shown in SEQ ID NO: 9 and SEQ ID NO: 13, respectively; or The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 14, respectively; or The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively; or The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 12, respectively; or The VH region and the VL region are or comprise the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 13, respectively; or The VH region and the VL region are or contain the amino acid sequences shown in SEQ ID NO: 10 and SEQ ID NO: 14, respectively. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that the anti-ROR1 antibody or antigen-binding fragment thereof is or comprises an amino acid sequence as shown in SEQ ID NO: 18, 20 or 25, or a heavy chain having at least about 85% identity thereto, and an amino acid sequence as shown in SEQ ID NO: 19, 21, 22, 23 or 24, or a light chain having at least about 85% identity thereto; Preferably, The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 18; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 19; or The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 21; or The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 22; or The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 23; or The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 24; or The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 21; or The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 22; or The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 23; or The heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 24. The anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that the anti-ROR1 antibody or antigen-binding fragment thereof is a murine antibody, a humanized antibody, a chimeric antibody or a fully human antibody. A polynucleotide encoding the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6. An immunoconjugate comprising: the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; and a drug linked thereto; Preferably, the chemical formula of the immunoconjugate is Ab-(L-D)y, wherein: Ab is the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; L is a linker; D is a drug; y is a decimal or integer from 1 to 10; more preferably, y is an integer or decimal from 1 to 8, more preferably an integer or decimal from 7 to 8; More preferably, the immunoconjugate has a structure as shown in Chemical Formula I:
wherein Ab is the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and y is an integer or decimal of 1 to 8, more preferably an integer or decimal of 7 to 8. The immunoconjugate of claim 8, wherein the drug comprises: a tumor-suppressing molecule, a molecule targeting a tumor surface marker, a molecule targeting a surface marker of an immune cell, a radioactive group, a detectable marker, or a combination thereof; Preferably, the molecule targeting a tumor surface marker is an antibody or ligand that binds to a tumor surface marker; or the molecule that inhibits tumors is an anti-tumor cytokine or an anti-tumor toxin; More preferably, The antibody that binds to the tumor surface marker is an antibody that recognizes antigens other than ROR1, and the other antigens include: 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 cytokines include: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha or variants thereof; and/or The anti-tumor toxins include: toxins that act on microtubules; toxins or their derivatives that act on DNA; compounds or their derivatives that act on intracellular metabolism, transcription, translation or signal transduction; more preferably include CPT2C, camptothecin, 20-deoxycamptothecin, 10-methoxycamptothecin, 9-methoxycamptothecin, 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, exatecan, deruxtecan (Dxd), exatecan (DXd), -8951f), rutotecan (GG-211), topotecan, irinotecan (CPT11), simmitecan, cilnotecan, ripotecan (TLC388), gimatidecan (ST1481), diflutecan (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), carninotecan (BNP-1350), or 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (AR-67); Maytansinoid; monomethyl auristatins DE and DF (MMAE and MMAF); dolastatin; calicheamicin or its derivatives; anthracycline, daunomycin, or doxorubicin; methotrexate; vindesine; taxanes, docetaxel, paclitaxel, larotaxel, tesetaxel, or ortataxel; trichothecenes; duocarmycin; pyrrolobenzodiazepines (PBDs); SN-38; GX-2; CC1065; Wherein, the CPT2C has a structure as shown in Chemical Formula II:
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 shown 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 shown in SEQ ID NO: 10 and SEQ ID NO: 13, respectively; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein: the heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 20; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 22; or the heavy chain is or comprises the amino acid sequence shown in SEQ ID NO: 25; and the light chain is or comprises the amino acid sequence shown in SEQ ID NO: 23. The immunoconjugate according to 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 photolabile linker, a dimethyl linker or a disulfide-containing linker; More preferably, the linker comprises a peptide portion, a reactive functional group, a benzoic acid or benzyloxycarbonyl group, or any combination thereof; wherein the peptide portion comprises SC, VC, VA, FK, EVC, GGFG; the reactive functional group comprises MC, SMCC, sulfo-SMCC, SPP, SPDP; the benzoic acid or benzyloxycarbonyl group comprises PABA, PAB, GABA; More preferably, the linker includes MC-VA, MC-VC, SC-VC-PAB, SPDP, SMCC, SuO-VA-PAB, SuO-VC-PAB, MC-VA-PAB, MC-VC-PAB, and MC-GGFG. The immunoconjugate of any one of claims 8 to 11, wherein the (L-D) portion 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-GGFG010-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-simmit ecan, MC-GGFG-sinotecan, MC-GGFG-TLC388, MC-GGFG-ST1481, MC-GGFG-BN-80915, MC-GGFG-CZ48, MC-GGFG-DLSFFZ93W4, MC-GGFG-NSC610457, MC-GG FG-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-am ino-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. A pharmaceutical composition comprising: the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the immunoconjugate according to any one of claims 8 to 12. Use of the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the immunoconjugate according to any one of claims 8 to 12, or the pharmaceutical composition according to claim 13 in the preparation of a preparation, a kit, or a drug kit for diagnosing or treating a tumor, wherein preferably, the tumor is a tumor expressing ROR1; 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, epithelial squamous cell carcinoma, 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 includes B-cell leukemia, B-cell chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphocytic leukemia, Burkitt's lymphoma, and mantle cell lymphoma; the breast cancer includes mammary ductal carcinoma and triple-negative breast cancer; the lung cancer includes non-small cell lung cancer and small cell lung cancer; more preferably, the non-small cell lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, lung large cell neuroendocrine carcinoma, and lung lymphoepithelioma-like carcinoma. A kit or a pharmaceutical kit comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the polynucleotide according to claim 7, or the immunoconjugate according to any one of claims 8 to 12, or the pharmaceutical composition according to claim 13.