Bispecific antibody targeting human ROR1 and CD3

Abstract

Provided are a bispecific antibody targeting ROR1 and CD3 and the use thereof. The bispecific antibody comprises a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is an antibody that specifically binds to ROR1 or an antigen-binding fragment thereof, and the second antigen-binding portion is an antibody that specifically binds to CD3 or an antigen-binding fragment thereof.

Description

Bispecific antibodies targeting human ROR1 and CD3

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number CN202311206180.4, filed on September 18, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention belongs to the fields of tumor immunotherapy and molecular immunology, and specifically relates to a high-affinity, high-specificity, and higher-safety human ROR1×CD3-targeted bispecific antibody and its application. Background Art

[0004] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein. As an orphan receptor tyrosine kinase-like surface antigen, it is expressed by many tissues during embryogenesis and plays a role in embryonic skeletal, cardiopulmonary, and neural development. ROR1 is primarily expressed in embryonic tissues; its expression levels in adult tissues are restricted, including the parathyroid glands, pancreatic islets, and the esophagus, stomach, and duodenum (Michael Y. et al., 2019). ROR1 is expressed in many B-cell malignancies and various cancer cell lines, including solid tumors such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and ovarian cancer. ROR1 is expressed on the surface of CLL cells and is an accurate and reliable marker for minimal residual disease in CLL. Higher ROR1 expression levels are associated with a higher number of circulating leukemic cells and disease aggressiveness, and ROR1 expression is retained on CLL cells during and after treatment. Therefore, ROR1 is an ideal drug target for cancer treatment (Balakrishnan et al., Clin Cancer Res. 2017 Jun 15; 23(12): 3061-3071, DOI: 10.1158 / 1078-0432.CCR-16-2083; Aghebati-Maleki et al., Biomedicine & Pharmacotherapy. 88(2017): 814-822, doi.org / 10.1016 / j.biopha.2017.01.070; De Propris et al., British Journal of Haematology, 2020, 190, e329–e363, DOI: 10.1111 / bjh.16910).

[0005] Currently, there are a variety of anticancer therapies targeting ROR1. These include monoclonal antibodies, CAR-T, antibody-drug conjugates (ADCs), and bispecific antibodies. Zilovertamab blocks the binding of ROR1 to Wnt-5a, inhibiting Wnt-5a-induced NF-κB activation, thereby inhibiting autocrine IL-6-dependent STAT3 activation in CLL (Chen et al, Blood (2019) 134(13):1084–1094, doi.org / 10.1182 / blood.2019001366). Zilovertamab is in a phase III clinical trial for the treatment of relapsed or refractory MCL. The clinical trial results of Zilovertamab Vedotin (MK-2140) showed significant therapeutic effects in both MCL and diffuse large B-cell lymphoma (DLBCL) (Michael L. Wang et al, 2021, NEJM Evid 2022; 1(1), DOI: 10.1056 / EVIDoa2100001).

[0006] T cell-redirecting bispecific antibodies (TCEs) have been widely studied and applied in cancer therapy, with significant progress particularly in the treatment of hematologic malignancies. For example, blinatumomab (CD19×CD3), the first FDA-approved drug in 2014, has demonstrated significant clinical efficacy in the treatment of refractory or relapsed B-cell acute lymphoblastic leukemia (Miguel J Franquiz et al, Biologics: Targets and Therapy 2020:14 23–34, DOI:10.2147 / BTT.S202746). The ROR1×CD3 antibody is constructed by combining the ROR1 antigen and the CD3ε subunit. It can recognize and bind to the ROR1 antigen on the surface of tumor cells and the CD3ε subunit on the surface of T cells, thereby bringing T cells into close contact with tumor cells and releasing a large number of cytotoxic molecules, such as perforin, targetin, and interferon-γ, to kill cancer cells. Compared to traditional monoclonal antibody therapies, ROR1×CD3 bispecific antibodies have higher specificity and stronger anti-tumor activity. Currently, the only ROR1×CD3 bispecific antibodies in clinical development are NVG-111 (NovalGen Ltd) and EMB-07 (EpimAb). While ROR1 is a very promising target in cancer treatment, the field still needs to develop multiple forms of ROR1×CD3 antibodies to improve tumor killing efficacy and reduce the risk of off-tumor.

[0007] Targeting ROR1 for tumor immunotherapy is a very useful treatment method. This patent develops a new bispecific antibody specifically targeting human ROR1×CD3, which has the characteristics of enhanced therapeutic window and safety. (1) The TCE distinguishes between tumor cells and normal cells through the binding valence of ROR1; (2) it decouples cytotoxicity from cytokine release, which manifests as lower cytokine secretion during target cell killing activity. It can be used to inhibit the growth of ROR1-positive tumor cells and treat cancer diseases that express ROR1.

[0008] Summary of the Invention

[0009] The present invention relates to a TCE molecule having a functional target site of human ROR1.

[0010] One aspect of the present invention provides a bispecific antibody targeting ROR1 and CD3, comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, and the second antigen-binding portion is an antibody or an antigen-binding fragment thereof that specifically binds to CD3.

[0011] In some embodiments, the first antigen binding portion includes a heavy chain variable region VH, which VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are selected from the following groups: (1) HCDR1 as shown in SEQ ID NO:19, HCDR2 as shown in SEQ ID NO:20 and HCDR3 as shown in SEQ ID NO:21; (2) HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:24 and HCDR3 as shown in SEQ ID NO:25; (3) HCDR1 as shown in SEQ ID NO:27, HCDR2 as shown in SEQ ID NO:28 and HCDR3 as shown in SEQ ID NO:29; (4) HCDR1 as shown in SEQ ID NO:31, HCDR2 as shown in SEQ ID NO:32 and HCDR3 as shown in SEQ ID NO:33; and (5) HCDR1 as shown in SEQ ID NO:35, HCDR2 as shown in SEQ ID NO:36 and HCDR3 as shown in SEQ ID NO:37.

[0012] In some embodiments, the VH of the first antigen binding moiety comprises the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 30, or 34, or a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 30, or 34. In some embodiments, the first antigen binding moiety is a single domain antibody.

[0013] In some embodiments, the second antigen binding portion includes a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises HCDR1 as shown in SEQ ID NO:39, HCDR2 as shown in SEQ ID NO:40, and HCDR3 as shown in SEQ ID NO:41, and the VL comprises LCDR1 as shown in SEQ ID NO:43, LCDR2 as shown in SEQ ID NO:44, and LCDR3 as shown in SEQ ID NO:45.

[0014] In some embodiments, the VH of the second antigen binding moiety comprises the amino acid sequence as set forth in SEQ ID NO: 38, or a sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 38, and the VL of the second antigen binding moiety comprises the amino acid sequence as set forth in SEQ ID NO: 42, or a sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 38. In some embodiments, the second antigen binding moiety is a Fab fragment.

[0015] In some embodiments, the bispecific antibody further comprises an Fc fragment. In some embodiments, the Fc fragment has a knob-hole structure. In some embodiments, the Fc fragment comprises mutations that reduce or eliminate binding of the antibody to Fcγ receptors, preferably the triple mutation L234S / L235T / G236R.

[0016] In some embodiments, the first antigen-binding moiety is fused to the N-terminus of the first Fc fragment, and the second antigen-binding moiety is fused to the N-terminus of the second Fc fragment. In some embodiments, the second antigen-binding moiety is a Fab fragment, and the heavy chain of the Fab fragment is fused to the N-terminus of the second Fc fragment. In some embodiments, the first and second Fc fragments may have mutations that form a Knob-Hole structure. In some embodiments, both the first and second Fc fragments have mutations that reduce or eliminate binding of the antibody to Fcγ receptors, preferably the L234S / L235T / G236R triple mutation.

[0017] In some embodiments, the bispecific antibody further comprises a third antigen-binding moiety, wherein the third antigen-binding moiety is an antibody or an antigen-binding fragment thereof that specifically binds to ROR1.

[0018] In some embodiments, the third antigen binding portion includes a heavy chain variable region VH, which VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are selected from the following groups: (1) HCDR1 as shown in SEQ ID NO:19, HCDR2 as shown in SEQ ID NO:20 and HCDR3 as shown in SEQ ID NO:21; (2) HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:24 and HCDR3 as shown in SEQ ID NO:25; (3) HCDR1 as shown in SEQ ID NO:27, HCDR2 as shown in SEQ ID NO:28 and HCDR3 as shown in SEQ ID NO:29; (4) HCDR1 as shown in SEQ ID NO:31, HCDR2 as shown in SEQ ID NO:32 and HCDR3 as shown in SEQ ID NO:33; and (5) HCDR1 as shown in SEQ ID NO:35, HCDR2 as shown in SEQ ID NO:36 and HCDR3 as shown in SEQ ID NO:37.

[0019] In some embodiments, the VH of the third antigen binding moiety comprises an amino acid sequence as shown in SEQ ID NO: 18, 22, 26, 30 or 34, or a sequence having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO: 18, 22, 26, 30 or 34. In some embodiments, the third antigen binding moiety is a single domain antibody.

[0020] In some embodiments, the third antigen binding moiety has the same HCDR1, HCDR2 and HCDR3 as the first antigen binding moiety. In some embodiments, the third antigen binding moiety has the same VH as the first antigen binding moiety.

[0021] In some embodiments, the third antigen binding moiety is fused to the N-terminus of the first antigen binding moiety or to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In some embodiments, the third antigen binding moiety is fused to the N-terminus of the first antigen binding moiety or to the N-terminus of the Fab heavy chain of the second antigen binding moiety via a peptide linker, preferably the sequence of the peptide linker is (GGGGS)n, wherein n is an integer not less than 1.

[0022] In some embodiments, the bispecific antibody is composed of a first chain, a second chain, and a third chain, wherein the first chain comprises a fusion protein of a first antigen-binding moiety and a first Fc fragment, the second chain comprises a fusion protein of the Fab heavy chain of the second antigen-binding moiety and a second Fc fragment, and the third chain comprises a Fab light chain of the second antigen-binding moiety. In some embodiments, the first chain comprises the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, the second chain comprises the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, and the third chain comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0023] In some embodiments, the bispecific antibody is composed of a first chain, a second chain, and a third chain, wherein the first chain comprises a third antigen-binding moiety, a fusion protein of the first antigen-binding moiety and a first Fc fragment, the second chain comprises a fusion protein of the Fab heavy chain of the second antigen-binding moiety and a second Fc fragment, and the third chain comprises a Fab light chain of the second antigen-binding moiety. In some embodiments, the first chain comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, or 12, the second chain comprises the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, and the third chain comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0024] In some embodiments, the bispecific antibody is composed of a first chain, a second chain, and a third chain, wherein the first chain comprises a fusion protein of a first antigen-binding moiety and a first Fc fragment, the second chain comprises a fusion protein of a third antigen-binding moiety, a Fab heavy chain of the second antigen-binding moiety, and a second Fc fragment, and the third chain comprises a Fab light chain of the second antigen-binding moiety. In some embodiments, the first chain comprises the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, the second chain comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, or 17, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, or 17, and the third chain comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the first chain, the second chain, and the third chain are selected from the group consisting of: (1) the first chain comprises the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, the second chain comprises the amino acid sequence of SEQ ID NO: 13 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13, and the third chain comprises the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3; (2) the first chain comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: NO:4, the second chain comprises the amino acid sequence set forth in SEQ ID NO:14 or a sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:14, and the third chain comprises the amino acid sequence set forth in SEQ ID NO:3 or a sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:3;(3) the first chain comprises the amino acid sequence of SEQ ID NO:5 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, the second chain comprises the amino acid sequence of SEQ ID NO:15 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:15, and the third chain comprises the amino acid sequence of SEQ ID NO:3 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3; (4) the first chain comprises the amino acid sequence of SEQ ID NO:6 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:6, the second chain comprises the amino acid sequence of SEQ ID NO:16 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:16, and the third chain comprises the amino acid sequence of SEQ ID NO:3 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3; and (5) the first chain comprises the amino acid sequence of SEQ ID NO:7 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:7, the second chain comprises the amino acid sequence of SEQ ID NO: The amino acid sequence shown in SEQ ID NO: 17 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, wherein the third chain comprises the amino acid sequence shown in SEQ ID NO: 3 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3.

[0025] Another aspect of the present invention provides a polynucleotide encoding any of the above-mentioned bispecific antibodies, a recombinant expression vector comprising the polynucleotide, and a host cell comprising the polynucleotide or the recombinant expression vector.

[0026] Another aspect of the present invention provides a pharmaceutical composition comprising any one of the aforementioned bispecific antibodies, the aforementioned polynucleotide, the aforementioned recombinant expression vector or the aforementioned host cell, and a pharmaceutically acceptable carrier.

[0027] Another aspect of the present invention provides use of any of the aforementioned bispecific antibodies, polynucleotides, recombinant expression vectors, or host cells in the preparation of a medicament for treating a ROR1-related disease. In some embodiments, the ROR1-related disease is a tumor or cancer.

[0028] Another aspect of the present invention provides a method for treating a ROR1-related disease, comprising administering any of the aforementioned bispecific antibodies, polynucleotides, recombinant expression vectors, host cells, or pharmaceutical compositions to a subject in need thereof. In some embodiments, the ROR1-related disease is a tumor or cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1: Configuration diagram of 1+1 bispecific antibody.

[0030] Figure 2: Configuration diagram of 2+1 bispecific antibody.

[0031] Figure 3: Configuration diagram of 1+1+1 bispecific antibody.

[0032] Figure 4: Binding test of 1+1 bispecific antibody to CHO-K1 / ROR1 overexpressing cell line.

[0033] Figure 5: Binding test of the 2+1 bispecific antibody to the CHO-K1 / ROR1 overexpressing cell line.

[0034] Figure 6: Binding test of the 1+1+1 bispecific antibody to the CHO-K1 / ROR1 overexpressing cell line.

[0035] Figure 7: Binding detection of 1+1 bispecific antibody to CD3-expressing cell line Jurkat.

[0036] Figure 8: Binding detection of 2+1 bispecific antibody to CD3-expressing cell line Jurkat.

[0037] Figure 9: Binding detection of 1+1+1 bispecific antibody to CD3-expressing cell line Jurkat.

[0038] FIG10A : SPR analysis of CD3×ROR1 bispecific antibody BsAb-12.

[0039] FIG10B : SPR analysis of CD3×ROR1 bispecific antibody BsAb-9.

[0040] FIG. 10C : SPR analysis of control antibody NVG-11.

[0041] Figure 11: Detection of cell binding ability of CD3×ROR1 bispecific antibody.

[0042] Figure 12: CD3×ROR1 bispecific antibody-mediated reporter gene-based T cell activation assay.

[0043] Figure 13: Detection of PBMC (Donor: NF0058) killing tumor cells mediated by CD3×ROR1 bispecific antibody.

[0044] Figure 14: Detection of PBMC (Donor: Y1701) killing tumor cells mediated by CD3×ROR1 bispecific antibody.

[0045] Figure 15: Experimental results of CD3×ROR1 bispecific antibody-mediated PBMC (Donor: NF0058) release of IFN-γ, TNF-α, IL-2 and IL-6.

[0046] Figure 16: Experimental results of CD3×ROR1 bispecific antibody-mediated IFN-γ and TNF-α release from PBMC (Donor: Y1701).

[0047] Figure 17: In vivo anti-tumor efficacy experimental results of CD3×ROR1 bispecific antibody in C-NKG mice transplanted with MDA-MB-231 breast cancer cells and human PBMCs. DETAILED DESCRIPTION

[0048] Unless otherwise noted, the terms used in the present invention have the meanings generally understood in the art and can be understood by reference to standard textbooks, reference books, and literature known to those skilled in the art. All publications mentioned herein are incorporated herein by reference in their entirety.

[0049] It should be understood that the specific methods and materials described in the embodiments of the present invention are for the purpose of describing specific embodiments only and are not intended to be limiting. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. The explanation of relevant theories or mechanisms in the present invention is merely to facilitate understanding of the invention and should not be construed as limiting the solutions protected by the present invention.

[0050] As used herein, the term "comprising," or other terms with similar meanings such as "including," "containing," or "having," should be understood to include the listed elements but not to exclude the presence of other elements. These terms also include situations where the content consists of only the listed elements. The term "consisting of..." means that the content consists of only the listed elements. The term "consisting essentially of..." indicates that elements that do not significantly affect the novel features of the solution in question are not excluded.

[0051] Unless otherwise specified, "a", "an" or "the" herein include both singular and plural forms. The meaning of the term "at least one" or "at least one" or other similar expressions is equivalent to the meaning of "one or more" or "one or more".

[0052] The numerical ranges described herein, such as temperature ranges, time ranges, composition or concentration ranges, or other numerical ranges, include the end values, all intermediate ranges, subranges (e.g., ranges between a certain intermediate value and a certain end value), and all individual values ​​within the range, especially intermediate ranges, subranges, and individual integer values ​​with integer values ​​as end values. Furthermore, any intermediate ranges, subranges, and all individual values ​​described in the numerical range may be excluded from the numerical range.

[0053] As used herein, “about” or “approximately” means a range of ±10% of the numerical value.

[0054] The term "and / or" used herein should be understood as including any one element or any combination of several elements among the multiple elements connected by the term.

[0055] Herein, “first” and “second” are only used for the purpose of distinction and do not imply a specific order.

[0056] In the present invention, unless otherwise indicated, nucleic acid sequences are generally described from the 5' end to the 3' end, and amino acid sequences are generally described from the N-terminus to the C-terminus.

[0057] Herein, when it is mentioned that a certain polypeptide is fused to the N-terminus of another polypeptide, it means that the C-terminus of the former is connected to the N-terminus of the latter via a peptide bond or a peptide linker.

[0058] Definition of terms

[0059] Unless otherwise indicated, the practice of the present disclosure will employ conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology within the skill of the art, many of which are described below for illustrative purposes. Such techniques are fully described in the literature, see, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY, 2009; Frederick M. Ausubel et al., Short Protocols in Molecule Biology, 4 th Edition, Wiley, 1999; Michael R. Green and Joseph Sambrook, Molecule Cloning: A Laboratory Manual, 4 th Edition, 2012 and other similar references.

[0060] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably to refer to a sequence composed of bases, sugars, and a phosphate backbone. The nucleic acid sequence of the present invention can be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and can include natural or non-natural bases, can be single-stranded or double-stranded, and can be a coding sequence or a non-coding sequence.

[0061] The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues. Such polymers may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers composed of amino acid residues. Full-length proteins as well as fragments thereof are encompassed by this definition. The term also includes polypeptides with post-expression modifications, such as glycosylation, sialylation, acetylation, phosphorylation, and the like.

[0062] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that: (1) is not associated with naturally associated components with which it is associated in its native state; (2) does not contain other proteins from the same species; (3) is expressed by cells from a different species; or (4) does not occur in nature. A polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell of its natural origin will be "isolated" from its naturally associated components. A protein can also be isolated by using protein purification techniques well known in the art to render it substantially free of naturally associated components. Proteins or polypeptides, such as antibodies or antigen-binding fragments thereof, referred to in the present invention are preferably isolated unless otherwise indicated.

[0063] The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA or synthetic origin, or a combination thereof, which is separated from other nucleic acid molecules present in the natural source of the nucleic acid. For genomic DNA, the term "isolated" includes nucleic acid molecules separated from the chromosomes naturally associated with the genomic DNA. Preferably, an "isolated" nucleic acid does not include sequences naturally flanking the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid of interest). The polynucleotides or nucleic acid sequences referred to herein are preferably isolated.

[0064] The term "CD3" refers to cluster of differentiation 3 protein, which is an important differentiation antigen on the T cell membrane. In mammals, CD3 is composed of four different chains, including a CD3γ chain, a CD3δ chain and two CD3ε chains, which bind to the T cell receptor (TCR) and CD3-zeta (ζ chain) to form a TCR complex, which generates activation signals in T lymphocytes and plays an extremely important role in the process of T cell antigen recognition and immune response generation. The term "CD3" also includes variants, isoforms, derivatives and homologs of different species of any CD3, which can be naturally expressed by cells or expressed in cells containing exogenous or heterologous CD3 encoding nucleic acid sequences by genetic recombination technology. CD3 herein may refer to CD3 proteins from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., macaques) and rodents (e.g., mice and rats), particularly human CD3.

[0065] The term "ROR1" refers to receptor tyrosine kinase-like orphan receptor 1. ROR1 is an evolutionarily conserved membrane protein that is widely expressed in embryonic development and a variety of human cancers. The molecular structure of ROR1 includes an extracellular domain composed of an Ig-like domain, a Frizzled domain, and a membrane-proximal Kringle domain; a transmembrane domain; and a cytoplasmic domain composed of a tyrosine kinase-like domain, two serine / threonine-rich domains, and a proline-rich domain (PRD). Due to the expression pattern of ROR1 and its function in tumor progression, it has become a promising target for tumor treatment. The term "ROR1" also includes any variants, isoforms, derivatives, and homologs of different species of ROR1, which can be naturally expressed by cells, or expressed by genetic recombination technology in cells containing exogenous or heterologous ROR1 encoding nucleic acid sequences. ROR1 herein may refer to ROR1 protein from any vertebrate source, including mammals such as primates (eg, humans), non-human primates (eg, macaques), and rodents (eg, mice and rats), particularly human ROR1.

[0066] The term "antigen" has the meaning commonly understood by those skilled in the art and includes polypeptides, polysaccharides, glycoproteins, polynucleotides, and the like. The term "epitope," also known as "antigenic determinant," refers to the chemical functional groups on the surface of an antigen that determine its specificity. Epitopes are recognized and bound by antigen-binding sites in antibodies.

[0067] The term "antibody" is used in its broadest sense and includes immunoglobulins or other types of molecules that contain one or more antigen-binding domains that specifically bind to an antigen, and are proteins or polypeptides that exhibit binding specificity for a specific antigen. Specific examples of antibodies may include (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, and the like. A complete antibody, which may also be referred to as a full-length antibody or classic antibody molecule in the present invention, is an immunoglobulin molecule (e.g., IgG) or a multimer thereof (e.g., IgA or IgM) composed of four polypeptide chains. The four polypeptide chains include two identical heavy chains (H) and two identical light chains (L), which are interconnected by disulfide bonds to form a tetramer. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (CH, including domains CH1, CH2, and CH3). Each light chain is composed of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The constant regions (CH and CL) of the heavy and light chains are not directly involved in the binding of antibodies to antigens, but exhibit a variety of effector functions, such as mediating the binding of antibodies to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The variable regions (VH and VL) of the heavy and light chains form the antigen binding site. VH and VL respectively have highly variable regions called complementarity determining regions (CDRs), which have a high degree of variation in amino acid composition and arrangement sequence. They are the key positions for antibody binding to antigens, and are interspersed with more conserved sequences called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to hydroxy terminus in the following order: FR1, CDR2, and FR3. DR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three heavy chain complementarity-determining regions are referred to as HCDR1 (CDR-H1), HCDR2 (CDR-H2), and HCDR3 (CDR-H3), and the four heavy chain framework regions are referred to as HFR1 (FR-H1), HFR2 (FR-H2), HFR3 (FR-H3), and HFR4 (FR-H4). The three light chain complementarity-determining regions are referred to as LCDR1 (CDR-L1), LCDR2 (CDR-L2), and LCDR3 (CDR-L3), and the four light chain framework regions are referred to as LFR1 (FR-L1), LFR2 (FR-L2), LFR3 (FR-L3), and LFR4 (FR-L4). The variable regions (VH and VL) of the heavy and light chains, respectively, form the antigen-binding site.

[0068] The term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of a CDR can be defined according to various numbering systems known in the art, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), the AbM numbering system, or the Contact numbering system. For a given antibody, one skilled in the art will readily identify the CDRs defined according to each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). Although the Kabat numbering system is used to define CDRs in the examples and descriptions of specific sequences of the present invention, it should be understood that the antibodies of the present invention may use any of these numbering systems to define CDRs.

[0069] The term "framework region" or "FR" refers to those amino acid sequences in the variable region of an antibody other than the CDR sequences defined above.

[0070] Based on the amino acid sequence of the constant region of its heavy chain, antibodies can be divided into five major classes: IgA, IgD, IgE, IgG, and IgM. These antibody classes are further divided into subclasses based on the size of the hinge region, the location of interchain disulfide bonds, and molecular weight, such as IgG1, IgG2a, IgG2b, IgG3, and IgG4. Based on the amino acid composition and arrangement of the constant region of its light chain, light chains can be divided into two types: kappa and lambda. The antibodies of the present invention encompass antibodies of any of the aforementioned classes or subclasses.

[0071] The term "monoclonal antibody" refers to a homogeneous antibody directed against a specific antigenic epitope. In contrast to conventional polyclonal antibody preparations, which typically include different antibodies directed against different antigenic epitopes, each monoclonal antibody is directed against a single antigenic epitope on the antigen. Although monoclonal antibodies can be obtained through hybridoma culture, the modifier "monoclonal" in the present invention indicates the homogeneous nature of the antibody and should not be construed as requiring the antibody to be produced by any specific method. The monoclonal antibodies of the present invention can be produced by recombinant DNA methods or obtained by other screening methods.

[0072] The term "antigen-binding fragment" refers to one or more portions of an intact antibody that retain the ability to specifically bind to the antigenic epitope to which the antibody is directed, for example, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd Edition, Raven Press, NY (1989). Examples of antigen-binding fragments include: (1) Fab fragment: an antigen-binding fragment produced by papain digestion of an intact antibody, consisting of the complete light chain (including the VL domain and the CL domain) of a full-length antibody and the heavy chain variable region VH and heavy chain constant region domain CH1 of a full-length antibody, without a hinge region; (2) F(ab')2 fragment: an antigen-binding fragment produced by pepsin digestion of an intact antibody, comprising the complete light chains (including the VL domain and the CL domain) of two full-length antibodies and the heavy chain variable region VH and heavy chain constant region domain CH1 of two full-length antibodies and a hinge region, which can be regarded as having two (1) a fragment formed by pairing two Fab' fragments with disulfide bonds; (2) a Fab' fragment formed by treating the hinge region of F(ab')2 with a reducing agent to break the disulfide bonds; (3) a Fab' fragment formed by treating the hinge region of F(ab')2 with a reducing agent to break the disulfide bonds; (4) an Fd fragment consisting of a VH domain and a CH1 domain; and (5) an Fv fragment consisting of a VL domain and a VH domain of a single antibody arm. The term "antigen-binding fragment" generally comprises at least one or both of the heavy chain variable region and the light chain variable region of an intact antibody, and may also comprise a portion or all of the light chain constant region and / or heavy chain constant region of an intact antibody, for example, one or two or more of the CL domain, CH1 domain, CH2 domain, and CH3 domain.

[0073] The term "Fc region" or "Fc fragment" refers to the C-terminal region of the immunoglobulin heavy chain, that is, the crystallizable fragment (Fc) that constitutes the handle region of the Y-shaped classic antibody molecule, including the second and third constant domains (CH2 and CH3 domains) of the heavy chain. The Fc region can be obtained by hydrolyzing the whole antibody molecule using papain. In some examples, the Fc region may include a hinge, CH2, and CH3. When the Fc region includes a hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment may be from IgG, IgM, IgD, IgE, or IgA. In some examples, the Fc region is from IgG1, IgG2, IgG3, or IgG4. "Fc fragment" also includes variant Fc fragments from a natural Fc fragment that have been modified but still retain their effector function. A "variant Fc fragment" comprises an amino acid sequence having at least one amino acid substitution in the amino acid sequence of a natural Fc fragment. In some instances, the variant Fc fragment has at least one amino acid substitution compared to the parent Fc fragment (native Fc fragment), for example, about 1 to about 10 amino acids are substituted in the parent Fc fragment, and preferably about 1 to about 5 amino acid substitutions. In some instances, the variant Fc fragment Fc region has at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the parent Fc fragment. The effector functions of the "Fc fragment" may include binding to Fc receptors, Clq binding and complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and the like.

[0074] The term "single domain antibody (sdAb)" refers to an antigen-binding polypeptide with a single variable domain that includes three complementary determining regions (CDRs). Single domain antibodies are able to bind to antigens alone without pairing with another polypeptide containing CDRs. Common single domain antibodies include heavy chains but lack light chains that are typically found in antibodies, such as camelid sdAbs (see eg, Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). Single domain antibodies can be artificially engineered from camel heavy chain antibodies, called V H H antibody, V H The H antibody has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single-domain antibodies can also be considered a special "antigen-binding fragment" of a full-length antibody.

[0075] The term "single-chain antibody (scFv)" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, 113, Roseburg & Moore, Springer-Verlag, New York, 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers may consist of repeated GGGGS amino acid sequences, i.e., (GGGGS)n or variants thereof, where n is an integer greater than or equal to 1. For example, a linker having the amino acid sequence (GGGGS) 3 , or a variant thereof ( Holliger et al., (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448 ) can be used. In some cases, a disulfide bond may also exist between the VH and VL of the scFv.

[0076] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and the other portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but still retains binding activity to the target antigen (Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). The term "chimeric antibody" may refer to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, for example, an antibody having mouse heavy and light chain variable regions linked to human constant regions.

[0077] The term "human antibody" refers to an antibody whose entire sequence (such as the variable and constant regions) is derived from human immunoglobulins. A "non-human antibody" refers to an antibody whose entire sequence (such as the variable and constant regions) is derived from immunoglobulins of non-human species. For example, an antibody derived from mouse immunoglobulins can be referred to as a murine antibody.

[0078] The term "humanized antibody" refers to a chimeric antibody obtained by modifying a non-human antibody, such as an antibody whose variable region and constant region (if any) are not derived from human immunoglobulins, to increase the homology with the human antibody sequence. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response). Humanized antibodies are particularly advantageous because they can retain the expected properties of non-human donor antibodies (e.g., mouse antibodies) while effectively reducing the immunogenicity of non-human donor antibodies (e.g., mouse antibodies) in human subjects. However, due to the matching problem between the CDRs of the donor antibody and the FRs of the recipient antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response) are generally lower than those of non-human donor antibodies (e.g., mouse antibodies).

[0079] The term "bispecific antibody" refers to an antibody that can specifically bind to two antigenic epitopes. The two antigenic epitopes can be different epitopes on the same antigen or different epitopes on different antigens. A bispecific antibody comprises two or more antigen-binding moieties, wherein one or more antigen-binding moieties target one antigenic epitope and the other antigen-binding moieties target another antigenic epitope. Each antigen-binding moiety can be independently selected from a monoclonal antibody (such as an IgG immunoglobulin), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a Fd fragment, a scFv, a single domain antibody, a chimeric antibody, a humanized antibody, and the like. An antigen-binding moiety can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent binding or other means) to one or more additional antigen-binding moieties to form a bispecific antibody. "ROR1×CD3 bispecific antibody" herein refers to a bispecific antibody that targets ROR1 and CD3.

[0080] As known to those skilled in the art, antibodies can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application No. 4,816,567), or phage antibody library technology (see, for example, Clackson et al., Nature, 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0081] As is known to those skilled in the art, antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G. Subsequently or alternatively, the specific antigen (the target molecule recognized by the antibody) or its antigenic epitope can be immobilized on a column and the immunospecific antibody can be purified by immunoaffinity chromatography. For purification of antibodies, reference can be made, for example, to D. Wilkinson, The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0082] For antibodies or their antigen-binding fragments, the terms "targeting," "directed at," or "specific binding" refer to that the antibody or its antigen-binding fragment forms a relatively stable complex with an antigen under physiological conditions, and preferably does not exhibit significant binding to other undesirable antigens. A molecule can target, direct at, or specifically bind to more than one molecule, e.g., a bispecific antibody can have a higher binding affinity for two different antigens relative to other molecules. The binding ability of an antibody or its antigen-binding fragment to an antigen or antigenic epitope can be measured by techniques such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or other protein interaction assays such as biofilm interferometry (BLI). Specific binding can be characterized by the equilibrium dissociation constant, KD, for the combination of antigen and antibody (a smaller KD represents a tighter combination), or by the EC50 value for the combination of antibody and antigen.

[0083] The term "EC50" (concentration for 50% of maximal effect) refers to the concentration that elicits 50% of the maximal effect. In enzyme-linked immunosorbent assays (ELISAs), when used to indicate the binding ability of an antibody molecule to its corresponding antigen, it refers to the antibody concentration that produces half the maximum detection signal (e.g., colorimetric or fluorescence intensity). The lower the EC50 value, the greater the binding affinity of the antibody to the antigen.

[0084] The term "ka" refers to the association rate constant of a specific antibody-antigen interaction, indicating the rate of binding of an antibody to its target antigen, or the rate of complex formation between an antibody and an antigen, and is expressed in M. -1 s -1 .

[0085] The term "kd" refers to the dissociation rate constant of a specific antibody-antigen interaction and indicates the rate at which an antibody dissociates from its target antigen, or the rate at which an antigen-antibody complex separates into free antibody and antigen over time, with units of s. -1 .

[0086] The term "KD" refers to the equilibrium dissociation constant for a specific antibody-antigen interaction, which is derived from the ratio of kd to ka (i.e., kd / ka), expressed as a molar concentration (M). KD can be used to measure the affinity of an antibody for binding to its binding ligand (e.g., antigen). A smaller KD indicates a tighter binding between the antibody and the antigen, or a higher affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) equilibrium dissociation constant will bind more tightly to a specific antigen than an antibody with a micromolar (μM) dissociation constant. The KD value of an antibody can be determined using methods well known in the art. One method for determining the ka, kd, and KD values ​​of an antibody is to use surface plasmon resonance, typically using a biosensor system such as Biacore. TM Another method for determining the KD value of an antibody is biolayer interferometry (BLI).

[0087] The term "fusion protein" refers to two or more polypeptide moieties covalently linked together to form a polypeptide. The two or more polypeptide moieties can be covalently linked by peptide bonds or linked together by a peptide linker.

[0088] The term "variant" or "mutant" refers to a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of a parent or reference polypeptide (including but not limited to a wild-type polypeptide) by at least one amino acid residue. A variant may have one or more amino acid sequence insertions, deletions, or substitutions compared to the parent polypeptide.

[0089] The term "sequence identity" refers to the percentage of identical nucleotides or amino acid residues at corresponding positions when two or more sequences are aligned in a manner that matches to the greatest extent, i.e., taking into account gaps and insertions. The comparison of sequences and the calculation of sequence identity percentages can be performed using suitable computer programs known in the art. Such programs include local alignment programs and global alignment programs, including but not limited to BLAST, ALIGN, ClustalW, EMBOSS Needle, etc. An example of a local alignment program is BLAST (Basic Local Alignment Search Tool) (see, e.g., Altschul et al., (1990) J. Mol. 215; 403-410), which can be obtained from the webpage of the National Center for Biotechnology Information (National Center for Biotechnology Information) at http: / / www.ncbi.nlm.nih.gov / . Examples of global alignment programs (which optimize alignments over the entire length of a sequence) are the EMBOSS Needle and EMBOSS Stretcher programs, which are based on the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970), “A general method applicable to the search for similarities in the amino acid sequence of two proteins”, Journal of Molecular Biology 48(3):443-53), both of which are available from http: / / www.ebi.ac.uk / Tools / psa / .

[0090] Variants of the present invention represent amino acid mutations in polypeptides using the format XnnnY, where X is the amino acid before mutation, Y is the amino acid after mutation, and nnn represents the position of the mutation, as determined by the EU index of Kabat. Amino acids are represented by conventional single-letter or three-letter designations. As is well known to those skilled in the art, the single-letter designations for amino acids are as follows: A for alanine; C for cysteine; D for aspartic acid; E for glutamic acid; F for phenylalanine; G for glycine; H for histidine; I for isoleucine; K for lysine; L for leucine; M for methionine; N for asparagine; P for proline; Q for glutamine; R for arginine; S for serine; T for threonine; V for valine; W for tryptophan; and Y for tyrosine.

[0091] The term "recombinant" refers to expressing the antibodies or antigen-binding fragments thereof of the present invention by recombinant DNA technology (which includes, for example, DNA splicing and transgenic expression), for example, using a recombinant expression vector expression system transfected into a non-human mammal (e.g., mouse) or cell (e.g., CHO cell) to express the antibody or antigen-binding fragment thereof, and the antibody can be an antibody isolated from a recombinant or combinatorial human antibody library.

[0092] The term "vector" refers to any molecule that can transport the heterologous nucleic acid contained therein into a host cell (existing in a free form or integrated into the host cell genome), such as a nucleic acid, a plasmid or a virus. After being introduced into a host cell, the vector can exist independently and replicate autonomously (e.g., a bacterial vector with a bacterial replication origin and an episomal mammalian vector), or be integrated into the host cell genome and replicate together with the host genome (e.g., a non-episomal mammalian vector). A vector used to express a target gene in a host cell is referred to as an "expression vector" or a "recombinant expression vector." The vector may contain regulatory elements operably linked to the target gene, such as an origin of replication, an expression regulatory sequence (e.g., a promoter and / or enhancer), and / or a selectable marker gene (e.g., an antibiotic resistance gene and a gene that can be used in colorimetric analysis, such as β-galactose).

[0093] In order to express the antibodies of the present invention or their antigen-binding fragments, expression vectors encoding heavy and light chains can be transfected into host cells by standard techniques. The term "transfection" is intended to include various techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although theoretically it is possible to express the antibodies of the present invention in prokaryotic or eukaryotic host cells, mammalian host cells are most preferred for expressing antibodies in eukaryotic cells because such eukaryotic cells, particularly mammalian cells, are more likely to assemble and secrete correctly folded immunologically active antibodies than prokaryotic cells.

[0094] The term "host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells, such as human cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical to the original parent cell, for example, in morphology or genomic DNA complement, due to natural, accidental, or deliberate mutations. The host cell can be an isolated cell or cell line, and also includes cells transfected in vivo with a nucleic acid molecule or expression vector provided herein.

[0095] Bispecific antibodies targeting ROR1 and CD3

[0096] The present invention provides bispecific antibodies targeting ROR1 and CD3, particularly bispecific antibodies targeting human ROR1 and human CD3. The bispecific antibodies comprise at least a first antigen-binding moiety and a second antigen-binding moiety, wherein the first antigen-binding moiety specifically binds to ROR1, particularly human ROR1, and the second antigen-binding moiety specifically binds to CD3, particularly human CD3.

[0097] The first antigen-binding portion can be an antibody or antigen-binding fragment thereof that specifically binds to the ROR1 protein (or its Frizzled domain or Kringle domain), for example, Fab, Fv, single-chain antibody (scFv) or single-domain antibody (sdAb), etc. In some embodiments, the first antigen-binding portion is a single-domain antibody.

[0098] In some embodiments, the first antigen binding portion comprises a heavy chain variable region VH, wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 can be selected from the following group:

[0099] (1) HCDR1 as set forth in SEQ ID NO: 19, HCDR2 as set forth in SEQ ID NO: 20, and HCDR3 as set forth in SEQ ID NO: 21;

[0100] (2) HCDR1 as shown in SEQ ID NO: 23, HCDR2 as shown in SEQ ID NO: 24, and HCDR3 as shown in SEQ ID NO: 25;

[0101] (3) HCDR1 as set forth in SEQ ID NO: 27, HCDR2 as set forth in SEQ ID NO: 28, and HCDR3 as set forth in SEQ ID NO: 29;

[0102] (4) HCDR1 as shown in SEQ ID NO:31, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:33; and

[0103] (5) HCDR1 as shown in SEQ ID NO: 35, HCDR2 as shown in SEQ ID NO: 36, and HCDR3 as shown in SEQ ID NO: 37.

[0104] In some embodiments, the VH of the first antigen binding portion comprises the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 30 or 34, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 30 or 34.

[0105] The second antigen binding moiety can be an antibody or antigen binding fragment thereof that specifically binds to CD3, for example, Fab, Fv, single chain antibody (scFv) or single domain antibody (sdAb), etc. In some embodiments, the second antigen binding moiety is Fab.

[0106] In some embodiments, the second antigen binding portion includes a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises HCDR1 as shown in SEQ ID NO:39, HCDR2 as shown in SEQ ID NO:40, and HCDR3 as shown in SEQ ID NO:41, and the VL comprises LCDR1 as shown in SEQ ID NO:43, LCDR2 as shown in SEQ ID NO:44, and LCDR3 as shown in SEQ ID NO:45.

[0107] In some embodiments, the VH of the second antigen binding portion comprises the amino acid sequence shown in SEQ ID NO:38, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO:38.

[0108] In some embodiments, the VL of the second antigen binding portion comprises the amino acid sequence shown in SEQ ID NO:42, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO:42.

[0109] In some embodiments, the second antigen-binding portion is a CD3 antigen-binding fragment in Fab form, comprising two chains, which may be referred to as Fab heavy chain and Fab light chain in the present invention, respectively. The Fab heavy chain comprises a heavy chain variable region VH and a heavy chain constant region CH1, and the Fab light chain comprises a light chain variable region VL and a light chain constant region CL. In some embodiments, the CH1 and CL in the second antigen-binding portion may be CH1 or CL of the constant region derived from IgG, IgM or IgA (e.g., human IgG, human IgM or human IgA). In some embodiments, the CH1 and CL in the second antigen-binding portion may be CH1 or CL of the constant region derived from IgG1, e.g., human IgG1.

[0110] In some embodiments, the bispecific antibodies of the present invention include an Fc fragment. In some embodiments, the first antigen binding portion and / or the second antigen binding portion are fused to the Fc fragment to form an Fc fusion protein. The Fc fusion protein comprising the first antigen binding portion and / or the second antigen binding portion can form a homologous or heterologous dimer. The Fc fragment can be an Fc fragment or a variant thereof derived from the constant region of IgG, IgM or IgA (e.g., human IgG or human IgM or human IgA). In some embodiments, the Fc fragment can be an Fc fragment or a variant thereof derived from the constant region of IgG1, such as human IgG1. The Fc fragment can be designed by mutation to include the insertion, substitution or deletion of one or more amino acids, so that the resulting Fc variants can form space-filling effects, electrostatic guidance, hydrogen bonding, hydrophobic interactions, etc., or so that the resulting variant Fc fragment has altered effector functions relative to those before mutation, such as binding to Fc receptors (such as Fcγ receptors, such as FcγRIIIA), Clq binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, etc., or a combination of these effects.

[0111] In some embodiments, the interaction between variant Fc fragments contributes to the formation of a stable heterodimeric protein. Variant Fc mutation design technology has been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion proteins. Representative examples include the "knob-into-hole" format proposed by Cater et al. (Protein Engineering, vol. 9, no. 7, pp. 617-621, 1996); the Fc-containing heterodimer format formed by electrostatic steering by Amgen technicians (US20100286374A1); the heterodimer format (SEEDbodies) formed by IgG / IgA chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection, pp. 1-8, 2010); the bispecific molecule formed by Genmab's DuoBody (Science, 2007, 317 (5844)) platform technology; and the heterodimer protein format formed by Xencor technicians using a combination of structural calculations and Fc amino acid mutations to integrate different modes of action (mAbs 3: 6, 546-557; November / December 2010). 2011); Suzhou Alphamab's charge network-based Fc modification method (CN201110459100.7) to produce a heterodimeric protein form; and other genetic engineering methods that achieve the formation of functional heterodimeric proteins based on Fc amino acid changes or functional modification. In some embodiments, the variant Fc fragment may have a knob-hole structure. In some embodiments, the variant Fc fragment fused to the first antigen-binding moiety and / or the second antigen-binding moiety may form a knob-hole structure to facilitate heterodimer formation. The knob-hole structure in the variant Fc fragment of the present invention refers to mutations in the two Fc fragments, which, after mutation, allow for binding in a "knob-into-hole" formation. For example, the "knob-into-hole" model of Cater et al. can be used to perform site-specific mutations in the Fc region, so that the resulting first and second variant Fc fragments can bind in a "knob-into-hole" formation to form a heterodimer. Selection of a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the skill of those skilled in the art. Preferably, the Fc region is from human IgG1, IgG2, IgG3, or IgG4, and more preferably, from human IgG1. Randomly select one of the first variant Fc fragment or the second variant Fc fragment to create a knob mutation and the other to create a hole mutation.

[0112] In some embodiments, the first antigen binding moiety is fused to a first Fc fragment, and the second antigen binding moiety is fused to a second Fc fragment.

[0113] The first antigen binding moiety and the first Fc fragment can be directly connected or connected through a peptide linker.In some embodiments, the first antigen binding moiety is fused to the N-terminus of the first Fc fragment.

[0114] The second antigen binding moiety and the second Fc fragment can be directly connected or connected through a peptide linker.In some embodiments, the second antigen binding moiety is fused to the N-terminus of the second Fc fragment.

[0115] In some embodiments, the peptide linker can be a flexible linker, such as a glycine-serine polymer, such as (GGGGS)n (SEQ ID NO: 50), wherein n is an integer not less than 1. In some embodiments, the peptide linker can be (GGGGS)3 (SEQ ID NO: 49).

[0116] In some embodiments, the first Fc fragment and / or the second Fc fragment is an Fc fragment of IgG1 or a variant thereof.

[0117] In some embodiments, the first Fc fragment and the second Fc fragment form a knob-hole structure, wherein one of the first Fc fragment and the second Fc fragment can form a knob structure and the other can form a hole structure, and the first Fc fragment and the second Fc fragment can be combined in a "knob-into-hole" form.

[0118] In some embodiments, the Fc fragment, such as the first Fc fragment and / or the second Fc fragment, may have mutations that alter Fc effector function, such as mutations that reduce or eliminate binding of the antibody to Fcγ receptors (such as FcγRIIIA), reduce CDC, and / or reduce ADCC. Such mutations are known to those skilled in the art and include, but are not limited to, the LALA double mutation (L234A / L235A) or the LALAPG triple mutation (L234A / L235A / P329G) in IgG1, the FES triple mutation (L234F / L235E / P331S), and the FQQ triple mutation (L234F / L235Q / K322Q), which can significantly reduce the affinity of the antibody for Fcγ receptors (FcγRs), thereby reducing ADCC. Such mutations also include the P329G mutation, which can significantly reduce the binding of the antibody to multiple Fcγ receptors (see, Schlothauer T, Herter S, Koller CE et al., Protein Eng Des Sel. 2016 Oct; 29(10): 457-466). Wilkinson I et al. (Fc-engineered antibodies with immune effector functions completely abolished.PLoS ONE, 2021, 16 (12): e0260954. https: / / doi.org / 10.1371 / journal.pone.0260954) describe that the immune effector function of antibodies can be reduced or eliminated, such as reducing or eliminating a variety of mutation combinations that bind to Fc fragments and Fcγ receptors (FcγRs), which can avoid systemic activation of T cells mediated by FcγRs, thereby allowing immune effector cells to be restrictedly activated only within target cell tissues, and the document is incorporated herein by reference in its entirety. The bispecific antibodies of the present invention can include mutations described in the document that reduce or eliminate the immune effector function of antibodies in Fc fragments (including the first Fc fragment and / or the second Fc fragment). In some embodiments, the Fc fragment, such as the first Fc fragment and / or the second Fc fragment, can include L234S / L235T / G236R triple mutations.

[0119] In some embodiments, the first and second Fc fragments form a Knob-Hole structure and both have the L234S / L235T / G236R triple mutation. In some embodiments, the first and second Fc fragments comprise mutations that can form a Knob-Hole structure and the L234S / L235T / G236R triple mutation.

[0120] In some embodiments, the second antigen-binding fragment is a CD3 antigen-binding fragment in Fab form, the Fab heavy chain of which is fused to a second Fc fragment. In some embodiments, the Fab heavy chain of the second antigen-binding moiety is fused to the N-terminus of the second Fc fragment.

[0121] In some embodiments, the bispecific antibody targeting ROR1 and CD3 of the present invention further comprises a third antigen-binding portion. The third antigen-binding portion can be an antibody or antigen-binding fragment thereof that specifically binds to ROR1 or an antibody or antigen-binding fragment thereof that specifically binds to CD3. The third antigen-binding portion can be the same as, different from, or partially identical to the first or second antigen-binding portion (e.g., the CDR portion is the same).

[0122] In some embodiments, the third antigen-binding moiety is an antibody or antigen-binding fragment thereof that specifically binds to the ROR1 protein (or its Frizzled domain or Kringle domain), such as Fab, Fv, single-chain antibody (scFv), or single-domain antibody (sdAb). In some embodiments, the third antigen-binding moiety is a single-domain antibody.

[0123] In some embodiments, the third antigen binding portion comprises a heavy chain variable region VH, wherein the VH comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 can be selected from the following group:

[0124] (1) HCDR1 as set forth in SEQ ID NO: 19, HCDR2 as set forth in SEQ ID NO: 20, and HCDR3 as set forth in SEQ ID NO: 21;

[0125] (2) HCDR1 as shown in SEQ ID NO: 23, HCDR2 as shown in SEQ ID NO: 24, and HCDR3 as shown in SEQ ID NO: 25;

[0126] (3) HCDR1 as set forth in SEQ ID NO: 27, HCDR2 as set forth in SEQ ID NO: 28, and HCDR3 as set forth in SEQ ID NO: 29;

[0127] (4) HCDR1 as shown in SEQ ID NO:31, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:33; and

[0128] (5) HCDR1 as shown in SEQ ID NO: 35, HCDR2 as shown in SEQ ID NO: 36, and HCDR3 as shown in SEQ ID NO: 37.

[0129] In some embodiments, the VH of the third antigen binding portion comprises an amino acid sequence as shown in SEQ ID NO: 18, 22, 26, 30 or 34, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 30 or 34.

[0130] In some embodiments, the third antigen binding moiety may have the same or different or partially identical CDR regions as the first antigen binding moiety, for example, the same or different or partially identical HCDR1, HCDR2 and HCDR3. In some embodiments, the third antigen binding moiety may have the same or different or partially identical variable regions as the first antigen binding moiety, for example, the same or different or partially identical VH.

[0131] In some embodiments, the third antigen binding moiety can be fused to the first antigen binding moiety, for example, directly or via a peptide linker. In some embodiments, the third antigen binding moiety can be fused to the N-terminus of the first antigen binding moiety.

[0132] In some embodiments, the third antigen binding moiety can be fused to the second antigen binding moiety, for example, directly or via a peptide linker. In the case where the second antigen binding moiety comprises two chains, the third antigen binding moiety can be fused to either chain of the second antigen binding moiety, for example, to the Fab heavy chain or the Fab light chain of the second antigen binding moiety, directly or via a peptide linker. In some embodiments, the third antigen binding moiety can be fused to the N-segment of either chain of the second antigen binding moiety, for example, the N-segment of the Fab heavy chain or the Fab light chain.

[0133] In some embodiments, the peptide linker may be (GGGGS)n (SEQ ID NO: 50), wherein n is an integer not less than 1. In some embodiments, the peptide linker is (GGGGS)3 (SEQ ID NO: 49).

[0134] In some embodiments, the bispecific antibody targeting ROR1 and CD3 comprises a first antigen binding portion and a second antigen binding portion, wherein the first antigen binding portion is a single domain antibody that specifically binds to ROR1, the second antigen binding portion is a Fab fragment that specifically binds to CD3, the Fab fragment comprises a Fab heavy chain and a Fab light chain, the first antigen binding portion is fused to the N-terminus of a first Fc fragment, and the Fab heavy chain of the second antigen binding portion is fused to the N-terminus of the second Fc fragment.

[0135] In some embodiments, the bispecific antibody targeting ROR1 and CD3 comprises a first antigen binding portion, a second antigen binding portion, and a third antigen binding portion, wherein the first antigen binding portion and the third antigen binding portion are single domain antibodies that specifically bind to ROR1, the second antigen binding portion is a Fab fragment that specifically binds to CD3, the Fab fragment comprises a Fab heavy chain and a Fab light chain, the first antigen binding portion is fused to the N-terminus of the first Fc fragment, the Fab heavy chain of the second antigen binding portion is fused to the N-terminus of the second Fc fragment, and the third antigen binding portion is fused to the N-terminus of the first antigen binding portion or the N-terminus of the Fab heavy chain of the second antigen binding portion.

[0136] In some embodiments, the bispecific antibody targeting ROR1 and CD3 consists of a first chain, a second chain, and a third chain, wherein the first chain comprises a fusion protein of a first antigen binding portion and a first Fc fragment, the second chain comprises a fusion protein of the Fab heavy chain of the second antigen binding portion and a second Fc fragment, and the third chain comprises the Fab light chain of the second antigen binding portion. In some embodiments, the first strand comprises the amino acid sequence of SEQ ID NO: 1, 4, 5, 6 or 7, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, 4, 5, 6 or 7, the second strand comprises the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and the third strand comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0137] In some embodiments, the bispecific antibody targeting ROR1 and CD3 consists of a first chain, a second chain, and a third chain, wherein the first chain comprises a third antigen binding portion, a fusion protein of the first antigen binding portion and a first Fc fragment, the second chain comprises a fusion protein of the Fab heavy chain of the second antigen binding portion and a second Fc fragment, and the third chain comprises the Fab light chain of the second antigen binding portion. In some embodiments, the first strand comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, 11 or 12, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8, 9, 10, 11 or 12, the second strand comprises the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and the third strand comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0138] In some embodiments, the bispecific antibody targeting ROR1 and CD3 consists of a first chain, a second chain, and a third chain, wherein the first chain comprises a fusion protein of a first antigen binding portion and a first Fc fragment, the second chain comprises a third antigen binding portion, a fusion protein of the Fab heavy chain of the second antigen binding portion and a second Fc fragment, and the third chain comprises the Fab light chain of the second antigen binding portion. In some embodiments, the first strand comprises the amino acid sequence of SEQ ID NO: 1, 4, 5, 6 or 7, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, 4, 5, 6 or 7, the second strand comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17, and the third strand comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0139] In some embodiments, the first strand, second strand, and third strand are selected from the group consisting of:

[0140] (1) the first strand comprises the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, the second strand comprises the amino acid sequence of SEQ ID NO: 13, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13, and the third strand comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3;

[0141] (2) the first strand comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4; the second strand comprises the amino acid sequence of SEQ ID NO:14, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:14; and the third strand comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3;

[0142] (3) the first strand comprises the amino acid sequence of SEQ ID NO:5, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:5, the second strand comprises the amino acid sequence of SEQ ID NO:15, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:15, and the third strand comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3;

[0143] (4) the first strand comprises the amino acid sequence of SEQ ID NO:6, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6, the second strand comprises the amino acid sequence of SEQ ID NO:16, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:16, and the third strand comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3; and

[0144] (5) the first chain comprises the amino acid sequence of SEQ ID NO:7, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:7; the second chain comprises the amino acid sequence of SEQ ID NO:17, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:17; and the third chain comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3.

[0145] The bispecific antibodies of the present invention have improved binding affinity to ROR1 protein (e.g., human ROR1 protein). In some embodiments, the binding affinity (KD) of the bispecific antibodies of the present invention to ROR1 protein (e.g., human ROR1 protein) increases with increasing antibody coupling concentration, for example, as can be measured by surface plasmon resonance (SPR). The bispecific antibodies of the present invention have improved binding affinity to human ROR1-expressing cell lines (e.g., CHO-K1 cell lines expressing human ROR1 or human ROR1 tumor cell lines) and / or human CD3-expressing cell lines (e.g., Jurkat cells expressing human CD3).

[0146] In some embodiments, the bispecific antibodies of the invention have enhanced ability to activate T cells, which can be determined, for example, by the EC50 of antibody activation of reporter T cells.

[0147] In some embodiments, the bispecific antibodies of the present invention have improved ability to mediate PBMC cell killing of target cells.

[0148] In some embodiments, the bispecific antibodies of the invention reduce cytokine secretion by PBMC cells, such as reduced secretion of cytokines IFN-γ, TNF-α, IL-2, and / or IL-6.

[0149] The present invention also provides a polynucleotide encoding the bispecific antibody targeting ROR1 and CD3 of the present invention. The polynucleotide can be DNA or RNA (eg, mRNA).

[0150] In some embodiments, the polynucleotides encode the first chain, the second chain, and the third chain of the bispecific antibody, respectively.

[0151] The present invention also provides a vector comprising the above-mentioned polynucleotide, which can be used to express the bispecific antibody of the present invention in a host cell. The vector can be an expression vector, such as a plasmid vector or a viral vector. The viral vector can be a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0152] The present invention also provides a host cell comprising the above-mentioned polynucleotide or the above-mentioned vector. The host cell can be used to express the bispecific antibody of the present invention. The host cell can be a mammalian cell, such as a mouse cell or a human cell, for example, a CHO cell, HEK-293 cell, BHK cell or PER-C6 cell, and its derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells.

[0153] Pharmaceutical compositions and treatments

[0154] The present invention also provides a pharmaceutical composition comprising the aforementioned bispecific antibody, the aforementioned polynucleotide, the aforementioned vector or the aforementioned host cell, and optionally a pharmaceutically acceptable carrier.

[0155] The bispecific antibodies, polynucleotides, vectors, host cells, and pharmaceutical compositions of the present invention can be used to treat ROR1-related diseases. The bispecific antibodies, polynucleotides, vectors, host cells, and pharmaceutical compositions of the present invention can be used to prepare medicaments for treating ROR1-related diseases. The present invention also provides methods for treating ROR1-related diseases, comprising administering the bispecific antibodies, polynucleotides, vectors, host cells, or pharmaceutical compositions of the present invention to a subject in need thereof.

[0156] The ROR1-related disease can be, for example, a ROR1-related cancer or tumor. The ROR1-related disease is characterized by the expression of ROR1, for example, by expressing ROR1 on the surface of its cells. In some embodiments, the cancer or tumor can be a hematological tumor or a solid tumor, such as acute myeloid leukemia (AML), myeloid dysplasia syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), lung cancer (such as non-small cell lung cancer NSCLC or small cell lung cancer SCLC), breast cancer, prostate cancer, pancreatic cancer, colon cancer, ovarian cancer, kidney cancer, uterine cancer, melanoma, etc.

[0157] The terms "subject" and "patient" are used interchangeably herein. The term "subject" as used herein refers to any organism to which the antibodies of the present invention or their antigen-binding fragments can be administered, for example, for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, such as chimpanzees and other apes, and humans). The subject can be a mammal, particularly a human, including female (female) or male (male), and includes newborns, infants, teenagers, youth, adults or the elderly, and further includes various races and ethnicities. In some instances, a subject refers to an individual in need of diagnosis, treatment or prevention of a disease or condition, and the subject may suffer from the disease or condition, or have the risk of suffering from the disease or condition.

[0158] The term "treatment" as used herein refers to providing a beneficial or desired clinical outcome to a disease, such as eliminating the disease, alleviating symptoms, reducing the extent of the disease, stabilizing, improving or alleviating the state of the disease, or slowing down the progression of the disease. Measurement of treatment outcome can be based on, for example, the results of a physical examination, pathological test, and / or diagnostic test known in the art. Treatment can also refer to extending survival compared to the expected survival of the subject if he or she is not receiving treatment. Treatment can also refer to reducing the incidence or morbidity of the disease, or its recurrence, compared to the disease that would occur if the measure were not taken. Clinically, this treatment can also be referred to as prevention.

[0159] The terms "pharmaceutical composition" and "pharmaceutical preparation" of the present invention can be used interchangeably. The pharmaceutical composition may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to any carrier included in the pharmaceutical composition as an inactive ingredient, which gives the pharmaceutical composition an appearance and properties suitable for administration. Pharmaceutically acceptable carriers have substantially no long-term or permanent adverse effects when administered to a subject, such as stabilizers, diluents, additives, adjuvants, excipients, etc. "Pharmaceutically acceptable carrier" should be a pharmaceutically inert material, substantially without biological activity, and constitute the main part of the formulation.

[0160] The pharmaceutical composition of the present invention can be formulated into various modes of administration according to known techniques. For example, see Remington, The Science and Practice of Pharmacy (9th Ed. 1995). When manufacturing a pharmaceutical composition, the active agent is usually mixed with a pharmaceutically acceptable carrier or the like. Of course, the pharmaceutically acceptable carrier must be acceptable, i.e., compatible with any other ingredients in the formulation and must be harmless to the subject. Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers, preservatives, wetting agents, surfactants, emulsifiers, or combinations thereof. Examples of buffers include, but are not limited to, acetic acid, citric acid, histidine, boric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline, etc.

[0161] The bispecific antibodies, polynucleotides, vectors, host cells and pharmaceutical compositions of the present invention can be administered in any manner suitable for the disease to be treated (or prevented) and the subject. In certain embodiments, the mode of administration may include, but is not limited to, parenteral or non-parenteral routes, including oral, sublingual, oral, transdermal, rectal, vaginal, intradermal, intranasal or parenteral routes, such as intravenous (iv), intraperitoneal, intradermal, subcutaneous, intramuscular, intracranial, intrathecal, intratumoral, transdermal, intramucosal, intraarticular, intrathecal, intrathecal, intrahepatic, intraneural or intracranial injection or infusion. The pharmaceutical composition can be injected directly into a tumor, lymph node, tissue, organ or site of infection.

[0162] Dosage forms suitable for oral administration include, but are not limited to, tablets, capsules, powders, pills, granules, suspensions, solutions or preconcentrates of solutions, emulsions or preconcentrates of emulsions. Pharmaceutically acceptable carriers for oral dosage forms include water, ethylene glycol, oils, alcohols, flavoring agents, preservatives, colorants, and the like. Carriers such as starch, sugar, microcrystalline cellulose, diluents, fillers, lubricants, granulating agents, lubricants, binders, stabilizers, disintegrants, and the like can be used to prepare oral solid dosage forms, such as powders, capsules, or tablets.

[0163] Dosage forms suitable for parenteral administration include, but are not limited to, sterile liquid preparations, such as isotonic aqueous solutions, emulsions, suspensions, dispersions, or viscous compositions, which may be buffered to a desired pH. Parenteral dosage forms may be ready for use or may be dry products to be dissolved or suspended in a pharmaceutically acceptable carrier. Parenteral dosage forms may be sterile formulations or capable of being sterilized prior to administration to a subject. Pharmaceutically acceptable carriers that may be used to provide parenteral dosage forms include, but are not limited to, water for injection; aqueous carriers such as, but not limited to, sodium chloride injection, Ringer's injection, and dextrose injection; water-soluble carriers such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; non-aqueous carriers such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate; and solubilizers such as cyclodextrins.

[0164] The bispecific antibodies, polynucleotides, vectors, host cells and pharmaceutical compositions of the present invention are administered to a subject in a therapeutically effective amount. As used herein, the terms "therapeutically effective amount" and "effective amount" are used interchangeably and refer to an amount that is effective at the necessary dosage and time period to achieve the desired therapeutic effect. A therapeutically effective amount is typically expressed as the amount of the active ingredient, for example, as the amount of the bispecific antibody. The therapeutically effective amount can vary depending on factors such as the disease state, age, sex and weight of the individual, as well as the ability of a treatment method or combination of treatment methods to elicit the desired response in the individual. An effective amount can refer to an amount that causes a detectable change in biological or chemical activity. Detectable changes can be detected and / or further quantified by those in the relevant field. In addition, an "effective amount" can specify an amount that maintains the desired physiological state, i.e., reduces or prevents a significant decline and / or promotes improvement in the condition. The amount and frequency of administration will be determined by factors such as the subject's condition (such as age, weight, sex and the subject's response to the drug) and the type and severity of the subject's disease, although the appropriate dosage can be determined by clinical trials.

[0165] The bispecific antibodies, polynucleotides, vectors, host cells, and pharmaceutical compositions of the invention can be administered to a subject in a therapeutically effective amount of about 0.5 to about 250 mg / kg, e.g., about 1 to about 250 mg / kg, about 2 to about 200 mg / kg, about 3 to about 120 mg / kg, about 5 to about 250 mg / kg, about 10 to about 200 mg / kg, or about 20 to about 120 mg / kg.

[0166] The bispecific antibodies, polynucleotides, vectors, host cells, and pharmaceutical compositions of the present invention can be administered once or twice a day; or once every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, once every 1, 2, 3, 4, 5, or 6 weeks, or once every 1, 2, 3, 4, 5, or 6 months or longer. The pharmaceutical composition can also be administered several times a week (e.g., 1, 2, 3, 4, or 5 times) or several times a month (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).

[0167] The bispecific antibodies, polynucleotides, vectors, host cells and pharmaceutical compositions of the present invention can be used in combination with other drugs and treatment methods, such as other anti-tumor drugs, chemotherapy or radiotherapy. "Used in combination" as mentioned herein refers to the administration of two (or more) different drugs and / or therapies to a subject during the treatment process. The two or more drugs and / or therapies in the combination can be administered by different routes and regimens. Two or more drugs and / or treatment methods can be administered to the subject simultaneously or sequentially. In some embodiments, when the administration of the second drug or therapy begins, the administration of one drug or therapy is still ongoing, so there is overlap in administration. Such a regimen may be referred to as "simultaneously" herein. When administered simultaneously, two or more drugs and / or therapies can be formulated together into a single dosage form, or formulated into two or more independent dosage forms.

[0168] The embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise specified, the methods and materials used in the examples described below are all commercially available products or can be prepared by known methods. The experimental methods in the following examples where no specific conditions are specified are generally based on conventional conditions such as those described in Michael R. Green and Joseph Sambrook, Molecule Cloning: A Laboratory Manual, 4 th The conditions described in SEQ ID NO: 1, SEQ ID NO: 2, or the conditions recommended by the manufacturer. It will be understood by those skilled in the art that the methods and materials described below are only exemplary and should not be considered as limiting the scope of the present invention.

[0169] In the following examples, the CDRs of the antibody heavy chain variable region and light chain variable region are defined according to the Kabat numbering scheme.

[0170] Example 1: Design of bispecific antibodies targeting ROR1 and CD3

[0171] Bispecific antibody configuration design: Based on the binding valence and position of ROR1 in the bispecific antibody, three bispecific antibody configurations targeting ROR1 and CD3 were designed for subsequent expression and activity verification. In order to reduce the production of bispecific antibody homodimers, different amino acid mutations were introduced into the constant regions of the two chains to form a knob-into-hole structure (the peptide chain with the hole mutation is subsequently referred to as the hole chain, and the peptide chain with the knob mutation is referred to as the knob chain). At the same time, in order to eliminate the effector function of the Fc region, the L234S / L235T / G236R triple mutation was introduced into the heavy chain constant region. The three specific configuration designs and the corresponding names are shown in Table 1 below.

[0172] Table 1 Configurations of bispecific antibodies targeting ROR1 and CD3

[0173] There are five antigen-binding units targeting ROR1, all derived from the discovery process of single-domain antibodies targeting human ROR1: AHP15662-H4-M21721, AHP15662-H4-MV2, AHP15580, AHP15485-H4-MV2, AHP15485-H4-MV2, and AHP15485-H4-M22013. The antigen-binding protein targeting CD3 is selected from the humanized antibody AED-2018. The human IgG1 Fc region and human IgG light chain constant region are used. HuIgG1Fc(L234S / L235T / G236R hole) and HuIgG1Fc(L234S / L235T / G236R knob) represent knob-into-hole mutations in the human IgG1 Fc fragment, respectively, to form a knob-into-hole structure. Both also incorporate the L234S / L235T / G236R triple mutation to silence Fc effector function. HuIgG1CH(L234S / L235T / G236R knob) features the human IgG1 heavy chain constant region CH1 and HuIgG1Fc(L234S / L235T / G236R knob). The peptide chain unit compositions and sequence numbers of the bispecific antibodies targeting ROR1 and CD3 are shown in Table 2.

[0174] Table 2 Composition and sequence numbers of each peptide chain unit of the bispecific antibody

[0175] Example 2. Recombinant production of bispecific antibodies

[0176] Codon-optimized expression fragments containing each peptide chain of the bispecific antibody were synthesized and inserted into the pcDNA3.4 expression vector to generate an expression plasmid. Bispecific molecules binding to ROR1 and CD3 were transiently transfected into ExpiCHO-S cells using the pcDNA3.4 vectors carrying the bispecific antibody encoding genes in combination. The three plasmids for each bispecific antibody were mixed at a 1:1:1 ratio for transfection. After 6 days of culture in a shake flask at 37°C, the supernatant was harvested for antibody purification. Prior to purification, the tubing and protein A column were depyrogenated with 0.2 M NaOH. The column was re-equilibrated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2× the above buffer and sterilized by filtration. The filtered supernatant was incubated with a protein A column at room temperature for 2 hours. After washing the column with 1× the above buffer, the IgG was eluted using sterile 0.1M sodium citrate (pH 3.5). The eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH = 9.0). Under sterile conditions, the product buffer was exchanged into PBS (pH 7.4) to remove any elution buffer and the sample was concentrated. After concentration, the antibody was quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%). The purified antibody was analyzed by SDS-PAGE using a BioRad electrophoresis system with a 10% precast gel (GenScript). The gel was stained with Estain 2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained bands with Protein Ladder (GenScript). Antibody samples were analyzed by SEC-HPLC using an Agilent Infinity Lab LC1260 system. Based on the principle that substances of different molecular weights have different retention times on a chromatographic column (TSkgel), molecular size was estimated by comparing the elution time of the main peak with that of a known molecular weight standard (STD, BIO-RAD). Sample purity was determined by the percentage of the main peak area. The yield and purity of the bispecific antibodies targeting ROR1 and CD3 are shown in Table 3. All 15 bispecific antibodies were of single composition and high purity.

[0177] Table 3 Bispecific antibody yield and purity information

[0178] Example 3: Binding detection of bispecific antibodies to human ROR1-expressing cell lines and human CD3-expressing Jurkat cell lines

[0179] Jurkat cells expressing human CD3 and CHO-K1 / ROR1 overexpressing cell lines for detection of human ROR1 were collected and washed three times with PBS. 2.5×10 5The cells were washed three times with PBS and 100 μl of 10 μg / ml purified antibody was added and incubated at 4°C for 1 hour. The cells were then washed three times with PBS, and 100 μl of iFluor-labeled goat anti-human IgG and Fcγ-specific fragment antibody were added and incubated at 4°C for 45 minutes. Finally, the cells were washed three times with PBS and the signal was read using FACS BD Calibur. The results of the cell binding assay are shown in Table 4 and Figures 4-10 (Figure 10 includes Figures 10A, 10B, and 10C). The results showed that the bispecific antibodies of different configurations can bind to cell lines expressing ROR1 and also to cell lines expressing CD3.

[0180] Table 4 EC of bispecific antibodies binding to ROR1 or CD3 cells 50

[0181] Example 4: SPR Binding Detection of Bis-antibodies to Human ROR1 Protein

[0182] The binding affinity or avidity of the bispecific antibodies to the human ROR1 protein was determined separately using the surface plasmon resonance (SPR) biosensor Biacore 8K (GE Healthcare). Detection method: Antigen proteins of different concentrations were immobilized on the sensor chip by amino coupling, and the bispecific antibody sample was used as the mobile phase analyte to detect the affinity level of the entire antibody molecule to the corresponding antigen. After the detection run was completed, the Biacore 8K evaluation software was used to obtain the data of the dissociation (kd) and association (ka) rate constants. The equilibrium dissociation constant (KD) was calculated by the ratio of kd to ka. The affinity differences under different antigen coupling conditions were compared to evaluate the affinity differences of the antibodies. The results of the SPR affinity determination are shown in Table 5 below, and the related sensor diagrams are shown in Figures 10A-10C. As shown in the test results, NVG-111, a monovalent ROR1 antibody, showed no significant change in KD values ​​with increasing ROR1 protein concentration. However, BsAb-09 and BsAb-12, bivalent ROR1 antibodies, showed significant decreases in KD values ​​with increasing ROR1 protein concentration, suggesting that higher protein concentrations increase affinity. The bivalent ROR1 design facilitates the TCE antibody's ability to differentiate ROR1 tumor cells from normal cells in vivo, resulting in stronger binding to ROR1 tumor cells.

[0183] Table 5 Affinity test data of bispecific antibodies and human ROR1 protein

[0184] Example 5: Binding of CD3×ROR1 Bispecific Antibody to Human ROR1 Cell Line

[0185] The human ROR1 tumor cell line for detection was collected and washed 3 times with PBS. 3×10 5The antibody was prepared by starting from a concentration of 100nM and then diluted 5-fold for a total of 8 concentrations. 100μl of the prepared antibody was transferred to a 96-well plate and incubated at 4°C for 1 hour. The cells were then washed twice with FACS buffer (DPBS + 1% FBS), and 100μl of iFluor-labeled goat anti-human IgG and Fcγ-specific fragment antibody (Jackson, 109-605-098) was added at a concentration of 1μg / mL and incubated at 4°C for 45 minutes. Finally, the cells were washed twice with FACS buffer and the signal was read using FACS BD Calibur. As shown in Figure 11 and Table 6, the bispecific antibody BsAb-12 bound to EC666 ROR1 tumor cell lines. 50 Both were smaller than that of Yangshen EMB-07, suggesting that BsAb-12 has stronger ROR1 binding activity.

[0186] Table 6 EC binding of CD3×ROR1 bispecific antibodies to ROR1 cell lines 50

[0187] Example 6: Antigen density detection of ROR1 tumor cell lines

[0188] Target cells were collected, washed three times with assay buffer A (99% DPBS + 1% FBS), and 2×10 5 Each test cell and 100 μl of PE anti-human ROR1 antibody (Biolegend, Cat#357804) were incubated at 4°C for 20 minutes. Finally, after washing three times with assay buffer A, 100 μL / well of assay buffer A was added to the test plate, and the cells were collected using a flow cytometer and the fluorescence signal value was read. A tube of BD Quantibrite PE (BD, Cat#340395) was taken out and resuspended with 0.5 mL of assay buffer B (99.5% DPBS + 0.5% BSA). After resuspension, the BD Quantibrite PE tube was run on the machine. All instrument settings such as PE channel voltage and compensation need to be the same as those for cell measurement. The antigen density of the ROR1 tumor cell line was calculated according to the instructions of the PE Fluorescence Quantitation Kit (BD, Cat#340395). By testing ROR1 cell lines with different antigen densities, the difference in ROR1 expression between tumor cells and normal cells in the in vivo environment was simulated. The results are shown in Table 7.

[0189] Table 7. Antigen density of ROR1 tumor cell lines

[0190] Example 7: CD3×ROR1 bispecific antibody-mediated reporter gene-based T cell activation detection

[0191] Target cells were collected and resuspended in RPMI 1640 medium (Gibco, Cat#22400-089) containing 10% FBS and the density was adjusted to 5×10 5 40 μL of target cell suspension was transferred to a 96-well plate. NVG-111, EMB-07, CD3×ROR1 dual antibody samples and negative control Human IgG1 were diluted 4-fold starting from 20 nM, for a total of 11 concentrations: 20 nM, 5 nM, 1.25 nM, 0.3125 nM, 0.07813 nM, 0.01953 nM, 0.004883 nM, 0.001221 nM, 0.0003052 nM, 0.00007629 nM, and 0.00001907 nM. 20 μL of samples and controls were transferred to a 96-well plate and incubated at room temperature for 30 minutes. The reporter gene T cells were collected and resuspended in RPMI 1640 medium (Gibco, Cat#22400-089) containing 10% FBS and the density was adjusted to 5×10 5 cells / mL, transfer 40μL of target cell suspension to a 96-well plate. Then transfer the culture plate to an incubator at 37°C and 5% CO2 and incubate for 6h. Prepare the luciferase detection reagent working solution, remove the incubated experimental plate from the cell culture incubator, transfer the luciferase detection reagent working solution to the corresponding wells of the experimental plate, and incubate at room temperature for 3-5 minutes. Use a PHERA star microplate reader to read the chemiluminescence value and record the data. MDA-MB-231, Jeko-1, and A375 simulate three different expression levels of ROR1, and K562 is a negative cell expressing ROR1 as a control. As shown in Figure 12 and Table 8, on MDA-MB-231 and Jeko-1 cells, the bispecific antibody BsAb-12 activates the EC of reporter gene T cells 50 The activation window of BsAb-12 for activating reporter gene T cells was lower than that of EMB-07 and NVG-111 on A375 cells with low ROR1 expression, and the EC 50 The difference was not much with NVG-111. The results suggested that BsAb-12 showed different activation intensities in cell lines with high and low expression of ROR1.

[0192] Table 8. CD3×ROR1 diabody-mediated reporter gene-based T cell activation experiment in vitro

[0193] Example 8: CD3×ROR1 diabody-mediated PBMC cell (Donor: NF0058) killing experiment in vitro

[0194] Target cells (Jeko-1 and PSN-1) were collected and resuspended in MEM-α medium (Gibco, Cat#41061-029) containing 1% FBS and the density was adjusted to 1×10 5 cells / mL, transfer 50μL of target cell suspension to 96-well plates. NVG-111, EMB-07, CD3×ROR1 dual antibody samples and negative control Human IgG1 were diluted 3-fold starting from 0.2nM concentration, with a total of 7 concentrations, namely 0.2nM, 0.066667nM, 0.022222nM, 0.0074074nM, 0.0024691nM, 0.00082305nM, 0.00027435nM, transfer 50μL of samples and controls to 96-well plates and incubate at room temperature for 30 minutes. After thawing, frozen PBMCs (Allcells, FPB004F-C, Donor: NF0058) were resuspended in MEM-α medium with 1% FBS and adjusted to a density of 2.5×10 6 cells / mL, transfer 100 μL of effector cell suspension, 100 μL of 2% Triton X-100 (Beijing Bailingwei Technology Co., Ltd., Cat#993361) (as the maximum killing signal value) or 100 μL of MEM-α medium containing 1% FBS (as the minimum killing signal value) to a 96-well plate. Then transfer the culture plate to an incubator at 37°C and 5% CO2 and incubate for 24 hours. After incubation for 24 hours, transfer the culture plate and centrifuge at 2000 rpm for 5 minutes. Pipette 50 μL of supernatant into another 96-well plate, add LDH detection reagent (Roche, Cat#11644793001), and read the absorbance at the detection wavelength OD492nm and the reference wavelength OD650nm using a microplate reader (PHERAstar FSX, BMG).

[0195] Killing calculation: Use the OD492nm absorbance value minus the background OD650nm absorbance value. The calculation formula is % target cell lysis rate = 100*(OD 样品 –OD 靶细胞+效应细胞 ) / (OD 最大杀伤信号 -OD 最小杀伤信号The experimental results are shown in Table 9 and Figure 13: The bispecific antibody samples BsAb-09 and BsAb-12 effectively mediated the killing of target cells Jeko-1 by PBMC cells. When the antibody concentration reached 0.2 nM, the killing rate could reach more than 60%. In addition, the killing rates of target cells PSN-1 mediated by PBMC cells by bispecific antibody samples BsAb-09 and BsAb-12 were both less than 30%. The negative control Human IgG1 did not play a role in mediating the killing of target cells by PBMC cells.

[0196] Table 9. CD3×ROR1 diabody-mediated PBMC cell (Donor: NF0058) killing experiment in vitro

[0197] Example 9: CD3×ROR1 diabody-mediated PBMC cell (Donor: Y1701) killing experiment in vitro

[0198] Target cells (Jeko-1 and PSN-1) were collected and resuspended in MEM-α medium (Gibco, Cat#41061-029) containing 1% FBS to adjust the density to 1×10 5 cells / mL, transfer 50 μL of the target cell suspension to a 96-well plate. Increase the EMB-07 concentration, starting from 4 nM, and perform a 5-fold serial dilution to a total of 7 concentrations: 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, 0.00128 nM, and 0.000256 nM. NVG-111, CD3 × ROR1 dual antibody samples and negative control Human IgG1 were diluted 3-fold starting from 0.2nM concentration, with a total of 7 concentrations, namely 0.2nM, 0.066667nM, 0.022222nM, 0.0074074nM, 0.0024691nM, 0.00082305nM, 0.00027435nM. 50μL of samples and controls were transferred to 96-well plates and incubated at room temperature for 30 minutes. After thawing, frozen PBMCs (Allcells, FPB004F-C, Donor: Y1701) were resuspended in MEM-α medium with 1% FBS and adjusted to a density of 2.5×10 6cells / mL, transfer 100 μL of effector cell suspension, 100 μL of 2% Triton X-100 (Beijing Bailingwei Technology Co., Ltd., Cat#993361) (as the maximum killing signal value) or 100 μL of MEM-α medium containing 1% FBS (as the minimum killing signal value) to a 96-well plate. Then transfer the culture plate to an incubator at 37°C and 5% CO2 and incubate for 24 hours. After incubation for 24 hours, transfer the culture plate and centrifuge at 2000 rpm for 5 minutes. Pipette 50 μL of supernatant into another 96-well plate, add LDH detection reagent (Roche, Cat#11644793001), and read the absorbance at the detection wavelength OD492nm and the reference wavelength OD650nm using a microplate reader (PHERAstar FSX, BMG).

[0199] Killing calculation: Use the OD492nm absorbance value minus the background OD650nm absorbance value. The calculation formula is % target cell lysis rate = 100*(OD 样品 –OD 靶细胞+效应细胞 ) / (OD 最大杀伤信号 -OD 最小杀伤信号 The experimental results are shown in Table 10 and Figure 14: The bispecific antibody samples BsAb-09 and BsAb-12 effectively mediated the killing of target cells Jeko-1 by PBMC cells. When the antibody concentration reached 0.2 nM, the killing rate could reach more than 60%. In addition, the killing rates of target cells PSN-1 mediated by PBMC cells by bispecific antibody samples BsAb-09 and BsAb-12 were both less than 50%. The negative control Human IgG1 did not play a role in mediating the killing of target cells by PBMC cells.

[0200] Table 10. CD3×ROR1 diabody-mediated PBMC cell (Donor: Y1701) killing experiment in vitro

[0201] Example 10: CD3×ROR1 diabody-mediated cytokine release experiment of PBMC cells (Donor: NF0058) in vitro

[0202] Target cells (Jeko-1) were collected and resuspended in MEM-α medium (Gibco, Cat#41061-029) containing 1% FBS and the density was adjusted to 1×10 5cells / mL, transfer 50μL of target cell suspension to 96-well plates. NVG-111, EMB-07, CD3×ROR1 dual antibody samples and negative control Human IgG1 were diluted 3-fold starting from 0.2nM concentration, with a total of 7 concentrations, namely 0.2nM, 0.066667nM, 0.022222nM, 0.0074074nM, 0.0024691nM, 0.00082305nM, 0.00027435nM, transfer 50μL of samples and controls to 96-well plates and incubate at room temperature for 30 minutes. After thawing, frozen PBMCs (Allcells, FPB004F-C, Donor: NF0058) were resuspended in MEM-α medium with 1% FBS and adjusted to a density of 2.5×10 6 cells / mL, and 100 μL of the effector cell suspension was transferred to a 96-well plate. The culture plates were then transferred to a 37°C, 5% CO2 incubator and incubated for 24 hours. After 24 hours of incubation, the plates were removed and centrifuged at 2000 rpm for 5 minutes. 50 μL of the supernatant was aspirated and the concentrations of IFN-γ and TNF-α in the supernatant were measured according to the instructions of the IFN-γ detection kit (Cisbio, Cat#62HIFNGPEH) and the TNF-α detection kit (Cisbio, Cat#62HTNFAPEH). The results are shown in Tables 11-14 and Figure 15: EMB-07 and the negative control Human IgG1 at a concentration of 0.2 nM did not significantly promote cytokine release from PBMCs. At a concentration of 0.2 nM, the bispecific antibody BsAb-12 significantly reduced the secretion of cytokines IFN-γ, TNF-α, IL-2, and IL-6 compared to NVG-111. The results suggest that BsAb-12 significantly reduced cytokine secretion while maintaining target cell killing activity.

[0203] Table 11. CD3×ROR1 diabody-mediated IFN-γ release experiment of PBMC cells (Donor: NF0058) in vitro

[0204] Table 12. CD3×ROR1 diabody-mediated TNF-α release experiment of PBMC cells (Donor: NF0058) in vitro

[0205] Table 13. CD3×ROR1 diabody-mediated IL-2 release from PBMC cells (Donor: NF0058) in vitro

[0206] Table 14. CD3×ROR1 diabody-mediated IL-6 release from PBMC cells (Donor: NF0058) in vitro

[0207] Example 11: CD3×ROR1 diabody-mediated cytokine release experiment of PBMC cells (Donor: Y1701) in vitro

[0208] Target cells (Jeko-1) were collected and resuspended in MEM-α medium (Gibco, Cat#41061-029) containing 1% FBS and the density was adjusted to 1×10 5 cells / mL, transfer 50 μL of the target cell suspension to a 96-well plate. Increase the EMB-07 concentration, starting from 4 nM, and perform a 5-fold serial dilution to a total of 7 concentrations: 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, 0.00128 nM, and 0.000256 nM. NVG-111, CD3 × ROR1 dual antibody samples and negative control Human IgG1 were diluted 3-fold starting from 0.2nM concentration, with a total of 7 concentrations, namely 0.2nM, 0.066667nM, 0.022222nM, 0.0074074nM, 0.0024691nM, 0.00082305nM, 0.00027435nM. 50μL of samples and controls were transferred to 96-well plates and incubated at room temperature for 30 minutes. After thawing, frozen PBMCs (Allcells, FPB004F-C, Donor: Y1701) were resuspended in MEM-α medium with 1% FBS and adjusted to a density of 2.5×10 6 cells / mL, and transfer 100 μL of the effector cell suspension to each 96-well plate. The culture plates were then transferred to a 37°C, 5% CO2 incubator and incubated for 24 hours. After 24 hours of incubation, the plates were removed and centrifuged at 2000 rpm for 5 minutes. 50 μL of the supernatant was aspirated and the concentrations of IFN-γ and TNF-α in the supernatant were determined according to the instructions of the IFN-γ detection kit (Cisbio, Cat#62HIFNGPEH) and the TNF-α detection kit (Cisbio, Cat#62HTNFAPEH). The results are shown in Tables 15, 16 and Figure 16: The negative control Human IgG1 did not promote the release of cytokines from PBMC cells. The bispecific antibody samples BsAb-09 (0.2 nM), NVG-111 (0.2 nM) and EMB-07 (4 nM) promoted the release of cytokines IFN-γ and TNF-α from PBMC cells at comparable concentrations. The bispecific antibody sample BsAb-12 significantly reduced the concentrations of cytokines IFN-γ and TNF-α released from PBMC cells.

[0209] Table 15. CD3×ROR1 diabody-mediated IFN-γ release experiment of PBMC cells (Donor: Y1701) in vitro

[0210] Table 16. CD3×ROR1 diabody-mediated TNF-α release experiment of PBMC cells (Donor: Y1701) in vitro

[0211] Example 12: In vivo anti-tumor efficacy study of CD3×ROR1 dual antibody in C-NKG mice transplanted with MDA-MB-231 breast cancer cells and human PBMCs

[0212] The antitumor efficacy was evaluated in C-NKG mice (Saiye (Suzhou) Biotechnology Co., Ltd.), a severely immunodeficient mouse model lacking T cells, B cells, and natural killer cells. 6 Individual PBMCs were transplanted into mice via tail vein injection. On day 4, 10x10 6 MDA-MB-231 cells (0.2 mL volume subcutaneous injection). On day 9, 100 μL of peripheral blood was collected from each mouse to detect the level of human CD45 positive cell reconstruction. On day 10, the reconstitution level of human CD45 positive cells was determined according to the tumor size (~100-120 mm 3 ) The animals were randomly divided into groups and treatment was started. Tumor growth was detected by caliper measurement. The experiment was terminated 18 days after the first dose, and the mice were euthanized if there were signs of severe GVHD. BsAb-12CD3×ROR1 (3 mg / kg, 1 mg / kg, 0.3 mg / kg), CD3×CD7 (3 mg / kg) or vehicle (PBS) were administered by tail vein injection twice a week for a total of five times. Among them, the CD7 VHH sequence of the control group CD3×CD7 control diabody was derived from clone AHP19509 in patent CN117362437A. The diabody and BsAb-12 of the present invention are both 1+1+1 configurations. Except for the CD7 sequence, the sequence and overall configuration are completely consistent with BsAb-12. As shown in Figure 17, mice in the CD3×ROR1 (3 mg / kg, 1 mg / kg, 0.3 mg / kg) treatment group showed a significant tumor growth inhibition effect compared to the CD3×CD7 (3 mg / kg) group, indicating that the dual antibody of the present invention has the effect of inhibiting tumor growth, and this effect is not produced by CD3 alone. ( **** P<0.0001, *** P<0.001, ** P<0.01, * P < 0.05; compared with the CD3 × CD7 (3 mg / kg) group, tested by One-way ANOVA)

Claims

1. A bispecific antibody targeting ROR1 and CD3, comprising a first antigen binding portion and a second antigen binding portion, wherein the first antigen binding portion is an antibody or an antigen binding fragment thereof that specifically binds to ROR1, and the second antigen binding portion is an antibody or an antigen binding fragment thereof that specifically binds to CD3, wherein the second antigen binding portion comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises HCDR1 as shown in SEQ ID NO: 39, HCDR2 as shown in SEQ ID NO: 40, and HCDR3 as shown in SEQ ID NO: 41, and the VL comprises LCDR1 as shown in SEQ ID NO: 43, LCDR2 as shown in SEQ ID NO: 44, and LCDR3 as shown in SEQ ID NO:

45.

2. The bispecific antibody of claim 1, wherein the first antigen binding portion comprises a heavy chain variable region VH, wherein the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are selected from the group consisting of: (1) HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 20, and HCDR3 as shown in SEQ ID NO: 21; (2) HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:24, and HCDR3 as shown in SEQ ID NO:25; (3) HCDR1 as shown in SEQ ID NO:27, HCDR2 as shown in SEQ ID NO:28, and HCDR3 as shown in SEQ ID NO:29; (4) HCDR1 as shown in SEQ ID NO:31, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:33; and (5) HCDR1 as shown in SEQ ID NO:35, HCDR2 as shown in SEQ ID NO:36 and HCDR3 as shown in SEQ ID NO:

37.

3. The bispecific antibody of claim 2, wherein the VH of the first antigen binding portion comprises an amino acid sequence as shown in SEQ ID NO: 18, 22, 26, 30 or 34, or a sequence having at least 80% sequence identity with the amino acid sequence as shown in SEQ ID NO: 18, 22, 26, 30 or 34.

4. The bispecific antibody of any one of claims 1 to 3, wherein the first antigen binding moiety is a single domain antibody.

5. The bispecific antibody of any one of claims 1 to 4, wherein the VH of the second antigen binding portion comprises the amino acid sequence as shown in SEQ ID NO: 38 or a sequence having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO: 38, and the VL of the second antigen binding portion comprises the amino acid sequence as shown in SEQ ID NO: 42 or a sequence having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO:

42. The bispecific antibody of claim 1 , wherein the second antigen binding moiety is a Fab fragment.

7. The bispecific antibody according to any one of claims 1 to 6, further comprising an Fc fragment. The bispecific antibody according to claim 7 , wherein the Fc fragment has a Knob-Hole structure. 9 . The bispecific antibody according to claim 7 , wherein the Fc fragment has a mutation that reduces or eliminates the binding of the antibody to the Fcγ receptor, preferably a triple mutation of L234S / L235T / G236R.

10. The bispecific antibody of any one of claims 1 to 9, wherein the first antigen binding moiety is fused to the N-terminus of the first Fc fragment, and the second antigen binding moiety is fused to the N-terminus of the second Fc fragment. The bispecific antibody of claim 10 , wherein the second antigen binding moiety is a Fab fragment, and the heavy chain of the Fab fragment is fused to the N-terminus of the second Fc fragment.

12. The bispecific antibody of any one of claims 1-11, further comprising a third antigen binding moiety, wherein the third antigen binding moiety is an antibody or an antigen binding fragment thereof that specifically binds to ROR1.

13. The bispecific antibody of claim 12, wherein the third antigen binding portion comprises a heavy chain variable region VH, wherein the VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 are selected from the group consisting of: (1) HCDR1 as shown in SEQ ID NO: 19, HCDR2 as shown in SEQ ID NO: 20, and HCDR3 as shown in SEQ ID NO: 21; (2) HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:24, and HCDR3 as shown in SEQ ID NO:25; (3) HCDR1 as shown in SEQ ID NO:27, HCDR2 as shown in SEQ ID NO:28, and HCDR3 as shown in SEQ ID NO:29; (4) HCDR1 as shown in SEQ ID NO:31, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:33; and (5) HCDR1 as shown in SEQ ID NO:35, HCDR2 as shown in SEQ ID NO:36 and HCDR3 as shown in SEQ ID NO:

37.

14. The bispecific antibody of claim 13, wherein the VH of the third antigen binding portion comprises an amino acid sequence as shown in SEQ ID NO: 18, 22, 26, 30 or 34, or a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 30 or 34.

15. The bispecific antibody of any one of claims 12-14, wherein the third antigen binding moiety is a single domain antibody.

16. The bispecific antibody of any one of claims 12-15, wherein the third antigen binding moiety has the same HCDR1, HCDR2 and HCDR3 as the first antigen binding moiety.

17. The bispecific antibody of any one of claims 12-16, wherein the third antigen binding moiety has the same VH as the first antigen binding moiety.

18. The bispecific antibody of any one of claims 12-17, wherein the third antigen binding moiety is fused to the N-terminus of the first antigen binding moiety or to the N-terminus of the Fab heavy chain of the second antigen binding moiety.

19. The bispecific antibody of claim 18, wherein the third antigen binding moiety is fused to the N-terminus of the first antigen binding moiety or to the N-terminus of the Fab heavy chain of the second antigen binding moiety via a peptide linker, preferably the sequence of the peptide linker is (GGGGS)n, wherein n is an integer not less than 1.

20. The bispecific antibody of any one of claims 1 to 19, which consists of a first chain comprising a fusion protein of a first antigen binding moiety and a first Fc fragment, a second chain comprising a fusion protein of a Fab heavy chain of a second antigen binding moiety and a second Fc fragment, and a third chain comprising a Fab light chain of the second antigen binding moiety.

21. The bispecific antibody of claim 20, wherein the first chain comprises the amino acid sequence shown in SEQ ID NO: 1, 4, 5, 6 or 7, or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 4, 5, 6 or 7, the second chain comprises the amino acid sequence shown in SEQ ID NO: 2, or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and the third chain comprises the amino acid sequence shown in SEQ ID NO: 3, or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

3.

22. The bispecific antibody of any one of claims 1 to 19, which consists of a first chain, a second chain and a third chain, wherein the first chain comprises a third antigen binding portion, a fusion protein of the first antigen binding portion and a first Fc fragment, the second chain comprises a fusion protein of the Fab heavy chain of the second antigen binding portion and a second Fc fragment, and the third chain comprises the Fab light chain of the second antigen binding portion.

23. The bispecific antibody of claim 22, wherein the first chain comprises the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11 or 12, or a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11 or 12, the second chain comprises the amino acid sequence shown in SEQ ID NO: 2, or a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and the third chain comprises the amino acid sequence shown in SEQ ID NO: 3, or a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

3.

24. The bispecific antibody of any one of claims 1 to 19, which consists of a first chain, a second chain, and a third chain, wherein the first chain comprises a fusion protein of a first antigen binding portion and a first Fc fragment, the second chain comprises a third antigen binding portion, a fusion protein of the Fab heavy chain of the second antigen binding portion and a second Fc fragment, and the third chain comprises the Fab light chain of the second antigen binding portion.

25. The bispecific antibody of claim 24, wherein the first chain comprises the amino acid sequence shown in SEQ ID NO: 1, 4, 5, 6 or 7, or a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, 4, 5, 6 or 7, the second chain comprises the amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16 or 17, or a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16 or 17, and the third chain comprises the amino acid sequence shown in SEQ ID NO: 3, or a sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

3.

26. The bispecific antibody of claim 25, wherein the first chain, the second chain and the third chain are selected from the group consisting of: (1) the first strand comprises the amino acid sequence of SEQ ID NO:1, or a sequence having at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1, the second strand comprises the amino acid sequence of SEQ ID NO:13, or a sequence having at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:13, and the third strand comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3; (2) the first chain comprises the amino acid sequence of SEQ ID NO:4 or a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:4, the second chain comprises the amino acid sequence of SEQ ID NO:14 or a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:14, and the third chain comprises the amino acid sequence of SEQ ID NO:3 or a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:3; (3) the first strand comprises the amino acid sequence of SEQ ID NO:5 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, the second strand comprises the amino acid sequence of SEQ ID NO:15 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:15, and the third strand comprises the amino acid sequence of SEQ ID NO:3 or a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3; (4) the first chain comprises the amino acid sequence shown in SEQ ID NO:6 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:6, the second chain comprises the amino acid sequence shown in SEQ ID NO:16 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:16, and the third chain comprises the amino acid sequence shown in SEQ ID NO:3 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:3; and (5) The first chain comprises the amino acid sequence shown in SEQ ID NO:7 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:7, the second chain comprises the amino acid sequence shown in SEQ ID NO:17 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:17, and the third chain comprises the amino acid sequence shown in SEQ ID NO:3 or a sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

3.

27. A polynucleotide encoding the bispecific antibody according to any one of claims 1 to 26.

28. A recombinant expression vector comprising the polynucleotide of claim 27.

29. A host cell comprising the polynucleotide of claim 27 or the recombinant expression vector of claim 28.

30. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 26, the polynucleotide of claim 27, the recombinant expression vector of claim 28 or the host cell of claim 29, and a pharmaceutically acceptable carrier.

31. Use of the bispecific antibody of any one of claims 1 to 26, the polynucleotide of claim 27, the recombinant expression vector of claim 28 or the host cell of claim 29 in the preparation of a medicament for treating a ROR1-related disease.

32. The use according to claim 31, wherein the ROR1-related disease is a tumor or cancer.

33. A method for treating a ROR1-related disease, comprising administering the bispecific antibody of any one of claims 1-26, the polynucleotide of claim 27, the recombinant expression vector of claim 28, the host cell of claim 29, or the pharmaceutical composition of claim 30 to a subject in need thereof.

34. The method of claim 33, wherein the ROR1-related disease is a tumor or cancer.

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