Anti-ROR1 antibodies, bispecific antibodies containing the same, and uses thereof

Abstract

Provided are novel anti-ROR1 antibodies, bispecific antibodies comprising the anti-ROR1 antibodies, and uses thereof.

Description

[Technical Field]

[0001] Provided are novel anti-ROR1 antibodies, bispecific antibodies comprising the anti-ROR1 antibodies, and uses thereof. [Background technology]

[0002] ROR (Receptor Tyrosine Kinase-Like Orphan Receptor) is a transmembrane protein of the RTK (Receptor Tyrosine Kinase) family, consisting of ROR1 and ROR2. ROR1 and ROR2 share 58% amino acid sequence identity, and the theoretical molecular weight of the two proteins is approximately 104 kDa. However, the molecular weight of ROR1 is approximately 130 kDa due to multiple N-glycosylation sites. The extracellular domain of the ROR family consists of Ig, cysteine-rich, and kringle domains, while the intracellular domain consists of tyrosine kinase, serine / threonine (Ser / Thr)-rich, and proline (Pro)-rich domains. Based on biological characteristics, the ligand for ROR2 is Wnt5a, while the ligand for ROR1 has not yet been identified.

[0003] ROR1 is expressed during embryonic and fetal development and regulates cell polarity, cell migration, and neurite outgrowth. Its expression gradually decreases with the progression of development, becoming almost nonexistent in adults. It is transiently expressed during B cell development, and only slight expression has been reported in adipocytes. However, overexpression of ROR1 has been observed in various cancer cells, leading to its classification as an oncofetal gene. ROR1, in particular, has been found to be overexpressed in chronic lymphocytic leukemia (CLL), and has begun to attract attention as a target for anticancer antibodies. In addition to chronic lymphocytic leukemia (CLL), ROR1 expression has been reported to be overexpressed in various solid tumors, including B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and marginal lymphoma (MZL), as well as breast cancer, kidney cancer, ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, non-small cell lung cancer (NSCLC), neuroblastoma, brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer. ROR1 expression in these cancers is associated with poor prognosis and is known to affect cancer metastasis. Injection of cancer cells with suppressed ROR1 expression into mice has been shown to increase survival and reduce the extent of metastasis.

[0004] Since ROR1 is expressed specifically in cancer cells, it could be an effective target for cancer therapy, and therefore the development of antibodies that specifically recognize it is highly desirable. Furthermore, when developing therapeutic antibodies, it is very important that they possess not only target specificity and binding strength, but also advantageous properties for production, storage, and administration, such as stability and developability.

[0005] Meanwhile, multispecific antibodies that target two or more antigens have been developed in various types and forms and are expected to be new therapeutic antibodies with superior therapeutic effects compared to single-target antibodies. Summary of the Invention [Problem to be solved by the invention]

[0006] The present specification provides a novel anti-ROR1 antibody or an antigen-binding fragment thereof, a bispecific antibody comprising the same, and uses thereof.

[0007] The anti-ROR1 antibody or antigen-binding fragment thereof is HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO:2; HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and LCDR3 comprising the amino acid sequence of SEQ ID NO:6 It may also include.

[0008] The anti-ROR1 antibody or antigen-binding fragment thereof is a heavy chain complementarity-determining region (CDR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a heavy chain variable region comprising the heavy chain complementarity-determining regions; and A light chain complementarity determining region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a light chain variable region comprising said light chain complementarity determining region. It may also include.

[0009] Specifically, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region of SEQ ID NO: 7 or SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 8 or SEQ ID NO: 126. More specifically, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise, but is not limited to, a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8, or a heavy chain variable region of SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 126.

[0010] Another example provides a bispecific antibody comprising the anti-ROR1 antibody or antigen-binding fragment thereof.

[0011] More specifically, a first targeting moiety that targets (specifically binds to and / or recognizes) a first antigen, and a second targeting moiety that targets (specifically binds to and / or recognizes) a second antigen; the first antigen and the second antigen are different from each other; one of the first targeting moiety and the second targeting moiety comprises the anti-ROR1 antibody or antigen-binding fragment thereof; Bispecific antibodies or antigen-binding fragments thereof are provided.

[0012] In one example, in the bispecific antibody or antigen-binding fragment thereof, one of the first antigen and the second antigen is ROR1, and the other antigen is selected from the group consisting of a cancer cell-expressed antigen, an immune checkpoint protein, and / or a T cell surface antigen, for example, but not limited to, 4-1BB and B7-H3.

[0013] Another example provides a conjugate comprising (1) the anti-ROR1 antibody or its antigen-binding fragment, or the bispecific antibody, and (2) one or more useful polymers, labeling substances, drugs, etc. The drug may be a cytotoxic drug, but may also be, for example, an anti-cancer drug.

[0014] Another example provides a pharmaceutical composition comprising the anti-ROR1 antibody or antigen-binding fragment thereof, the bispecific antibody, and / or the conjugate. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutical composition may have anti-cancer activity.

[0015] Another example provides a pharmaceutical composition for preventing and / or treating cancer, comprising the anti-ROR1 antibody or antigen-binding fragment thereof, the bispecific antibody, and / or the conjugate. The cancer may be a cancer that expresses ROR1.

[0016] Another example provides a method for preventing and / or treating cancer, comprising administering a pharmaceutically effective amount of the anti-ROR1 antibody or antigen-binding fragment thereof, the bispecific antibody, and / or a conjugate to a subject in need of cancer prevention and / or treatment. The method may further comprise, prior to the administering step, identifying (diagnosing) the subject in need of cancer prevention and / or treatment.

[0017] Other examples provide uses of the anti-ROR1 antibody or its antigen-binding fragment; the bispecific antibody; and / or the complex for the prevention and / or treatment of cancer and / or for the production of drugs (anticancer drugs) for the prevention and / or treatment of cancer.

[0018] Another example provides a composition for detecting ROR1, which comprises the anti-ROR1 antibody or an antigen-binding fragment thereof.

[0019] Another example provides a method for detecting ROR1 in a biological sample, comprising contacting the anti-ROR1 antibody or antigen-binding fragment thereof with a biological sample in which ROR1 expression needs to be detected.

[0020] Another example provides a composition for diagnosing cancer, comprising the anti-ROR1 antibody or its antigen-binding fragment. The cancer may be a cancer that expresses ROR1.

[0021] Another example provides a method for diagnosing cancer or a method for providing information on cancer diagnosis in a biological sample or a subject from which the biological sample was derived (separated), comprising contacting the anti-ROR1 antibody or antigen-binding fragment thereof with the biological sample. [Means for solving the problem]

[0022] Provided are novel anti-ROR1 antibodies, bispecific antibodies comprising the same, and anti-cancer uses thereof.

[0023] Definition of Terms As used herein, a polynucleotide (which may be used interchangeably with "gene or nucleic acid molecule") or a polypeptide (which may be used interchangeably with "protein" and may refer to a CDR, variable region, heavy chain, light chain, and / or antibody depending on the context) "comprises a specific nucleic acid sequence or amino acid sequence" or "consists of or is expressed as a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide essentially contains the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including "substantially equivalent sequences" in which mutations (deletions, substitutions, modifications, and / or additions) have been made to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained (or as not excluding the mutations).

[0024] In one example, as used herein, a polynucleotide or polypeptide "comprising a specific nucleic acid sequence or amino acid sequence" or "consisting of or expressed as a specific nucleic acid sequence or amino acid sequence" can mean that the polynucleotide or polypeptide (i) essentially contains the specific nucleic acid sequence or amino acid sequence, or (ii) consists of or essentially contains a nucleic acid sequence or amino acid sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more identity to the specific nucleic acid sequence or amino acid sequence, and maintains its original and / or intended function.

[0025] As used herein, the term "sequence homology" or "sequence identity" refers to the degree of correspondence between a given nucleic acid sequence or amino acid sequence, and is expressed as a percentage (%). Homology to nucleic acid sequences can be determined, for example, using the literature-based algorithm BLAST or Pearson's FASTA. Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed (see http: / / www.ncbi.nlm.nih.gov).

[0026] As used herein, a protein or polypeptide "comprising, consisting of, or expressed as a specific amino acid sequence" may mean that the protein or polypeptide essentially contains the amino acid sequence, and / or that the amino acid sequence contains meaningless mutations (e.g., substitution, deletion, and / or addition of amino acid residues) that do not affect the original activity and / or the intended activity (e.g., antigen binding ability, anti-cancer activity (e.g., cancer cell growth inhibition, cancer cell killing, etc.)).

[0027] The amino acid positions described herein are calculated from the N-terminus of the reference amino acid sequence unless otherwise specified.

[0028] As used herein, the term "antibody" collectively refers to a protein that specifically binds to a specific antigen. It may be a protein produced in the immune system upon antigen stimulation, or a chemically synthesized or recombinantly produced protein; the type is not particularly limited. An antibody may refer to an intact immunoglobulin of any isotype, or an antigen-binding fragment that can compete with the intact antibody for binding to a target antigen. An antibody is a type of antigen-binding protein in itself. An intact antibody typically contains at least two full-length heavy chains and two full-length light chains. As used herein, an antibody or its antigen-binding fragment may be non-naturally produced, e.g., recombinantly or synthetically produced. For example, an antibody or its antigen-binding fragment may be produced by hybridomas, recombinant DNA technology, or enzymatic or chemical cleavage of an intact antibody. Unless otherwise specified, the term "antibody" as used herein includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and / or mutants thereof. The antibody may be an animal antibody (e.g., a mouse antibody), a chimeric antibody (e.g., a mouse-human antibody), a humanized antibody, or a human antibody. The antibody may be a monoclonal antibody or a polyclonal antibody.

[0029] In the antibodies or antigen-binding fragments thereof provided herein, the defined heavy chain CDRs and light chain CDRs, or the remaining regions excluding the heavy chain variable region and light chain variable region, may be derived from immunoglobulins of any isotype (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), and may be derived from, for example, framework regions and / or light chain constant regions and / or heavy chain constant regions of immunoglobulins of any of the above isotypes. In one example, the antibodies provided herein may be human IgG-type antibodies, for example, IgG1, IgG2, IgG3, or IgG4-type antibodies, but are not limited thereto.

[0030] A complete antibody (e.g., IgG type) has two full-length light chains and two full-length heavy chains, each connected to a heavy chain by a disulfide bond. The constant regions of antibodies are divided into heavy-chain and light-chain constant regions. The heavy-chain constant regions have gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) types, with gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2) subclasses. The light-chain constant regions have kappa (κ) or lambda (λ) types.

[0031] In one specific example, the constant region of the IgG (e.g., human IgG1) may be wild-type. In another specific example, the constant region of the IgG may be one into which a mutation has been introduced for a predetermined purpose. For example, the constant region of the IgG may be a mutant type based on human IgG1 that contains a known mutation, such as N297A (the 297th amino acid residue, N, is replaced with A), and such a mutant type may have reduced ADCC (antibody dependent cellular cytotoxicity) compared to the wild-type type.

[0032] The term "heavy chain" is intended to include all full-length heavy chains and fragments thereof, including a variable region domain VH containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, three constant region domains CH1, CH2, and CH3, and a hinge. The VH domain is located at the N-terminus of the heavy chain, the CH domain is located at the C-terminus, and the CH3 domain is located closest to the C-terminus. Heavy chains are selected from the following isotypes: IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), and IgM and IgE.

[0033] The term "light chain" is also intended to encompass full-length light chains and fragments thereof, including a variable region domain (VL) and a constant region domain (CL) that contain sufficient amino acid sequence to confer specificity to an antigen. The variable region domain of the light chain is located at the amino terminus of the light chain. Types of light chains can include kappa or lambda chains.

[0034] The term "CDR (complementarity determining region)" refers to the portion of an antibody variable region that confers antigen-binding specificity, and refers to the amino acid sequence of the hypervariable region of an immunoglobulin heavy or light chain. Each heavy or light chain can contain three CDRs: HCDR1 (or CDRH1), HCDR2 (or CDRH2), HCDR3 (or CDRH3), LCDR1 (or CDRL1), LCDR2 (or CDRL2), and LCDR3 (or CDRL3). The CDRs can provide the main contact residues for antibody binding to an antigen or epitope. Meanwhile, the terms "specifically bind" and "specifically recognize" as used herein have the same meaning as commonly known to those skilled in the art, meaning that an antigen and an antibody specifically interact to cause an immunological reaction.

[0035] The complementarity determining regions (CDRs) described herein may be determined based on the CDR definition according to the Kabat system.

[0036] Unless otherwise specified, the term "antibody" as used herein can be understood to include antigen-binding fragments of antibodies that have antigen-binding ability.

[0037] The term "antigen-binding fragment" refers to any form of polypeptide that contains a portion capable of binding to an antigen (e.g., six CDRs as defined herein). For example, it may be one or more selected from the group consisting of antibody scFv, scFv-Fc, scFv-Fc-scFv, (scFv)2, Fab, Fab', Fab-FcFab'-Fc, F(ab')2, diabody, and minibody, but is not limited thereto.

[0038] Among the antigen-binding fragments, Fab has one antigen-binding site in a structure comprising the variable regions of the light chain and heavy chain, the constant region of the light chain, and the first constant region (CH1) of the heavy chain.

[0039] Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.

[0040] F(ab')2 antibodies are produced by forming disulfide bonds between cysteine residues in the hinge region of Fab'.

[0041] Fv is the smallest antibody fragment containing only the heavy-chain variable region and the light-chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. Two-chain Fvs (two-chain Fvs) have a non-covalent linkage between the heavy-chain variable region and the light-chain variable region. Single-chain Fvs (scFvs) can form a single-chain dimer structure in which the heavy-chain variable region and the single-chain variable region are linked covalently (peptide bond) via a conventional peptide linker or directly (without a linker). scFv-Fc is a dimer in which Fc is linked to scFv. scFv-Fc-scFv is a dimer in which one scFv is linked to each end (N-terminus and C-terminus) of Fc. Minibodies are dimers in which CH3 is linked to scFv. Diabodies contain two scFv molecules.

[0042] The antigen-binding fragment can be produced by chemical cleavage of a whole antibody or enzymatic cleavage using protease (for example, limited cleavage of a whole antibody with papain to obtain Fab fragments, or cleavage with pepsin to obtain F(ab')2 fragments), or by genetic recombination techniques.

[0043] The term "hinge region" refers to a region contained in an antibody heavy chain, which is located between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site within the antibody.

[0044] As used herein, "Fc" region refers to an antibody heavy chain fragment comprising the CH2 and CH3 domains, and may optionally include a hinge region at the N-terminus of the CH2 and CH3 domains (i.e., the N-terminus of the CH2 domain). Two Fc regions can be bound to each other by two or more disulfide bonds and / or hydrophobic interactions of the CH3 domains.

[0045] As used herein, a "bivalent antibody or bivalent antigen-binding fragment" comprises two antigen-binding sites each containing six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3). The two antigen-binding sites contained in the bivalent antibody or bivalent antigen-binding fragment may have the same antigen specificity or may have dual specificity, binding to different antigens. As used herein, a "monovalent antibody or monovalent antigen-binding fragment" comprises one antigen-binding site containing six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3).

[0046] As used herein, a "bispecific" antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment thereof that targets two antigens, i.e., a hybrid antibody that binds to two different epitopes. The two epitopes of the bispecific antibody or antigen-binding fragment can be located on the same or different target proteins (antigens).

[0047] The antibodies provided herein may be monoclonal antibodies. Monoclonal antibodies can be produced by methods well known in the art, such as phage display technology. Alternatively, the antibodies can be produced as monoclonal antibodies derived from animals (e.g., mice, rats, rabbits, etc.) by conventional methods, or as humanized antibodies by conventional methods.

[0048] As used herein, "antigen" or "immunogen" refers to a molecule or portion of a molecule that can be bound by, for example, an antigen-binding protein (e.g., an antibody or an immunologically functional antigen-binding fragment thereof) and can be used to generate antibodies in an animal that can bind to the antigen. The antigen can contain one or more epitopes that can interact with another antibody or fragment thereof.

[0049] As used herein, "epitope" refers to the portion of a molecule that is bound by or recognized by an antibody or antigen-binding fragment, and includes any determinant capable of specifically binding to an antigen-binding protein, such as an antibody or T cell receptor. The epitope may be contiguous or discontinuous, e.g., amino acid residues that are not contiguous in the polypeptide sequence but are spaced apart from one another by a single antigen-binding protein, such as a conformational epitope. The epitope determinant may be a chemically activated group formed on the surface by a molecule such as an amino acid, a sugar side chain, a phosphoryl group, and / or a sulfonyl group, or may have specific three-dimensional structural and / or charge characteristics.

[0050] As used herein, an antibody (e.g., a CDR, a variable region, or a heavy chain / light chain, an antigen-binding fragment, etc.) "comprises, consists of, or is expressed as a specific amino acid sequence" can mean both cases where the antibody essentially contains the amino acid sequence, and cases where the amino acid sequence contains meaningless mutations (e.g., substitution, deletion, and / or addition of amino acid residues) that do not significantly affect antibody activity (e.g., antigen affinity, pharmacological activity, etc.).

[0051] Summary of the Invention One example provides an anti-ROR1 antibody or antigen-binding fragment thereof, comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO:2; HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0052] Another example provides an anti-ROR1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region of SEQ ID NO: 7 or 127 and a light chain variable region of SEQ ID NO: 8 or 126.

[0053] Other examples provide nucleic acid molecules encoding the aforementioned anti-ROR1 antibodies or antigen-binding fragments thereof, recombinant vectors containing the same, and / or recombinant cells containing the recombinant vectors.

[0054] Another example provides a pharmaceutical composition for preventing or treating cancer, comprising the anti-ROR1 antibody or its antigen-binding fragment and a pharmaceutically acceptable excipient. Another example provides a method for preventing or treating cancer, comprising administering a pharmaceutically effective amount of the anti-ROR1 antibody or its antigen-binding fragment to a subject in need of cancer prevention or treatment. Another example provides a use of the anti-ROR1 antibody or its antigen-binding fragment for preventing or treating cancer, or for producing a pharmaceutical composition for preventing or treating cancer.

[0055] In the pharmaceutical compositions, methods and / or uses, the cancer may be a cancer that expresses ROR1.

[0056] Other examples provide a composition for detecting ROR1 containing the above-mentioned anti-ROR1 antibody or its antigen-binding fragment, a method for detecting ROR1 using the same, and uses thereof for detecting ROR1.

[0057] Another example provides a cancer diagnostic composition comprising the above-described anti-ROR1 antibody or antigen-binding fragment thereof.

[0058] In the composition for cancer diagnosis, the cancer may be a cancer that expresses ROR1.

[0059] Other examples include: an anti-ROR1 antibody or antigen-binding fragment thereof, and Anti-4-1BB antibody or antigen-binding fragment thereof Including, The anti-ROR1 antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6. Anti-ROR1 / anti-4-1BB bispecific antibodies are provided.

[0060] In the above-mentioned anti-ROR1 / anti-4-1BB bispecific antibody, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region of SEQ ID NO: 7 or SEQ ID NO: 127, and a light chain variable region of SEQ ID NO: 8 or SEQ ID NO: 126.

[0061] In the aforementioned anti-ROR1 / anti-4-1BB bispecific antibody, the anti-4-1BB antibody or antigen-binding fragment thereof is HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, 14 or 15; HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17, or 18; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, or 23; LCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or 25; LCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 27; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 or 29 It may also include.

[0062] In the aforementioned anti-ROR1 / anti-4-1BB bispecific antibody, the anti-4-1BB antibody or antigen-binding fragment thereof is (1) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 19; (b) HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 20; (c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 21; (d) HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 22; and (e) HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 23 a heavy chain complementarity determining region selected from (2) (a) LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; and (b) LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 a light chain complementarity determining region selected from It may also include.

[0063] In the aforementioned anti-ROR1 / anti-4-1BB bispecific antibody, the anti-4-1BB antibody or antigen-binding fragment thereof is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, or 47 It may also include.

[0064] Other examples provide nucleic acid molecules encoding the aforementioned anti-ROR1 / anti-4-1BB bispecific antibodies or antigen-binding fragments thereof, recombinant vectors comprising the same, and / or recombinant cells comprising the recombinant vectors.

[0065] Another example provides a pharmaceutical composition for preventing or treating cancer, comprising the above-described anti-ROR1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable excipient.

[0066] Another example provides a method for preventing or treating cancer, comprising administering a pharmaceutically effective amount of the anti-ROR1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof to a subject in need thereof. Another example provides a use of the anti-ROR1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof for the prevention or treatment of cancer, or for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.

[0067] In the pharmaceutical compositions, methods and / or uses, the cancer may be a cancer that expresses ROR1.

[0068] Other examples include: an anti-ROR1 antibody or antigen-binding fragment thereof, and Anti-B7-H3 antibody or antigen-binding fragment thereof Including, The anti-ROR1 antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6. Anti-ROR1 / anti-B7-H3 bispecific antibodies are provided.

[0069] In the above-mentioned anti-ROR1 / anti-B7-H3 bispecific antibody, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region of SEQ ID NO: 7 or SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 8 or SEQ ID NO: 126.

[0070] In the aforementioned anti-ROR1 / anti-B7-H3 bispecific antibody, the anti-B7-H3 antibody or antigen-binding fragment thereof is HCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 48 to 51; HCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 52 to 57; HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 58 to 62; LCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 63 to 67; LCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 68 to 73; and LCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 74 to 79 It may also include.

[0071] In the aforementioned anti-ROR1 / anti-B7-H3 bispecific antibody, the anti-B7-H3 antibody or antigen-binding fragment thereof is (i) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 48, HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; (b) HCDR1 comprising the amino acid sequence of SEQ ID NO: 49, HCDR2 comprising the amino acid sequence of SEQ ID NO: 53, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 59; (c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 50, HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 60; (d) HCDR1 comprising the amino acid sequence of SEQ ID NO: 48, HCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 61; (e) HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 62; and (f) HCDR1 comprising the amino acid sequence of SEQ ID NO: 50, HCDR2 comprising the amino acid sequence of SEQ ID NO: 57, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 60 a heavy chain complementarity determining region selected from (ii) (a) LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 74; (b) LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, LCDR2 comprising the amino acid sequence of SEQ ID NO: 69, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 75; (c) LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, LCDR2 comprising the amino acid sequence of SEQ ID NO: 70, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 77; (e) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 67, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 78; and (f) LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, LCDR2 comprising the amino acid sequence of SEQ ID NO: 73, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76 a light chain complementarity determining region selected from It may also include.

[0072] In the aforementioned anti-ROR1 / anti-B7-H3 bispecific antibody, the anti-B7-H3 antibody or antigen-binding fragment thereof is A heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 80 to 91; and A light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 92 to 103 It may also include.

[0073] Other examples provide nucleic acid molecules encoding the aforementioned anti-ROR1 / anti-B7-H3 bispecific antibodies or antigen-binding fragments thereof, recombinant vectors comprising the same, and / or recombinant cells comprising the recombinant vectors.

[0074] Another example provides a pharmaceutical composition for preventing or treating cancer, comprising the anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient. Another example provides a method for preventing or treating cancer, comprising administering a pharmaceutically effective amount of the anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof to a subject in need thereof. Another example provides use of the anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof for preventing or treating cancer, or for producing a pharmaceutical composition for preventing or treating cancer.

[0075] In the pharmaceutical compositions, methods and / or uses, the cancer may be a cancer that expresses ROR1, B7-H3, or both.

[0076] Another example provides an antibody-drug conjugate comprising a cytotoxic drug linked to the aforementioned anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof.

[0077] The antibody-drug conjugate may have an antibody-linker-drug structure.

[0078] In the antibody-drug conjugate, the cytotoxic drug may be an anti-cancer drug.

[0079] Another example provides a pharmaceutical composition for preventing or treating cancer, comprising the aforementioned antibody-drug conjugate and a pharmaceutically acceptable excipient. Another example provides a method for preventing or treating cancer, comprising administering a pharmaceutically effective amount of the aforementioned anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof to a subject in need thereof. Another example provides a use of the aforementioned anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof for the prevention or treatment of cancer, or for the manufacture of a pharmaceutical composition for the prevention or treatment of cancer.

[0080] In the pharmaceutical compositions, methods and / or uses, the cancer may be a cancer that expresses ROR1, B7-H3, or both.

[0081] The present invention will now be described in more detail: Anti-ROR1 antibody or antigen-binding fragment thereof One example provides an anti-ROR1 antibody or antigen-binding fragment thereof that binds to ROR1. The anti-ROR1 antibody or antigen-binding fragment thereof provided herein may target (specifically recognize and / or bind to) human ROR1 (e.g., the extracellular domain). The anti-ROR1 antibody or antigen-binding fragment thereof may not only have inhibitory activity against ROR1, but also have improved stability and ease of development, thereby offering the advantages of enabling the economical production of antibody pharmaceuticals that are more effective and safer than conventional therapeutic antibodies.

[0082] The anti-ROR1 antibody or antigen-binding fragment thereof is HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO:2; HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and LCDR3 comprising the amino acid sequence of SEQ ID NO:6 It may also include.

[0083] In the present specification, the complementarity determining region (CDR) is determined based on the CDR definition according to the Kabat system.

[0084] In one specific example, the amino acid sequences (N→C) of the CDRs of the six possible CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) contained in the anti-ROR1 antibody or antigen-binding fragment thereof provided herein are summarized below: HCDR1: NYDMS (SEQ ID NO: 1) HCDR2: AIYHSGSSKYYADSVKG (SEQ ID NO: 2) HCDR3: GGSGAWDTGFDY (SEQ ID NO: 3) LCDR1:SGSSSNIGSNDVS (SEQ ID NO: 4) LCDR2: YENNRPS (SEQ ID NO: 5) LCDR3:GAWDDSLSGYV (SEQ ID NO: 6) (HCDR1, HCDR2, and HCDR3 refer to heavy chain complementarity determining regions, and LCDR1, LCDR2, and LCDR3 refer to light chain complementarity determining regions.) The anti-ROR1 antibody or antigen-binding fragment thereof is a heavy chain complementarity-determining region (CDR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a heavy chain variable region comprising the heavy chain complementarity-determining regions; and A light chain complementarity determining region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a light chain variable region comprising said light chain complementarity determining region. It may also include.

[0085] The heavy and light chain variable regions of the anti-ROR1 antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment derived from IgG, or may be an antibody or antigen-binding fragment having a wild-type sequence, e.g., an antibody or antigen-binding fragment having mutations introduced into the framework regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and / or LFR4). In one example, the mutations may be for stabilization and / or disulfide bridge formation. For example, the antigen-binding fragment may have one or more (e.g., one) G (Gly) to C (Cys) mutations (replacement of G with C) introduced into the framework regions of the antibody variable region, but is not limited thereto. In one specific example, when the ROR1 antibody is a complete IgG antibody or a portion thereof (e.g., Fab or Fab-Fc), the heavy and light chain variable regions contained therein may be derived from wild-type IgG. In another specific example, when the anti-ROR1 antibody is an antigen-binding fragment of an anti-ROR1 antibody (e.g., scFv, etc.), the heavy chain variable region and light chain variable region contained therein may have G→C mutations introduced into the framework regions.

[0086] Specifically, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region of SEQ ID NO: 7 or 127 and a light chain variable region of SEQ ID NO: 8 or 126. More specifically, the anti-ROR1 antibody or antigen-binding fragment thereof may comprise, but is not limited to, a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 8, or a heavy chain variable region of SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 126.

[0087] Optionally (eg, when recombinantly produced), the heavy chain variable region and / or light chain variable region can further comprise a suitable signal sequence at the N-terminus.

[0088] Examples of amino acid sequences of heavy chain variable regions and light chain variable regions that can be included in the anti-ROR1 antibodies or antigen-binding fragments thereof provided herein are shown in Table 1 below:

[0089] [Table 1] (In Table 1, the underlined regions represent CDR1, CDR2, and CDR3 of the heavy and light chains, respectively.)

[0090] ROR1 (Receptor Tyrosine Kinase-Like Orphan Receptor), the antigen of which the anti-ROR1 antibody or antigen-binding fragment provided herein is, is a transmembrane protein belonging to the RTK (Receptor Tyrosine Kinase) family. The anti-ROR1 antibody or antigen-binding fragment provided herein may recognize (bind to) the extracellular domain of the ROR1 protein, whether present or absent from the cell membrane. The ROR1 protein may be a mammalian ROR1 protein, for example, a human ROR1 protein. The amino acid sequence of the human ROR1 protein may be, but is not limited to, NCBI Accession No. NP_005003.2937aa, and the nucleic acid sequence encoding it may be, but is not limited to, NCBI Accession No. NM_005012.3. In another example, the ROR1 protein is represented by the mouse ROR1 protein (GenBank Accession No. BAA75480.1).

[0091] The antibodies or antigen-binding fragments thereof provided herein have binding affinity to an antigen (e.g., a human antigen protein) as measured, for example, by surface plasmon resonance (SPR) (K D ) or based on the EC50 measured by ELISA, it may be 5 nM or less, 1 nM or less, 0.5 nM or less, 0.2 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, 0.005 nM or less, or 0.001 nM or less, for example, 0.001 nM to 5 nM, or 0.001 nM to 1 nM, but is not limited to these.

[0092] The anti-ROR1 antibodies or antigen-binding fragments thereof provided herein have functions such as ROR1 inhibition (e.g., suppression of ROR1 expression and / or activity), cancer cell targeting by specific binding to cancer cells (e.g., ROR1-expressing cancer cells), and anti-cancer activity (e.g., cancer cell death, suppression of cancer cell proliferation, etc.). Thus, the anti-ROR1 antibodies or antigen-binding fragments thereof can be usefully applied to the prevention and / or treatment of cancer.

[0093] Another example provides a composition or kit for detecting ROR1, which comprises one or more members selected from the group consisting of the anti-ROR1 antibody or its antigen-binding fragment, and the nucleic acid molecule encoding the same.

[0094] Another example provides a method for detecting ROR1 in a biological sample, comprising contacting a biological sample in which ROR1 expression needs to be detected with one or more of the anti-ROR1 antibodies or antigen-binding fragments thereof and nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof. The method may further comprise measuring binding in the biological sample treated (contacted) with the antibodies, antigen-binding fragments, and / or nucleic acid molecules after the contacting step. In one embodiment, the method may be performed in vitro or in vivo.

[0095] Another example provides a cancer diagnostic composition or a cancer diagnostic kit comprising one or more members selected from the group consisting of the anti-ROR1 antibody or its antigen-binding fragment and its encoding nucleic acid molecule. The cancer may be a cancer that expresses ROR1.

[0096] Another example provides a method for diagnosing cancer in a biological sample or a subject from which the biological sample was derived (separated), or a method for providing information regarding cancer diagnosis, comprising contacting the biological sample with one or more selected from the group consisting of the anti-ROR1 antibody or its antigen-binding fragment and its encoding nucleic acid molecule. The method may further include, after the contacting step, measuring binding in the biological sample treated (contacted) with the antibody or antigen-binding fragment and / or nucleic acid molecule and / or, if binding is detected in the treated (contacted) biological sample, determining (confirming) that the biological sample or the subject from which the biological sample was derived (separated) is a cancer patient, or additionally determining that such subject will respond to treatment with the anti-ROR1 antibody or its antigen-binding fragment. In one embodiment, the method can be performed in vitro or in vivo. The cancer may be a cancer that expresses ROR1.

[0097] In the compositions and / or methods for ROR1 detection and / or cancer diagnosis provided herein, the biological sample may be cells, tissues, body fluids (e.g., serum), transformed cells, or cultures thereof derived from any animal selected from mammals including all animals, for example, humans, primates such as monkeys, rodents such as rats and mice, etc., and may be, for example, cells, tissues, body fluids (e.g., serum) isolated from humans or cells, tissues, or body fluids (e.g., serum) isolated from humans.

[0098] Another example provides a nucleic acid molecule encoding the anti-ROR1 antibody or antigen-binding fragment thereof.

[0099] Specifically, one example provides a nucleic acid molecule encoding the heavy chain complementarity determining regions, heavy chain variable region, or heavy chain of an anti-ROR1 antibody.

[0100] Other examples provide nucleic acid molecules encoding the light chain complementarity determining regions, light chain variable regions, or light chains of anti-ROR1 antibodies.

[0101] Other examples include: (1) a nucleic acid molecule encoding a heavy chain complementarity-determining region, a heavy chain variable region, or a heavy chain of the anti-ROR1 antibody; and (2) a nucleic acid molecule encoding the light chain complementarity determining region, light chain variable region, or light chain of an anti-ROR1 antibody; The recombinant vector may be an expression vector for expressing the nucleic acid molecule.

[0102] Another example provides a recombinant cell comprising the recombinant vector.

[0103] One example of the present application provides a bispecific antibody or antigen-binding fragment thereof comprising the anti-ROR1 antibody or antigen-binding fragment thereof.

[0104] More specifically, a first targeting moiety that targets (specifically binds to and / or recognizes) a first antigen, and a second targeting moiety that targets (specifically binds to and / or recognizes) a second antigen; the first antigen and the second antigen are different from each other; one of the first targeting moiety and the second targeting moiety comprises the anti-ROR1 antibody or antigen-binding fragment thereof; Bispecific antibodies or antigen-binding fragments thereof are provided.

[0105] For example, in the bispecific antibody or antigen-binding fragment thereof, one of the first antigen and the second antigen may be ROR1, and the other may be selected from 4-1BB and B7-H3, but is not limited thereto, and may be selected from, for example, other cancer cell-expressed antigens, immune checkpoint proteins, and / or T cell surface antigens.

[0106] The explanations given herein regarding anti-ROR1 antibodies or antigen-binding fragments thereof can be similarly applied to the anti-ROR1 / 4-1BB bispecific antibodies, anti-ROR1 / B7-H3 bispecific antibodies, or antigen-binding fragments thereof described below. Similarly, the explanations given herein below regarding anti-ROR1 / 4-1BB bispecific antibodies, anti-ROR1 / B7-H3 bispecific antibodies, or antigen-binding fragments thereof can be similarly applied to anti-ROR1 antibodies or antigen-binding fragments thereof.

[0107] Anti-ROR1 / anti-4-1BB bispecific antibody An example is: (1) an anti-ROR1 antibody or antigen-binding fragment thereof as the ROR1 targeting moiety; and (2) an anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB targeting moiety; The present invention provides an anti-ROR1 / anti-4-1BB bispecific antibody comprising: The anti-ROR1 / anti-4-1BB bispecific antibody targets (specifically binds to and / or recognizes) ROR1 and 4-1BB.

[0108] The anti-ROR1 antibody or antigen-binding fragment thereof as the ROR1 targeting moiety may be a protein that targets (specifically binds to and / or recognizes) ROR1. More specifically, the ROR1 targeting moiety may be the aforementioned anti-ROR1 antibody or antigen-binding fragment thereof, and the explanations provided for the aforementioned anti-ROR1 antibody or antigen-binding fragment thereof also apply to the anti-ROR1 antibody or antigen-binding fragment thereof contained in the anti-ROR1 / anti-4-1BB bispecific antibody.

[0109] The anti-4-1BB antibody or antigen-binding fragment thereof as the 4-1BB targeting moiety may be a protein that targets (specifically binds to and / or recognizes) 4-1BB.

[0110] 4-1BB, also known as CD137 or TNFRSF9 (TNF Receptor Superfamily Member 9), is a member of the TNF-receptor superfamily (TNFRSF) and is a costimulatory molecule expressed after immune cell activation, both innate and acquired immune cells. 4-1BB plays an important role in regulating the activities of various immune cells. The 4-1BB antigen of the anti-4-1BB antibody or antigen-binding fragment thereof provided herein may be mammalian 4-1BB, such as human 4-1BB. The amino acid sequence of human 4-1BB may be, but is not limited to, NCBI Accession No. NP_001552.2.

[0111] For example, the anti-4-1BB antibody or antigen-binding fragment thereof may be CDR (complementarity determining region)-H1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 13, 14 or 15; HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17, or 18; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, or 23; LCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or 25; LCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 27; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 or 29 may include:

[0112] The amino acid sequences of the CDRs of anti-4-1BB antibodies or antigen-binding fragments are exemplified in Table 2:

[0113] [Table 2]

[0114] For example, the anti-4-1BB antibody or antigen-binding fragment thereof may include: HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, HCDR3 comprising the amino acid sequence of SEQ ID NO: 20, LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 29; HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, HCDR3 comprising the amino acid sequence of SEQ ID NO: 20, LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 29; HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 29; HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, HCDR3 comprising the amino acid sequence of SEQ ID NO: 22, LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, HCDR3 comprising the amino acid sequence of SEQ ID NO: 22, LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 29; an HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; or HCDR1 having the amino acid sequence of SEQ ID NO: 15, HCDR2 having the amino acid sequence of SEQ ID NO: 18, HCDR3 having the amino acid sequence of SEQ ID NO: 23, LCDR1 having the amino acid sequence of SEQ ID NO: 25, LCDR2 having the amino acid sequence of SEQ ID NO: 27, and LCDR3 having the amino acid sequence of SEQ ID NO: 29.

[0115] In yet another example, the anti-4-1BB antibody or antigen-binding fragment thereof is The heavy chain variable region may include an HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, 14 or 15, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22 or 23; and a light chain variable region including an LCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or 25, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 27, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 or 29.

[0116] The heavy and light chain variable regions of the anti-4-1BB antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment derived from IgG, or may be an antibody or antigen-binding fragment having a wild-type sequence, e.g., an antibody or antigen-binding fragment having mutations introduced into the framework regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and / or LFR4). In one example, the mutations may be for stabilization and / or disulfide bridge formation. For example, the antigen-binding fragment may have one or more (e.g., one) G (Gly) to C (Cys) mutations (replacement of G with C) introduced into the framework regions of the antibody variable region, but this is not limiting. In one specific example, when the 4-1BB targeting moiety is a complete IgG antibody or a portion thereof (e.g., Fab or Fc-Fab), the heavy and light chain variable regions contained therein may be derived from wild-type IgG. In another specific example, when the 4-1BB targeting moiety is an antigen-binding fragment of an anti-4-1BB antibody (e.g., scFv, etc.), the heavy chain variable region and light chain variable region contained therein may have G→C mutations introduced into the framework regions.

[0117] In one example, the anti-4-1BB antibody or antigen-binding fragment thereof is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, or 47.

[0118] The amino acid sequences (N→C) of the variable regions of anti-4-1BB antibodies or antigen-binding fragments are exemplified in Table 3:

[0119] [Table 3]

[0120] In another embodiment, the anti-4-1BB antibody or antigen-binding fragment thereof may be an scFv (single chain variable fragment) comprising: a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, 14, or 15, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17, or 18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, or 23; and A light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or 25, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 27, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 or 29.

[0121] In one example, the anti-4-1BB scFv can comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 (more specifically, SEQ ID NO: 36, 37, 38, 39, 40, or 41); and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, or 47 (more specifically, SEQ ID NO: 45, 46, or 47).

[0122] In this case, the heavy chain variable region and the light chain variable region are linked to each other in any order, either directly or via a peptide linker.

[0123] In the anti-4-1BB scFv, the heavy chain variable region and the light chain variable region can be linked in any order directly (e.g., without a linker) or via a peptide linker. More specifically, the anti-4-1BB scFv can comprise, in order from N-terminus to C-terminus, a light chain variable region and a heavy chain variable region; a heavy chain variable region and a light chain variable region; a light chain variable region, a peptide linker, and a heavy chain variable region; or a heavy chain variable region, a peptide linker, and a light chain variable region.

[0124] Anti-ROR1 / anti-B7-H3 bispecific antibody An example is: (1) an anti-ROR1 antibody or antigen-binding fragment thereof as the ROR1 targeting moiety; and (2) an anti-B7-H3 antibody or an antigen-binding fragment thereof as the B7-H3 targeting moiety; The present invention provides an anti-ROR1 / anti-B7-H3 bispecific antibody comprising: The anti-ROR1 / anti-B7-H3 bispecific antibody targets (specifically binds to and / or recognizes) ROR1 and B7-H3.

[0125] The anti-ROR1 antibody or antigen-binding fragment thereof as the ROR1 targeting moiety may be a protein that targets (specifically binds to and / or recognizes) ROR1. More specifically, the ROR1 targeting moiety may be the aforementioned anti-ROR1 antibody or antigen-binding fragment thereof, and the explanations provided for the aforementioned anti-ROR1 antibody or antigen-binding fragment thereof also apply to the anti-ROR1 antibody or antigen-binding fragment thereof contained in the anti-ROR1 / anti-B7-H3 bispecific antibody.

[0126] The anti-B7-H3 antibody or antigen-binding fragment thereof as the B7-H3 targeting moiety may be a protein that targets (specifically binds to and / or recognizes) B7-H3.

[0127] B7-H3 (B7 Homolog 3, CD276) is a transmembrane protein belonging to the immunoglobulin (Ig) superfamily and can contain an extracellular region, a transmembrane region, and / or an intracellular region. The B7-H3 antigen recognized by the anti-B7-H3 antibodies or antigen-binding fragments thereof provided herein may be an extracellular region that is present or absent from the cell membrane. B7-H3 may be, but is not limited to, mammalian B7-H3, such as human B7-H3 (e.g., NCBI Accession No. NP_001019907.1 (534 aa)), monkey B7-H3 (e.g., NCBI Accession No. XP_005560056.1), or mouse B7-H3 (e.g., NCBI Accession No. NP_598744.1).

[0128] For example, the anti-B7-H3 antibody or antigen-binding fragment thereof may be HCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 48 to 51; HCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 52 to 57; HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 58 to 62; LCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 63 to 67; LCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 68 to 73; and LCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 74 to 79 may include:

[0129] The amino acid sequences of the CDRs of anti-B7-H3 antibodies or antigen-binding fragments are exemplified in Table 4:

[0130] [Table 4]

[0131] The heavy and light chain variable regions of the anti-B7-H3 antibody or antigen-binding fragment thereof may be an antibody or antigen-binding fragment derived from IgG, or may be an antibody or antigen-binding fragment having a wild-type sequence, e.g., an antibody or antigen-binding fragment having mutations introduced into the framework regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and / or LFR4). In one example, the mutations may be for stabilization and / or disulfide bridge formation. For example, the antigen-binding fragment may have one or more (e.g., one) G (Gly) to C (Cys) mutations (replacement of G with C) introduced into the framework regions of the antibody variable region, but this is not limiting. In one specific example, when the B7-H3 targeting moiety is a complete IgG antibody or a portion thereof (e.g., Fab or Fc-Fab), the heavy and light chain variable regions contained therein may be derived from wild-type IgG. In another specific example, when the B7-H3 targeting moiety is an antigen-binding fragment of an anti-B7-H3 antibody (e.g., scFv, etc.), the heavy chain variable region and light chain variable region contained therein may have G→C mutations introduced into the framework regions.

[0132] In one embodiment, the anti-B7-H3 antibody or antigen-binding fragment thereof is It may comprise a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 80 to 91; and a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 92 to 103.

[0133] The amino acid sequences (N→C) of the variable regions of anti-B7-H3 antibodies or antigen-binding fragments are exemplified in Table 5:

[0134] [Table 5] TIFF2025525538000007.tif22170

[0135] For example, the anti-B7-H3 antibody or antigen-binding fragment thereof can include: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 80 or 86 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 92 or 98; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81 or 87 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 93 or 99; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 82 or 88 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 94 or 100; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 83 or 89 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 95 or 101; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 84 or 90 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 96 or 102; or A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 85 or 91 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 97 or 103.

[0136] In another embodiment, the anti-B7-H3 antibody or antigen-binding fragment thereof can be an scFv (single chain variable fragment) comprising: a heavy chain variable region comprising an HCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 48 to 51, an HCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 52 to 57, and an HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 58 to 62; and A light chain variable region comprising LCDR1 having an amino acid sequence selected from SEQ ID NOs: 63 to 67, LCDR2 having an amino acid sequence selected from SEQ ID NOs: 68 to 73, and LCDR3 having an amino acid sequence selected from SEQ ID NOs: 74 to 79.

[0137] In one example, the anti-B7-H3 scFv can comprise: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 80 to 91 (more specifically, SEQ ID NOs: 86 to 91); and A light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 92 to 103 (more specifically, SEQ ID NOs: 98 to 103).

[0138] In this case, the heavy chain variable region and the light chain variable region are linked to each other in any order, either directly or via a peptide linker.

[0139] In one example, the anti-B7-H3 scFv can comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 80 or 86 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 92 or 98; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 81 or 87 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 93 or 99; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 82 or 88 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 94 or 100; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 83 or 89 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 95 or 101; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 84 or 90 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 96 or 102; or A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 85 or 91 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 97 or 103.

[0140] In the anti-B7-H3 scFv, the heavy chain variable region and the light chain variable region can be linked in any order directly (e.g., without a linker) or via a peptide linker. More specifically, the anti-B7-H3 scFv can comprise, in order from N-terminus to C-terminus, a light chain variable region and a heavy chain variable region; a heavy chain variable region and a light chain variable region; a light chain variable region, a peptide linker, and a heavy chain variable region; or a heavy chain variable region, a peptide linker, and a light chain variable region.

[0141] Bispecific antibodies containing anti-ROR1 antibodies In the anti-ROR1 / anti-4-1BB bispecific antibody and / or anti-ROR1 / anti-B7-H3 bispecific antibody provided herein, the anti-ROR1 antibody or antigen-binding fragment thereof as the ROR1 targeting moiety may be any of the above-described anti-ROR1 antibodies or antigen-binding fragments thereof that target (specifically bind to and / or recognize) ROR1.

[0142] In one specific example, when the ROR1 targeting moiety is a complete IgG antibody or a portion thereof (e.g., Fab or Fab-Fc), the heavy and light chain variable regions contained therein may be derived from wild-type IgG. In another specific example, when the anti-ROR1 antibody is an antigen-binding fragment of an anti-ROR1 antibody (e.g., scFv, etc.), the heavy and light chain variable regions contained therein may have mutations introduced into framework regions.

[0143] In one example, the anti-ROR1 antibody or antigen-binding fragment thereof included in the bispecific antibody provided herein may be an antibody in the form of a complete immunoglobulin (e.g., an IgG form containing two heavy chains and two light chains), a portion thereof (e.g., Fab or Fab-Fc), or an antigen-binding fragment derived therefrom (e.g., scFv, etc.). The anti-ROR1 scFv may further comprise a peptide linker connecting the heavy chain variable region and the light chain variable region.

[0144] The anti-ROR1 / anti-4-1BB bispecific antibody or anti-ROR1 / anti-B7-H3 bispecific antibody provided herein may contain one or more ROR1 targeting moieties and one or more B7-H3 or 4-1BB targeting moieties as described above, and there are no particular limitations on the bond form or structure between them.

[0145] In one specific example, in the anti-ROR1 / anti-4-1BB bispecific antibody or anti-ROR1 / anti-B7-H3 bispecific antibody, the ROR1 targeting moiety and the 4-1BB or B7-H3 targeting moiety may each be a bivalent antibody or bivalent antigen-binding fragment having two antigen-binding sites (e.g., an intact antibody (e.g., IgG), a dimer of two Fab-Fc or Fab'-Fc linked by an Fc), or an scFv-Fc-scFv). For example, the bispecific antibody comprises an anti-ROR1 IgG antibody linked to an anti-4-1BB scFv or anti-B7-H3 scFv, and the scFvs may be linked to the N- or C-termini of the two heavy chains or two light chains of the IgG antibody via peptide linkers or directly without a linker.

[0146] In another specific example, in the anti-ROR1 / anti-4-1BB bispecific antibody or anti-ROR1 / anti-B7-H3 bispecific antibody, one of the ROR1 targeting moiety and the 4-1BB or B7-H3 targeting moiety is a bivalent antibody or bivalent antigen-binding fragment having two antigen-binding sites (e.g., an intact antibody (e.g., IgG), a dimer of two Fab-Fc or Fab'-Fc linked via an Fc), or scFv-Fc-scFv), and the other is a monovalent antibody or monovalent antigen-binding fragment having one antigen-binding site (e.g., scFv, scFv-Fc, Fab, Fab-Fc, Fab'-Fc, etc.), and the monovalent antibody or monovalent antigen-binding fragment may be linked to the N-terminus and / or C-terminus of the heavy chain or light chain of one or both of the bivalent antibody or bivalent antigen-binding fragment via a peptide linker or directly without a linker, or linked via the Fc. For example, the bispecific antibody comprises an anti-ROR1 IgG antibody and an anti-4-1BB scFv or an anti-B7-H3 scFv linked thereto, and the scFv may be linked to the N-terminus or C-terminus of either one of the two heavy chains or two light chains of the IgG antibody via a peptide linker or directly without a linker.

[0147] In another embodiment, the bispecific antibody can comprise an anti-ROR1 Fab-Fc linked to an anti-B7-H3 Fab-Fc, Fab-Fc-scFv, scFv-Fc-scFv, or scFv-Fc, where the scFv can be linked via a peptide linker or directly without a linker.

[0148] For example, in one embodiment, in the anti-ROR1 / anti-4-1BB bispecific antibody, the ROR1 targeting moiety may be an anti-ROR1 IgG antibody and the 4-1BB targeting moiety may be an anti-4-1BB scFv.

[0149] In the case of B7-H3 antibodies, excessively high antigen-binding avidity may result in toxicity. Therefore, in the case of antibodies against B7-H3 contained in the anti-ROR1 / anti-B7-H3 bispecific antibodies provided herein, it may be preferable to use clones that have an appropriate level of binding avidity and little risk of toxicity.

[0150] In another embodiment, in the anti-ROR1 / anti-4-1BB bispecific antibody or anti-ROR1 / anti-B7-H3 bispecific antibody, the ROR1 targeting moiety and the 4-1BB or B7-H3 targeting moiety may each be a monovalent antibody or antigen-binding fragment having one antigen-binding site (e.g., scFv, scFv-Fc, Fab, Fab-Fc, Fab'-Fc, etc.). The antibodies or antigen-binding fragments may be directly linked via a peptide linker or without a linker, and may be linked to the Fc, the terminus of the light or heavy chain constant region, within the light or heavy chain constant region, or the terminus of the light or heavy chain variable region, or linked between the Fc.

[0151] In one embodiment, the scFv can comprise a heavy chain variable region, a variable region, and optionally a linker, in any order. For example, the scFv can comprise, from N- to C-terminus, a light chain variable region and a heavy chain variable region; a heavy chain variable region and a light chain variable region; a light chain variable region, a peptide linker, and a heavy chain variable region; or a heavy chain variable region, a peptide linker, and a light chain variable region.

[0152] In the anti-ROR1 / anti-4-1BB bispecific antibody or anti-ROR1 / anti-B7-H3 bispecific antibody provided herein, the light chain variable region and heavy chain variable region, the scFv and Fc, and / or the ROR1 targeting moiety and the 4-1BB or B7-H3 targeting moiety are linked via a peptide linker. In one example, a peptide linker that can be used may refer to an oligopeptide containing 1 to 100 amino acids, particularly 2 to 50 amino acids, each of which may contain any type of amino acid without limitation. Any conventional peptide linker can be used with or without appropriate modifications for a specific purpose. In certain embodiments, the peptide linker may contain, for example, Gly, Asn, and / or Ser residues, and / or neutral amino acids such as Thr and / or Ala. Amino acid sequences suitable for peptide linkers are publicly available. The length of the peptide linker can be appropriately determined within the limits that do not affect the function of the polypeptide and / or scFv. For example, the peptide linker can be formed of a total of about 1 to about 100 amino acids, about 2 to about 50 amino acids, or about 5 to about 25 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids, each of which may be independently selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker is m S l ) n (m, l, and n are "G", "S", and "(G m Sl )" which are each independently selected from the integers of about 1 to about 10, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker may be, but is not limited to, the amino acids (GGGGS)2, (GGGGS)3, (GGGGS)4, or (GS)9.

[0153] Uses of anti-ROR1 antibodies and / or bispecific antibodies containing the same The anti-ROR1 antibodies provided herein have ROR1 inhibitory activity (e.g., inhibition of ROR1 expression and / or activity). Furthermore, anti-ROR1 / anti-4-1BB bispecific antibodies comprising these antibodies simultaneously possess ROR1 inhibitory activity and T cell activation effects, and anti-ROR1 / anti-B7-H3 bispecific antibodies have functions such as cancer cell targeting through specific binding to cancer cells (ROR1- and / or B7-H3-expressing cancer cells) and anti-cancer activity (e.g., cancer cell death, inhibition of cancer cell proliferation, etc.). Therefore, the anti-ROR1 antibodies, anti-ROR1 / anti-4-1BB bispecific antibodies, and anti-ROR1 / anti-B7-H3 bispecific antibodies can be usefully applied to the prevention and / or treatment of cancer. Furthermore, the anti-ROR1 / anti-B7-H3 bispecific antibodies provided herein have excellent cellular internalization function (see Example 3.4), and when used in conjunction with a cytotoxic drug such as an anticancer agent (e.g., antibody-drug conjugate (ADC)), can achieve excellent cancer cell killing (anticancer) effects.

[0154] As used herein, the term "conjugate" refers to a cell-binding agent covalently attached to one or more molecules of a cytotoxic compound. Here, the "cell-binding agent" is a molecule having affinity for a biological target, such as a ligand, protein, antibody, or antigen-binding fragment thereof, and the binding agent functions to direct a biologically active compound to the biological target. In one embodiment of the present invention, the conjugate is designed to target cancer cells via a cell surface antigen. The antigen may be a cell surface antigen that is overexpressed or expressed on abnormal cell types. Specifically, the target antigen may be one that is predominantly expressed on proliferating cells (e.g., cancer cells). The target antigen may be selected based on differential expression between normal proliferating tissues and normal tissues.

[0155] The anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody, or antigen-binding fragment thereof provided herein can be used in the form of a conjugate conjugated with one or more useful substances selected from the group consisting of polymers, labeling substances, drugs, etc.

[0156] The useful polymer may be, for example, a polymer that increases the in vivo half-life of a polypeptide, antibody, and / or antigen-binding fragment, and examples thereof include, but are not limited to, one or more hydrophilic polymers selected from the group consisting of polyethylene glycol (PEG) (e.g., PEG having a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa), dextrin, monomethoxypolyethylene glycol (mPEG), etc.

[0157] The labeling substance can be selected from fluorescent or chemiluminescent small chemicals, radioisotopes, etc. commonly used in the art, but is not limited thereto.

[0158] In the present invention, the drug may be one or more selected from the group consisting of cytotoxic drugs (e.g., anticancer drugs, antibiotics, etc.) and contrast agents. The cytotoxic drug may be any drug that has cytotoxic activity, particularly cancer cytotoxicity or bacterial / viral cytotoxicity, such as a tubulin inhibitor, a DNA alkylating agent, a topoisomerase inhibitor, an RNA polymerase inhibitor, a ribosome inactivating protein, a NAMPT inhibitor, or an immunomodulator.Examples of the drug include maytansine drugs (e.g., mertansine (DM1), ravtansine (DM4), auristatin drugs (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.), calicheamicin drugs, pyrrolobenzodiazepine drugs, duocarmycin, docetaxel, doxorubicin, carboplatin (paraplatin), cyclophosphamide, ifosfamide, Nidran, nitrogen mustard, mechlorethamine hydrochloride, HCl, bleomycin, mitomycin C, cytarabine, flurouracil, gemcitabine, trimetrexate, methotrexate, etoposide, vinblastine, pinorelbine, alimta, altretamine, procarbazine, paclitaxel (taxol), taxotere, topotecan, irinotecan, exatecan, or analogs thereof. In this case, antibody-drug conjugates (antibody-drug conjugates) The drug may be used in the form of an ADC (anticoagulant conjugate), and the drug may preferably be a maytansine drug, an auristatin drug, a calicheamicin drug, a pyrrolobenzodiazepine drug, a duocarmycin, an irinotecan, or an exatecan.In the conjugate, the antibody and cytotoxic drug (e.g., an anticancer drug) may be linked by a chemical bond, for example, a covalent bond. For example, the antibody and cytotoxic drug may be linked by thiol coupling (SH coupling) or amine coupling (NH coupling). To this end, any amino acid residue in the heavy and / or light chain (e.g., in the constant region) of the antibody may be modified or substituted (or mutated) with cysteine to have a functional group capable of thiol coupling or amine coupling, and / or the drug may be derivatized to have a functional group capable of thiol coupling or amine coupling. Alternatively, the antibody and cytotoxic drug may be linked by glycosylation of an asparagine (N) residue in the Fc.

[0159] In the present invention, the term "drug-to-antibody ratio (DAR)" refers to the average number of therapeutic moieties, e.g., drugs, conjugated to a conjugate of the present invention. The drug-to-antibody ratio is measured by liquid chromatography-mass spectrometry or the like. The drug-to-antibody ratio of the conjugate of the present invention is 1 to 10, specifically 2 to 8, but is not limited thereto and can be appropriately adjusted depending on the type and properties of the drug and linker used.

[0160] As used herein, an "antibody-drug conjugate" (ADC) is a conjugate that can deliver a drug to cells to which an antibody can specifically bind. For example, by conjugating a drug with an antibody of the present invention that can specifically bind to ROR1 and / or B7-H3, the drug can be selectively delivered to cells that express ROR1 and / or B7-H3.

[0161] In the present invention, the conjugate between the antibody and the drug may be a direct chemical bond, or an indirect bond via a linker or a secondary antibody bound to the antibody, but is not limited thereto.

[0162] The antibody-drug conjugate may include a linker, which is a compound that covalently binds an active agent to a ligand. For example, the linker may be cleavable, non-cleavable, hydrophilic, or hydrophobic, but is not limited thereto. For example, a linker sequence may be attached to the Fc sequence of the antibody of the present invention, and the linker sequence may be linked to a drug to prepare and use the antibody-linker-drug form.

[0163] Another example provides a pharmaceutical composition comprising the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody, or antigen-binding fragment thereof; and / or the antibody-drug conjugate. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutical composition may have anti-cancer activity.

[0164] Another example provides a pharmaceutical composition for preventing and / or treating cancer, comprising the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof; and / or the complex.

[0165] Another example provides a method for preventing and / or treating cancer, comprising administering a pharmaceutically effective amount of the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof, and / or the conjugate to a subject in need of cancer prevention and / or treatment. The method may further comprise the step of identifying (diagnosing) the subject in need of cancer prevention and / or treatment prior to the administering step.

[0166] Other examples provide uses of the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof; and / or the antibody-drug conjugate for the prevention and / or treatment of cancer and / or for the production of drugs (anticancer drugs) for the prevention and / or treatment of cancer.

[0167] The cancer may be a solid cancer or a blood cancer, and may be, for example, a cancer that has the characteristic of expressing ROR1 and / or B7-H3 (e.g., expressed (or overexpressed) on the cell surface), but is not limited thereto. For example, the cancer may be, but is not limited to, breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, colorectal cancer, colon cancer, colorectal cancer, cervical cancer, endometrial cancer, uterine cancer, kidney cancer, nephroblastoma, skin cancer, oral squamous cell carcinoma, epidermal cancer, nasopharyngeal cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, lymphatic cancer (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma), gastric cancer, pancreatic cancer, testicular cancer, thyroid cancer, follicular thyroid carcinoma, hepatocellular carcinoma, oral cancer, renal cell carcinoma, adrenal cancer, melanoma, bone Examples of tumors include myeloma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, hepatic lobar tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal sarcoma, malignant peripheral nerve sheath tumor (MPNST), Ewing's sarcoma, leiomyosarcoma, hepatic lobar chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratocarcinoma, neuroblastoma, stromal cell carcinoma, glioma, skin tumor, leukemia, Burkitt's lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, etc. Lung cancer may be, for example, small cell lung carcinoma (SCLC) or non-small cell lung carcinoma (NSCLC). Leukemia may be, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), or chronic lymphocytic leukemia (CLL). The cancer may be a primary cancer or a metastatic cancer.

[0168] The anti-cancer activity or cancer prevention and / or treatment effect may mean any anti-cancer and / or anti-tumor effect, such as an effect of removing (destroying) cancer cells, an effect of suppressing the occurrence and / or growth of cancer cells, an effect of suppressing the progression of cancer due to migration, invasion, metastasis, etc., or alleviating or improving symptoms, and / or an effect of partially or completely eliminating cancer.

[0169] The subjects for administration of the anti-ROR1 antibodies, anti-ROR1 / anti-4-1BB bispecific antibodies, anti-ROR1 / anti-B7-H3 bispecific antibodies, antigen-binding fragments thereof, antibody-drug conjugates, and / or pharmaceutical compositions containing the same provided herein may be any animal or cell, for example, an animal selected from mammals including humans, primates such as monkeys, and rodents such as rats and mice, or cells, tissues, body fluids (e.g., serum) derived (isolated) from such animals, or cultures thereof, for example, humans or cells, tissues, body fluids (e.g., serum) isolated from humans.

[0170] The pharmaceutical composition may further comprise, in addition to the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody, or antigen-binding fragment or complex thereof as the active ingredient, a pharmaceutically acceptable excipient and / or carrier, and one or more of the excipients and / or carriers may be appropriately selected by a person of ordinary skill in the art from those typically used in formulating protein drugs.

[0171] The administration of the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody, antigen-binding fragment thereof, conjugate, and / or pharmaceutical composition is via oral or parenteral routes.

[0172] The content of the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody, or antigen-binding fragment or complex thereof, which is the active ingredient in the pharmaceutical composition, may be variously formulated depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration interval, administration route, excretion rate, and reaction sensitivity.

[0173] For more details on excipients and / or carriers, administration routes, dosages, etc. of the pharmaceutical composition, reference may be made to Korean Patent Registration No. 10-2551365, etc.

[0174] Other examples provide nucleic acid molecules encoding anti-ROR1 antibodies, anti-ROR1 / anti-4-1BB bispecific antibodies or anti-ROR1 / anti-B7-H3 bispecific antibodies.

[0175] Another example provides a recombinant vector comprising the nucleic acid molecule. The recombinant vector may be an expression vector for expressing the nucleic acid molecule.

[0176] Another example provides a recombinant cell comprising the recombinant vector.

[0177] When the antibodies or antigen-binding fragments thereof provided herein are recombinantly produced, they may be in a form to which a conventional signal peptide, cleavage site, tag, etc. is attached for purification. Thus, in a non-limiting example, the antibodies or antigen-binding fragments thereof provided herein may further comprise one or more selected from the group consisting of signal peptides, cleavage sites, tags (e.g., His tag, GST (glutathione-S-transferase) tag, MBP (maltose binding protein) tag, etc.) that are commonly used in recombinant protein production processes, or may be in a purified form in which these have been removed.

[0178] The term "vector" refers to a means for expressing a target gene in a host cell. Examples include plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors such as lentivirus vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. Vectors that can be used as the recombinant vector include plasmids commonly used in the art (e.g., pBR series, pUC series, pBluescript II series, pGEM series, pGEX series, pTZ series, pCL, pcDNA series, pET series, etc.; more specifically, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, etc.). , pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pcDNA3.1, pcDNA3.3, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.), but are not limited thereto.

[0179] In the recombinant vector, the nucleic acid molecule is operably linked to a promoter. The term "operably linked" refers to a functional connection between a nucleotide expression control sequence (e.g., a promoter sequence) and another nucleotide sequence. The control sequence is "operably linked" so as to regulate the transcription and / or translation of the other nucleotide sequence.

[0180] The recombinant vector is typically constructed as a vector for cloning or expression. The expression vector may be any vector commonly used in the art for expressing foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed by various methods known in the art.

[0181] The recombinant vector is constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, a strong promoter capable of promoting transcription (e.g., pL λ Typically, the vector contains a promoter (such as a CMV promoter, trp promoter, lac promoter, tac promoter, or T7 promoter), a ribosome binding site for the initiation of transcription, and a transcription / transcription termination sequence. When a eukaryotic cell is used as the host, the replication origin operable in eukaryotic cells contained in the vector includes, but is not limited to, the f1 origin, SV40 origin, pMB1 origin, adenovirus origin, AAV origin, and BBV origin. In addition, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter) are used, and generally contain a polyadenylation sequence as a transcription termination sequence.

[0182] The recombinant cell may be obtained by introducing the recombinant vector into a suitable host cell. The host cell may be any host cell known in the art as a cell capable of stably and continuously cloning or expressing the recombinant vector. Examples of prokaryotic cells include Bacillus strains such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, and Bacillus thuringiensis, as well as enteric bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. When transforming eukaryotic cells, examples of host cells include yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO Cell lines that can be used include, but are not limited to, GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK.

[0183] The nucleic acid molecule or a recombinant vector containing the same can be delivered (introduced) into a host cell using a delivery method well known in the art, such as, but not limited to, the CaCl2 method or electroporation when the host cell is a prokaryotic cell, or microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene bombardment when the host cell is a eukaryotic cell.

[0184] The transformed host cells can be easily selected by a method well known in the art using the phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformants can be easily selected by culturing them in a medium containing the antibiotic.

[0185] Another example provides a method for producing an anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, or anti-ROR1 / anti-B7-H3 bispecific antibody, comprising expressing the nucleic acid molecule in a host cell. Expressing the nucleic acid molecule in a host cell may include culturing cells containing the nucleic acid molecule or a recombinant vector containing the same. The production method may optionally further comprise, after the culturing step, isolating and / or purifying the antibody or antigen-binding fragment from the culture medium. [Effects of the Invention]

[0186] The present disclosure provides improved anti-ROR1 antibodies, bispecific antibodies comprising the ROR1 antibodies and a T cell engager, and / or bispecific antibodies comprising the anti-ROR1 antibodies and an antibody specific for a tumor-associated antigen (TAA; e.g., B7-H3), thereby enabling more effective anti-cancer effects.

[0187] In particular, the present application provides ROR1 antibodies and bispecific antibodies containing the same that have not only excellent binding strength but also improved stability and ease of development, thereby enabling the economical production of antibody pharmaceuticals that are more effective and safer than conventional therapeutic antibodies. [Brief explanation of the drawings]

[0188] [Figures 1A-1C] 1 is a graph showing the binding affinity of an anti-ROR1 antibody to ROR1 according to one example. [Figure 2A-2B] 1 is a graph showing the binding affinity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example to 4-1BB and / or ROR1. [Figure 2C] 1 is a graph showing the binding affinity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example to cells expressing 4-1BB on their surface. [Figure 2D] 1 is a graph showing the binding affinity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example to cells expressing ROR1 on their surface. [Figures 3A-3D] 1 is a graph showing the serum stability of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example (A and B: human serum; C and D: monkey serum). [Figure 4A] 1 is a graph showing activation of 4-1BB signaling in the presence of ROR1 by an anti-ROR1 / anti-4-1BB bispecific antibody according to one example. [Figure 4B] 1 is a graph showing ROR1-dependent 4-1BB signaling activation by an anti-ROR1 / anti-4-1BB bispecific antibody according to one example. [Figure 4C] 1 is a graph showing the correlation between anti-ROR1 / anti-4-1BB bispecific antibody-dependent 4-1BB signaling activation and ROR1 expression according to one example. [Figure 4D] 1 is a graph showing FcγRI-dependent 4-1BB signaling activation by an anti-ROR1 / anti-4-1BB bispecific antibody according to one example. [Figure 5A] 1 is a graph showing the PBMC activation and cytokine (interferon-gamma) release-inducing activity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example in the presence of ROR1-overexpressing CHOK1 cells. [Figure 5B] 1 is a graph showing the PBMC activation and cytokine (interferon-gamma) release-inducing activity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example in the presence of gastric cancer cell line cells expressing ROR1. [Figure 5C] 1 is a graph showing the PBMC activation and cytokine (interferon-gamma) release-inducing activity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example in the presence of gastric cancer cell line cells expressing ROR1. [Figures 6A-6B] 1 is a graph showing the anti-tumor effect (in vivo) of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example. [Figure 6C]1 is a graph showing the long-lasting antitumor memory response of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example. [Figure 6D] 1 is a graph showing the results of measuring the immunomodulatory activity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example in tumor, blood, and liver. [Figure 6E] 1 is a graph showing the results of measuring the immunomodulatory activity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one example using non-ROR1-expressing PBMCs. [Figures 7A-7D] 1 is a graph showing the binding affinity of an anti-ROR1 / anti-B7-H3 bispecific antibody according to one example to ROR1 or B7-H3. [Figure 8] 1 is a graph showing the binding affinity of an anti-ROR1 / anti-B7-H3 bispecific antibody according to one example to cells expressing ROR1 and B7-H3 on the cell surface. [Figure 9] 1 is a graph showing the degree of cellular internalization of an anti-ROR1 / anti-B7-H3 bispecific antibody according to an example. [Figures 10A-10C] 10A to 10C are graphs showing the results of measuring the purity of an antibody-drug conjugate (ADC) containing an anti-ROR1 / anti-B7-H3 bispecific antibody according to one example by size-exclusion high-performance liquid chromatography (SE-HPLC). The peak values (min.) shown in Figures 10A to 10C are as follows: 10A: 12.441, 14.677; 10B: 13.564, 16.170; 10C: 13.752, 16.314). [Figure 11] 1 is a graph showing the results of measuring the drug-to-antibody ratio (DAR) in an ADC comprising an anti-ROR1 / anti-B7-H3 bispecific antibody according to one example, using liquid chromatography mass spectrometry (LC / MS). [Figure 12] 1 is a graph showing the antigen (ROR1) binding ability of an ADC comprising an anti-ROR1 / anti-B7-H3 bispecific antibody according to one example. [Figure 13]1 is a graph showing the cytotoxicity of an ADC comprising an anti-ROR1 / anti-B7-H3 bispecific antibody according to one example against cancer cells expressing ROR1 and B7-H3. MODE FOR CARRYING OUT THE INVENTION

[0189] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[0190] Example 1. Anti-ROR1 antibody 1.1. Production of Improved Anti-ROR1 Antibodies Posttranslational modification (PTM) sites were identified through peptide mapping (Accurate Determination of Succinimide Degradation Products Using High Fidelity Trypsin Digestion Peptide Map Analysis, Anal. Chem. 2011, 83(15):5912-5919) from the anti-ROR1 antibody clone sequence disclosed in International Publication No. WO2019-225992A1, and mutations to remove these sites were introduced to produce antibodies with improved properties, including physical properties and developability. Among the anti-ROR1 antibodies disclosed in WO2019-225992A1, the BA6 antibody (WT) (IgG1-based) was selected as the antibody for which PTM sites were to be removed. The heavy chain CDR1 (HCDR1), CDR2 (HCDR2), and CDR3 (HCDR3), and the light chain CDR1 (LCDR1), CDR2 (LCDR2), and CDR3 (LCDR3) of the BA6 antibody are summarized in Table 6 below.

[0191] [Table 6]

[0192] Peptide mapping confirmed PTM in LCDR2 and HCDR3. To identify the hotspots within the BA6 antibody sequence where PTM actually occurs, peptide mapping was performed using the BA6 antibody. After storing the antibody at 4°C and 37°C for two weeks, each sample was digested and fragmented with IdeS (Immunoglobulin G-degrading enzyme of Streptococcus pyogenes; Genovis) and DTT (Dithiothreitol). Analysis by LC / MS confirmed that PTM actually occurred in CDRL2 and CDRH3. Because there were two PTM hotspots, the two groups were divided into two groups and single mutations were induced. If the binding activity was confirmed to be similar to that of the wild-type antibody, multiple mutations were combined to create double or triple mutants.

[0193] A total of 10 PTM mutants were constructed and their ligand binding activity was confirmed. Specifically, to obtain the IgG of each anti-ROR1 antibody clone with PTM-removing mutations, we first synthesized a mutagenesis primer set (Macrogen, Korea) to introduce mutation sequences into the PTM hotspots CDRL2 and CDRH3 of the clone. We then performed site-directed mutagenesis using PCR to obtain the nucleic acid sequences encoding the light and heavy chains.

[0194] For transfection, 6.0 × 10 ExpiCHO-S cells (Thermo Fisher) were placed in a 500 mL flask. 6The heavy and light chain expression vectors (the light and heavy chains were cloned into separate pcDNA 3.4 vectors (Thermo Fisher) for plasmid preparation before transfection) and ExpiFectamine (Thermo Fisher) were diluted to 1 μg / mL and 3.2 μL / mL, respectively, in OptiPRO medium (Thermo Fisher). The diluted vector and ExpiFectamine mixture was mixed and incubated at room temperature for 5 minutes. The mixture was then added to the flask containing the cells, shaken vigorously, and cultured at 8% CO2, 37°C, and 120 rpm. The day after transfection, 600 μL of enhancer (Thermo Fisher) and 24 mL of feed (Thermo Fisher) were added, and the cells were cultured for 7 days at 8% CO2, 37°C, and 120 rpm. For detailed experimental procedures, please refer to the manufacturer's guide for the ExpiCHO expression system provided by Thermo Fisher.

[0195] A total of 10 mutants (M1 to M10) were obtained in this manner and used as candidate clones for anti-ROR1 antibodies. Each of these clones was confirmed to contain a mutation in CDRL2 or CDRH3, where the PTM site was identified. M1 to M4 clones contained a mutation at L51 in CDRL2, M5 to M10 clones contained a mutation at CDRH3, and M11 contained the M3 mutation (at L51 in CDRL2) and M9 mutation (at H97 in CDRH3), which were selected as lead candidate clones among M1 to M10. Specific information about these candidate clones is summarized in Table 7 below.

[0196] [Table 7] (deD:Asp Deamidation, deN:Asn Deamidation, isoD:Aspartate Isomerization; Pass: Evaluated based on ligand (antigen) binding activity

[0197] 1.2. Analysis of binding specificity of candidate clones to the extracellular domain of ROR1 (ELISA) Because the PTM hotspot of the BA6 antibody was confirmed to be located within the CDR region, even if the PTM was removed, if altering the CDR sequence resulted in a decrease in antigen-binding activity compared to the WT (wild-type, BA6 antibody), it could result in a decrease in antibody potency. Therefore, the ultimate goal of PTM engineering is to find clones with binding activity equal to or greater than that of the WT. Therefore, the specific binding ability of the candidate clones M1 to M11 prepared in Example 1.1 to the ROR1 antigen was analyzed as follows.

[0198] Anti-ROR1 antibody-antigen binding affinity was assessed using an ELISA-based solution binding assay. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with 1 μg / ml ROR1 protein (ROR1-His; Sino Biological, Catalog #13968-H08H) in PBS at 4°C for 16 hours, and then treated with 1% (v / v) bovine serum albumin (BSA) for 2 hours to block nonspecific binding sites.

[0199] Anti-ROR1 antibodies were then added to a 96-well microtiter plate at the concentrations shown in Figures 1A-1C, and their binding activity was analyzed by ELISA as follows. Specifically, after incubation at 37°C for 2 hours, the plate was washed five times with PBS containing 0.05% (v / v) Tween 20. HRP-conjugated Fab multiclonal antibody reagent (Pierce, Catalog #31414) was diluted 1:30,000 and added to the washed microtiter plate. The plate was incubated at 37°C for 1 hour to detect plate-bound anti-ROR1 antibodies. After incubation, color development was performed using TMB (Tetramethylbenzidine, Sigma, T0440). The enzyme reaction was terminated with 0.5 mol / L sulfuric acid, and the absorbance was measured at 450 nm and 650 nm using a microplate reader (Molecular Device).

[0200] The results are shown in Figures 1A-1C. As shown in Figures 1A-1C, the ELISA analysis results showed that M1 and M3 exhibited similar ligand (antigen) binding activity compared to WT. However, M1 was excluded from final clone selection because its saturation point was lower than that of WT. Furthermore, among M7, M8, and M10, no clones exhibited comparable binding activity compared to WT. M3 and M9 exhibited comparable binding activity compared to WT. Therefore, the M11 clone (D51E(LC) + N97S(HC)), a double mutant combining M3 and M9, was produced using the same method as in the previous example. The M11 clone thus produced was confirmed to exhibit similar binding activity compared to WT and M3, and was selected as the final candidate clone.

[0201] 1.3. Developability analysis of candidate clones The BA6M11 (also referred to as BA6M11) clone (PTM-engineered to remove PTM liability from BA6), whose efficacy was confirmed in Example 1.2, was compared with BA6 to determine whether its stability under stressed conditions was increased. More specifically, the BA6 and BA6M11 antibodies were stored at 4°C (normal conditions) and 37°C (stress conditions) for two weeks, and then SE-HPLC, icIEF, and LC / MS analyses were performed on each sample to confirm purity and impurity content. Furthermore, to determine whether changes in substance content affect efficacy, an antigen-antibody binding assay (ELISA; see Example 1.2) was performed.

[0202] The results obtained are shown in Table 8 below:

[0203] [Table 8] (*N / A:Not applicable;**N / D:Not detected)

[0204] As shown in Table 8, the BA6M11 clone maintained high levels of antibody purity, comparable to that of BA6, when stored at 4°C and 37°C. Furthermore, compared to BA6, the BA6M11 clone showed significantly reduced charge variant formation during the production process under both basic and acidic conditions, and the formation of charge variants under harsh conditions was also significantly reduced (ΔMajor%). Peptide mapping analysis of the BA6M11 clone confirmed the elimination of succinimide formation at problematic residues, confirming that this change resulted in a reduction in charge variants. The reduction in charge variants reduces the risk of antibody quality fluctuations during production, storage, and distribution and enhances the reliability of the purification process using ion exchange resins. It also reduces the risk of increased immunogenicity during human administration, preventing the hydrolysis of the succinimide intermediate, which can lead to antibody chain cleavage and reduced titer, resulting in increased immunogenicity. Furthermore, when the antigen-antibody binding ability was examined using human ROR1 recombinant protein (ROR1-His; sinobiological, Catalog #13968-H08H) (ELISA; Protein Binding), it was confirmed that there was no significant difference in binding ability between samples stored for two weeks at 4°C and 37°C. These results confirmed that BA6M11 maintained its potency compared to BA6, and that its physical properties were improved in terms of development potential.

[0205] Finally, we confirmed that the BA6M11 clone not only exhibited comparable binding to WT but also had clearly improved development potential through PTM engineering.

[0206] The amino acid sequence of the thus prepared novel anti-ROR1 antibody BA6M11 in IgG form is listed in Tables 9 and 10:

[0207] [Table 9] TIFF2025525538000012.tif47170

[0208] [Table 10]

[0209] Example 2. Anti-ROR1 / anti-4-1BB bispecific antibody 2.1. Production of Improved Anti-ROR1 / Anti-4-1BB Bispecific Antibodies An anti-ROR1 / 4-1BB bispecific antibody was prepared based on BA6M11, an improved anti-ROR1 antibody clone prepared and selected in Example 1, and 41B01.03 (also referred to as 1A10M12), one of the anti-4-1BB antibody clones disclosed in International Publication No. WO2020 / 111913A1.

[0210] In this example, a bispecific antibody comprising an anti-ROR1 IgG (full length) antibody and 4-1BB scFv linked to the C-terminus of the heavy chain of the anti-ROR1 IgG (full length) antibody was prepared and used.

[0211] To construct the anti-ROR1 / anti-4-1BB bispecific antibody, DNA segment 1, containing the nucleotide sequence encoding the heavy chain of the anti-ROR1 IgG antibody (BA6M11), was inserted into pcDNA 3.4 (Invitrogen, A14697; Plasmid 1), and DNA segment 2, containing the nucleotide sequence encoding the light chain of the IgG antibody of the anti-ROR1 / anti-4-1BB bispecific antibody, was inserted into pcDNA 3.4 (Invitrogen, A14697; Plasmid 2). DNA segment 3, encoding the scFv, was then fused to the portion of DNA segment 1 corresponding to the C-terminus of the Fc region of the IgG antibody inserted into Plasmid 1 using DNA segment 4, encoding a 20-amino acid peptide linker composed of (GGGGS)4 (SEQ ID NO: 114), or DNA segment 5, encoding an 18-amino acid linker peptide composed of (GS)9 (SEQ ID NO: 113), to construct a vector for expressing the bispecific antibody.

[0212] The anti-4-1BB scFv (designated 1A10M12) was constructed by fusing VL103-VH44 (VL103: a VL with a G (Gly) to C (cys) mutation at position 103; VH44: a VH with a G to C mutation at position 44) to the C-terminus of the light chain variable region (VL) and the C-terminus of the heavy chain variable region (VH) via a linker ((GGGGS)4 (SEQ ID NO: 114)). These mutations stabilize the scFv and enable the formation of disulfide bridges.

[0213] The amino acid sequences of the bispecific antibodies thus prepared are listed in Table 11 below.

[0214] [Table 11] TIFF2025525538000015.tif155170 (Linker1: BA6M11 (IgG) and 1A10M12 (scFv) linked (the C-terminus of the heavy chain of BA6M11 and the N-terminus of the VL of 1A10M12 linked); Linker 2: VL and VH of 1A10M12 (scFv) linked (C-terminus of VL linked to N-terminus of VH)

[0215] The coding nucleic acid sequence (5'→3') of the bispecific antibody is as follows: [Heavy chain_BA6M11(WT)x1A10M12] (SEQ ID NO: 118) TIFF2025525538000016.tif161170

[0216] [Light chain_BA6M11] (SEQ ID NO: 119) TIFF2025525538000017.tif50170

[0217] The DNA encoding the cloned bispecific antibodies was expressed using the ExpiCHO system (Thermo Fisher) according to the manufacturer's protocol. The culture medium in which the antibodies were expressed was centrifuged and filtered to remove the supernatant, and the antibody was then purified using Mabselect Sure. TM The antibody was purified using affinity chromatography using resin and size exclusion chromatography using Superdex 200 resin. The purity of the purified antibody was analyzed by HPLC using TSKgel SuperSW3000, and was excellent, exceeding 95%.

[0218] 2.2. Evaluation of anti-ROR1 / anti-4-1BB bispecific antibodies 2.2.1. ELISA-based Solution Binding Assay Using Double Antigens (Dual Antigen Capture ELISA, DACE) The binding avidity of anti-ROR1 / anti-4-1BB bispecific antibodies containing the BA6M11 clone (PTM-removed clone, test group) or the BA6 clone (parental clone, control group) to ROR1 and 4-1BB was compared and analyzed by ELISA. Two versions of the BA6M11 clone used as the test group were BA6M11(NA), which contains the N297A (NA) mutation that reduces ADCC, and BA6M11(WT), which does not contain such a mutation.

[0219] The anti-ROR1 / anti-4-1BB bispecific antibody sequence containing the BA6 clone used as a control is as follows: Light chain BA6 (SEQ ID NO: 124) QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYDNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS Heavy chain BA6x1A10M12 (SEQ ID NO: 125) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS Briefly, a 96-well microtiter plate (NUNC, 446612) was coated with 10 ng / well of human ROR1-Fc protein (Sinobio, 13968-H02H1) in PBS overnight at 4°C. The plate was then incubated with 200 μl / well of 1% BSA buffer (1x PBS, 1% (v / v) BSA) at 37°C for 2 hours to block nonspecific binding. After blocking nonspecific binding, the plate contents were shaken off, and 4-fold dilutions (starting at 100 nM) of each test substance (bispecific antibody) were added to each well. The plate was then incubated at 37°C for 2 hours and then washed with PBS-T (1x PBS / 0.05% (v / v) Tween-20). Human 4-1BB-His protein (Sinobio, 10041-H08H) dissolved in 1% (v / v) BSA was then added to each well at 100 ng / well and incubated at 37°C for 1 hour. The plate was washed with PBS-T and then incubated with Anti-His HRP (Roche, Cat: 11965085001) at 37°C for 1 hour. After washing, the plate was developed with TMB (Tetramethylbenzidine, Sigma, T0440). The color reaction was stopped with 0.5 M sulfuric acid (SAMCHUN Pure Chemical, S1410). The plate was then analyzed by measuring the absorbance (OD) at 450-650 nm using a spectrophotometer. Four-parameter logistic curve analysis was performed using GraphPad software.

[0220] The results obtained through the above experiments are shown in Table 12 and Figures 2A and 2B.

[0221] [Table 12]

[0222] As shown in FIG. 2A and Table 12, the bispecific antibody comprising BA6M11 did not exhibit reduced binding activity compared to the bispecific antibody comprising its parent clone, BA6; rather, the measured EC50 was lower, confirming that the binding activity was improved.

[0223] Furthermore, when the presence or absence of a difference in binding ability depending on whether or not the NA mutation was present was measured in Figure 2B, it was shown that there was no difference in binding activity between BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 for human ROR1 and 4-1BB.

[0224] Considering that it is difficult to maintain the binding ability of the parent clone when the CDR sequence is altered from that of the parent clone, the result that the antigen binding ability of BA6M11, whose sequence was altered by removing post-translational modifications (PTMs), was equal to or greater than that of the parent clone BA6, indicates the favorable properties of BA6M11 and the potential and advantages of its future development as a therapeutic antibody.

[0225] 2.2.2. Cell surface antigen binding test (flow cytometry) Using various cell lines expressing human ROR1 or human 4-1BB on the cell surface, the binding affinities of anti-ROR1 / anti-4-1BB bispecific antibodies, including the BA6M11(WT) or BA6M11(NA) clones, were measured and compared using a flow cytometry system (BD).

[0226] Briefly, Jurkat cells (Promega) overexpressing human 4-1BB and NCI-N87 cells (ATCC), a human ROR1-positive cell line, were placed in a V-bottom 96-well plate (96 Well Plate-RV, Bioneer, 910D09) and washed with 200 μl / well of 1% BSA buffer. Four-fold dilutions (starting at 100 nM) of each test substance (bispecific antibody) were added to each well and incubated at 4°C for 1 hour. The plate was then washed with 1% BSA buffer. FITC-conjugated anti-human IgG (Fc specific) (Sigma, F9512) in 1% BSA buffer was added to each well and incubated at 4°C for 1 hour. The plate was washed with 1% BSA buffer. The mean fluorescence intensity (MFI) of FITC was measured by flow cytometry. The obtained MFI values were subjected to four-parameter logistic curve analysis using GraphPad software.

[0227] As shown in Figures 2C and 2D, both BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 bound to Jurkat cells overexpressing human 4-1BB and ROR1-positive NCI-N87 cells at similar levels, confirming that there was no difference in their binding activity to human 4-1BB and human ROR1.

[0228] 2.2.3. Binding affinity testing using surface plasmon resonance (SPR) The binding affinities of the anti-ROR1 / anti-4-1BB doublets, including the BA6M11 clone, BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 clones, were measured by surface plasmon resonance. The antigens used were human ROR1 protein 13968-H02H1 (Fc tag) from Sino biological company and human 4-1BB protein 9220-4B-100 (His tag) from R&D systems. Binding affinity measurements were performed using a Biacore T200 (Cytiva).

[0229] The binding affinity of candidate compounds to human ROR1 was measured as follows. Approximately 80 RU of 5 μg / mL human ROR1 protein diluted with Acetate 4.0 (Cytiva, BR100349) was immobilized on a CM5chip (Cytiva, BR100530) using the amine coupling method (Cytiva, BR100050). BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 were serially diluted 2-fold in HBS-EP (1x) (Cytiva, BR100669) buffer to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 0 nM. The diluted antibody sample was flowed over a CM5 chip with immobilized human ROR1 protein (antigen) for approximately 60 seconds to allow the sample to bind to the antigen. Then, HBS-EP (1x) buffer was flowed over the sample-antigen complex for 180 seconds to dissociate the sample from the antigen. The chip surface was regenerated by flowing Glycine-HCl pH 1.5 (Cytiva, BR100354) for 30 seconds. Analysis was performed using HBS-EP (1x) as the running buffer at a flow rate of 30 μL / min and a temperature of 25°C. The binding affinity was calculated using a bivalent analyte model to calculate the first-order binding rate constant (Ka1) and the first-order dissociation rate constant (Kd1). The equilibrium dissociation constant (KD) was calculated by dividing the first-order dissociation rate constant by the first-order binding rate constant, and this was used as the final binding affinity.

[0230] The binding affinity of candidate substances for human 4-1BB was measured as follows. Anti-Fc antibody (Invitrogen, 31125) diluted at 10 μg / mL with Acetate 5.0 (Cytiva, BR100351) in CM5 (Cytiva, BR100530) was immobilized at approximately 15,000 RU using the amine coupling method. BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 diluted in HBS-EP (1x) buffer were coupled to the anti-Fc antibody-immobilized CM5 chip at approximately 300 RU. Human 4-1BB antigen was serially diluted in HBS-EP (1x) buffer at 2-fold dilutions to 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, 7.8125 nM, and 0 nM. Diluted human 4-1BB was flowed onto a CM5 chip for approximately 60 seconds to allow binding to the sample. Then, HBS-EP (1x) buffer was flowed over the sample (antibody)-antigen complex for 180 seconds to dissociate the antigen from the sample. The chip surface was regenerated by flowing Glycine-HCl pH 1.5 (Cytiva, BR100354) for 30 seconds. Analysis was performed using HBS-EP (1x) as the running buffer at a flow rate of 30 μL / min and a temperature of 25°C. The binding affinity was calculated using a 1:1 binding model to calculate the association rate constant (Ka) and dissociation rate constant (Kd). The equilibrium dissociation constant (KD) was calculated by dividing the dissociation rate constant by the association rate constant, and this was used as the final binding affinity.

[0231] The results obtained are shown in Table 13:

[0232] [Table 13]

[0233] As shown in Table 13, both BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 bound to human ROR1 and human 4-1BB at similar levels, confirming that there was no difference in antigen binding activity between the WT and NA cell lines.

[0234] 2.2.4. Developability Analysis of Anti-ROR1 / 4-1BB Bispecific Antibodies The possibility of developing an anti-ROR1 / 4-1BB bispecific antibody containing the BA6 clone or the BA6M11 clone was analyzed.

[0235] To analyze the development potential, we evaluated capillary electric focusing (cIEF) and imaged capillary electric focusing (icIEF). Both analytical methods use isoelectric point (pI) values to analyze changes in charge variants.

[0236] Samples for developability analysis were prepared by exposure to temperatures of 4°C and 37°C (or 40°C) for 2 weeks. Storage temperatures were 4°C or 37°C for the BA6x1A10M12 clone, and 4°C or 40°C for BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12.

[0237] cIEF analysis was performed using a Beckman Coulter PA800plus instrument equipped with a UV detector. A capillary cartridge (neutral capillary 50 μm I.D.) was fabricated to a total length of 30.2 cm and attached to the instrument.

[0238] The bispecific antibody samples used in the analysis were diluted with formulation buffer (20 mM histidine, 7% (w / v) trehalose, pH 6.0) to a final concentration of 5 mg / mL. The master mix solution was prepared using 3 M urea gel (Urea: GE Healthcare, 17-1319-01; cIEF Gel: Beckman Coulter, 477497), cathodic electrolyte (Ampholyte 3-10) (Pharmalyte 3-10; GE Healthcare, 17-0456-01), anodic stabilizer solution (200 mM iminodiacetic acid; Sigma, 220000), cathodic stabilizer solution (500 mM L-arginine; Sigma, A5006), and synthetic peptide pI markers (10, 9.5, 7.0, 5.5, 4.1; cIEF Peptide Marker Kit; Beckman Coulter, A58481). Ten μL of the sample (bispecific antibody) was mixed with 239 μL of the master mix solution and centrifuged. 200 μL of this mixture was transferred to a PCR tube and placed in a plastic vial for preparation in a Sampletray. A buffer tray was prepared for analysis by placing distilled water, anolyte solution (200 mM phosphoric acid; Sigma, 345-245), catholyte solution (300 mM sodium hydroxide; Sigma, 79724), capillary cleaning solution (4.3 M urea; GE Healthcare, 17-1319-01), cIEF gel solution (Beckman Coulter, 477497), and chemical mobilizer solution (350 mM acetic acid; Sigma, 695092) in individual plastic vials.

[0239] For icIEF analysis, we used a Maurice cIEF cartridge (Proteinsimple, PS-MC02-C) with a capillary cartridge. 2 mL each of the anode solution (Catholyte solution, 100 mM sodium hydroxide in 0.1% methyl cellulose; Proteininsimple) and cathode solution (Anolyte solution, 0.08 M phosphoric acid in 0.1% methyl cellulose; Proteininsimple) was added to the cartridge and then inserted into the instrument.

[0240] The bispecific antibody samples used in the analysis were diluted with distilled water to a final concentration of 1 mg / mL. The master mix solution consisted of 8 M urea solution (Sigma, U6504), 1% methyl cellulose (Proteinsimple), a positive electrolyte (Ampholyte, pH 3-10) (Pharmalyte pH 3-10; Proteinsimple, 042-684 or GE Healthcare, 17-0456-01), and distilled water (UltraPure TM The assay was prepared using DNase / RNase-free distilled water (Invitrogen, 10977015) and peptide isoelectric point markers (5.85, 9.50; Proteinsimple). 20 μL of the assay sample (bispecific antibody) was mixed with 80 μL of the master mix solution, centrifuged, and transferred to a 96-well plate.

[0241] Distilled water, 0.5% methyl cellulose (Proteinsimple), fluorescence calibration standard (Proteinsimple), and air were placed in individual vials and prepared at each vial position for analysis.

[0242] The analytical results are shown in Table 14 below.

[0243] [Table 14]

[0244] As a result of the developability evaluation shown in Table 14, the main peak content of BA6x1A10M12 was about 52% when stored at 4°C. When stored at 37°C compared to 4°C, the main peak content of this clone decreased by about 17% and the basic peak content increased by about 14%.

[0245] The results of the developability of two BA6M11 clones, which were mutated from the above BA6 clone, were as follows: the main peak content was approximately 75% for BA6M11(WT)x1A10M12 and approximately 78% for BA6M11(NA)x1A10M12 at 4°C. The main peak content of the two clones decreased by approximately 16-17% when stored at 40°C compared to 4°C, and the basic peak content increased by approximately 2-3%.

[0246] The tested BA6M11 clone showed improved content characteristics of basic charge variants compared to BA6. Analysis of samples stored at 4°C confirmed that the content of basic charge variants decreased during production, and the rate of change was also reduced under high-temperature (40°C) storage conditions. The two mutant clones showed similar main peak content in the WT and NA clones based on samples stored at 4°C. This confirms that the BA6M11 clone has superior development potential compared to its parent clone, BA6.

[0247] 2.2.5. Serum stability analysis of anti-ROR1 / anti-4-1BB bispecific antibody To evaluate the serum stability of the anti-ROR1 / anti-4-1BB bispecific antibody containing the BA6 clone (parental clone) or the BA6M11 clone (PTMs-removed clone), the antibody was stored in human serum and monkey serum, and then analyzed and compared by ELISA.

[0248] In this analysis, the anti-ROR1 / anti-4-1BB bispecific antibody was stored in human and monkey serum at 37°C for 2, 7, and 14 days.

[0249] A 96-well microtiter plate (NUNC, 446612) was coated with 100 ng / well of human ROR1-Fc protein (Sinobio, 13968-H02H1) in PBS overnight at 4°C. The plate was incubated with 200 μl / well of 1% BSA buffer (1× PBS, 1% (v / v) BSA) at 37°C for 2 hours to block nonspecific binding. After blocking nonspecific binding, the plate contents were shaken off, and 4-fold dilutions (starting at 100 nM) of each test substance (bispecific antibody) were added to each well. The plate was incubated at 37°C for 2 hours and then washed with PBS-T (1× PBS / 0.05% (v / v) Tween-20). Human 4-1BB-His protein (Sinobio, 10041-H08H) dissolved in 1% BSA buffer was then added to each well at 100 ng / well and incubated at 37°C for 1 hour. The plate was washed with PBS-T and then incubated with Anti-His HRP (Roche, Cat: 11965085001) at 37°C for 1 hour. After washing, color development was performed using TMB (Tetramethylbenzidine, Sigma, T0440). The color reaction was stopped with 0.5 M sulfuric acid (SAMCHUN Pure Chemical, S1410). The plate was analyzed by measuring the optical density (OD) at 450-650 nm using a spectrophotometer. Four-parameter logistic curve analysis was performed using GraphPad software.

[0250] The results obtained are shown in Table 15 and Figures 3A to 3D.

[0251] [Table 15]

[0252] As shown in Table 15 and Figures 3A to 3D, the anti-ROR1 / anti-4-1BB bispecific antibody containing BA6M11 maintained stability for a longer period in human and monkey serum than the bispecific antibody containing its parent clone, BA6, demonstrating improved serum stability. Serum stability is a key physical property to consider in antibody development and production because it has a significant impact on the efficacy of antibody pharmaceuticals upon infusion, particularly binding strength and specificity. Therefore, these results confirm that BA6M11 significantly contributes to antibody serum stability compared to BA6.

[0253] 2.2.6. 4-1BB signal activation of anti-ROR1 / anti-4-1BB bispecific antibody (4-1BB reporter bioanalysis) 2.2.6.1.4-1BB signal activation depending on the presence or absence of ROR1 expression To measure, compare, and analyze 4-1BB signal activation of the anti-ROR1 / 4-1BB bispecific antibody, including the BA6 clone or BA6M11 clone, we performed a 4-1BB NFκB luciferase reporter bioassay. For the 4-1BB reporter bioassay, we used the human ROR1-positive gastric cancer cell line AGS and the hamster ovary epithelial cell line CHOK1 stably overexpressing human ROR1 (CHOK1-hROR1).

[0254] In this assay, GloResponse, a gene expression vector genetically engineered to stably express human 4-1BB and luciferase downstream of a response element, was used. TM The NFκB-luc2 / 4-1BB Jurkat cell line (Promega, cat#CS196004) was used as effector cells.

[0255] In summary, AGS (ROR1-positive gastric cancer cell line, ATCC, CRL-1739, 2.5 × 10 4 , or CHOK1-hROR1 (ROR1-overexpressing hamster ovary epithelial cells, BPS Bioscience, 79609-H, 2.5 × 10 4 ) were dispensed into a white 96-well assay plate containing 100 μl of culture medium (10% (v / s) FBS, RPMI 1640) and cultured overnight at 37°C in a 5% CO2 humidified incubator. After overnight culture, 100 μl of culture medium was removed, and 25 μl of assay medium (1% (v / v) FBS, RPMI 1640) was dispensed onto pre-attached target cells (AGS or CHOK1-hROR1) as described above. 25 μl of bispecific antibody (5-fold dilutions starting from 40 nM) was added to the plate. 25 μl of GLORESPONSE suspended in assay medium was added. TM NFκB-luc2 / 4-1BB Jurkat cell line (Promega, CS196004) at 2.5 × 10 cells per well 4 The cells were plated onto plates at 37°C in a 5% CO2 humidified incubator for 6 hours. TM Bio-Glo Luciferase Assay System (Promega, G7940) was used according to the manufacturer's instructions. TM The reagent was reconstituted. After a 6-hour incubation, 75 μl of Bio-Glo per well was added to the assay plate. TM The reagent was added. After waiting for 5 minutes, luminescence was measured using a microplate reader. A four-parameter logistic curve was calculated using GraphPad software. The experimental results using AGS and CHOK1-hROR1 cells are shown in Table 16 and Figure 4A, respectively.

[0256] [Table 16]

[0257] As shown in Table 16 and Figure 4A, the anti-ROR1 / 4-1BB bispecific antibody containing BA6M11 exhibited a lower EC50 (nM) value in ROR1-expressing cells compared to the bispecific antibody containing its parent clone, BA6, indicating that the anti-ROR1 / 4-1BB bispecific antibody containing BA6M11 induced stronger 4-1BB signal activation in the presence of ROR1 antigen. In particular, the anti-ROR1 / 4-1BB bispecific antibody containing BA6M11 exhibited a superior 4-1BB signal activation effect in both ROR1-positive tumor cells (AGS) and human ROR1-overexpressing cells (CHOK1-hROR1), and these results were observed regardless of the presence or absence of NA mutations.

[0258] This indicates that anti-ROR1 / 4-1BB bispecific antibodies, including BA6M11, act specifically on ROR1-expressing cancer cells.

[0259] To further verify that the 4-1BB signaling mediated by BA6M11(NA)x1A10M12 and BA6M11(WT)x1A10M12 is dependent on ROR1 expression, we measured 4-1BB signaling activity using a mixture of parental CHOK1 cells and CHOK1 cells overexpressing human ROR1 (CHOK1-hROR1) as described above. For comparison, we used the anti-4-1BB antibody urelumab (BMS-663513).

[0260] The results are shown in Table 17 and Figure 4B.

[0261] [Table 17]

[0262] As shown in Figure 4B, both BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 showed higher Maxfold induction as the proportion of CHOK1-ROR1 cells increased. This indicates that the greater the number of ROR1-expressing cells, the greater the 4-1BB activation induced. As shown in Figure 4B and Table 17, BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 induced 4-1BB activation at similar levels.

[0263] 2.2.6.2. 4-1BB signal activation of anti-ROR1 / anti-4-1BB bispecific antibodies according to ROR1 expression levels ROR1 cell surface expression levels in various cancer cell lines were quantified using the QIFIKIT quantification kit (Dako, K0078) according to the manufacturer's recommendations. Briefly, various cancer cells used in the study were stained with unlabeled anti-ROR1 mouse monoclonal antibody (BD, 564464) or purified mouse IgG2b isotype control antibody (BD, 555740) at saturating concentrations. After washing, the stained cells and calibration beads were simultaneously labeled with FITC-conjugated goat-anti-mouse IgG secondary antibody. The calibration beads and secondary antibody were included in the kit. The labeled cells and calibration beads were analyzed by flow cytometry. Linear regression analysis was performed using the MFI values of the calibration beads. The antibody binding capacity (ABC) of the anti-ROR1 antibody and the isotype control antibody was calculated using the regression line derived by linear regression analysis, and the sABC (specific ABC) value was obtained by subtracting the value of the isotype control antibody from the antigen binding capacity (ABC) value of the anti-ROR1 antibody.

[0264] The results obtained are shown in Table 18.

[0265] [Table 18]

[0266] The correlation between the obtained ROR1 expression level (ROR1 sABC) and antibody 4-1BB-induced NF-kB signaling was examined.

[0267] The ROR1 levels measured in Table 18 were normalized to the ROR1 expression level in NCI-N87, and the 4-1BB activation levels by the bispecific antibodies BA6M11(WT)x1A10M12, BA6M11(NA)x1A10M12, and the control antibody urelumab were determined as the maximum fold change compared to the control group in the 4-1BB NF-kB luciferase reporter assay. The method used was the same as in Example 2.2.6.1. The common area indicates the confidence interval for the linear fit.

[0268] The results are shown in Figure 4C. As shown in Figure 4C, compared with the control antibody, 4-1BB activation by the anti-ROR1 / 4-1BB bispecific antibodies BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 showed a high correlation with ROR1 cell surface expression.

[0269] 2.2.6.3.4-1BB signal activation by FcγRI engagement CHO-K1 cells (Promega) expressing FcγRI, FcγRIIb, or FcγRIIIa were used to assess the 4-1BB activation signal. The method used was similar to that described in Example 2.2.6.1 above. Four-parameter logistic curve analysis was performed using GraphPad Prism® software.

[0270] As shown in Figure 4D, only CHO-K1 cells overexpressing FcγRI induced 4-1BB signaling from BA6M11(WT)x1A10M12. This suggests that BA6M11(NA)x1A10M12, which has an NA mutation in the Fc region, binds weakly to FcγRI and fails to induce 4-1BB signaling. However, BA6M11(WT)x1A10M12, which has no mutation in the Fc region, induced strong 4-1BB signaling in the presence of FcγRI. CHO-K1 cells overexpressing FcγRIIb or FcγRIIIa, which bind weaker to the Fc region than FcγRI, did not induce 4-1BB signaling.

[0271] 2.2.7. T Cell Immune Response Induction and Cytokine Secretion Activity of Anti-ROR1 / Anti-4-1BB Bispecific Antibody Against ROR1-Expressing Cells (In Vitro Efficacy Test) 2.2.7.1. PBMC activation test using CHOK1 cells artificially overexpressing ROR1 Anti-ROR1 / 4-1BB bispecific antibodies, including the BA6 clone or BA6M11 clone, were co-cultured with hamster ovary epithelial cells stably expressing human ROR1 (CHOK1-hROR1) to assess whether the 4-1BB signal activation of these bispecific antibodies could activate human peripheral blood mononuclear cells (PBMCs). Interferon gamma (IFN-gamma), a cytokine primarily produced by natural killer (NK) cells, CD4+ accessory T cells (Th1), and CD8+ cytotoxic T cells, was measured to assess PBMC activation.

[0272] Briefly, 96-well plates were coated with 5 μg / mL anti-human CD3 (Biolegend, 300438 in PBS) for 2 hours at 37°C. After removing the anti-human CD3 solution, 3 × 10 human PBMCs (Peripheral Blood Mononuclear Cells; CTL-UP-1) were added per well. 4CHOK1-hROR1 cells were added at a concentration of 3 × 10 per well. 4 The cells were added in the same amount as the cells. Test antibodies (starting at 50 nM, 5-fold dilutions) were added to the plate wells. The plates were then incubated at 37°C in a 5% CO2 humidification incubator for 72 hours. After incubation, the concentration of interferon gamma (IFN-gamma) released from the supernatant was measured using the Human IFN-gamma Duoset ELISA Kit (R&D Systems, DY285) according to the manufacturer's protocol. Four-parameter logistic curves were evaluated using GraphPad software.

[0273] As shown in Figure 5A, the anti-ROR1 / 4-1BB bispecific antibody BA6M11(NA)x1A10M12 induced IFN-gamma release at lower concentrations but at higher concentrations than BA6x1A10M12, suggesting that the BA6M11-containing bispecific antibody exhibited superior PBMC activation potency compared to the BA6 parent clone. Furthermore, the BA6M11-containing bispecific antibodies BA6M11(NA)x1A10M12 and BA6M11(NA)x1A10M12 induced similar levels of IFN-gamma release.

[0274] 2.2.7.2.PBMC activation test using gastric cancer cell lines expressing ROR1 NCI-N87 is a gastric cancer cell line that expresses human ROR1. Human PBMCs were co-cultured with these human ROR1-expressing gastric cancer cells to evaluate whether the 4-1BB signal activation activity of the bispecific antibody of the present invention could activate human peripheral blood mononuclear cells (PBMCs). Briefly, 96-well plates were coated with 5 μg / mL of anti-human CD3 (Biolegend, 300438) dissolved in PBS for 2 hours at 37°C. Human PBMCs (CTLs) from various donors and NCI-N87 (ATCC) cells were then added to the plates. Four-fold dilutions of each test antibody (starting at 20 nM) were added to each well and the cells were cultured for 72 hours. After 72 hours of culture, the IFN-gamma concentration in the supernatant was measured using a Human IFN-gamma Duoset ELISA Kit (R&D system, DY285B). The viability of NCI-N87 was measured using cell counting kit-8 (Dojindo, CK04-20).

[0275] The results are shown in Figures 5B and 5C. As shown in Figures 5B and 5C, the anti-ROR1 / 4-1BB bispecific antibody containing BA6M11 had a lower EC50 (nM) value compared to the control antibody (urelumab), induced IFN-gamma secretion at lower concentrations, and induced NCI-N87 killing at lower concentrations than the control antibody. This indicates that it exhibited better PBMC activation ability than the control antibody in the presence of human ROR1 antigen. Furthermore, the anti-ROR1 / 4-1BB bispecific antibody containing BA6M11 exhibited superior PBMC activation ability compared to the combination of BA6M11 and 1A10M12 monoclonal antibodies (BA6M11 + 1A10M12) in both WT and NA forms. These results confirm that the anti-ROR1 / 4-1BB bispecific antibody of the present invention exhibits superior synergistic effects compared to the simple combination of monoclonal antibodies.

[0276] 2.3. In vivo efficacy of anti-ROR1 / anti-4-1BB bispecific antibodies 2.3.1. Antitumor Efficacy The in vivo antitumor efficacy of the anti-ROR1 / anti-4-1BB bispecific antibodies BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 was evaluated using human 4-1BB4 knock-in mice (Biocytogen). Briefly, human ROR1 was stably expressed in MC38 cells, a mouse colon cancer cell line, and hROR1-expressing MC38 cells (5 × 10 5 The tumor-bearing humanized mice were divided into three groups based on tumor size (average tumor size: approximately 89 mm). 3 (n=5 / group). After grouping, on day 0, a single dose of human IgG1 antibody (hIgG1 antibody) (3 mg / kg), BA6M11(WT)x1A10M12 (4 mg / kg), or BA6M11(NA)x1A10M12 (4 mg / kg) was intravenously administered. Tumor size was measured twice weekly using a digital caliper, and tumor volume was calculated using the following formula: V (mm 3 =a*b 2 / 2 (a and b represent the long and short dimensions of the tumor). The tumor growth inhibition (TGI) ratio of tumor volume was calculated using the following formula: TGI%(Dayi)=[1-(T Day(i) -T Day0 ) / (V Day(i) -V Day0 )]×100%(T: tumor volume of the treated group; V: tumor volume of the control group) The antitumor efficacy results are shown in FIG. 6A and Table 19.

[0277] [Table 19]

[0278] As shown in Figure 6A and Table 19, BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 demonstrated significant tumor-inhibitory effects. On day 23 after grouping, the TGIs in the BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12-treated groups were 98.6% and 69.9%, respectively. On day 44, the survival rates in the BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12-treated groups were 100% and 80%, respectively, whereas there were no survivors in the hIgG1 control group. These results confirm that both BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 bispecific antibodies exhibited excellent antitumor efficacy and high survival rates even with a single administration.

[0279] 2.3.2. Dose-dependent antitumor efficacy To confirm the dose-dependent effect of the bispecific antibody BA6M11(WT)x1A10M12, the in vivo antitumor efficacy was evaluated in hROR1-expressing MC38-bearing mice. Specifically, tumor cells (hROR1-expressing MC38 cells, 5 × 10) were inoculated into the right flank of human 4-1BB4 mice. 5 Human ROR1-expressing MC38-bearing mice were prepared by subcutaneous injection of 10 ... 3 On day 0 of grouping, mice were intravenously injected with a single dose of hIgG1 antibody (10 mg / kg) or BA6M11(WT)x1A10M12 (0.4 mg / kg, 1 mg / kg, or 4 mg / kg). Antitumor efficacy was measured as described in Example 2.3.1 above.

[0280] As shown in Figure 6B, BA6M11(WT)x1A10M12 demonstrated dose-dependent antitumor efficacy. BA6M11(WT)x1A10M12 treatment also increased the number of mice with complete tumor regression (50% in the BA6M11(WT)x1A10M12 4 mg / kg treatment group compared to 0% in the hIgG1 treatment group), and these mice survived for more than 100 days after initial tumor inoculation without any signs of tumor recurrence.

[0281] 2.3.3. Memory response upon serial tumor re-challenge To evaluate the long-lasting memory response, mice whose tumors had been cured by BA6M11(WT)x1A10M12 were rechallenged with tumors 100 days after the last administration of the antibody. Specifically, hROR1-expressing MC38 cells (2.5 × 10 6 The recovered mice and control naive mice (mice that had not received tumor inoculation or antibody administration) were subcutaneously injected with 2.5 × 10 cells (5 times the initial inoculation) (second inoculation). 59 days after the second inoculation, the surviving mice were reinoculated with tumors (2.5 × 10 cells). 6 The antitumor efficacy was measured with reference to Example 2.3.1 above.

[0282] As shown in Figure 6C, mice previously treated with the bispecific antibody of the present application were resistant to repeated secondary and tertiary tumor rechallenge, whereas no tumor regression was observed in naive control mice, confirming that treatment with BA6M11(WT)x1A10M12 induces a sustained anti-cancer memory response.

[0283] 2.3.4. In vivo immune modulation To investigate the effect of the bispecific antibody on immune cells, tumor and peripheral tissues (blood and liver) were analyzed by flow cytometry.

[0284] More specifically, the human ROR1-expressing MC38 tumor-bearing humanized mice used in the above-mentioned examples were cultured in a tumor volume (approximately 100 mm 3 The mice were randomly grouped based on their tumor size and tumor type. hIgG1 antibody (7.5 mg / kg), urelumab (7.5 mg / kg), or the bispecific antibody BA6M11(WT)x1A10M12 (10 mg / kg) was administered intraperitoneally on days 0 and 4. After grouping, immune cell analysis was performed by flow cytometry on day 7. Leukocytes (CD45 + ) and Treg cells (CD45 + CD3 + CD4 + FOXP3 + ) and T cells (CD45 + CD3 + ) were analyzed. In the liver, T cells, CD8+ T cells (CD45 + CD3 + CD8 + ), and macrophages (CD45 + CD11b + F4 / 80 + The results are shown in Figure 6D (A: tumor; B: peripheral blood; C: liver).

[0285] As shown in Figure 6D, a significant increase in tumor-infiltrating leukocytes (CD45+ / Live) was observed in the BA6M11(WT)x1A10M12-treated group. Furthermore, unlike urelumab, BA6M11(WT)x1A10M12 specifically induced depletion of immunosuppressive Treg cells (Treg / CD45+) in tumors but not in the systemic circulation. Expression of 4-1BB (4-1BB+ / CD3+) on T cells was measured at higher levels in tumors compared to blood, and this level was increased by BA6M11(WT)x1A10M12 treatment. These results confirmed the tumor-specific T cell activation effect of the bispecific antibody of the present application. Furthermore, while systemic activation by urelumab was accompanied by massive immune cell infiltration in the liver, the bispecific antibody of the present application did not show an increase in T cells or macrophages in the liver. These results confirm that the bispecific antibody of the present application exerts a potent antitumor effect through tumor-specific immune regulation while minimizing the risk of peripheral toxicity.

[0286] 2.3.5. Further Demonstration of Tumor-Specific T Cell Activation by Anti-ROR1 / Anti-4-1BB Bispecific Antibodies To demonstrate that the bispecific antibody of the present application has a T cell activation effect specifically on cells that express ROR1 (e.g., tumor cells) and does not act on normal cells and tissues that do not express ROR1, the cytokine secretion activity of the anti-ROR1 / anti-4-1BB bispecific antibody was confirmed using human PBMCs in the absence of cells that express ROR1.

[0287] More specifically, five types of human PBMCs (CTL, CTL-UP1) were thawed and then cultured at 2 x 10 in 10% RPMI medium. 5The cells were seeded into a 96-well plate at a concentration of 0.01 μg / well. For the positive control group, 0.01 μg, 0.1 μg, 1 μg, 25 μg, and 50 μg of anti-human CD3 antibody (Biolegend, 317326) was diluted in RPMI containing 10% FBS and added to each well. For the test group, 1 μg, 25 μg, and 50 μg of a bispecific antibody (BA6M11(WT)x1A10M12) was added to each well.

[0288] The plates were incubated for 48 hours in a 37°C, 5% CO2 incubator. After 48 hours, the plates were centrifuged to separate the supernatant. ELISA assays were performed according to the manufacturer's protocol to measure the concentrations of IFN-gamma (R&D Systems, DY285B), IL-2 (R&D Systems, DY202), IL-6 (R&D Systems, DY206), and TNF-alpha (R&D Systems, DY210) in the culture supernatant. The results are shown in Figure 6E.

[0289] As shown in Figure 6E, compared to the positive control group, BA6M11(WT)x1A10M12 did not induce cytokine release in the absence of ROR1. In other words, the bispecific antibody of the present application activated T cells only in tumor tissues expressing ROR1, but not in normal cells and tissues, and was therefore assessed to have little risk of unexpected toxicity when administered in vivo.

[0290] Example 3. ROR1xB7-H3 bispecific antibody and antibody-drug conjugates (ADCs) containing the same 3.1. Design and Production of Improved Anti-ROR1 / Anti-B7-H3 Bispecific Antibodies An anti-ROR1 / anti-B7-H3 bispecific antibody containing an anti-ROR1 antibody and an anti-B7-H3 antibody was produced based on the anti-ROR1 antibody clone BA6M11 selected in Example 1 and B5, one of the anti-B7-H3 antibody clones disclosed in International Publication WO2019-226017A1.

[0291] An anti-ROR1 / anti-B7-H3 bispecific antibody was prepared in the form of an IgG-scFv fusion in which an scFv antibody fragment against one of the two antigens (ROR1 and B7-H3) was fused to the C-terminus (heavy chain C-terminus) of a full-length IgG antibody against the other antigen. In this example, a bispecific antibody was prepared comprising an anti-ROR1 IgG antibody and a B5 scFv linked to the C-terminus of the heavy chain of each of the anti-ROR1 IgG antibodies.

[0292] To construct the anti-ROR1 / anti-B7-H3 bispecific antibody, DNA segment 1, containing the nucleotide sequence encoding the heavy chain of the anti-ROR1 IgG antibody (BA6M11), was inserted into pcDNA 3.4 (Invitrogen, A14697; Plasmid 1), and DNA segment 2, containing the nucleotide sequence encoding the light chain of the IgG antibody of the anti-ROR1 / anti-B7-H3 bispecific antibody, was inserted into pcDNA 3.4 (Invitrogen, A14697; Plasmid 2). DNA segment 3, encoding the scFv, was then fused to the portion of DNA segment 1 corresponding to the C-terminus of the Fc region of the IgG antibody inserted into Plasmid 1 using DNA segment 4, encoding a 20-amino acid peptide linker consisting of (GGGGS)3 (SEQ ID NO: 120), to construct a vector for expression of the bispecific antibody.

[0293] The anti-B7-H3 scFv (designated B5) is constructed by fusing VL103-VH44 (VL103: a VL with a G (Gly) to C (cys) mutation at position 103; VH44: a VH with a G to C mutation at position 44) to the C-terminus of the light chain variable region (VL) and the C-terminus of the heavy chain variable region (VH) via the linker ((GGGGS)4) (SEQ ID NO: 114). These mutations stabilize the scFv and enable disulfide bridge formation.

[0294] The amino acid sequence of the bispecific antibody (BA6M11xB5) thus produced is shown in Table 20 below.

[0295] [Table 20] (Linker 1: BA6M11 (IgG) and B5 (scFv) linked together (the C-terminus of the heavy chain of BA6M11 linked to the N-terminus of the VL of B5); Linker 2: VL and VH of B5 (scFv) linked (C-terminus of VL linked to N-terminus of VH)

[0296] The coding nucleic acid sequence of the bispecific antibody (BA6M11xB5) is as follows: [Heavy chain_BA6M11xB5] (SEQ ID NO: 122) TIFF2025525538000027.tif164170

[0297] [Light chain_BA6M11] (SEQ ID NO: 123) TIFF2025525538000028.tif51170

[0298] The DNA encoding the cloned bispecific antibodies was expressed using the ExpiCHO system (Thermo Fisher) according to the manufacturer's protocol. The culture medium in which the antibodies were expressed was centrifuged and filtered to remove the supernatant, and the antibodies were purified using affinity chromatography using Mabselec Sure resin and size exclusion chromatography using Superdex 200 resin. The purity of the purified antibodies was analyzed by HPLC using TSKgel SuperSW3000 and was found to be greater than 95%.

[0299] 3.2. Stability Assessment of Anti-ROR1 / B7-H3 Bispecific Antibodies Containing Improved Anti-ROR1 Clones (1) Binding specificity analysis of anti-ROR1 / B7-H3 bispecific antibodies under harsh conditions (ELISA) The control group was the BA6xB5 bispecific antibody, which contains the parent clone anti-ROR1 antibody BA6, whose PTM hotspot had not been removed. The experimental group was the BA6M11xB5 bispecific antibody. After storage at 4°C (normal conditions) and 40°C (harsh conditions) for 2 weeks, the antibody-antigen binding affinity of each sample to human ROR1 and human B7H3 recombinant proteins was evaluated using an ELISA-based solution binding test. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with 1 μg / ml human ROR1 protein (ROR1-His; Sino Biological, Catalog #13968-H08H) and human B7-H3 protein (B7H3-His; Sino Biological, Catalog #11188-H08H) in PBS at 4°C for 16 hours, and then treated with 1% (v / v) bovine serum albumin (BSA) at 37°C for 2 hours to block nonspecific binding sites.

[0300] The BA6xB5 and BA6M11xB5 bispecific antibodies stored for two weeks on a 96-well microtiter plate were then added to the microtiter plate at the concentrations shown in Figures 7A-7D, and their binding activity was analyzed by ELISA as follows. Specifically, after incubation at 37°C for two hours, the plate was washed five times with PBS containing 0.05% (v / v) Tween 20. An HRP-conjugated Fc multiclonal antibody reagent (Pierce, Catalog #31413) was diluted 1:30,000 in 1% (v / v) bovine serum albumin (BSA) and added to the washed microtiter plate. The plate was incubated at 37°C for one hour to detect the bispecific antibodies bound to the plate. After incubation, color development was performed using tetramethylbenzidine (TMB, Sigma, T0440). The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the absorbance was measured at 450 nm and 650 nm using a microplate reader (molecular device).

[0301] The results are shown in Figures 7A-7D. As can be seen in Figures 7A-7D, antigen-antibody binding activity was assessed using human ROR1 protein and human B7-H3 protein (ELISA; Protein Binding). We confirmed that there was no significant difference in binding activity between samples stored at 4°C and 40°C for two weeks. These results confirmed that BA6M11xB5 maintained potency compared to BA6xB5 while exhibiting improved physical properties in terms of developability. Finally, we confirmed that the BA6M11 clone not only exhibited binding comparable to WT, but also demonstrated clear improvements in developability through PTM engineering.

[0302] (2)Developability analysis of anti-ROR1 / B7-H3 bispecific antibody We selected the BA6M11 clone, which was derived by removing PTM liability from BA6 through PTM engineering, and constructed BA6M11xB5 bispecific antibodies and BA6xB5 bispecific antibodies containing BA6M11 to confirm whether they had increased stability under stressed conditions.

[0303] More specifically, the BA6xB5 bispecific antibody and BA6M11xB5 bispecific antibody were stored at 4°C (normal conditions) and 40°C (harsh conditions) for two weeks, and then each sample was analyzed by SE-HPLC, icIEF, and LC / MS to confirm purity and impurity content. Additionally, to determine whether changes in substance content affect efficacy, an antigen-antibody binding assay (ELISA; see Example 3.21) was performed.

[0304] The results obtained are shown in Table 21 below.

[0305] [Table 21] (*N / A: Not applicable)

[0306] As shown in Table 21, the BA6M11xB5 clone maintained high levels of antibody purity, comparable to that of BA6xB5, when stored at 4°C and 40°C. Regarding charge variants, the BA6M11xB5 clone exhibited an improved pattern of an increased basic variant peak associated with succinimide production compared to the BA6xB5 clone. LC-MS analysis also confirmed that, compared to BA6xB5, BA6M11xB5 did not exhibit an increased level of succinimide in the Fd and LC under 40°C stress conditions, nor did it produce fragments suggestive of cleavage within the Fd site. This reduction in charge variants indicates improved stability of antibody quality and reliability of the purification process, as described in Example 1.3. Furthermore, bispecific antibodies are expected to maintain low immunogenicity and potency when administered to humans.

[0307] 3.3. Analysis of Binding Specificity of Anti-ROR1 / B7-H3 Bispecific Antibody to Cell Surface-Expressed ROR1 and B7-H3 (FACS) To evaluate the cell-binding ability of the anti-ROR1 / B7-H3 bispecific antibody, cells co-expressing ROR1 and B7-H3 were used to compare the cell-binding ability of the bispecific antibody with that of the single antibody.

[0308] The cell lines used for cell binding comparison were the CHO-huROR1-huB7-H3 cell line, which was stably transfected with human ROR1 and human B7-H3 genes to artificially overexpress both human ROR1 and human B7-H3 proteins (obtained by transfecting the ROR1-CHO recombinant cell line (BPS Bioscience) with the human B7-H3 gene (Origene) and selecting single cells carrying the desired gene using G418). Cell binding was evaluated using flow cytometry (LSR Fortessa X-20, BDBiosciences). Specifically, the overexpressing cell lines were dissociated and washed with PBS. After counting, the cells were plated at 2 x 10 per well in a V-bottom 96-well plate (96Well Plate-RV, Bioneer, 910D09). 5 Cells were aliquoted. Antibodies were diluted 4-fold starting from 100 nM in 1% BSA solution, and 100 μL of each was added to the centrifuged cells. The cells were incubated at 4°C for 1 hour. After 1 hour, the cells were washed twice with the same buffer, and 100 μL of an FITC-labeled Fc-specific antibody (Goat anti-human IgG-FITC antibody produced in goat, Sigma, F9512) was diluted 500-fold in 1% BSA and added to each well. The incubation was continued at 4°C for 1 hour. After incubation, the cells were washed twice with the same buffer, resuspended in 100 μL of PBS, and analyzed by flow cytometry using a FACS instrument. Ten thousand cells were detected per assay, and the data were analyzed using FlowJo software. The fold change values in the graphs were calculated by dividing the MFI (Mean Fluorescence Intensity) of the antibody-treated experimental group by the MFI of the control group treated with only the secondary antibody.

[0309] As shown in Figure 8, for cell lines co-expressing ROR1 and B7-H3, the anti-ROR1 / B7-H3 bispecific antibody, which has more antigen-binding sites, showed higher binding affinity than the monospecific antibodies.

[0310] These results confirmed that the anti-ROR1 / B7-H3 bispecific antibody provided herein exhibits superior binding avidity by simultaneously targeting two antigens. This improved binding avidity confirmed superior selectivity for cells expressing two targets (antigens) at the same time compared to cells expressing a single target (antigen). This may be a key feature in developing tumor treatments, achieving the goals of reduced toxicity and increased efficacy through selectivity.

[0311] 3.4. Comparison of Cellular Internalization of Monospecific and Bispecific Antibodies To compare the internalization rate of the anti-ROR1 / B7-H3 bispecific antibody, we evaluated the internalization of the bispecific antibody using cells co-expressing ROR1 and B7-H3. The experiment was performed using Incucyte® FabFluor-pH Red antibody labeling reagent (Satorius, 4722), which fluoresces at low pH. The same CHO-huROR1-huB7-H3 cell line used in Example 3.3 was plated at 1 × 10 cells per well in a 96-well plate (96-well Clear Flat Bottom TC-treated Culture Microplate, Falcon, 353072). 4 The cells were then placed in a 96-well plate and incubated for 24 hours. The antibody and IncuCyte® FabFluor-pH Red antibody labeling reagent were mixed at a 1:1 molar ratio, incubated at 37°C for 15 minutes, and then placed in the 96-well plate containing the cells. The plate was placed in an IncuCyte® real-time cell analysis system (IncuCyte S3, Satorius) and analyzed for phase contrast and fluorescence every hour using the software. As a result, as shown in Figure 9, the fluorescent signal increased over time, confirming the internalization process in which the antibody entered the cells and reached the lysosomes.

[0312] That is, as shown in Figure 9, in a cell line co-expressing ROR1 and B7-H3, the anti-ROR1 / B7-H3 bispecific antibody, which has more antigen-binding sites, increased the fluorescence signal more rapidly than the monospecific antibody, confirming that the bispecific antibody is internalized into cells more rapidly than the monospecific antibody.

[0313] These results confirmed that the anti-ROR1 / B7-H3 bispecific antibody provided herein promotes efficient cellular internalization by simultaneously targeting two antigens.

[0314] Based on the above results, when the bispecific antibody is developed into an ADC, it is expected that the bispecific antibody will have the property of selectively increasing the efficacy of the ADC against cells that simultaneously express two targets (antigens), and this was confirmed in the Examples described below.

[0315] 3.5. Production of anti-ROR1 / anti-B7-H3 bispecific antibody ADC (1) Production of bispecific antibody ADCs To conjugate a drug to amino acid 205 of the antibody light chain, Kabat 205 valine in the antibody light chain constant region was mutated to cysteine (V205C), and a thiol group was generated at V205C of the antibody light chain by reaction with a reducing agent such as dithiothreitol (DTT). The drug was then conjugated to the antibody via Michael addition to form thiosuccinimide. Specifically, antibody was prepared at a concentration of 5 mg / ml or more by ultrafiltration / diafiltration (UF / DF), and 1 M tris(hydroxymethyl)aminomethane (Tris-HCl), pH 8.8, and 500 mM ethylenediaminetetraacetic acid (EDTA) were added to the antibody to achieve a final concentration of 5 mg / ml, 75 mM Tris-HCl, and 2 mM EDTA. The prepared antibody was added with 100 mM dithiothreitol (DTT) at an antibody:DTT molar ratio of 1:20 and incubated at 25°C for 16.5 hours to cleave the free cysteine linked to cysteine 205 of the antibody light chain via a disulfide bond. This process is called decapping, and cation exchange chromatography (CEX) was used to isolate the decapped antibody. The reaction product was applied to a Hitrap SPHP column (GE Healthcare) equilibrated with SPHP-A buffer (10 mM succinate, pH 5.0) and eluted with SPHP-B buffer (50 mM Tris-HCl, pH 7.5, 0.5 M sodium chloride).To reconjugate the decapped antibody, the antibody was oxidized using 1 M tris(hydroxymethyl)aminomethane (Tris-HCl, pH 7.5). The antibody was prepared in 75 mM Tris-HCl (pH 7.5). Dihydroascorbic acid (DHAA), an oxidized form of vitamin C, was added at an antibody:DHAA ratio of 1:20 and reoxidized for 2 hours at 25°C in the dark. The reoxidized antibody was then purified using cation exchange chromatography (CEX) and eluted with SPHP-C buffer (10 mM succinate, pH 5.0, 0.5 M sodium chloride). The purified antibody was concentrated to over 5 mg / ml by ultrafiltration / diafiltration (UF / DF). To prepare the antibody-drug conjugate, the antibody was prepared at a final concentration of 5 mg / ml in 100 mM Tris(hydroxymethyl)aminomethane (Tris-HCl, pH 7.0) at a 1:10 antibody:drug molar ratio and incubated at 25°C for 16.5 hours. To isolate the antibody-drug conjugate, cation exchange chromatography (CEX) was used for purification and elution with SPHP-C buffer (10 mM succinate, pH 5.0, 0.5 M sodium chloride). Hydrophobic interaction chromatography (HIC) was then used to isolate the antibody-drug conjugate, DAR2.To this end, a Hitrap Butyl HP column (GE Healthcare) was equilibrated with HIC-A buffer (50 mM potassium phosphate, pH 7.0, 1.0 M ammonium sulfate) and eluted with HIC-B buffer (50 mM potassium phosphate, pH 7.0, 30% isopropyl alcohol (2-propanol)). The antibody was then exchanged into HS buffer (20 mM histidine, pH 6.0, 240 mM sucrose) by ultrafiltration / diafiltration (UF / DF) to remove the isopropyl alcohol (2-propanol). The final antibody-drug conjugate was then prepared. ADCs were also prepared using pyrrolobenzodiazepine (PBD) as the drug. The resulting antibodies were designated "Antibody (V205C)-T-PBD."

[0316] (2) Purity and DAR Measurement of the Produced Bispecific Antibody ADC The purity of the ADCs was determined by size-exclusion high-performance liquid chromatography (SE-HPLC). Measurements were performed using an Agilent 1200 series HPLC system with a Tosoh TSKgel G3000SWxl column (Tosoh Bioscience). The purity of each sample was determined by comparing the elution position and area under the curve (AUC) of each sample. The purity of the main ADC peak was 99.0% or higher (see Table 23 and Figures 10A-10C).

[0317] The drug-to-antibody ratio (DAR) of the ADC was determined by liquid chromatography-mass spectrometry (LC / MS). N-glycans were removed by adding 1 unit of PNGaseF (NEB) per 100 μg of antibody at 1 mg / mL in PBS and incubating at 37°C for 15 hours. An Acquity UPLC BEH200 SEC 1.7 μm (4.6 × 150 mm) column was installed on an LC / MS system consisting of a Waters UPLC I-class system and a Waters SynaptG2-S column, and equilibrated with a mobile phase of 30% (v / v) acetonitrile, 0.1% (v / v) formic acid, and 0.05% trifluoroacetic acid (TFA). Five μg of the N-glycan-removed sample was loaded onto the column and analyzed by LC / MS. The drug-to-antibody ratio (DAR) of the bispecific antibody ADC was measured by LC / MS, confirming that two drugs were conjugated per antibody (see Tables 22 and 23 and Figures 11A to 11C).

[0318] [Table 22]

[0319] [Table 23]

[0320] (3) Ligand binding activity test using bispecific antibody ADC The antibody-antigen binding affinity for human ROR1 recombinant protein was evaluated using the BA6M11(V205C)-T-PBD monoantibody and BA6xB5(M11(V205C)-T-PBD bispecific antibody, both manufactured in ADC form, using an ELISA-based solution binding assay. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with 1 μg / ml ROR1 protein (ROR1-His; Sino Biological, Catalog #13968-H08H) in PBS solution at 4°C for 16 hours, and then treated with 1% (v / v) bovine serum albumin (BSA) at 37°C for 2 hours to block nonspecific binding sites.

[0321] Then, the BA6M11(V205C)-T-PBD monoantibody and the BA6xB5(M11(V205C)-T-PBD bispecific antibody were added to a 96-well microtiter plate at the concentrations shown in Figure 12, and the binding ability was analyzed by ELISA as follows. Specifically, after incubation at 37°C for 2 hours, the plate was washed five times with PBS containing 0.05% (v / v) Tween 20. Then, HRP-conjugated Fc multiclonal antibody reagent (Pierce, Catalog #31413) was added to PBS containing 1% (v / v) BSA (bovine serum albumin). The bispecific antibody was diluted 1:30,000 with PBS (Primer's albumin) and added to the washed microtiter plate. The plate-bound bispecific antibody was detected by incubation at 37°C for 1 hour. After incubation, color development was performed using TMB (Tetramethylbenzidine, Sigma, T0440). The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the absorbance was measured at 450 nm and 650 nm using a microplate reader (Molecular Device).

[0322] The results are shown in FIG. 12. From the results of confirming the antigen-antibody binding ability using human ROR1 protein, it was confirmed that the ligand binding ability of the prepared BA6xB5(M11(V205C)-T-PBD bispecific antibody was at the same level as the prepared BA6M11(V205C)-T-PBD monospecific antibody. In other words, it was confirmed that the excellent ROR1 antigen-binding ability of BA6M11 was maintained even after it was prepared in the form of a bispecific antibody ADC.

[0323] 3.6. Comparative evaluation of cytotoxicity of bispecific antibody ADCs The cancer cell killing effect of ADCs prepared using the bispecific antibodies of the present invention was evaluated in comparison with that of single-antibody ADCs. Two cancer cell lines, calu-3 (KCLB, Catalog No. 30055) and calu-6 (ATCC, Catalog No. HTB-56), which express both ROR1 and B7-H3, were used. Cell killing ability was compared by adding ADCs to the cells and measuring their metabolic activity. To confirm whether the effect of the bispecific antibodies was merely due to the combination of single-antibody targets, the effect was also evaluated in a control group treated with both anti-ROR1 and anti-B7-H3 single antibodies (combination treatment, indicated as +). Cancer cell lines were cultured using the recommended culture media. 5 x 10 cells per well of a 96-well culture plate were cultured. 3 Cells were seeded in 50 μl of a 96-well plate and cultured for 4–6 hours. After incubation, 50 μl of variously diluted ADCs was dispensed into the wells of the plate. The cells were then cultured at 37°C and 5% CO for approximately 6 days. Viable cell counts were quantified using WST-8 (Dojindo, CK04).

[0324] Figure 13 shows the cytotoxicity of mono- and bispecific antibodies against cancer cell lines using B5 as the anti-B7-H3 antibody and BA6M11 as the anti-ROR1 antibody. We confirmed that the cytotoxicity of the bispecific antibody (BA6M11xB5) ADC was greater than that of the combination of mono-antibody (BA6M11 and B5) ADCs in cancer cell lines (calu-3 and calu-6) that co-express ROR1 and B7-H3. On the other hand, in the KATO III cell line (ATCC, Catalog No. HTB-103), which barely expresses ROR1 or B7-H3, the ADC showed little cytotoxicity, and there was no difference in cytotoxicity between the mono- and bispecific antibody ADCs. This confirms that the higher cytotoxicity of the bispecific antibody ADC compared to the mono-antibody ADC is antigen-dependent. The results showed that treatment with the bispecific antibody induced higher cytotoxicity than treatment with the combination of each of the individual antibodies, confirming that the superior cytotoxicity of the bispecific antibody ADC of the present invention is an effect that goes beyond that of simply combining the individual antibodies.

[0325] Although certain aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific aspects are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

Claims

1. HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO:2; HCDR3 comprising the amino acid sequence of SEQ ID NO:3; LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and LCDR3 comprising the amino acid sequence of SEQ ID NO:6 An anti-ROR1 antibody or antigen-binding fragment thereof comprising:

2. 2. The anti-ROR1 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region of SEQ ID NO: 7 or 127 and a light chain variable region of SEQ ID NO: 8 or 126.

3. A nucleic acid molecule encoding the anti-ROR1 antibody or antigen-binding fragment thereof of claim 1 or 2.

4. A pharmaceutical composition for preventing or treating cancer, comprising the anti-ROR1 antibody or its antigen-binding fragment according to claim 1 or 2, and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition for preventing or treating cancer according to claim 4, wherein the cancer is a cancer that expresses ROR1.

6. A composition for detecting ROR1, comprising the anti-ROR1 antibody or its antigen-binding fragment according to claim 1 or 2.

7. A composition for cancer diagnosis comprising the anti-ROR1 antibody or its antigen-binding fragment according to claim 1 or 2.

8. The cancer diagnostic composition according to claim 7 , wherein the cancer is a cancer that expresses ROR1.

9. an anti-ROR1 antibody or antigen-binding fragment thereof, and Anti-4-1BB antibody or antigen-binding fragment thereof Including, The anti-ROR1 antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

6. Anti-ROR1 / anti-4-1BB bispecific antibody.

10. The anti-ROR1 / anti-4-1BB bispecific antibody of claim 9, wherein the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 7 or SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 8 or SEQ ID NO:

126.

11. The anti-4-1BB antibody or antigen-binding fragment thereof is HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, 14 or 15; HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17, or 18; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, or 23; LCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or 25; LCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 27; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 or 29 The anti-ROR1 / anti-4-1BB bispecific antibody of claim 9 or 10, comprising:

12. The anti-4-1BB antibody or antigen-binding fragment thereof is (1) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 19; (b) HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 20; (c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 21; (d) HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 22; and (e) HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 23 a heavy chain complementarity determining region selected from (2) (a) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; and (b) LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 a light chain complementarity determining region selected from The anti-ROR1 / anti-4-1BB bispecific antibody of any one of claims 9 to 11, comprising:

13. The anti-4-1BB antibody or antigen-binding fragment thereof is a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, or 47 The anti-ROR1 / anti-4-1BB bispecific antibody of any one of claims 9 to 12, comprising:

14. A nucleic acid molecule encoding the anti-ROR1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to any one of claims 9 to 13.

15. A pharmaceutical composition for preventing or treating cancer, comprising the anti-ROR1 / anti-4-1BB bispecific antibody or antigen-binding fragment thereof according to any one of claims 9 to 13, and a pharmaceutically acceptable excipient.

16. The pharmaceutical composition for preventing or treating cancer according to claim 15, wherein the cancer is a cancer that expresses ROR1.

17. an anti-ROR1 antibody or antigen-binding fragment thereof, and Anti-B7-H3 antibody or antigen-binding fragment thereof Including, The anti-ROR1 antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

6. Anti-ROR1 / anti-B7-H3 bispecific antibody.

18. 18. The anti-ROR1 / anti-B7-H3 bispecific antibody of claim 17, wherein the anti-ROR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region of SEQ ID NO: 7 or SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 8 or SEQ ID NO:

126.

19. The anti-B7-H3 antibody or antigen-binding fragment thereof comprises: HCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 48-51; HCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 52-57; HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 58-62; LCDR1 comprising an amino acid sequence selected from SEQ ID NOs: 63-67; LCDR2 comprising an amino acid sequence selected from SEQ ID NOs: 68-73; and LCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 74-79 19. The anti-ROR1 / anti-B7-H3 bispecific antibody of claim 17 or 18, comprising:

20. The anti-B7-H3 antibody or antigen-binding fragment thereof comprises: (i) (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 48, HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 58; (b) HCDR1 comprising the amino acid sequence of SEQ ID NO: 49, HCDR2 comprising the amino acid sequence of SEQ ID NO: 53, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 59; (c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 50, HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 60; (d) HCDR1 comprising the amino acid sequence of SEQ ID NO: 48, HCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 61; (e) HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, HCDR3 comprising the amino acid sequence of SEQ ID NO: 62; and (f) HCDR1 comprising the amino acid sequence of SEQ ID NO: 50, HCDR2 comprising the amino acid sequence of SEQ ID NO: 57, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 60 a heavy chain complementarity determining region selected from (ii) (a) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 63, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 68, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 74; (b) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 64, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 69, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 75; (c) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 70, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 76; (d) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 66, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 71, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 77; (e) an LCDR1 comprising the amino acid sequence of SEQ ID NO: 67, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 72, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 78; and (f) LCDR1 comprising the amino acid sequence of SEQ ID NO: 65, LCDR2 comprising the amino acid sequence of SEQ ID NO: 73, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 76 a light chain complementarity determining region selected from The anti-ROR1 / anti-B7-H3 bispecific antibody of any one of claims 16 to 19, comprising:

21. The anti-B7-H3 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 80-91; and A light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 92 to 103 The anti-ROR1 / anti-B7-H3 bispecific antibody of any one of claims 16 to 20, comprising:

22. A nucleic acid molecule encoding the anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof of any one of claims 16 to 21.

23. A pharmaceutical composition for preventing or treating cancer, comprising the anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 16 to 21, and a pharmaceutically acceptable excipient.

24. The pharmaceutical composition for preventing or treating cancer according to claim 23, wherein the cancer is a cancer that expresses ROR1, B7-H3, or both.

25. 22. An antibody-drug conjugate comprising a cytotoxic drug linked to the anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof according to any one of claims 16 to 21.

26. The antibody-drug conjugate of claim 25, wherein the antibody-drug conjugate has an antibody-linker-drug structure.

27. 27. The antibody-drug conjugate of claim 25 or 26, wherein the cytotoxic drug is an anti-cancer drug.

28. A pharmaceutical composition for preventing or treating cancer, comprising the antibody-drug conjugate of any one of claims 25 to 27 and a pharmaceutically acceptable excipient.

29. The pharmaceutical composition for preventing or treating cancer according to claim 28, wherein the cancer is a cancer that expresses ROR1, B7-H3, or both.

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