Anti-ROR1 antibody, bispecific antibody comprising same and use thereof

Abstract

Provided are: a novel anti-ROR1 antibody; a bispecific antibody comprising an anti-ROR1 antibody; and uses thereof.

Description

Technical Field

[0001] Provided are a novel anti-ROR1 antibody, a bispecific antibody comprising the anti-ROR1 antibody, and uses thereof. Background Art

[0002] Receptor tyrosine kinase-like orphan receptors (RORs) are transmembrane proteins in the receptor tyrosine kinase (RTK) family, including ROR1 and ROR2. ROR1 and ROR2 have 58% amino acid sequence homology to each other, and both have a theoretical molecular weight of about 104 kDa; however, due to multiple N-glycosylation sites, ROR1 has a molecular weight of about 130 kDa. The extracellular domain of the ROR family consists of an Ig domain, a cysteine-rich domain, and a kringle domain, while the intracellular domain consists of a tyrosine kinase domain, a serine / threonine (Ser / Thr)-rich domain, and a proline (Pro)-rich domain. In terms of biological characteristics, the ligand of ROR2 is Wnt5a, while the ligand of ROR1 has not yet been elucidated.

[0003] ROR1 is expressed during embryonic and fetal development, regulating cell polarity, cell migration, and neurite outgrowth. The expression of ROR1 gradually decreases with development, and it is barely expressed in adults, except for transient expression during B cell development and slight expression in adipocytes. However, overexpression of ROR1 in various cancer cells has led to its classification as an oncofetal gene. In particular, the discovery that ROR1 is overexpressed in chronic lymphocytic leukemia (CLL) has attracted increased attention as a target for cancer antibodies. In addition to CLL, overexpression of ROR1 has been reported in various hematological malignancies, such as B-cell leukemia, lymphoma, acute myeloid leukemia (AML), Burkitt's lymphoma, mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), as well as in a variety of solid tumors, including 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, epithelial squamous cell carcinoma, melanoma, multiple myeloma, cervical cancer, thyroid cancer, head and neck cancer, and adrenal cancer. The expression of ROR1 in such cancers is associated with poor prognosis in cancer patients and is known to affect cancer metastasis. It has been shown that mice injected with cancer cells that inhibit ROR1 expression have increased survival time and reduced metastasis.

[0004] Therefore, ROR1, which is specifically expressed in cancer cells, can be an effective target for cancer therapy, and it is necessary to develop antibodies that specifically recognize ROR1. In addition, when developing therapeutic antibodies, it is also very important to have not only target specificity and binding affinity, but also favorable properties related to production, storage, and administration, such as physicochemical properties (such as stability and developability).

[0005] Multispecific antibodies targeting two or more antigens are being developed in various types and forms, and are expected to be superior therapeutic antibodies in terms of therapeutic effects compared to single-target antibodies. Summary of the invention

[0006] Technical issues

[0007] Provided herein is a novel anti-ROR1 antibody or an antigen-binding fragment thereof, a bispecific antibody comprising the same, and uses thereof.

[0008] The anti-ROR1 antibody or antigen-binding fragment thereof may comprise:

[0009] HCDR1, comprising the amino acid sequence of SEQ ID NO: 1;

[0010] HCDR2, comprising the amino acid sequence of SEQ ID NO: 2;

[0011] HCDR3, comprising the amino acid sequence of SEQ ID NO: 3;

[0012] LCDR1, comprising the amino acid sequence of SEQ ID NO:4;

[0013] LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and

[0014] LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0015] The anti-ROR1 antibody or antigen-binding fragment thereof may comprise:

[0016] a heavy chain complementarity determining region (CDR) comprising: HCDR1 having the amino acid sequence of SEQ ID NO: 1, HCDR2 having the amino acid sequence of SEQ ID NO: 2, and HCDR3 having the amino acid sequence of SEQ ID NO: 3, or a heavy chain variable region comprising the heavy chain CDR; and

[0017] A light chain complementarity determining region (CDR) comprising: LCDR1 having the amino acid sequence of SEQ ID NO:4, LCDR2 having the amino acid sequence of SEQ ID NO:5 and LCDR3 having the amino acid sequence of SEQ ID NO:6, or a light chain variable region comprising the light chain CDR.

[0018] In one embodiment, 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 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, but is not limited thereto.

[0019] In another aspect, a bispecific antibody comprising the anti-ROR1 antibody or an antigen-binding fragment thereof is provided.

[0020] More specifically, provided is a bispecific antibody or an antigen-binding fragment thereof, which comprises: a first targeting portion that targets (specifically binds to and / or recognizes) a first antigen; and a second targeting portion that targets (specifically binds to and / or recognizes) a second antigen.

[0021] wherein the first antigen and the second antigen are different from each other, and

[0022] One of the first targeting moiety and the second targeting moiety comprises the anti-ROR1 antibody or an antigen-binding fragment thereof.

[0023] In the bispecific antibody or antigen-binding fragment thereof according to one embodiment, one of the first antigen and the second antigen may be ROR1, and the other may be selected from the group consisting of cancer cell-expressing antigens, immune checkpoint proteins and / or T cell surface antigens, such as, but not limited to, the group consisting of 4-1BB and B7-H3.

[0024] In another aspect, a conjugate is provided, comprising (1) an anti-ROR1 antibody or an antigen-binding fragment thereof, or a bispecific antibody, and (2) at least one selected from the group consisting of a useful polymer, a label, a drug, etc. The drug may be a cytotoxic drug, such as an anticancer drug.

[0025] In another aspect, a pharmaceutical composition is provided, comprising: an anti-ROR1 antibody or an antigen-binding fragment thereof; a bispecific antibody; and / or a conjugate. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutical composition may have anti-cancer activity.

[0026] In another aspect, a pharmaceutical composition is provided, comprising: an anti-ROR1 antibody or an antigen-binding fragment thereof; a bispecific antibody; and / or a conjugate, for preventing and / or treating cancer. The cancer may be a cancer expressing ROR1.

[0027] On the other hand, a method for preventing and / or treating cancer is provided, comprising administering a pharmaceutically effective amount of the anti-ROR1 antibody or its antigen-binding fragment; the bispecific antibody; and / or the conjugate to a subject in need thereof. The method may also include identifying (diagnosing) a subject in need of preventing and / or treating cancer before the administering step.

[0028] In another aspect, provided is a use of an anti-ROR1 antibody or an antigen-binding fragment thereof; a bispecific antibody; and / or a conjugate for preventing and / or treating cancer and / or for preparing a medicament (anticancer agent) for preventing and / or treating cancer.

[0029] In another aspect, provided is a composition for detecting ROR1, comprising an anti-ROR1 antibody or an antigen-binding fragment thereof.

[0030] In another aspect, a method for detecting ROR1 in a biological sample is provided, the method comprising the step of contacting the biological sample with an anti-ROR1 antibody or an antigen-binding fragment thereof.

[0031] In another aspect, a composition for diagnosing cancer is provided, comprising the anti-ROR1 antibody or antigen-binding fragment thereof. The cancer may be a cancer expressing ROR1.

[0032] In another aspect, a method for diagnosing cancer in a biological sample or a subject derived from the biological sample or for providing cancer diagnosis information is provided, the method comprising the step of contacting an anti-ROR1 antibody or an antigen-binding fragment thereof with the biological sample.

[0033] Technical Solution

[0034] Provided are a novel anti-ROR1 antibody, a bispecific antibody comprising the same, and anti-cancer use thereof.

[0035] Definition of terms

[0036] Throughout the present specification, the expression that a polynucleotide (which can be used interchangeably with a gene or a nucleic acid molecule) or a polypeptide (which can be used interchangeably with a protein and can refer to a CDR, a variable region, a heavy chain, a light chain and / or an antibody depending on the context) "comprises a specific nucleic acid sequence or an amino acid sequence" or "consists of or is represented by a specific nucleic acid sequence or an amino acid sequence" is intended to indicate that the polynucleotide or polypeptide must contain a specific nucleic acid or amino acid sequence, and may contain a sequence substantially equivalent to the specific nucleic acid or amino acid sequence, which has changes (deletions, substitutions, modifications and / or additions) that are consistent with maintaining the original and / or intended function of the polynucleotide or polypeptide (or does not exclude the possibility of such variations).

[0037] In one embodiment, the expression that a polynucleotide or polypeptide "comprises a specific nucleic acid sequence or amino acid sequence" or "consists of or is represented by a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide (i) substantially comprises the specific nucleic acid or amino acid sequence, or (ii) consists of or substantially comprises a nucleic acid or amino acid sequence having 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 sequence identity with the specific nucleic acid or amino acid sequence, and retains the original function and / or intended function.

[0038] As used herein, the term "sequence homology" or "sequence identity" refers to the degree of match between given nucleic acid or amino acid sequences and can be expressed as a percentage (%). For nucleic acid sequence homology, for example, algorithms such as BLAST or Pearson's FASTA can be used to determine homology or identity. Based on these algorithms, programs called BLASTN or BLASTX have been developed (see: http: / / www.ncbi.nlm.nih.gov).

[0039] Herein, the statement that a protein or polypeptide "comprises a specific amino acid sequence, consists of specific amino acids, or is represented by specific amino acids" may mean a situation where the amino acid sequence is substantially comprised and / or insignificant changes (e.g., substitution, deletion and / or addition of amino acid residues) that do not affect the original activity and / or expected activity (e.g., binding affinity to an antigen, anti-cancer activity such as inhibiting cancer cell growth, inducing cancer cell apoptosis, etc.) are introduced into the amino acid sequence.

[0040] Unless otherwise defined, amino acid positions referred to in the present specification are counted from the N-terminus of the reference amino acid sequence.

[0041] As used herein, the term "antibody" generally refers to a protein that specifically binds to a specific antigen, and is intended to cover proteins produced by the immune system in response to antigenic stimulation or proteins that are chemically synthesized or recombinantly produced, and the type is not particularly limited. Antibodies can be any isotype or antigen-binding fragment of a complete immunoglobulin that can compete with complete antibodies for binding to target antigens. The antibody itself is also an antigen-binding protein. A complete antibody can generally include at least two full-length heavy chains and two full-length light chains. In the present specification, antibodies or their antigen-binding fragments can be non-naturally produced, such as recombinantly or synthetically produced. For example, antibodies or their antigen-binding fragments can be produced by hybridoma technology, recombinant DNA technology, or enzymatic or chemical cleavage of complete antibodies. Unless otherwise specified in the present specification, the term "antibody" covers antibodies including two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments and / or mutants thereof. Antibodies can be animal antibodies (e.g., mouse antibodies), chimeric antibodies (e.g., mouse-human antibodies), humanized antibodies or human antibodies. Antibodies can be monospecific (or monoclonal antibodies) or multispecific (or polyclonal antibodies).

[0042] In the antibodies or antigen-binding fragments thereof provided herein, the portions other than the defined heavy chain CDR and light chain CDR or heavy chain variable region and light chain variable region can be derived from all immunoglobulin isotypes (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3 or IgG4), IgM, etc.), for example, derived from the framework regions of all these isotypes of immunoglobulins, and / or derived from the constant regions of light and heavy chains. In one embodiment, the antibodies provided in this specification can be human IgG type, such as IgG1, IgG2, IgG3 or IgG4, but are not limited thereto.

[0043] A complete antibody (e.g., an IgG-type antibody) has a structure with two full-length light chains and two full-length heavy chains with disulfide bonds therebetween. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is of γ, μ, α, δ, or ε type, and the subclasses include γ1, γ2, γ3, γ4, α1, or α2. The constant region of a light chain is of κ or λ type.

[0044] In one embodiment, the constant region of IgG (e.g., human IgG1) can be wild type. In another embodiment, the constant region of IgG can have mutations introduced for a specific purpose. For example, the constant region of IgG based on human IgG1 can include known mutations, such as N297A mutation (wherein the 297th amino acid residue N is replaced by A), and such variants can exhibit reduced antibody-dependent cellular cytotoxicity (ADCC) compared to the wild type.

[0045] The term "heavy chain" is to be interpreted as including a full-length heavy chain and fragments thereof, wherein a full-length heavy chain includes a variable region domain VH having sufficient variable region sequence to confer specificity for an antigen, three constant region domains C H1 , C H2 and C H3 The VH domain is located at the N-terminus of the heavy chain, the CH domain is located at the C-terminus, and the C H3 The domain is closest to the C-terminus. The heavy chain may be selected from the isotypes of IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), and IgM and IgE.

[0046] In addition, the term "light chain" is interpreted as including full-length light chains and fragments thereof, wherein the full-length light chain includes a variable region domain VL and a constant region domain CL having sufficient variable region 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. The type of light chain may include a kappa or lambda chain.

[0047] The term "complementarity determining region" (CDR) refers to a portion of an antibody variable region that confers binding specificity to an antigen, representing the amino acid sequence of the hypervariable region of the heavy and light chains of an immunoglobulin. Both the heavy and light chains may contain three CDRs (HCDR1 (or CDRH1), HCDR2 (or CDRH2), HCDR3 (or CDRH3), LCDR1 (or CDRL1), LCDR2 (or CDRL2), and LCDR3 (or CDRL3)). CDRs may provide key contact residues for antibody binding to an antigen or epitope. Throughout the specification, the term "specific binding" or "specific recognition" is understood to have the same meaning as is generally known to those skilled in the art, meaning that antigens and antibodies specifically interact to elicit an immune response.

[0048] The complementarity determining regions (CDRs) described herein can be determined according to the definition of the Kabat system.

[0049] Unless specifically stated otherwise, the term "antibody" will be understood to encompass antigen-binding fragments of antibodies that retain the ability to bind to the antigen.

[0050] The term "antigen-binding fragment" as used herein refers to any type of polypeptide that includes a portion capable of binding to an antigen (e.g., the 6 CDRs defined herein), and for example, may be selected from the group consisting of scFv, scFv-Fc, (scFv)2, Fab, Fab', F(ab')2, bispecific antibodies and small antibodies, but is not limited thereto.

[0051] In the antigen-binding fragment, Fab has the variable regions of the light chain and the heavy chain, the constant region of the light chain and the first constant region of the heavy chain (CH1 ) structure, retaining an antigen binding site.

[0052] Fab' differs from Fab in that the former includes the heavy chain C H1 The C-terminus of the domain has a hinge region with at least one cysteine ​​residue.

[0053] F(ab')2 antibodies are formed by disulfide bridges of cysteine ​​residues in the hinge region of Fab'.

[0054] Fv is the smallest antibody fragment consisting only of the heavy chain variable region and the light chain variable region. Recombinant techniques for producing Fv fragments are well known in the art. Double-chain Fv includes a heavy chain variable region and a light chain variable region connected to each other by non-covalent bonds. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region, which are connected to each other by covalent bonds via a peptide linker or directly (without a linker) at the C-terminus to have a dimer structure. scFv-Fc is a conjugate in which Fc is connected to scFv. scFv-Fc-scFv is a structure in which scFv is connected to both ends (N-terminus and C-terminus) of Fc. Miniantibodies are constructed to have a C-terminus. H3 Linked scFv. A bispecific antibody contains two scFv molecules.

[0055] Antigen-binding fragments can be obtained by chemical or enzymatic cleavage using proteases (e.g., Fab can be obtained by restricted cleavage of whole antibodies with papain, and F(ab′)2 fragments can be obtained by cleavage with pepsin), or can be prepared by using genetic recombination techniques.

[0056] The term "hinge region" refers to the C H1 and C H2 The region between domains that serves to provide flexibility to the antigen-binding site in an antibody.

[0057] As used herein, the term "Fc" region refers to the region of an antibody that includes the C H2 and C H3 domain, and in some cases, may include a heavy chain fragment of the C H2 and C H3 The N-terminal end of the domain (i.e., C H2 The two Fc regions can be linked by two or more disulfide bonds and / or C H3 The domains are connected to each other by hydrophobic interactions.

[0058] Herein, "bivalent antibodies or bivalent antigen-binding fragments" refer to those that include two antigen-binding sites, each of which includes six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3). The two antigen-binding sites included in a bivalent antibody or bivalent antigen-binding fragment can have the same antigen specificity, or they can have dual specificity, binding to two different antigens. Herein, a "monovalent antibody or monovalent antigen-binding fragment" includes one antigen-binding site that includes six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3).

[0059] As used herein, a "bispecific" antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment thereof that targets two antigens, and is a hybrid antibody that binds to two different epitopes. The two epitopes of a bispecific antibody or antigen-binding fragment can be located on the same or different target proteins (antigens).

[0060] The antibodies provided herein can be monoclonal antibodies. Monoclonal antibodies can be manufactured using methods well known in industry, such as using phage display technology. Alternatively, antibodies can be produced using conventional methods as monoclonal antibodies of animal origin (e.g., from mice, rats, rabbits, etc.), or manufactured as humanized antibodies by standard methods.

[0061] As used herein, the term "antigen" or "immunogen" refers to a molecule or a portion of a molecule that can bind to an antigen binding protein (e.g., an antibody or an immunologically functional antigen binding fragment thereof) and can be used to generate antibodies that can bind to an antigen in an animal. An antigen can include one or more epitopes that can interact with other antibodies or fragments thereof.

[0062] Herein, "epitope" is a part of a molecule that is bound or recognized by an antibody or antigen binding fragment, and includes any determinant that can specifically bind to an antigen binding protein (e.g., an antibody or a T cell receptor). The amino acid residues responsible for the epitope can be continuous or discontinuous. For example, they may not be continuous in the polypeptide sequence, but, in one aspect of the whole antibody molecule, they can be located in adjacent positions on different conformations, and are therefore bound by an antigen binding protein (e.g., a conformational epitope). Epitope determinants can be chemically activated groups present on the surface by molecules, such as amino acids, sugar chains, phosphoryl and / or sulfonyl groups, or they can have specific three-dimensional structural features and / or specific charge characteristics.

[0063] Herein, an antibody (e.g., CDR, variable region or heavy chain / light chain, antigen-binding fragment, etc.) "comprising (including) or consisting of a specific amino acid sequence" may refer to all cases in which the amino acid sequence is substantially contained, and / or insignificant mutations (e.g., substitution, deletion and / or addition of amino acid residues) that do not affect the activity of the antibody (e.g., antigen-binding affinity, pharmacological activity, etc.) are introduced into the amino acid sequence. SUMMARY OF THE INVENTION

[0065] According to one embodiment, an anti-ROR1 antibody or an antigen-binding fragment thereof is provided, comprising:

[0066] HCDR1, comprising the amino acid sequence of SEQ ID NO: 1;

[0067] HCDR2, comprising the amino acid sequence of SEQ ID NO: 2;

[0068] HCDR3, comprising the amino acid sequence of SEQ ID NO: 3;

[0069] LCDR1, comprising the amino acid sequence of SEQ ID NO:4;

[0070] LCDR2 comprising the amino acid sequence of SEQ ID NO:5; and

[0071] LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0072] In another aspect, an anti-ROR1 antibody or an antigen-binding fragment thereof is provided, 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.

[0073] In another aspect, provided is a nucleic acid molecule encoding the anti-ROR1 antibody or antigen-binding fragment thereof, a recombinant vector carrying the same, and / or a recombinant cell comprising the recombinant vector.

[0074] On the other hand, a pharmaceutical composition for preventing or treating cancer is provided, the composition comprising the above-mentioned anti-ROR1 antibody or its antigen-binding fragment and a pharmaceutically acceptable excipient. On the other hand, a method for preventing or treating cancer is provided, the method comprising the step of administering a pharmaceutically effective amount of the above-mentioned anti-ROR1 antibody or its antigen-binding fragment to a subject in need of preventing or treating cancer. On the other hand, a use of the above-mentioned anti-ROR1 antibody or its antigen-binding fragment for preventing or treating cancer or for preparing a pharmaceutical composition for preventing or treating cancer is provided.

[0075] In the pharmaceutical composition, method and / or use, the cancer may be a cancer expressing ROR1.

[0076] In another aspect, provided is a composition for detecting ROR1, comprising the anti-ROR1 antibody or antigen-binding fragment thereof; a method for detecting ROR1 using the same; and use of the composition for detecting ROR1.

[0077] In another aspect, a composition for diagnosing cancer is provided, the composition comprising the anti-ROR1 antibody or the antigen-binding fragment thereof.

[0078] In the composition for diagnosing cancer, the cancer may be a cancer expressing ROR1.

[0079] In another aspect, an anti-ROR1 / anti-4-1BB bispecific antibody is provided, comprising:

[0080] an anti-ROR1 antibody or an antigen-binding fragment thereof; and

[0081] Anti-4-1BB antibody or antigen-binding fragment thereof,

[0082] Wherein, the anti-ROR1 antibody or its antigen-binding fragment comprises: HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5 and LCDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0083] In the above-mentioned anti-ROR1 / anti-4-1BB bispecific antibody, the anti-ROR1 antibody or its antigen-binding fragment 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.

[0084] In the above-mentioned anti-ROR1 / anti-4-1BB bispecific antibody, the anti-4-1BB antibody or its antigen-binding fragment may comprise:

[0085] HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, 14 or 15,

[0086] HCDR2 comprises the amino acid sequence of SEQ ID NO: 16, 17 or 18,

[0087] HCDR3 comprises the amino acid sequence of SEQ ID NO: 19, 20, 21, 22 or 23,

[0088] LCDR1, comprising the amino acid sequence of SEQ ID NO:24 or 25,

[0089] LCDR2 comprises the amino acid sequence of SEQ ID NO: 26 or 27, and

[0090] LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 or 29.

[0091] In the above-mentioned anti-ROR1 / anti-4-1BB bispecific antibody, the anti-4-1BB antibody or its antigen-binding fragment may comprise:

[0092] (1) a heavy chain complementarity determining region selected from the following:

[0093] (a) a HCDR1 comprising the amino acids of SEQ ID NO: 13, a HCDR2 comprising the amino acids of SEQ ID NO: 16, and a HCDR3 comprising the amino acids of SEQ ID NO: 19,

[0094] (b) a HCDR1 comprising the amino acids of SEQ ID NO: 13, a HCDR2 comprising the amino acids of SEQ ID NO: 16, and a HCDR3 comprising the amino acids of SEQ ID NO: 20,

[0095] (c) a HCDR1 comprising the amino acids of SEQ ID NO: 13, a HCDR2 comprising the amino acids of SEQ ID NO: 16, and a HCDR3 comprising the amino acids of SEQ ID NO: 21,

[0096] (d) a HCDR1 comprising the amino acids of SEQ ID NO: 14, a HCDR2 comprising the amino acids of SEQ ID NO: 17, and a HCDR3 comprising the amino acids of SEQ ID NO: 22, and

[0097] (e) a HCDR1 comprising the amino acids of SEQ ID NO: 15, a HCDR2 comprising the amino acids of SEQ ID NO: 18, and a HCDR3 comprising the amino acids of SEQ ID NO: 23, and

[0098] (2) a light chain complementary determining region selected from the following:

[0099] (a) LCDR1 comprising the amino acids of SEQ ID NO: 24, LCDR2 comprising the amino acids of SEQ ID NO: 26, and LCDR3 comprising the amino acids of SEQ ID NO: 28, and

[0100] (b) LCDR1 comprising the amino acids of SEQ ID NO:24, LCDR2 comprising the amino acids of SEQ ID NO:26, and LCDR3 comprising the amino acids of SEQ ID NO:28.

[0101] In the above-mentioned anti-ROR1 / anti-4-1BB bispecific antibody, the anti-4-1BB antibody or its antigen-binding fragment may comprise:

[0102] 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

[0103] The light chain variable region comprises the amino acid sequence of SEQ ID NO:42, 43, 44, 45, 46 or 47.

[0104] On the other hand, provided are a nucleic acid molecule encoding the anti-ROR1 / anti-4-1BB bispecific antibody or an antigen-binding fragment thereof, a recombinant vector carrying the same, and / or a recombinant cell comprising the recombinant vector.

[0105] On the other hand, a pharmaceutical composition for preventing or treating cancer is provided, the composition comprising the above-mentioned anti-ROR1 / anti-4-1BB bispecific antibody or its antigen-binding fragment and a pharmaceutically acceptable excipient. On the other hand, a method for preventing or treating cancer is provided, the method comprising administering a pharmaceutically effective amount of the above-mentioned anti-ROR1 / anti-4-1BB bispecific antibody or its antigen-binding fragment to a subject in need of preventing or treating cancer. On the other hand, a use of the above-mentioned anti-ROR1 / anti-4-1BB bispecific antibody or its antigen-binding fragment for preventing or treating cancer or for preparing a pharmaceutical composition for preventing or treating cancer is provided.

[0106] In the pharmaceutical composition, method and / or use, the cancer may be a cancer expressing ROR1.

[0107] In another aspect, an anti-ROR1 / anti-B7-H3 bispecific antibody is provided, comprising:

[0108] an anti-ROR1 antibody or an antigen-binding fragment thereof; and

[0109] an anti-B7-H3 antibody or an antigen-binding fragment thereof,

[0110] Wherein, the anti-ROR1 antibody or its antigen-binding fragment comprises: HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5 and LCDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0111] In the above anti-ROR1 / anti-B7-H3 bispecific antibody, the anti-ROR1 antibody or its antigen-binding fragment 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.

[0112] In the above anti-ROR1 / anti-B7-H3 bispecific antibody, the anti-B7-H3 antibody or antigen-binding fragment thereof may comprise:

[0113] HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 51;

[0114] HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 57;

[0115] HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 62;

[0116] LCDR1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 to 67;

[0117] LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 73; and

[0118] LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74 to 79.

[0119] In the above anti-ROR1 / anti-B7-H3 bispecific antibody, the anti-B7-H3 antibody or antigen-binding fragment thereof may comprise:

[0120] (i) a heavy chain complementarity determining region selected from the group consisting of:

[0121] (a) a HCDR1 comprising the amino acids of SEQ ID NO: 48, a HCDR2 comprising the amino acids of SEQ ID NO: 52, and a HCDR3 comprising the amino acids of SEQ ID NO: 58,

[0122] (b) a HCDR1 comprising the amino acids of SEQ ID NO: 49, a HCDR2 comprising the amino acids of SEQ ID NO: 53, and a HCDR3 comprising the amino acids of SEQ ID NO: 59,

[0123] (c) a HCDR1 comprising the amino acids of SEQ ID NO: 50, a HCDR2 comprising the amino acids of SEQ ID NO: 54, and a HCDR3 comprising the amino acids of SEQ ID NO: 60,

[0124] (d) a HCDR1 comprising the amino acids of SEQ ID NO: 48, a HCDR2 comprising the amino acids of SEQ ID NO: 55, and a HCDR3 comprising the amino acids of SEQ ID NO: 61, and

[0125] (e) a HCDR1 comprising the amino acids of SEQ ID NO: 51, a HCDR2 comprising the amino acids of SEQ ID NO: 56, and a HCDR3 comprising the amino acids of SEQ ID NO: 62, and

[0126] (f) a HCDR1 comprising the amino acids of SEQ ID NO: 50, a HCDR2 comprising the amino acids of SEQ ID NO: 57, and a HCDR3 comprising the amino acids of SEQ ID NO: 60, and

[0127] (ii) a light chain complementarity determining region selected from the group consisting of:

[0128] (a) LCDR1 comprising the amino acids of SEQ ID NO: 63, LCDR2 comprising the amino acids of SEQ ID NO: 68, and LCDR3 comprising the amino acids of SEQ ID NO: 74,

[0129] (b) LCDR1 comprising the amino acids of SEQ ID NO:64, LCDR2 comprising the amino acids of SEQ ID NO:69, and LCDR3 comprising the amino acids of SEQ ID NO:75;

[0130] (c) LCDR1 comprising the amino acids of SEQ ID NO:65, LCDR2 comprising the amino acids of SEQ ID NO:70, and LCDR3 comprising the amino acids of SEQ ID NO:76;

[0131] (d) LCDR1 comprising the amino acids of SEQ ID NO:66, LCDR2 comprising the amino acids of SEQ ID NO:71, and LCDR3 comprising the amino acids of SEQ ID NO:77;

[0132] (e) LCDR1 comprising the amino acids of SEQ ID NO:67, LCDR2 comprising the amino acids of SEQ ID NO:72, and LCDR3 comprising the amino acids of SEQ ID NO:78; and

[0133] (f) LCDR1 comprising the amino acids of SEQ ID NO:65, LCDR2 comprising the amino acids of SEQ ID NO:73, and LCDR3 comprising the amino acids of SEQ ID NO:76.

[0134] In the above anti-ROR1 / anti-B7-H3 bispecific antibody, the anti-B7-H3 antibody or antigen-binding fragment thereof may comprise:

[0135] A heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 80 to 91; and

[0136] The light chain variable region comprises the amino acid sequence of SEQ ID NO:92 to 103.

[0137] On the other hand, provided are a nucleic acid molecule encoding the anti-ROR1 / anti-B7-H3 bispecific antibody or an antigen-binding fragment thereof, a recombinant vector carrying the same, and / or a recombinant cell comprising the recombinant vector.

[0138] On the other hand, a pharmaceutical composition for preventing or treating cancer is provided, the composition comprising the above-mentioned anti-ROR1 / anti-B7-H3 bispecific antibody or its antigen-binding fragment and a pharmaceutically acceptable excipient. On the other hand, a method for preventing or treating cancer is provided, the method comprising the step of administering a pharmaceutically effective amount of the above-mentioned anti-ROR1 / anti-B7-H3 bispecific antibody or its antigen-binding fragment to a subject in need of preventing or treating cancer. On the other hand, a use of the above-mentioned anti-ROR1 / anti-B7-H3 bispecific antibody or its antigen-binding fragment for preventing or treating cancer or for preparing a pharmaceutical composition for preventing or treating cancer is provided.

[0139] In the pharmaceutical composition, method and / or use, the cancer may be a cancer expressing ROR1, B7-H3, or both.

[0140] In another aspect, provided is an antibody-drug conjugate (ADC) comprising the anti-ROR1 / anti-B7-H3 bispecific antibody or an antigen-binding fragment thereof linked to a cytotoxic drug.

[0141] The antibody-drug conjugate may have an antibody-linker-drug structure.

[0142] In the antibody-drug conjugate, the cytotoxic drug may be an anticancer agent.

[0143] On the other hand, a pharmaceutical composition for preventing or treating cancer is provided, the composition comprising the above-mentioned antibody-drug conjugate and a pharmaceutically acceptable excipient. On the other hand, a method for preventing or treating cancer is provided, the method comprising the step of administering a pharmaceutically effective amount of the above-mentioned anti-ROR1 / anti-B7-H3 bispecific antibody or its antigen-binding fragment to a subject in need of preventing or treating cancer. On the other hand, a use of the above-mentioned anti-ROR1 / anti-B7-H3 bispecific antibody or its antigen-binding fragment for preventing or treating cancer or for preparing a pharmaceutical composition for preventing or treating cancer is provided.

[0144] In the pharmaceutical composition, method and / or use, the cancer may be a cancer expressing ROR1, B7-H3, or both.

[0145] Hereinafter, a detailed description of the present disclosure will be given:

[0146] Anti-ROR1 antibodies or antigen-binding fragments thereof

[0147] On the one hand, an anti-ROR1 antibody or an antigen-binding fragment thereof that binds to ROR1 is provided. The anti-ROR1 antibody or antigen-binding fragment thereof provided herein can target (specifically recognize and / or bind) human ROR1 (e.g., extracellular domain). The antibody or antigen-binding fragment thereof not only has inhibitory activity against ROR1, but also enhances stability and developability, thereby enjoying the advantage of allowing economical production of more effective and safer antibody drugs compared to conventional therapeutic antibodies.

[0148] The anti-ROR1 antibody or antigen-binding fragment thereof may comprise:

[0149] HCDR1, comprising the amino acid sequence of SEQ ID NO: 1,

[0150] HCDR2 comprises the amino acid sequence of SEQ ID NO: 2,

[0151] HCDR3 comprises the amino acid sequence of SEQ ID NO: 3,

[0152] LCDR1, comprising the amino acid sequence of SEQ ID NO:4,

[0153] LCDR2 comprises the amino acid sequence of SEQ ID NO:5, and

[0154] LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0155] Here, the complementarity determining region (CDR) can be determined based on the CDR definition according to the Kabat system.

[0156] In one embodiment, the amino acid sequences (N→C) of the six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) that may be included in the anti-ROR1 antibodies or antigen-binding fragments thereof provided herein are summarized as follows:

[0157] HCDR1:NYDMS (SEQ ID NO:1)

[0158] HCDR2:AIYHSGSSKYYADSVKG(SEQ ID NO:2)

[0159] HCDR3:GGSGAWDTGFDY(SEQ ID NO:3)

[0160] LCDR1:SGSSSSNIGSNDVS(SEQ ID NO:4)

[0161] LCDR2:YENNRPS (SEQ ID NO:5)

[0162] LCDR3:GAWDDSLSGYV(SEQ ID NO:6)

[0163] (HCDR1, HCDR2 and HCDR3 represent heavy chain complementarity determining regions, and LCDR1, LCDR2 and LCDR3 represent light chain complementarity determining regions.)

[0164] The anti-ROR1 antibody or antigen-binding fragment thereof may comprise:

[0165] A heavy chain complementarity determining region (CDR) or a heavy chain variable region comprising the heavy chain CDR, wherein the heavy chain CDR comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0166] A light chain complementary determining region (CDR) or a light chain variable region comprising the light chain CDR, wherein the light chain CDR comprises: 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.

[0167] The heavy chain variable region and light chain variable region of the anti-ROR1 antibody or antigen-binding fragment thereof may be derived from an IgG-derived antibody or antigen-binding fragment thereof. They may comprise wild-type sequences, such as framework regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and / or LFR4) into which mutations are introduced.

[0168] In an embodiment, mutations can be used to stabilize and / or form disulfide bonds. For example, the antigen binding fragment may have one or more (e.g., one) G(Gly)→C(Cys) mutations (wherein G is replaced by C) introduced into the framework region of the variable domain in the antibody, but is not limited thereto. In a specific embodiment, if the ROR1 antibody is a complete IgG antibody or a portion thereof (e.g., Fab or Fab-Fc), the heavy chain variable region and the light chain variable region contained therein may be derived from wild-type IgG. In another embodiment, if the anti-ROR1 antibody is an antigen binding fragment of an anti-ROR1 antibody (e.g., scFv, etc.), the heavy chain variable region and the light chain variable region contained therein may have a G→C mutation introduced into its framework region.

[0169] In one embodiment, 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 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, but is not limited thereto.

[0170] In certain cases (eg, when recombinantly produced), the heavy chain variable region and / or the light chain variable region may further comprise an appropriate signal sequence at the N-terminus.

[0171] The amino acid sequences of the heavy chain variable region and the light chain variable region that may be included in the anti-ROR1 antibodies or antigen-binding fragments thereof provided herein are exemplified in Table 1 below:

[0172] Table 1

[0173]

[0174]

[0175] (In Table 1, CDR1, CDR2 and CDR3 of the heavy and light chains are underlined in this order)

[0176] The antigen receptor tyrosine kinase-like orphan receptor (ROR1) targeted by the anti-ROR1 antibody or its antigen-binding fragment provided herein is a transmembrane protein belonging to the receptor tyrosine kinase (RTK) family. The anti-ROR1 antibody or its antigen-binding fragment can recognize (bind) a portion of the ROR1 protein present on the cell membrane or an extracellular domain that is not present on the cell membrane. The ROR1 protein can be a mammalian ROR1 protein, such as a human ROR1 protein. The amino acid sequence of the human ROR1 protein can be found in NCBI Accession No. NP_005003.2 (937aa), and the nucleic acid sequence encoding it can be found in NCBI Accession No. NM_005012.3, but is not limited thereto. In another embodiment, the ROR1 protein can be represented by a mouse ROR1 protein (GenBank Accession No. BAA75480.1).

[0177] The antibodies or antigen-binding fragments thereof provided herein can have an antigen-binding affinity of 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, e.g., 0.001 nM-5 nM, or 0.001 nM-1 nM, as measured, e.g., by surface plasmon resonance (SPR) (K D ) or EC measured by ELISA 50 , but not limited to this.

[0178] The anti-ROR1 antibodies or antigen-binding fragments thereof provided herein have functions such as ROR1 inhibition (e.g., inhibition of ROR1 expression and / or activity), targeting of cancer cells by specific binding to cancer cells (e.g., cancer cells expressing ROR1), and anti-cancer activity (e.g., inducing apoptosis of cancer cells, inhibiting proliferation of cancer cells, etc.). Therefore, the anti-ROR1 antibodies or antigen-binding fragments thereof may have advantageous findings in the prevention and / or treatment of cancer.

[0179] In another aspect, provided is a composition or a kit for detecting ROR1, the composition or the kit comprising at least one selected from the group consisting of an anti-ROR1 antibody or an antigen-binding fragment thereof, and a nucleic acid molecule encoding the same.

[0180] On the other hand, a method for detecting ROR1 in a biological sample is provided, the method comprising contacting the biological sample in which ROR1 is to be detected with at least one selected from the group consisting of an anti-ROR1 antibody or its antigen-binding fragment and a nucleic acid molecule encoding it. After the contacting step, the method may further comprise a step of measuring the binding in the biological sample treated with (contacted with) the antibody, antigen-binding fragment and / or nucleic acid molecule. In one embodiment, the method may be performed in vitro or in vivo.

[0181] In another aspect, a composition or kit for cancer diagnosis is provided, the composition or kit comprising at least one selected from the group consisting of an anti-ROR1 antibody or an antigen-binding fragment thereof and a nucleic acid molecule encoding the same. The cancer may be a cancer expressing ROR1.

[0182] On the other hand, a method for diagnosing cancer in a biological sample or in a subject from which the biological sample is derived or separated, or for providing cancer diagnostic information, is provided, the method comprising contacting the biological sample with one or more selected from the group consisting of an anti-ROR1 antibody or its antigen-binding fragment and a nucleic acid molecule encoding it. After the contact step, the method may include measuring the binding in a biological sample treated (contacted) with an antibody, an antigen-binding fragment and / or a nucleic acid molecule. In addition, if binding is detected in a treated (contacted) biological sample, the method may include determining (confirming) that the biological sample or the subject from which the biological sample is derived (or separated) is a cancer patient, or further assessing that such a subject may respond to treatment with an anti-ROR1 antibody or its antigen-binding fragment. The method may be performed in vitro or in vivo. The cancer may be a cancer that expresses ROR1.

[0183] In the compositions and / or methods provided herein for detecting ROR1 and / or diagnosing cancer, the biological sample can be derived from (or isolated from) an animal selected from a mammal including primates (e.g., humans, monkeys, etc.) and rodents (e.g., rats, mice, etc.), and for example, the biological sample can be a cell, tissue, body fluid (e.g., serum), transformed cell, or culture thereof derived from a human, or can be a human-derived cell, tissue, body fluid (e.g., serum).

[0184] In another aspect, a nucleic acid molecule encoding an anti-ROR1 antibody or an antigen-binding fragment thereof is provided.

[0185] Specifically, one embodiment provides a nucleic acid molecule encoding a heavy chain complementarity determining region, a heavy chain variable region or a heavy chain of an anti-ROR1 antibody.

[0186] In another aspect, a nucleic acid molecule encoding a light chain complementarity determining region, a light chain variable region, or a light chain of an anti-ROR1 antibody is provided.

[0187] In another aspect, there is provided a recombinant vector, carrying together or individually:

[0188] (1) a nucleic acid molecule encoding the heavy chain complementary determining region, heavy chain variable region or heavy chain of the anti-ROR1 antibody, and

[0189] (2) Nucleic acid molecules encoding the light chain complementary determining region, light chain variable region or light chain of the anti-ROR1 antibody. The recombinant vector can be an expression vector designed to express a nucleic acid molecule.

[0190] In another aspect, a recombinant cell comprising a recombinant vector is provided.

[0191] In one aspect of the present application, a bispecific antibody or an antigen-binding fragment thereof is provided, which comprises an anti-ROR1 antibody or an antigen-binding fragment thereof.

[0192] More specifically, a bispecific antibody or an antigen-binding fragment thereof is provided, comprising:

[0193] a first targeting moiety that targets (specifically binds to and / or recognizes) a first antigen, and

[0194] a second targeting moiety that targets (specifically binds to and / or recognizes) a second antigen,

[0195] wherein the first antigen and the second antigen are different from each other, and

[0196] One of the first targeting moiety and the targeting moiety comprises the anti-ROR1 antibody or an antigen-binding fragment thereof.

[0197] For example, in a bispecific antibody or antigen-binding fragment thereof, one of the antigens may be ROR1 and the other may be selected from 4-1BB and B7-H3, but is not limited thereto. For example, one of the antigens may be selected from an antigen expressed by a cancer cell, an immune checkpoint protein, or a T cell surface antigen expressed in different types of cancer cells.

[0198] The description of the anti-ROR1 antibody or its antigen-binding fragment provided herein can also be applied to the anti-ROR1 / 4-1BB bispecific antibody, anti-ROR1 / B7-H3 bispecific antibody or its antigen-binding fragment described below. Similarly, the description of the anti-ROR1 / 4-1BB bispecific antibody, anti-ROR1 / B7-H3 bispecific antibody or its antigen-binding fragment can also be applied to the anti-ROR1 antibody or its antigen-binding fragment.

[0199] Anti-ROR1 / anti-4-1BB bispecific antibody

[0200] In one aspect, an anti-ROR1 / anti-4-1BB bispecific antibody is provided, comprising:

[0201] (1) an anti-ROR1 antibody or an antigen-binding fragment thereof as a ROR1 targeting moiety; and

[0202] (2) Anti-4-1BB antibodies or antigen-binding fragments thereof as 4-1BB targeting moieties. Anti-ROR1 / anti-4-1BB bispecific antibodies target (specifically bind to and / or recognize) both ROR1 and 4-1BB.

[0203] The anti-ROR1 antibody or its antigen binding fragment used as a ROR1 targeting portion is a protein that targets (specifically binds and / or recognizes) ROR1. More specifically, the ROR1 targeting portion can be the above-mentioned anti-ROR1 antibody or its antigen binding fragment. The description of the anti-ROR1 antibody or its antigen binding fragment provided above can also be applied to the anti-ROR1 antibody or its antigen binding fragment contained in the anti-ROR1 / anti-4-1BB bispecific antibody.

[0204] The anti-4-1BB antibody or antigen-binding fragment thereof as a 4-1BB targeting moiety may be a protein that targets (specifically binds to and / or recognizes) 4-1BB.

[0205] 4-1BB, also known as CD137 or tumor necrosis factor receptor superfamily member 9 (TNFRSF9), is a member of the TNF-receptor superfamily (TNFRSF), used as a co-stimulatory molecule expressed after activation of innate and adaptive immune cells. 4-1BB plays a key role in regulating the activity of various immune cells. The antigen targeted by the anti-4-1BB antibody or its antigen-binding fragment provided herein can be mammalian 4-1BB, such as human 4-1BB. The amino acid sequence of human 4-1BB can be found under NCBI accession number NP_001552.2, but is not limited thereto.

[0206] For example, an anti-4-1BB antibody or antigen-binding fragment thereof may comprise:

[0207] CDR-H1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 13, 14 or 15;

[0208] HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17 or 18;

[0209] HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, 20, 21, 22 or 23;

[0210] LCDR1, comprising the amino acid sequence of SEQ ID NO: 24 or 25;

[0211] LCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or 27; and

[0212] LCDR3 comprising the amino acid sequence of SEQ ID NO: 28 or 29.

[0213] The amino acid sequences of the CDRs of the anti-4-1BB antibodies or antigen-binding fragments are exemplified in Table 2.

[0214] Table 2

[0215]

[0216]

[0217] For example, an anti-4-1BB antibody or antigen-binding fragment thereof may comprise:

[0218] a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 28;

[0219] a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 28;

[0220] a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 28;

[0221] a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 29;

[0222] a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 29;

[0223] a HCDR1 comprising the amino acid sequence of SEQ ID NO: 13, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 29;

[0224] a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 22, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 24, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 28;

[0225] a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 17, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 22, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 25, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 29;

[0226] HCDR1 comprising the amino acid sequence of SEQ ID NO: 15, HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, HCDR3 comprising the amino acid sequence of SEQ ID NO: 23, 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; or

[0227] HCDR1 comprising the amino acid sequence of SEQ ID NO:15, HCDR2 comprising the amino acid sequence of SEQ ID NO:18, HCDR3 comprising the amino acid sequence of SEQ ID NO:23, 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.

[0228] In another embodiment, the anti-4-1BB antibody or antigen-binding fragment thereof may comprise:

[0229] a heavy chain variable region comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 13, 14 or 15, a HCDR2 having an amino acid sequence of SEQ ID NO: 16, 17 or 18, and a HCDR3 having an amino acid sequence of SEQ ID NO: 19, 20, 21, 22 or 23; and

[0230] The light chain variable region comprises LCDR1 having the amino acid sequence of SEQ ID NO: 24 or 25, LCDR2 having the amino acid sequence of SEQ ID NO: 26 or 27, and LCDR3 having the amino acid sequence of SEQ ID NO: 28 or 29.

[0231] The heavy chain variable region and light chain variable region of anti-4-1BB antibody or its antigen binding fragment can be derived from IgG antibody or its antigen binding fragment.Antibody or its antigen binding fragment may include wild-type sequence, such as framework region (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and / or LFR4), wherein mutation is introduced to stabilize and / or produce disulfide bond.For example, the antigen binding fragment may have one or more (e.g., one) G (Gly) to C (Cys) mutations (G is replaced by C) introduced in the framework region of the variable domain in the antibody, but is not limited thereto.In one embodiment, if the 4-1BB targeting portion is a complete IgG antibody or a portion thereof (e.g., Fab or Fc-Fab), the heavy chain variable region and light chain variable region contained therein may be derived from wild-type IgG.In another embodiment, if the 4-1BB targeting portion is an antigen binding fragment (e.g., scFv) of an anti-4-1BB antibody, the heavy chain variable region and light chain variable region contained therein may have a G to C mutation introduced into the framework region.

[0232] In one embodiment, the anti-4-1BB antibody or antigen-binding fragment thereof may comprise:

[0233] 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

[0234] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46 or 47.

[0235] The amino acid sequences (N→C) of the variable regions of the anti-4-1BB antibodies or antigen-binding fragments thereof are exemplified in Table 3.

[0236] Table 3

[0237]

[0238]

[0239]

[0240] In another embodiment, the anti-4-1BB antibody or antigen-binding fragment thereof may be a single-chain variable fragment (scFv) comprising:

[0241] a heavy chain variable region comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 13, 14 or 15, a HCDR2 having an amino acid sequence of SEQ ID NO: 16, 17 or 18, and a HCDR3 having an amino acid sequence of SEQ ID NO: 19, 20, 21, 22 or 23; and

[0242] The light chain variable region comprises LCDR1 having the amino acid sequence of SEQ ID NO: 24 or 25, LCDR2 having the amino acid sequence of SEQ ID NO: 26 or 27, and LCDR3 having the amino acid sequence of SEQ ID NO: 28 or 29.

[0243] In one embodiment, an anti-4-1BB scFv may comprise:

[0244] 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

[0245] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46 or 47 (more specifically, SEQ ID NO: 45, 46 or 47).

[0246] In this regard, the heavy chain variable region and the light chain variable region may be linked directly or via a peptide linker in any order.

[0247] In anti-4-1BB scFv, the heavy chain variable region and the light chain variable region can be directly connected (e.g., without a joint) or connected in any order through a peptide joint. More specifically, anti-4-1BB scFv can include a light chain variable region and a heavy chain variable region in the order from N-terminus to C-terminus; a heavy chain variable region and a light chain variable region; a light chain variable region, a peptide joint and a heavy chain variable region; or a heavy chain variable region, a peptide joint and a light chain variable region.

[0248] Anti-ROR1 / anti-B7-H3 bispecific antibody

[0249] In one aspect, an anti-ROR1 / anti-B7-H3 bispecific antibody is provided, comprising:

[0250] (1) an anti-ROR1 antibody or an antigen-binding fragment thereof as a ROR1 targeting moiety, and

[0251] (2) Anti-B7-H3 antibodies or antigen-binding fragments thereof as B7-H3 targeting moieties. Anti-ROR1 / anti-B7-H3 bispecifically targets (specifically binds to and / or recognizes) both ROR1 and B7-H3.

[0252] The anti-ROR1 antibody or antigen-binding fragment thereof as a ROR1 targeting portion can be a protein that targets (specifically binds to and / or recognizes) ROR1. More specifically, the ROR1 targeting portion can be the above-mentioned anti-ROR1 antibody or antigen-binding fragment. The description of the anti-ROR1 antibody or antigen-binding fragment thereof provided above can also be applied to the anti-ROR1 antibody or antigen-binding fragment thereof contained in the anti-ROR1 / anti-B7-H3 bispecific antibody.

[0253] The anti-B7-H3 antibody or antigen-binding fragment thereof as a B7-H3 targeting moiety may be a protein that targets (specifically binds to and / or recognizes) B7-H3.

[0254] B7-H3 (B7 homolog 3, CD276) is a transmembrane protein belonging to the immunoglobulin (Ig) superfamily and may include an extracellular domain, a transmembrane domain and / or an intracellular domain. The B7-H3 targeted by an anti-B7-H3 antibody or an antigen-binding fragment thereof may be an extracellular domain present on the cell membrane or not on the cell membrane. B7-H3 may be mammalian B7-H3, such as human B7-H3 (e.g., NCBI Accession No. NP_001019907.1 (534aa)), monkey B7-H3 (e.g., NCBI Accession No. XP_005560056.1), or mouse B7-H3 (e.g., NCBI Accession No. NP_598744.1), but is not limited thereto.

[0255] For example, an anti-B7-H3 antibody or antigen-binding fragment thereof may comprise:

[0256] HCDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 51;

[0257] HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 57;

[0258] HCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 62;

[0259] LCDR1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 to 67;

[0260] LCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 73; and

[0261] LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 74 to 79.

[0262] The amino acid sequences of the CDRs of the anti-B7-H3 antibodies or antigen-binding fragments thereof are exemplified in Table 4.

[0263] Table 4

[0264]

[0265]

[0266] The heavy chain variable region and light chain variable region of the anti-B7-H3 antibody or its antigen-binding fragment can be derived from an IgG-derived antibody or its antigen-binding fragment. They can contain wild-type sequences, such as framework regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and / or LFR4), which have mutations introduced therein. In one embodiment, mutations can be used for stabilization and / or disulfide bond formation. For example, the antigen-binding fragment can have one or more (e.g., one) G (Gly) → C (Cys) mutations (wherein G is replaced by C) introduced into the framework region of the antibody variable domain, but is not limited thereto. In a specific embodiment, if the B7-H3 targeting portion is a complete IgG antibody or a portion thereof (e.g., Fab or Fab-Fc), the heavy chain variable region and light chain variable region contained therein can be derived from wild-type IgG. In another embodiment, if the B7-H3 targeting moiety is an antigen-binding fragment of an anti-ROR1 antibody (eg, scFv, etc.), the heavy chain variable region and light chain variable region contained therein may have a G→C mutation introduced into the framework region thereof.

[0267] In one embodiment, the anti-B7-H3 antibody or antigen-binding fragment thereof comprises:

[0268] a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 80 to 91; and

[0269] A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 92 to 103.

[0270] The sequences of the variable regions of the anti-B7-H3 antibodies or antigen-binding fragments thereof (N→C) are exemplified in Table 5.

[0271] Table 5

[0272]

[0273]

[0274]

[0275] For example, an anti-B7-H3 antibody or antigen-binding fragment thereof may comprise:

[0276] 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;

[0277] 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;

[0278] 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;

[0279] 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;

[0280] 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

[0281] 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;

[0282] In another embodiment, the anti-B7-H3 antibody or antigen-binding fragment thereof may be a single chain variable fragment (scFv) comprising:

[0283] a heavy chain variable region comprising a HCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 51, a HCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 57, and a HCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 58 to 62; and

[0284] A light chain variable region comprising a LCDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 to 67, a LCDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 73, and a LCDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 74 to 79.

[0285] In one embodiment, the anti-B7-H3 scFv may comprise:

[0286] a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 80 to 91 (more specifically, SEQ ID NOs: 86 to 91); and

[0287] A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 92 to 103 (more specifically, SEQ ID NOs: 98 to 103).

[0288] In this regard, the heavy chain variable region and the light chain variable region may be linked directly or via a peptide linker in any order.

[0289] In an embodiment, an anti-B7-H3 scFv may comprise:

[0290] 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;

[0291] 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;

[0292] 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;

[0293] 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;

[0294] 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

[0295] A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 85 or 91 and a light chain variable region having the amino acid sequence of SEQ ID NO: 97 or 103.

[0296] In the anti-B7-H3 scFv, the heavy chain variable region and the light chain variable region can be directly connected (e.g., without a linker) or connected in any order through 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.

[0297] Bispecific antibodies comprising anti-ROR1 antibodies

[0298] In the anti-ROR1 / anti-4-1BB bispecific antibodies and / or anti-ROR1 / anti-B7-H3 bispecific antibodies provided herein, the anti-ROR1 antibody or antigen-binding fragment thereof used as a ROR1 targeting moiety is capable of targeting (specifically binding to and / or recognizing) ROR1.

[0299] In one embodiment, if the ROR1 targeting moiety is a complete IgG antibody or a portion thereof (e.g., Fab or Fab-Fc), the heavy chain variable region and the light chain variable region contained therein may be derived from wild-type IgG. In another embodiment, if the ROR1 targeting moiety is an antigen-binding fragment of an anti-ROR1 antibody (e.g., scFv), the heavy chain variable region and the light chain variable region contained therein may have mutations introduced in the framework region.

[0300] In one embodiment, the anti-ROR1 antibody or antigen-binding fragment thereof contained in the bispecific antibody provided herein can be an antibody in the form of a complete immunoglobulin (e.g., in the form of an IgG comprising 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 can further comprise a peptide linker connecting the heavy chain variable region and the light chain variable region.

[0301] The anti-ROR1 / anti-4-1BB bispecific antibody or anti-ROR1 / anti-B7-H3 bispecific antibody provided herein may comprise at least one copy of a ROR1 targeting moiety and at least one copy of a B7-H3 or 4-1BB targeting moiety, with no particular limitation on the connection form and structure therebetween.

[0302] In one embodiment, the ROR1 targeting moiety and the 4-1BB or B7-H3 targeting moiety within the anti-ROR1 / anti-4-1BB bispecific antibody or the anti-ROR1 / anti-B7-H3 bispecific antibody can both be bivalent antigen-binding antibodies or bivalent antigen-binding fragments (e.g., complete antibodies (e.g., IgG), Fab-Fc or Fab'-Fc dimers connected at Fc, or scFv-Fc-scFv). For example, the bispecific antibody can comprise an anti-ROR1 IgG antibody and an anti-4-1BB or anti-B7-H3 scFv grafted thereto, wherein the scFv is directly connected to the N-terminus or C-terminus of one or both of the heavy chain or light chain of the IgG antibody through a peptide linker or without a linker.

[0303] In another embodiment, one of the ROR1 targeting moiety and the 4-1BB or B7-H3 targeting moiety in the anti-ROR1 / anti-4-1BB or anti-ROR1 / anti-B7-H3 bispecific antibody can be a bivalent antigen or antigen binding fragment (e.g., a complete IgG, a dimer of Fab-Fc or Fab'-Fc connected at the Fc, or a scFv-Fc-scFv), and the other can be a monovalent antibody or antigen binding fragment (e.g., scFv, scFv-Fc, Fab, Fab-Fc, Fab'-Fc, etc.). The monovalent antibody or antigen binding fragment can be directly connected to one or both of the N-terminus and C-terminus of one or both of the heavy or light chains of the bivalent antibody or antigen binding fragment through a peptide linker or without any linker, or connected between the Fc regions. For example, the bispecific antibody may comprise an anti-ROR1 IgG antibody and an anti-4-1BB or anti-B7-H3 scFv grafted thereto, wherein the scFv is directly linked to the N-terminus or C-terminus of one of the two heavy chains or two light chains of the IgG antibody via a peptide linker or without a linker.

[0304] In another embodiment, the bispecific antibody may comprise an anti-ROR1 Fab-Fc and an anti-B7-H3 Fab-Fc, Fab-Fc-scFv, scFv-Fc-scFv or scFv-Fc linked thereto. The scFv may be linked via a peptide linker or directly linked without any linker.

[0305] For example, in one embodiment, the ROR1 targeting moiety in the anti-ROR1 / anti-4-1BB bispecific antibody can be an anti-ROR1 IgG antibody, and the 4-1BB targeting moiety can be an anti-4-1BB scFv.

[0306] For B7-H3 antibodies, when the binding affinity to the antigen is too high, there is a possibility of toxicity. Therefore, for the anti-ROR1 / anti-B7-H3 bispecific antibodies provided herein, it may be preferred to use clones with appropriate binding affinity levels while also having a reduced risk of toxicity targeting B7-H3.

[0307] In another embodiment, the ROR1 targeting moiety and the 4-1BB or B7-H3 targeting moiety in the anti-ROR1 / anti-4-1BB or anti-ROR1 / anti-B7-H3 bispecific antibody can each be a monovalent antibody or antigen binding fragment (e.g., scFv, scFv-Fc, Fab, Fab-Fc, Fab'-Fc, etc.) having one antigen binding site. These antibodies or antigen binding fragments can be connected by a peptide linker or directly connected without any linker, and can be connected to the end of the Fc, or the heavy chain constant region or the light chain constant region, the interior of the heavy chain constant region or the light chain constant region, or the end of the heavy chain variable region or the light chain variable region, or connected between Fc regions.

[0308] In one embodiment, scFv can comprise the variable region of the heavy chain and the variable region of the light chain and an optional linker in any order. For example, scFv can comprise, in order from N-terminus to C-terminus: the variable region of the light chain and the variable region of the heavy chain; the variable region of the heavy chain and the variable region of the light chain; the variable region of the light chain, a peptide linker and the variable region of the heavy chain; or the variable region of the heavy chain, a peptide linker and the variable region of the light chain.

[0309] In the anti-ROR1 / anti-4-1BB bispecific antibody or anti-ROR1 / anti-B7-H3 bispecific antibody provided herein, the light chain variable region, the heavy chain variable region, the scFv and Fc and / or the ROR1 targeting moiety and the 4-1BB or B7-H3 targeting moiety can be connected by a peptide linker. In one embodiment, the peptide linker can be an oligopeptide comprising 1-100 amino acids, particularly 2-50 amino acids, and each amino acid can be any type of amino acid without restriction. Any conventional peptide linker can be used, with or without appropriate modification according to a specific purpose. In certain embodiments, the peptide linker may include residues such as Gly, Asn and / or Ser, and may also include neutral amino acids such as Thr and / or Ala. Suitable amino acid sequences of peptide linkers are well known in the relevant art. The length of the peptide linker can be appropriately determined within the limits of not affecting the function of the polypeptide and / or scFv. For example, the peptide linker can be composed of a total of about 1 to about 100 amino acids, about 2 to about 50 amino acids, or about 5 to about 25 amino acids (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 amino acid independently selected from Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker can be represented by (G m S l )n(where m, l and n represent "G", "S" and "(G m S l)" units are each independently selected from an integer of about 1 to about 10, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). In one embodiment, the peptide linker can be an amino acid, such as (GGGGS)2, (GGGGS)3, (GGGGS)4 or (GS)9, but is not limited thereto.

[0310] Use of anti-ROR1 antibodies and / or bispecific antibodies comprising the same

[0311] The anti-ROR1 antibodies provided herein exhibit inhibitory activity against ROR1 (e.g., inhibition of ROR1 expression and / or activity inhibition). In addition, the anti-ROR1 / anti-4-1BB bispecific antibodies comprising it have ROR1 inhibitory activity and T cell activation, while the anti-ROR1 / anti-B7-H3 bispecific antibodies have the functions of targeting cancer cells (cancer cells expressing ROR1 and / or B7-H3) by specifically binding to cancer cells, anti-cancer activity (e.g., inhibiting cancer cell proliferation and inducing cancer cell death), etc. Therefore, these anti-ROR1 antibodies, anti-ROR1 / anti-4-1BB bispecific antibodies or anti-ROR1 / anti-B7-H3 bispecific antibodies can be effectively used to prevent and / or treat cancer. In addition, the anti-ROR1 / anti-B7-H3 bispecific antibodies provided herein have excellent cellular internalization ability (see Example 3.4), and when used in combination with cytotoxic drugs, such as in antibody-drug conjugates (ADCs), the anti-ROR1 / anti-B7-H3 bispecific antibodies provided herein can achieve excellent cancer cell killing (anti-cancer) effects.

[0312] As used herein, the term "conjugate" refers to a cell binding agent covalently bonded to one or more cytotoxic compound molecules. Here, a "cell binding agent" is a molecule with affinity to a biological target (e.g., a ligand, protein, antibody, or its antigen binding fragment), and is used to guide a bioactive compound to a biological target. In an embodiment of the present disclosure, a conjugate can be designed to target cancer cells by cell surface antigens. Antigens can be cell surface antigens that are overexpressed or expressed on abnormal cell types. Specifically, target antigens can be expressed primarily on proliferating cells (e.g., cancer cells). Target antigens can be selected based on their differential expression between proliferating tissues and normal tissues.

[0313] The anti-ROR1 antibodies, anti-ROR1 / anti-4-1BB bispecific antibodies, anti-ROR1 / anti-B7-H3 bispecific antibodies or antigen-binding fragments thereof provided herein can be used in the form of conjugates, wherein one or more selected from the group consisting of useful polymers, markers, drugs, etc. are conjugated.

[0314] Useful polymers can be, for example, polymers that can increase the half-life of a polypeptide, antibody and / or antigen-binding fragment thereof in vivo, and can be selected from the group consisting of polyethylene glycol (PEG) (having a molecular weight of, for example, 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and monomethoxy polyethylene glycol (mPEG), but are not limited thereto.

[0315] The label may be selected from fluorescent or chemiluminescent small molecule compounds, radioactive isotopes, etc. commonly used in industry, but is not limited thereto.

[0316] In the present disclosure, the drug can be at least one selected from the group consisting of cytotoxic drugs (e.g., anticancer agents, antibiotics, etc.) and contrast agents. These cytotoxic drugs may have cytotoxicity for cells (especially cancer cells) and toxicity for bacteria / viruses, and may include, for example, tubulin inhibitors, DNA alkylating agents, topoisomerase inhibitors, RNA polymerase inhibitors, ribosome inactivating proteins, NAMPT inhibitors, and immunomodulators. The example of the drug includes maytansine drugs (e.g., maytansine (DM1), ravtansine (ravtansine) (DM4)), auristatins (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) etc.), calicheamicin drugs, pyrrolobenzodiazepines The invention relates to a kind of drug, duocarmycin, docetaxel, doxorubicin, carboplatin (Beldin), cyclophosphamide, ifosfamide, nimustine hydrochloride (nidran), nitrogen mustard, nitrogen mustard hydrochloride, bleomycin, mitomycin C, cytarabine, fluorouracil, gemcitabine, trimetrexate, methotrexate, etoposide, vinblastine, vinorelbine, pemetrexed, hexamethylmelamine, procarbazine, paclitaxel (tacozo), taxotere, topotecan, irinotecan, exatecan and the like. In this case, it can be used in the form of antibody-drug conjugate (ADC), wherein the drug can preferably be maytansine drug, auristatin drug, calicheamicin drug, pyrrolobenzodiazepine In the conjugate, the antibody and the cytotoxic drug (e.g., anticancer agent) can be linked to each other via a chemical bond (e.g., a covalent bond). For example, the antibody and the cytotoxic drug can be linked by thiol coupling (SH coupling) or amine coupling (NH2 coupling). To this end, any amino acid residue in the heavy chain and / or light chain (e.g., in the constant region) of the antibody can be modified or substituted by cysteine ​​to achieve thiol or amine coupling, and / or the drug can be derivatized to have a functional group that allows thiol or amine coupling. Alternatively, the antibody and the cytotoxic drug can be linked to each other by glycosylation of asparagine (N) residues in the Fc portion.

[0317] As used herein, the term "drug to antibody ratio" (DAR) refers to the average number of therapeutic moieties (e.g., drugs) bound per conjugate according to the present disclosure. The drug to antibody ratio can be measured by methods such as liquid chromatography-mass spectrometry. The drug to antibody ratio of the conjugate according to the present disclosure can be 1:10, particularly 2:8, but is not limited thereto, and can be appropriately adjusted according to the type and characteristics of the drug or linker used.

[0318] As used herein, an "antibody-drug conjugate" (ADC) can deliver a drug to cells to which an antibody can specifically bind; for example, because the drug is conjugated to an antibody of the present disclosure that can specifically bind to ROR1 and / or B7-H3, the conjugate can selectively deliver the drug to cells expressing ROR1 and / or B7-H3.

[0319] In the present disclosure, the conjugation between the antibody and the drug can be achieved through a direct chemical bond or an indirect bond through a linker or a secondary antibody bound to the antibody, but is not limited thereto.

[0320] The antibody-drug conjugate may include a linker, which refers to 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 linked to the Fc sequence of an antibody according to the present disclosure, and the linker and the drug may be linked to prepare an antibody-linker-drug format.

[0321] On the other hand, a pharmaceutical composition is provided, comprising an anti-ROR1 antibody, an anti-ROR1 / anti-4-1BB bispecific antibody, an anti-ROR1 / anti-B7-H3 bispecific antibody or an antigen-binding fragment thereof; and / or an antibody-drug conjugate. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutical composition may have anti-cancer activity.

[0322] On the other hand, provided is a pharmaceutical composition for preventing and / or treating cancer, the composition comprising an anti-ROR1 antibody, an anti-ROR1 / anti-4-1BB bispecific antibody, an anti-ROR1 / anti-B7-H3 bispecific antibody or an antigen-binding fragment thereof; and / or a conjugate.

[0323] On the other hand, a method for preventing and / or treating cancer is provided, the method comprising administering an effective amount of an anti-ROR1 antibody, an anti-ROR1 / anti-4-1BB bispecific antibody, an anti-ROR1 / anti-B7-H3 bispecific antibody or its antigen-binding fragment to a subject in need of preventing and / or treating cancer; and / or an antibody-drug conjugate. The method may also include a step of identifying (diagnosing) a subject in need of preventing and / or treating cancer before the administration step.

[0324] On the other hand, provided is an anti-ROR1 antibody, an anti-ROR1 / anti-4-1BB bispecific antibody, an anti-ROR1 / anti-B7-H3 bispecific antibody or an antigen-binding fragment thereof; and / or an antibody-drug conjugate for preventing and / or treating cancer, and / or for preparing a drug (anticancer drug) for preventing and / or treating cancer.

[0325] The cancer may be a solid cancer or a blood cancer, and may be characterized, for example, by expression (or overexpression) of ROR1 and / or B7-H3 on the cell surface, but is not limited thereto. Examples of cancer include, but are not limited to, breast cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, colorectal cancer, cervical cancer, endometrial cancer, kidney cancer, skin cancer, nasopharyngeal cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, stomach cancer, pancreatic cancer, testicular cancer, thyroid cancer, oral cancer, renal cell carcinoma, adrenal cancer, melanoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, soft tissue sarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing's sarcoma, leiomyosarcoma, chondrosarcoma, lymphoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, glioma, leukemia, Burkitt's lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, etc. For example, the lung cancer can be small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). Leukemias can include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL). These cancers can be primary or metastatic.

[0326] Anticancer activity or preventive and / or therapeutic effects on cancer refers to any anticancer and / or antitumor effect that eliminates (kills) cancer cells, inhibits the occurrence and / or growth of cancer cells, prevents or reduces cancer progression due to migration, invasion, metastasis, relieves or alleviates symptoms and / or partially or completely eradicates cancer.

[0327] The subject to which the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody, antigen-binding fragment thereof, antibody-drug conjugate and / or pharmaceutical composition comprising the same are administered herein can be any animal or cell. Specifically, the subject can include an animal selected from mammals, including primates (e.g., humans and monkeys), rodents (e.g., rats and mice), etc., or cells, tissues, body fluids (e.g., serum), or cultures derived from (isolated from) these animals, especially humans, or cells, tissues, body fluids (e.g., serum) isolated from humans.

[0328] The pharmaceutical composition may contain, in addition to active ingredients such as anti-ROR1 antibodies, anti-ROR1 / anti-4-1BB bispecific antibodies, anti-ROR1 / anti-B7-H3 bispecific antibodies, antigen-binding fragments or conjugates thereof, a pharmaceutically acceptable excipient and / or carrier, and the excipient and / or carrier may be selected by those skilled in the art from those conventionally used in the formulation of protein drugs, and one or more types may be appropriately selected for use.

[0329] Administration of the anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody, antigen-binding fragments thereof, conjugates and / or pharmaceutical compositions can be performed by oral or parenteral routes.

[0330] The content of the active ingredient anti-ROR1 antibody, anti-ROR1 / anti-4-1BB bispecific antibody, anti-ROR1 / anti-B7-H3 bispecific antibody, antigen-binding fragments thereof and conjugates in the pharmaceutical composition can be variably specified according to factors including formulation method, administration mode, patient's age, body weight, sex, pathological condition, food intake, administration time, administration interval, administration route, excretion rate and reaction sensitivity.

[0331] For more detailed information on excipients and / or carriers in the pharmaceutical composition, administration routes, dosages, etc., please refer to Korean Patent No. 10-2551365

[0332] In another aspect, a nucleic acid molecule is provided, which encodes the anti-ROR1 antibody, the anti-ROR1 / anti-4-1BB bispecific antibody or the anti-ROR1 / anti-B7-H3 bispecific antibody.

[0333] In another aspect, a recombinant vector carrying a nucleic acid molecule is provided. The recombinant vector may be an expression vector for expressing the nucleic acid molecule.

[0334] In another aspect, a recombinant cell comprising a recombinant vector is provided.

[0335] In the case of recombinantly produced antibodies or antigen-binding fragments thereof, for the purpose of purification, they may be linked to conventional signal peptides, cleavage sites, tags, etc. Thus, in a non-limiting example, the antibodies or antigen-binding fragments thereof provided herein may comprise one or more elements selected from the group consisting of signal peptides, cleavage sites, tags commonly used in protein recombinant production processes (e.g., His tags, GST (glutathione S-transferase) tags, MBP (maltose binding protein) tags, etc.), or they may be in a purified form with these elements removed.

[0336] The term "vector" refers to a means for expressing a target gene in a host cell. Examples include plasmid vectors, cosmid vectors, phage vectors, lentiviral vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. Vectors that can be used as recombinant vectors can be obtained by manipulating 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, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pcDNA3.1, pcDNA3.3, etc.), bacteriophage (for example, λgt4λB, λ-Charon, λΔz1, M13, etc.) or virus (for example, SV40), but not limited thereto.

[0337] In a recombinant vector, a nucleic acid molecule can be operably linked to a promoter. The term "operably linked" is intended to refer to a functional connection between a target nucleotide sequence and an expression regulatory sequence (e.g., a promoter sequence). When "operably linked", a regulatory element can control transcription and / or translation of a target polynucleotide.

[0338] Recombinant vectors can generally be constructed as cloning vectors or expression vectors. For recombinant expression vectors, vectors that are generally used in the relevant art to express foreign proteins in plants, animals or microbial cells can be used. Various methods well known in the art can be used to construct recombinant vectors.

[0339] Prokaryotic or eukaryotic cells can be used as hosts to construct recombinant vectors. For example, when the vector is constructed as an expression vector for prokaryotic cells as a host, the vector generally includes a strong promoter for transcription (e.g., pL λPromoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. On the other hand, the expression vector for eukaryotic cells used as a host generally includes a replication origin operable in eukaryotic cells, such as f1 replication origin, SV40 replication origin, pMB1 replication origin, adenovirus replication origin, AAV replication origin, and BBV replication origin, but is not limited thereto. In addition, the expression vector generally includes a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.), and a polyadenylation sequence as a transcription termination sequence.

[0340] Recombinant cells can be prepared by introducing the recombinant vector into a suitable host cell. As long as it allows the recombinant vector to be cloned and expressed continuously in a stable manner, any host cell known in the art can be used for the present disclosure. Examples of prokaryotic host cells that can be used for the present disclosure can be selected from Escherichia coli (E.coli), such as Escherichia coli JM109, Escherichia coli BL21, Escherichia coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X1776, Escherichia coli W3110, Bacillus spp., such as Bacillus subtilis and Bacillus thuringiensis, and Enterobacteriaceae strains, such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species. Eukaryotic host cells that can be used for transformation can be selected from, but are not limited to, Saccharomyces cerevisiae, insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO (Chinese Hamster Ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK, etc.

[0341] The nucleic acid molecule or the recombinant vector carrying it can be delivered (introduced) into the host cell using methods known in the relevant art. For example, when the host cell is a prokaryotic cell, CaCl or electroporation methods can be used but not limited to carry out such delivery, while for eukaryotic host cells, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection or particle bombardment can be used but not limited to achieve gene introduction.

[0342] In order to select transformed host cells, the phenotype associated with the selection marker can be utilized according to methods well known in the art. For example, when the selection marker is a gene that confers resistance to a certain antibiotic, the host cells can be grown in the presence of the antibiotic in a culture medium to select the desired transformant.

[0343] On the other hand, a method for producing an anti-ROR1 antibody, an anti-ROR1 / anti-4-1BB bispecific antibody or an anti-ROR1 / anti-B7-H3 bispecific antibody is provided, the method comprising the step of expressing a nucleic acid molecule in a host cell. The step of expressing a nucleic acid molecule in a host cell can be a step of culturing a cell comprising a nucleic acid molecule or a recombinant vector thereof. The method may further include a step of separating and / or purifying an antibody or an antigen binding fragment from a culture medium after the culturing step.

[0344] Beneficial Effects

[0345] The present disclosure provides an improved anti-ROR1 antibody, a bispecific antibody comprising a ROR1 antibody and a T cell engaging antibody (T cell engager), and / or a bispecific antibody comprising an anti-ROR1 antibody and a tumor-associated antigen-specific antigen (TAA, such as B7-H3), thereby achieving excellent anti-cancer effects.

[0346] Specifically, designed to provide anti-ROR1 antibodies and bispecific antibodies comprising the same having excellent binding ability and enhanced stability and developability, the present disclosure allows for more efficient and safer production of antibody drugs in a cost-effective manner compared to conventional therapeutic antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0347] Figure 1a to Figure 1c is a graph showing the binding affinity of an anti-ROR1 antibody to ROR1 according to one embodiment.

[0348] Figure 2a and Figure 2b is a graph showing the binding affinity of an anti-ROR1 / anti-4-1BB bispecific antibody to 4-1BB and / or ROR1 according to one embodiment.

[0349] Figure 2cis a graph showing the binding affinity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one embodiment to cells expressing 4-1BB on their surface.

[0350] Figure 2d is a graph showing the binding affinity of an anti-ROR1 / anti-4-1BB bispecific antibody according to one embodiment to cells expressing ROR1 on their surface.

[0351] Figure 3a to Figure 3d are graphs showing the serum stability of the anti-ROR1 / anti-4-1BB bispecific antibody according to one embodiment (a and b: in human serum; c and d: in monkey serum).

[0352] Figure 4a is a graph showing activation of 4-1BB signaling by an anti-ROR1 / anti-4-1BB bispecific antibody in the presence of ROR1 according to one embodiment.

[0353] Figure 4b is a graph showing ROR1-dependent 4-1BB signaling activation by an anti-ROR1 / anti-4-1BB bispecific antibody according to one embodiment.

[0354] Figure 4c is a graph showing the correlation between ROR1-dependent 4-1BB signaling activation and ROR1 expression of an anti-ROR1 / anti-4-1BB bispecific antibody according to one embodiment.

[0355] Figure 4d is a diagram showing FcγRI-dependent 4-1BB signaling activation by an anti-ROR1 / anti-4-1BB bispecific antibody according to one embodiment.

[0356] Figure 5a is a diagram showing confirmation that the anti-ROR1 / anti-4-1BB bispecific antibody activates PBMC and induces cytokine (interferon-γ) release in CHO-K1 cells overexpressing ROR1 according to one embodiment.

[0357] Figure 5b is a graph showing that an anti-ROR1 / anti-4-1BB bispecific antibody activates PBMC and induces cytokine (interferon-γ) release in a gastric cancer cell line expressing ROR1 according to one embodiment.

[0358] Figure 5c is a graph showing that an anti-ROR1 / anti-4-1BB bispecific antibody activates PBMC and induces cytokine (interferon-γ) release in a gastric cancer cell line expressing ROR1 according to one embodiment.

[0359] Figure 6a and Figure 6b is a graph showing the anti-tumor effect (in vivo) of the anti-ROR1 / anti-4-1BB bispecific antibody according to one embodiment.

[0360] Figure 6c is a graph depicting the long-term anti-tumor memory response of an anti-ROR1 / anti-4-1BB bispecific antibody according to one embodiment.

[0361] Figure 6d is a graph presenting the results of measuring the immunomodulatory activity of the anti-ROR1 / anti-4-1BB bispecific antibody in tumor, blood, and liver according to one embodiment.

[0362] Figure 6e is a graph showing the results of measuring the immunomodulatory activity of an anti-ROR1 / anti-4-1BB bispecific antibody in PBMCs that do not express ROR1 according to one embodiment.

[0363] Figure 7a-7d is a graph showing the binding affinity of the anti-ROR1 / anti-B7-H3 bispecific antibody to ROR1 or B7-H3 according to one embodiment.

[0364] Figure 8 is a graph demonstrating the binding affinity of the anti-ROR1 / anti-B7-H3 bispecific antibody according to one embodiment to cells expressing ROR1 and B7-H3 on their surface.

[0365] Fig. 9 is a graph illustrating the extent of cellular internalization of an anti-ROR1 / anti-B7-H3 bispecific antibody according to one embodiment.

[0366] Figures 10a to 10c is a graph confirming the results of measuring the purity of an antibody-drug conjugate (ADC) comprising an anti-ROR1 / anti-B7-H3 bispecific antibody using size exclusion high performance liquid chromatography (SE-HPLC) according to one embodiment. Figures 10a to 10c The peak values ​​(min.) shown in are as follows: 10a: 12.441, 14.677; 10b: 13.564, 16.170; 10c: 13.752, 16.314.

[0367] 11 is a graph showing the results of measuring the drug to antibody ratio (DAR) in ADCs comprising an anti-ROR1 / anti-B7-H3 bispecific antibody using size exclusion liquid chromatography-mass spectrometry (LC / MS) according to one embodiment.

[0368] Fig.12is a graph demonstrating the ability of an ADC comprising an anti-ROR1 / anti-B7-H3 bispecific antibody according to one embodiment to bind to an antigen (ROR1).

[0369] Fig.13 is a graph illustrating the cytotoxicity of an ADC comprising an anti-ROR1 / anti-B7-H3 bispecific antibody against cancer cells expressing ROR1 and B7-H3 according to one embodiment. DETAILED DESCRIPTION

[0370] Hereinafter, the following examples will be described in detail to help understand the present invention. However, these embodiments are provided to illustrate the present invention and should not be construed as limiting the scope of the present invention. The embodiments of the present application are provided to more fully describe the application to those skilled in the art.

[0371] Example 1. Anti-ROR1 Antibodies

[0372] 1.1. Preparation of anti-ROR1 antibodies

[0373] Starting from the anti-ROR1 antibody clone sequence disclosed in International Publication No. WO2019-225992A1, peptide mapping analysis (Accurate Determination of Succinimide Degradation Products Using High Fidelity Trypsin Digestion Peptide Map Analysis, Anal. Chem. 2011, 83 (15): 5912-5919) was used to identify post-translational modification (PTM) sites, and mutations were introduced to remove these sites, thereby producing antibodies with improved physicochemical properties and developability. Among the anti-ROR1 antibodies disclosed in WO2019-225992A1, BA6 antibody (WT) (based on IgG1) was selected as the target for PTM site removal, and the amino acid sequences of the complementarity determining regions (CDRs) of 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.

[0374] Table 6

[0375]

[0376] Peptide mapping identified PTMs in LCDR2 and HCDR3. To pinpoint the actual hotspots of PTMs within the BA6 antibody sequence, peptide mapping was performed using the BA6 antibody. After storing the antibodies for two weeks at 4°C and 37°C, the samples were digested and fragmented with immunoglobulin G degrading enzyme (IdeS; Genovis) from Streptococcus pyogenes and dithiothreitol (DTT), followed by LC / MS analysis, which confirmed the presence of PTMs in CDRL2 and CDRH3. Since there are two PTM hotspots, the method involves dividing them into two groups, inducing single mutations, and then, if the binding activity is found to be similar to that of the wild-type antibody, combining mutations to produce double, triple mutants, and so on.

[0377] A total of 10 PTM mutants were constructed to verify the ligand binding activity. Specifically, in order to obtain IgG of each anti-ROR1 antibody clone with PTM removal mutation, mutagenic primer sets targeting CDRL2 and CDRL3 of the above cloned PTM hotspots were synthesized (Macrogen, South Korea) to introduce mutant sequences, and the nucleic acid sequences encoding the light and heavy chains were obtained by site-directed mutagenesis using PCR technology.

[0378] For transfection, 6.0 × 10 6 ExpiCHO-S cells (ThermoFisher) were prepared at a concentration of 1 μg / mL and 2.5 μL / mL. The heavy and light chain-expressing vectors (cloned into separate pcDNA3.4 vectors (Thermo Fisher) for plasmid preparation before transfection) and ExpiFectamine (Thermo Fisher) were diluted in OptiPRO medium (ThermoFisher) to form concentrations of 1 μg / mL and 3.2 μL / mL, respectively. Then, the diluted vector and ExpiFectamine mixture were mixed and allowed to stand at room temperature for 5 minutes, then added to the flask containing the prepared cells, and then the mixture was fully stirred and cultivated under the conditions of 8% CO2, 37°C and 120rpm. On the day after transfection, 600 μL of enhancer (Thermo Fisher) and 24mL of feed (Thermo Fisher) were added respectively, and continued to be cultivated for 7 days under the conditions of 8% CO2, 37°C and 120rpm. For detailed experimental schemes, the manufacturer's instructions for the ExpiCHO expression system provided by Thermo Fisher were referenced.

[0379] By this method, a total of 10 mutants (M1-M10) were generated and used as candidate clones for anti-ROR1 antibodies. Each of these clones contained a mutation confirmed in the PTM site CDRL2 or CDRH3. Clones M1 to M4 had mutations introduced at L51 in CDRL2, clones M5-M10 had mutations introduced in CDRH3, and clone M11 introduced mutations from M3 (mutation at L51 in CDRL2) and M9 (mutation at H97 in CDRH3), which were selected as the main clones in M1 to M10. The specific information of these candidate clones is summarized in Table 7 below.

[0380] Table 7

[0381]

[0382] (deD: aspartate deamidation, deN: asparagine deamidation, isoD: aspartate isomerization;

[0383] By: Based on ligand (antigen) binding activity assessment)

[0384] 1.2 Determination of Binding Specificity of Candidate Clones to the Extracellular Domain of ROR1 (ELISA)

[0385] Given that PTM hotspots in the BA6 antibody were identified within the CDR region, removal of the PTMs may reduce antigen binding activity compared to WT (wild type, BA6 antibody) due to sequence changes in the CDR region, which may in turn reduce the potency of the antibody. Therefore, the ultimate goal of PTM engineering is to identify clones with the same or better binding activity than WT. Therefore, the specific binding ability of candidate clones M1 to M11 produced in Example 1.1 to the ROR1 antigen was analyzed as follows.

[0386] The binding affinity of anti-ROR1 antibody-antigen was evaluated using an ELISA-based solution binding assay. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with ROR1 protein (ROR1-His; sino biological, catalog number 13968-H08H) in PBS solution at a concentration of 1 μg / ml at 4°C for 16 hours, and nonspecific binding sites were blocked with 1% (v / v) BSA (bovine serum albumin) for 2 hours.

[0387] Then, in a 96-well microtiter plate, Figure 1a to Figure 1cAnti-ROR1 antibodies at the concentrations described in were added to microtiter plates, and their binding ability was analyzed by ELISA, as shown below. Specifically, after incubation at 37°C for 2 hours, the plate was washed 5 times with PBS containing 0.05% (v / v) Tween 20, and then the HRP-conjugated Fab polyclonal antibody reagent (Pierce, catalog number 31414) was diluted at a ratio of 1:30,000 and added to the washed microtiter plate, followed by reaction at 37°C for 1 hour, and then the bound anti-ROR1 antibody was detected. After the reaction, TMB (tetramethylbenzidine, Sigma, T0440) was used for color development. The enzymatic reaction was terminated with 0.5 mol / L sulfuric acid, and the absorbance was read at 450 nm and 650 nm using a microplate reader (Molecular Devices).

[0388] The results obtained are shown in Figure 1a to Figure 1c In. From Figure 1a to Figure 1c The data can be understood that, based on ELISA analysis, M1 and M3 show similar ligand (antigen) binding activity compared with WT, however, due to the saturation point of M1 being lower than WT, M1 is excluded from the final clone selection. In addition, M7, M8 and M10 do not show binding activity comparable to WT. M3 and M9 show binding activity comparable to WT. Therefore, the double mutant M11 clone (D51E (LC) + N97S (HC)) combining M3 and M9 was produced using the same method as in Example. The M11 clone thus produced was confirmed to show similar binding activity compared with WT and M3, so that it was selected as the final candidate clone.

[0389] 1.3 Developability analysis of candidate clones

[0390] The stability of the clone BA6M11 (also indicated as BA6M11) clone (derived from BA6 by PTM engineering to remove PTM tendency) under stress conditions compared with BA6 was determined. More specifically, after storing both BA6 antibody and BA6M11 antibody at 4°C (normal conditions) and 37°C (stress conditions) for two weeks, each sample was analyzed by SE-HPLC, icIEF and LC / MS to check purity and impurity content. In addition, in order to determine whether the change in substance content also affects efficacy, an antigen-antibody binding capacity analysis (ELISA; see Example 1.2) was performed.

[0391] The results obtained are summarized in Table 8 below.

[0392] Table 8

[0393]

[0394] (*N / A: Not Applicable; **N / D: Not Detected)

[0395] As shown in Table 8, when stored at 4°C and 37°C, it was observed that the purity of the BA6M11 clone remained high and comparable to that of BA6. In addition, compared with BA6, during the production process, the formation of charge variants in the BA6M11 clone was significantly reduced under alkaline and acidic conditions, and the formation of charge variants was also significantly reduced under stress conditions (Δ major %). In the BA6M11 clone, peptide mapping confirmed the disappearance of succinimide formation at problematic residues, resulting in a reduction in charge variants. The reduction in charge variants indicates that the risk of antibody quality changes during production and storage / distribution is reduced, and the reliability of the purification process using ion exchange resins is improved. It also reduces the risk of increased immunogenicity when administered to humans by preventing the hydrolysis of succinimide intermediates, which may lead to antibody chain cleavage, reduced efficacy and increased immunogenicity. In addition, the antigen-antibody binding capacity verified using human recombinant ROR1 protein (ROR1-His; Sino biological, catalog number 13968-H08H) did not show significant differences in binding capacity between samples stored for two weeks at 4°C and 37°C. These results demonstrate that BA6M11 maintains comparable potency to BA6 while improving physicochemical properties with regard to developability.

[0396] Ultimately, the BA6M11 clone not only showed comparable binding to WT but also significantly improved developability through PTM engineering.

[0397] The amino acid sequence of the novel anti-ROR1 antibody BA6M11 in IgG format is listed in Tables 9 and 10.

[0398] Table 9

[0399]

[0400]

[0401]

[0402] Table 10

[0403]

[0404]

[0405] Example 2. Anti-ROR1 / anti-4-1BB bispecific antibody

[0406] 2.1 Preparation of improved anti-ROR1 / anti-4-1BB bispecific antibodies

[0407] An anti-ROR1 / anti-4-1BB bispecific antibody was produced based on the improved anti-ROR1 antibody clone BA6M11 produced and selected in Example 1 and one of the anti-4-1BB antibody clones disclosed in International Publication No. WO2020 / 111913A1, namely 41B01.03 (also known as 1A10M12).

[0408] In this example, a bispecific antibody comprising an anti-ROR1 IgG (full length) antibody and a 4-1BB scFv linked to the C-terminus of the heavy chain of the anti-ROR1 IgG (full length) antibody was constructed.

[0409] To construct the anti-ROR1 / anti-4-1BB bispecific antibody, DNA fragment 1 having a 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 fragment 2 having a nucleotide sequence encoding the light chain of the IgG antibody in the anti-ROR1 / anti-4-1BB bispecific antibody was inserted into pcDNA3.4 (Invitrogen, A14697; plasmid 2).

[0410] Subsequently, DNA segment 4 (SEQ ID NO: 114) encoding a peptide linker of 20 amino acids consisting of (GGGGS)4, or DNA segment 5 (SEQ ID NO: 113) encoding an 18 amino acid linker peptide consisting of (GS)9, was used to fuse DNA fragment 3 encoding scFv at the portion corresponding to the C-terminus of the IgG antibody Fc region inserted in plasmid 1 to construct a vector for expressing a bispecific antibody.

[0411] In addition, anti-4-1BB scFv (named 1A10M12) takes such a form, wherein VL103-VH44 (VL103: VL with G (Gly) → C (Cys) mutation introduced at position 103; VH44: VH with G → C mutation introduced at position 44) are fused by a linker ((GGGGS) 4, SEQ ID NO: 114) at the C-terminus of the light chain variable region (VL) and the heavy chain variable region (VH), respectively. These mutations stabilize scFv and enable disulfide bond formation.

[0412] The amino acid sequences of the bispecific antibodies thus prepared are listed in Table 11 below.

[0413] Table 11

[0414]

[0415]

[0416]

[0417]

[0418] (Linker 1: BA6M11 (IgG) linked to 1A10M12 (scFv) (C-terminus of the heavy chain of BA6M11 linked to the N-terminus of the VL of 1A10M12);

[0419] Linker 2: VL linked to VH in 1A10M12 (scFv) (the C-terminus of VL is linked to the N-terminus of VH)

[0420] The encoding nucleic acid sequence of the bispecific antibody (5'→3') is as follows:

[0421] [Heavy chain_BA6M11 (WT) x 1A10M12] (SEQ ID NO: 118)

[0422]

[0423]

[0424] [Light chain_BA6M11] (SEQ ID NO: 119)

[0425]

[0426] The DNA encoding the bispecific antibody cloned as described above was transiently expressed using the ExpiCHO system (Thermo Fisher) according to the manufacturer's protocol. The suspension of the antibody-expressing culture was cleared by centrifugation and filtration and the culture was purified using Mabselect Sure TM The antibody was purified by affinity chromatography with resin and size exclusion chromatography using Superdex200 resin. The purity of the purified antibody was analyzed using HPLC with TSKgel SuperSW3000 and was found to be excellent, exceeding 95%.

[0427] 2.2 Evaluation of anti-ROR1 / anti-4-1BB bispecific antibodies

[0428] 2.2.1. Double Antigen Capture ELISA (DACE)

[0429] ELISA method was used to compare and analyze the binding affinity of anti-ROR1 / anti-4-1BB bispecific antibodies to ROR1 and 4-1BB comprising BA6M11 clones (PTM removal clones, test group) or BA6 clones (parental clones, control group). Two versions of the test group BA6M11 clones were used: BA6M11 (NA), which included the N297A (NA) mutation that reduced ADCC, and BA6M11 (WT) that did not include this mutation.

[0430] The sequence of the anti-ROR1 / anti-4-1BB bispecific antibody comprising the BA6 clone used as a control is as follows:

[0431] Light chain BA6 (SEQ ID NO: 124)

[0432] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYYDNNRPSGVPDRFSGSKSG TSASLAISGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECS

[0433] Heavy chain BA6x1A10M12 (SEQ ID NO: 125)

[0434] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSAIYHSGSSKYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCARGGNGAWDTGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVLGGGGSGGGGSGGGGSGGGGS EVQLLESGG GLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS

[0435] Briefly, a 96-well microtiter plate (NUNC, 446612) was coated with human ROR1-Fc protein (Sinobio, 13968-H02H1) in PBS at 10 ng / well overnight at 4°C, and the plate was then incubated with 1% BSA buffer (1×PBS, 1% (v / v) BSA) at 200 μl / well at 37°C for 2 hours to prevent nonspecific binding. After blocking nonspecific binding, the wells were washed and serial dilutions of the bispecific antibody test material (starting at 100 nM) were added to each well. After incubation at 37°C for 2 hours, the plate was washed with PBS-T (1×PBS / 0.05% (v / v) Tween-20). Then, human 4-1BB-His protein (Sinobio, 10041-H08H) dissolved in 1% (v / v) BSA was added to each well at 100 ng / well and incubated at 37°C for 1 hour. After washing the plate with PBS-T, anti-His HRP (Roche, catalog number: 11965085001) was added and incubated at 37°C for 1 hour. After washing, color development was performed with TMB (tetramethylbenzidine, Sigma, T0440), and the reaction was terminated with 0.5M sulfuric acid (Samchun Chemicals, S1410), and then the absorbance (OD) at 450-650nm was read in a spectrophotometer. Four-parameter logistic curve analysis was performed using GraphPad software.

[0436] The results obtained by the experiment are shown in Table 12 and Figure 2a and Figure 2b middle.

[0437] Table 12

[0438] ROR1x4-1BB BA6x1A10M12 BA6M11(NA)x1A10M12 EC50(nM) 0.386 0.268

[0439] like Figure 2a As shown in Table 12, the bispecific antibody comprising BA6M11 did not show any decrease in binding affinity compared to the parental clone BA6, but rather an improvement in binding affinity was observed, as shown by a lower EC50.

[0440] In addition, when the difference in binding affinity due to the presence or absence of the NA mutation was measured, BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 showed no difference in binding activity to human ROR1 and 4-1BB.

[0441] Considering the fact that it is often challenging to maintain the binding affinity of the parental clone when the CDR sequence is changed, it was found that the antigen binding affinity of BA6M11, whose sequence was altered by PTM removal, was equivalent to or better than that of the parental clone BA6, demonstrating the desirable characteristics of BA6M11 and its potential and advantages for further development as a therapeutic antibody.

[0442] 2.2.2. Binding assay of cell surface antigens (flow cytometry)

[0443] Using various cells expressing human ROR1 or human 4-1BB on their surface, the binding affinity of the anti-ROR1 / anti-4-1BB bispecific antibody comprising BA6M11 (WT) or BA6M11 (NA) clone was compared by measuring with a flow cytometry apparatus (BD).

[0444] In particular, Jurkat cells overexpressing human 4-1BB (Promega) and human ROR1 positive cell line (ATCC) NCI-N87 cells were inoculated into V-bottom 96-well plates (96-well plates-RV, Bioneer, 910D09) and washed with 200 μl / well 1% BSA buffer. Four-fold dilutions of the test material (bispecific antibody) starting from 100nM were added to each well and incubated at 4°C for 1 hour. The plate was washed with 1% BSA buffer. FITC-conjugated anti-human IgG (Fc specificity) (Sigma, F9512) dissolved 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 again. The mean fluorescence intensity (MFI) of FITC was measured by flow cytometry. The obtained MFI values ​​were analyzed by four-parameter logistic curves using GraphPad software.

[0445] like Figure 2c and Figure 2d As shown, BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 bound similarly to Jurkat cells overexpressing human 4-1BB and ROR1-positive NCI-N87 cells, indicating that there was no difference in binding activity to human 4-1BB and human ROR1.

[0446] 2.2.3. Binding affinity determination using surface plasmon resonance (SPR)

[0447] Surface plasmon resonance (SPR) was used to assess the binding affinity of BA6M11 (WT) x1A10M12 and BA6M11 (NA) x1A10M12 clones (anti-ROR1 / anti-4-1BB bispecific antibodies comprising BA6M11 clones). Antigens used for the assessment were human ROR1 proteins (13968-H02H1, Fc tags) from Sino Biological and human 4-1BB proteins (9220-4B-100, his tags) from R&D Systems. Measured on Biacore T200 instruments (Cytiva).

[0448] The binding affinity of the candidate materials to human ROR1 was measured as follows.

[0449] A CM5 chip (Cytiva, BR100530) was coated with 5 μg / mL human ROR1 protein dilution (in acetate 4.0, Cytiva, BR100349) using the amine coupling method (Cytiva, BR100050) to a surface density of approximately 80 RU. BA6M11 (WT) x 1A10M12 and BA6M11 (NA) x 1A10M12 samples were serially diluted two-fold in HBS-EP buffer (1x) (Cytiva, BR100669) to form concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 0 nM. These diluted antibody samples were then passed through the CM5 chip with human ROR1 protein fixed for approximately 60 seconds to allow the sample to bind to the antigen, and then HBS-EP buffer (1x) was passed through the sample-antigen complex for 180 seconds to separate the sample from the antigen. The chip surface was regenerated by flowing glycine-HCl pH 1.5 (Cytiva, BR100354) over the chip for 30 seconds. HBS-EP (1x) was used as the running buffer at a flow rate of 30 μL / min and a temperature of 25°C. Binding affinity was analyzed using a bivalent analyte model to derive the primary association rate constant (Ka1) and the primary dissociation rate constant (Kd1). The equilibrium dissociation constant (KD) was calculated by dividing the primary dissociation rate constant by the primary association rate constant, and this value was used to represent the final binding affinity.

[0450] The binding affinity of candidate materials to human 4-1BB is measured as follows: 10 μg / mL anti-Fc antibodies (Invitrogen, 31125) diluted in 5.0 (Cytiva, BR100351) are fixed to CM5 chips (Cytiva, BR100530) with about 15000RU using an amine coupling method. Then BA6M11 (WT) x1A10M12 and BA6M11 (NA) x1A10M12 diluted in HBS-EP (1x) buffer are bound to the CM5 chips fixed with anti-Fc antibodies with 300RU. Human 4-1BB antigen is diluted twice in HBS-EP (1x) buffer continuously to form concentrations of 125nM, 62.5nM, 31.25nM, 15.625nM, 7.8125nM and 0nM. Then, these diluted human 4-1BB samples are flowed through the CM5 chip for about 60 seconds to bind the sample to the antigen, and then HBS-EP buffer (1x) is flowed through the sample-antigen complex for 180 seconds to separate the sample from the antigen. The chip surface is regenerated by flowing glycine-HCl pH1.5 (Cytiva, BR100354) on the chip for 30 seconds. HBS-EP (1x) is used as a running buffer and measured at a flow rate of 30 μL / min and a temperature of 25 ° C. Binding affinity is measured using a 1:1 binding model to obtain an association rate constant (Ka) and a dissociation rate constant (Kd). The equilibrium dissociation constant (KD) is calculated by dividing the dissociation rate constant by the association rate constant, and this value is used as the final measurement value of the binding affinity.

[0451] The results thus obtained are summarized in Table 13 below:

[0452] Table 13

[0453]

[0454] As shown in Table 13, both BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 exhibited similar binding levels to human ROR1 and human 4-1BB, confirming that there was no difference in antigen binding activity between the WT and NA cell lines.

[0455] 2.2.4. Determination of the developability of anti-ROR1 / 4-1BB bispecific antibodies

[0456] The developability of anti-ROR1 / 4-1BB bispecific antibodies comprising the BA6 clone or the BA6M11 clone was determined.

[0457] To analyze developability, capillary isoelectric focusing (cIEF) or imaged capillary isoelectric focusing (icIEF) is performed. Both methods use isoelectric point (pI) values ​​to analyze changes in charge variants.

[0458] Samples for developability analysis were prepared by exposing them to temperatures of 4°C and 37°C (or 40°C) for two weeks. The storage temperature was 4°C or 37°C for the BA6x1A10M12 clone, and 4°C or 40°C for the BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 clones.

[0459] The cIEF analysis was performed using a PA 800plus instrument from Beckman Coulter equipped with a UV detector. Neutral capillary cartridges (50 μm ID) were fabricated to a total length of 30.2 cm and installed in the instrument.

[0460] The bispecific antibody samples for analysis were diluted to a final concentration of 5 mg / mL with formulation buffer (20 mM histidine, 7% (w / v) trehalose, pH 6.0). A master mix solution was prepared using 3 M urea solution (including gel (urea: GE Healthcare, 17-1319-01; cIEF gel: Beckman Coulter, 477497), ampholytes 3-10 (Pharmalyte 3-10; GE Healthcare, 17-0456-01), anode stabilizer solution (200 mM iminodiacetic acid; Sigma, 220000), cathode stabilizer solution (500 mM L-arginine; Sigma, A5006) and synthetic peptide isoelectric point markers (10, 9.5, 7.0, 5.5, 4.1; cIEF peptide labeling kit; Beckman Coulter, A58481)). 10 μL of analysis sample (bispecific antibody) was mixed with 239 μL of master mixture solution and centrifuged. 200 μL of mixture was transferred to a PCR tube, placed in a plastic vial, and prepared on a sample tray. Distilled water, anolyte (200 mM phosphoric acid; Sigma, 345-245), catholyte (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 migration agent solution (350 mM acetic acid; Sigma, 695092) were prepared in separate plastic bottles on a buffer tray for analysis.

[0461] For icIEF analysis, a Maurice instrument and capillary cartridge from ProteinSimple were used. A Maurice icIEF cartridge (ProteinSimple, PS-MC02-C) was filled with 2 mL of catholyte (100 mM sodium hydroxide in 0.1% methylcellulose; ProteinSimple) and 2 mL of anolyte (0.08 M phosphoric acid in 0.1% methylcellulose; ProteinSimple) and then installed in the instrument.

[0462] The bispecific antibody samples for analysis were diluted in distilled water to a final concentration of 1 mg / mL. 8 M urea solution (Sigma, U6504), 1% methylcellulose (methylcellulose; ProteinSimple), ampholytes (pH 3-10) (Pharmacyte pH 3-10; ProteinSimple, 042-684 or GE Healthcare, 17-0456-01), distilled water (UltraPure TM A master mix solution was prepared with 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 then transferred to a 96-well plate for analysis.

[0463] Distilled water, 0.5% methylcellulose (ProteinSimple), fluorescence calibration standards (ProteinSimple), and air were prepared in individual vials and placed in their respective locations for analysis.

[0464] The results of this analysis are shown in Table 14 below:

[0465] Table 14

[0466]

[0467] The developability evaluation results presented in Table 14 show that the main peak content of BA6x1A10M12 was about 52% under storage conditions at 4° C. Compared with storage conditions at 4° C., the main peak content of this clone under storage conditions at 37° C. decreased by about 17%, while the basic peak content increased by about 14%.

[0468] The results of the developability determination of two BA6M11 clone variants with modified BA6 clones are as follows: the main peak content of BA6M11 (WT) x 1A10M12 at 4°C was about 75%, and the main peak content of BA6M11 (NA) x 1A10M12 was about 78%. Compared with the storage condition at 4°C, both clones showed a decrease of about 16-17% in the main peak content and an increase of about 2-3% in the basic peak content under the storage condition of 40°C.

[0469] Compared with BA6, the tested BA6M11 clone showed improved characteristics in the content of basic charge variants. The reduction in the content of basic charge variants was confirmed by analyzing samples stored at 4°C, indicating that it was also reduced during production, and the rate of change under high temperature storage conditions (40°C) was also reduced, and based on samples stored at 4°C, two mutant clones showed that WT and NA forms had similar main peak contents. From these results, it was confirmed that the BA6M11 clone had excellent developability compared to the parental BA6 clone.

[0470] 2.2.5. Serum stability analysis of anti-ROR1 / anti-4-1BB bispecific antibodies

[0471] To evaluate the serum stability of the anti-ROR1 / anti-4-1BB bispecific antibodies comprising the BA6 clone (parental clone) or the BA6M11 clone (PTM removed), they were stored in human serum and monkey serum and then analyzed and compared using the ELISA method.

[0472] In this analysis, anti-ROR1 / anti-4-1BB bispecific antibodies were stored in human and monkey serum at 37°C for 2, 7, and 14 days.

[0473] A 96-well microtiter plate (NUNC, 446612) was coated with human ROR1-Fc protein (Sinobio, 13968-H02H1) in PBS at 4°C overnight at 100ng / well. Then, the plate was incubated with 1% BSA buffer (1×PBS, 1% (v / v) BSA) at 200μl / well at 37°C for 2 hours to block nonspecific binding. Thereafter, the blocking solution was discarded, and a four-fold dilution (starting from 100nM) of each bispecific antibody test material was added to the 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). Subsequently, human 4-1BB-His protein (Sinobio, 10041-H08H) dissolved in 1% BSA buffer was added at 100ng / well and incubated at 37°C for 1 hour. After washing with PBS-T, the plate was incubated with anti-His HRP (Roche, catalog number 11965085001) at 37°C for 1 hour. After washing, color development was performed with TMB (tetramethylbenzidine, Sigma, T0440), color development was stopped with 0.5M sulfuric acid (Samchun Chemicals, S1410), and the absorbance (OD) at 450-650nm was read using a spectrophotometer. Four-parameter logistic curve analysis was performed using GraphPad software.

[0474] The results obtained are presented in Table 15 and Figure 3a to Figure 3d middle.

[0475] Table 15

[0476]

[0477]

[0478] As shown in Table 15 and Figure 3a to Figure 3d As shown, compared with the bispecific antibody comprising the parental BA6 clone, the anti-ROR1 / anti-4-1BB bispecific antibody comprising BA6M11 maintains longer stability in human serum and monkey serum, indicating that serum stability is improved. Serum stability is a key property that significantly affects the efficacy of antibody drugs when administered in vivo, especially in terms of binding affinity and specificity, and should be considered in antibody development and production. Therefore, these results confirm that, compared with BA6, BA6M11 significantly contributes to enhancing the serum stability of antibodies.

[0479] 2.2.6. Activation of 4-1BB Signaling by Anti-ROR1 / Anti-4-1BB Bispecific Antibodies (4-1BB Reporter Gene Bioassay)

[0480] 2.2.6.1. Activation of 4-1BB signaling depends on ROR1 expression

[0481] To measure, compare and analyze 4-1BB signaling activation by anti-ROR1 / 4-1BB bispecific antibodies containing BA6 clone or BA6M11 clone, a 4-1BB NFκB luciferase reporter gene bioassay was performed. The 4-1BB reporter gene bioassay used human ROR1-positive cell lines, gastric cancer cell line AGS, and hamster ovarian epithelial cells CHOK1 stably overexpressing human ROR1 (CHOK1-hROR1).

[0482] In this analysis, GloResponse TM The NFκB-luc2 / 4-1BB Jurkat cell line (Promega, catalog number CS196004) was used as the effector cell, which was genetically engineered to stably express human 4-1BB and luciferase downstream of the response element.

[0483] Briefly, AGS (ROR1-positive gastric cancer cell line, ATCC, CRL-1739, 2.5×10 4 ) or CHOK1-hROR1 (ROR1-overexpressing hamster ovarian epithelial cells, BPS Bioscience, 79609-H, 2.5×10 4 ) were each dispensed into a white 96-well assay plate containing 100 μL of culture medium (10% (v / v) FBS, RPMI1640) and incubated overnight at 37°C in a 5% CO2 humidified incubator. After overnight incubation, 100 μL of culture medium was removed and 25 μL of assay medium (1% (v / v) FBS, RPMI 1640) was dispensed onto the pre-attached target cells (AGS or CHOK1-hROR1) as described previously. 25 μL of bispecific antibody (starting at 40 nM, serially diluted 5-fold) was added to the plate. 25 μL of GLORESPONSE TM NFκB-luc2 / 4-1BB Jurkat cell suspension (Promega, CS196004) was dispensed into the plate to obtain 2.5×10 4 Cells / well were added and incubated at 37°C in a 5% CO2 humidified incubator for 6 hours. During the incubation period, BIO-GLO was reconstituted according to the manufacturer's instructions. TM Luciferase Assay System (Promega, G7940). After 6 hours of incubation, 75 μL of BIO-GLO TM The reagent was added to each well of the assay plate. After 5 minutes, luminescence was measured using a microplate reader. Four-parameter logistic curves were evaluated using GraphPad software. The experimental results using AGS and CHOK1-hROR1 cells are shown in Tables 16 and 17, respectively. Figure 4a middle.

[0484] Table 16

[0485]

[0486] As shown in Table 16 and Figure 4a As shown, compared with the bispecific antibody comprising the parental clone BA6, the anti-ROR1 / 4-1BB bispecific antibody comprising BA6M11 exhibited a lower EC50 (nM) value in ROR1-expressing cells, indicating that the anti-ROR1 / 4-1BB bispecific antibody comprising BA6M11 induced stronger 4-1BB signal activation in the presence of ROR1 antigen. Specifically, the anti-ROR1 / 4-1BB bispecific antibody comprising BA6M11 showed excellent 4-1BB signal activation effects 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.

[0487] The data demonstrate that the anti-ROR1 / 4-1BB bispecific antibody comprising BA6M11 specifically targets ROR1-expressing cancer cells.

[0488] To further verify that BA6M11(NA)x1A10M12 and BA6M11(WT)x1A10M12 activation of 4-1BB depends on ROR1 expression, a mixture of parental CHOK1 cells and CHOK1 cells overexpressing human ROR1 (CHOK1-hROR1) was used to measure 4-1BB signaling activation as described previously. For comparison, the anti-4-1BB antibody Urelumab (BMS-663513) was used.

[0489] The results are shown in Table 17 and Figure 4b middle.

[0490] Table 17

[0491]

[0492] like Figure 4b As shown, when the proportion of CHOK1-ROR1 cells increased, both BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 showed higher maximum fold induction. This suggests that the more cells express ROR1, the more 4-1BB activation is induced. Figure 4b As shown in Table 17, BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 induced 4-1BB activation at similar levels.

[0493] 2.2.6.2. Anti-ROR1 / anti-4-1BB bispecific antibodies 4-1BB signaling activation depends on ROR1 expression levels

[0494] According to the manufacturer's recommendations, the cell surface expression level of ROR1 in various cancer cell lines was quantified using the QIFIKIT quantitative kit (Dako, K0078). In short, the cancer cells used in the test were saturated with unlabeled anti-ROR1 mouse monoclonal antibody (BD, 564464) or purified mouse IgG2b isotype control antibody (BD, 555740). After washing, the stained cells and calibration beads were simultaneously labeled with the same FITC-conjugated goat anti-mouse IgG secondary antibody. Calibration beads and secondary antibodies are included in the kit. Labeled cells and calibration beads were analyzed by flow cytometry. Linear regression analysis was performed using the MFI values ​​of the calibration beads. Using the regression line derived from the linear regression analysis, the antibody binding capacity (ABC) of the anti-ROR1 antibody and the isotype control antibody was calculated, and the specific ABC (sABC) value was obtained by subtracting the value of the isotype control antibody from the ABC value of the anti-ROR1 antibody.

[0495] The results obtained are shown in Table 18.

[0496] Table 18

[0497]

[0498]

[0499] The correlation between ROR1 expression levels obtained by antibodies (ROR1 sABC) and 4-1BB-induced NF-kB signaling was tested.

[0500] The ROR1 levels measured in Table 18 are normalized to the ROR1 expression levels in NCI-N87, and the 4-1BB activation levels induced by bispecific antibodies BA6M11 (WT) x1A10M12, BA6M11 (NA) x1A10M12 and control antibody Urerumab are determined as the maximum fold change in the 4-1BB NF-kB luciferase reporter gene assay compared to the control group. The method used is the same as described in Example 2.2.6.1. The shaded area represents the confidence interval of the linear fit.

[0501] The results obtained are shown in Figure 4c In. Figure 4c As shown, 4-1BB activation induced by the anti-ROR1 / 4-1BB bispecific antibodies BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12 showed a high correlation with ROR1 cell surface expression compared to the control antibody.

[0502] 2.6.3. Activation of 4-1BB signaling based on FcγRI binding

[0503] CHO-K1 cells expressing FcγRI, FcγRIIb or FcγRIIIa (Promega) were used to evaluate 4-1BB activation signal. The method used was the same as described in Example 2.2.6.1. GraphPad The software performs four-parameter logistic curve analysis.

[0504] like Figure 4d As shown, only CHO-K1 cells overexpressing FcγRI induced 4-1BB signaling of BA6M11(WT)x1A10M12. From this data, it can be determined that for BA6M11(NA)x1A10M12 with NA mutation in Fc, binding to FcγRI becomes weak, and thus 4-1BB signaling activation cannot be induced, however, BA6M11(WT)x1A10M12 (no mutation introduced in Fc) was found to induce strong 4-1BB signaling activation in the presence of FcγRI. CHO-K1 cells overexpressing FcγRIIb or FcγRIIIa have weaker binding to the Fc region than FcγRI and do not induce 4-1BB signaling activation.

[0505] 2.2.7. Anti-ROR1 / anti-4-1BB bispecific antibodies induce T cell immune responses and cytokine secretion activity in ROR1-expressing cells (in vitro efficacy test)

[0506] 2.2.7.1. PBMC activation test using CHOK1 cells artificially overexpressing ROR1

[0507] By co-culturing the anti-ROR1 / 4-1BB bispecific antibody comprising BA6 clone or BA6M11 clone with hamster ovarian epithelial cells (CHOK1-hROR1) stably expressing human ROR1, the 4-1BB signal activation ability of these bispecific antibodies to activate human peripheral blood mononuclear cells (PBMC) is evaluated. Interferon gamma (IFN-γ), called cytokine, is mainly produced by natural killer (NK) cells, CD4+ helper T cells (Th1) and CD8+ cytotoxic T cells. Interferon gamma is measured in the present embodiment to evaluate PBMC activation.

[0508] Briefly, 96-well plates were coated with 5 μg / mL anti-human CD3 (Biolegend, 300438) (in PBS) at 37°C for 2 hours. After removing the anti-human CD3 solution, 3×10 4 Human PBMC (peripheral blood mononuclear cells; CTL-UP-1) were added at a density of 3×10 cells per well. 4CHOK1-hROR1 cells were added at a density of 10 cells. Test antibodies (starting from 50nM, diluted 5 times) were added to the wells of the plate. The plate was then incubated at 37°C in a 5% CO2 humidified incubator for 72 hours. After incubation, the concentration of interferon gamma (IFN-γ) released into the supernatant was measured using a human IFN-γ Duoset ELISA kit (R&D Systems, DY285) according to the manufacturer's protocol. Four-parameter logistic curves were evaluated using GraphPad software.

[0509] like Figure 5a As shown, compared with BA6x1A10M12, the anti-ROR1 / 4-1BB bispecific antibody BA6M11 (NA) x1A10M12 induces the release of IFN-γ at a lower concentration, while inducing a higher concentration of IFN-γ release at the same concentration. Therefore, compared with the parental clone BA6, the bispecific antibody containing BA6M11 exhibits excellent PBMC activation efficacy. In addition, bispecific antibodies containing BA6M11 (e.g., BA6M11 (NA) x1A10M12 and BA6M11 (NA) x1A10M12) induce IFN-γ release at similar levels.

[0510] 2.2.7.2. PBMC activation assay using ROR1-expressing gastric cancer cell lines

[0511] NCI-N87 (gastric cancer cell line expressing human ROR1) is used to evaluate whether the 4-1BB signal activation activity of the bispecific antibody disclosed herein activates human peripheral blood mononuclear cells (PBMC) when co-cultured with these gastric cancer cells expressing human ROR1. In brief, 96-well plates were coated with 5 μg / mL anti-human CD3 (Biolegend, 300438) dissolved in PBS for 2 hours at 37°C. Subsequently, human PBMC (CTL) and NCI-N87 (ATCC) were added to the plate. A four-fold dilution solution of each test antibody (starting from 20nM) was added to each well and incubated for 72 hours. After incubation for 72 hours, the concentration of IFN-γ in the supernatant was measured using a human IFN-γ Duoset ELISA kit (R&D System, DY285B). The activity of NCI-N87 was measured with a cell counting kit-8 (Dojindo, CK04-20).

[0512] The results obtained are shown in Figure 5b and Figure 5c In. Figure 5b and Figure 5cAs shown, compared with the control antibody (Urelumab), the anti-ROR1 / 4-1BB bispecific antibody comprising BA6M11 has a lower EC50 (nM) value, induces IFN-γ secretion at lower concentrations, and induces cell death in NCI-N87 at lower concentrations. The data show that compared with the control antibody, it has excellent PBMC activation ability in the presence of human ROR1 antigen. In addition, compared with the combination (BA6M11+1A10M12) of BA6M11 alone and 1A10M12 alone, the anti-ROR1 / 4-1BB bispecific antibody comprising WT and NA forms of BA6M11 shows excellent PBMC activation ability. It is confirmed from this observation that compared with the simple combination of monospecific antibodies, the anti-ROR1 / 4-1BB bispecific antibody disclosed in the present invention shows an excellent synergistic effect.

[0513] 2.3. In vivo efficacy of anti-ROR1 / anti-4-1BB bispecific antibodies

[0514] 2.3.1. Anti-tumor efficacy

[0515] Human 4-1BB knock-in mice (Biocytogen) were used to evaluate the in vivo antitumor efficacy of the anti-ROR1 / anti-4-1BB bispecific antibodies BA6M11(WT)x1A10M12 and BA6M11(NA)x1A10M12. Briefly, MC38 colon cancer cells stably expressing human ROR1 (MC38 cells expressing hROR1, 5×10 5 , Biocytogen) was injected subcutaneously into the right flank of the mice. Based on tumor size (average tumor size: about 89 mm 3 , n = 5 mice / group) were divided into three groups of tumor-bearing humanized mice. After grouping, on day 0, a single intravenous dose of human IgG1 antibody (hIgG1 antibody, 3 mg / kg), BA6M11 (WT) x 1A10M1211 (4 mg / kg) or BA6M11 (NA) x 1A10M1211 (4 mg / kg) was administered. Tumor size was measured twice a week using a digital caliper and the formula: V (mm 3 )=a*b 2 / 2 (where a and b represent the long and short dimensions of the tumor, respectively) to calculate the tumor volume. The tumor growth inhibition (TGI) percentage was calculated based on the tumor volume using the following formula: TGI% (day i) = [1-(T 第(i)天 -T 第0天 ) / (V 第(i)天 -V 第0天 )]×100% (T: tumor volume of the treatment group; V: tumor volume of the control group).

[0516] The results of antitumor efficacy are presented in Figure 6a and Table 19.

[0517] Table 19

[0518]

[0519] like Figure 6a As shown in Table 19, BA6M11 (WT) x1A10M12 and BA6M11 (NA) x1A10M12 showed significant tumor inhibition. On the 23rd day after grouping, the TGI of the groups treated with BA6M11 (WT) x1A10M12 and BA6M11 (NA) x1A10M12 were 98.6% and 69.9%, respectively. On the 44th day, the survival rates of the groups treated with BA6M11 (WT) x1A10M12 and BA6M11 (NA) x1A10M12 were 100% and 80%, respectively, while there were no survivors in the hIgG1 control group. This confirms that the bispecific antibodies BA6M11 (WT) x1A10M12 and BA6M11 (NA) x1A10M12 exhibit excellent anti-tumor efficacy and high survival rate even with a single dose.

[0520] 2.3.2. Dose-dependent antitumor efficacy

[0521] To confirm the dose-dependent effect of the bispecific antibody BA6M11(WT)x1A10M12, the in vivo antitumor efficacy was evaluated in MC38-bearing mice expressing human ROR1. 5 10 cells) were injected subcutaneously into the right flank of human 4-1BB knock-in mice to prepare MC38-bearing mice expressing human ROR1. These prepared mice were based on tumor volume (approximately 80 mm 3 ) were randomly divided into groups. On day 0 of grouping, hIgG1 antibody (10 mg / kg) or BA6M11 (WT) x 1A10M12 (0.4 mg / kg, 1 mg / kg or 4 mg / kg) was injected intravenously once. The anti-tumor efficacy was measured with reference to Example 2.3.1.

[0522] like Figure 6b As shown, BA6M11 (WT) x 1A10M12 exhibited dose-dependent antitumor efficacy. In addition, treatment with BA6M11 (WT) x 1A10M12 increased the number of mice with complete tumor regression (0% in the hIgG1-treated group vs. 50% in the BA6M11 (WT) x 1A10M12 4 mg / kg-treated group), and mice with complete regression survived for more than 100 days after initial tumor implantation with no signs of tumor recurrence.

[0523] 2.3.3. Memory response to serial tumor rechallenge

[0524] To evaluate the long-lasting memory response, tumor re-challenge was performed in mice with cured tumors by BA6M11 (WT) x 1A10M12. In cured mice, tumor re-challenge was performed 100 days after the last dose of antibody. Specifically, MC38 cells expressing hROR1 (2.5 × 10 6 The secondary inoculation was performed by subcutaneous injection of 5 times the initial inoculation of cells into cured mice and control naive mice (mice that did not undergo tumor inoculation and antibody treatment). 59 days after the secondary inoculation, the tumor (2.5×10 6 The surviving mice were challenged again with 100 cells per immunization (three times). The anti-tumor efficacy was measured with reference to Example 2.3.1.

[0525] like Figure 6c As shown, mice previously treated with the bispecific antibody exhibited resistance to repeated secondary and tertiary tumor rechallenges, whereas control naive mice exhibited no tumor reduction. This demonstrates that treatment with BA6M11 (WT) x 1A10M12 induces a persistent anti-cancer memory response.

[0526] 2.3.4. In vivo immune regulation

[0527] To understand the effects of the bispecific antibodies on immune cells, tumor and peripheral tissues (blood and liver) were analyzed using flow cytometry.

[0528] Specifically, the humanized mice expressing human ROR1 and bearing MC38 tumors used in the previous examples were based on approximately 100 mm 3 The mice were randomly divided into groups according to the tumor volume. On days 0 and 4 after grouping, hIgG1 antibody (7.5 mg / kg), Urelumab (7.5 mg / kg) or bispecific antibody BA6M11 (WT) x 1A10M121 (10 mg / kg) was intraperitoneally administered to the mice. On day 7 after grouping, immune cells were analyzed by flow cytometry. In tumors and peripheral blood, leukocytes (CD45 + ) and Treg cells (CD45 + CD3 + CD4 + FOXP3 + ) and T cells (CD45 + CD3 + ) expression of 4-1BB. In the liver, T cells, CD8 + T cells (CD45 + CD3 + CD8 + ) and macrophages (CD45 + CD11b+ F4 / 80 + ) ratio. The results are shown in Figure 6d (A: tumor; B: peripheral blood; C: liver).

[0529] like Figure 6d As shown, tumor infiltrating leukocytes (CD45 + / Live) significantly increased. In addition, unlike Urecitabine, BA6M11 (WT) x1A10M12 specifically induces the consumption of immunosuppressive Treg cells (Treg / CD45+) in tumors rather than in systemic circulation. Compared with blood, the expression of 4-1BB on T cells (4-1BB+ / CD3+) is measured at a higher level in tumors, and its level is increased by treatment with BA6M11 (WT) x1A10M12. These results confirm the tumor-specific T cell activation effect of the bispecific antibody of the present application. In addition, although the systemic activation of Urecitabine is accompanied by a large amount of infiltration of immune cells into the liver, the bispecific antibody of the present application does not show an increase in T cells and macrophages in the liver. These results confirm that the bispecific antibody of the present application exerts a powerful anti-tumor effect through tumor-specific immunomodulation while minimizing the risk of peripheral toxicity.

[0530] 2.3.5. Additional Demonstration of Tumor-Specific T Cell Activation by Anti-ROR1 / Anti-4-1BB Bispecific Antibodies

[0531] In order to prove that the bispecific antibody of the present application specifically activates T cells only in cells expressing ROR1 (e.g., tumor cells) and does not act on normal cells and tissues that do not express ROR1, the cytokine secretion activity induced by the anti-ROR1 / anti-4-1BB bispecific antibody was verified using human PBMC in the absence of cells expressing ROR1.

[0532] Specifically, five types of human PBMCs (CTL, CTL-UP1) were thawed and then cultured at 2×10 5 The concentration of 1 μg / well was inoculated in a 96-well plate. As a positive control, anti-human CD3 antibody (Biolegend, 317326) was diluted to 0.01 μg, 0.1 μg, 1 μg, 25 μg and 50 μg in RPMI with 10% FBS and added to each well. For the test group, 1 μg, 25 μg and 50 μg of bispecific antibody (BA6M11 (WT) x 1A10M12) were added to each well.

[0533] The plates were incubated in a 37°C, 5% CO2 incubator for 48 hours. After 48 hours, the plates were centrifuged to separate the supernatant. ELISA analysis was performed according to the manufacturer's protocol to measure the concentrations of IFN-γ (R&D Systems, DY285B), IL-2 (R&D Systems, DY202), IL-6 (R&D Systems, DY206), and TNF-α (R&D Systems, DY210) in the culture supernatant. The results are shown in Figure 6e middle.

[0534] like Figure 6e As shown, BA6M11 (WT) x 1A10M12 did not induce cytokine release in the absence of ROR1 compared to the positive control. This means that the bispecific antibody of the present application activates T cells only in tumor tissues expressing ROR1 but not in normal cells and tissues, indicating that the risk of unexpected toxicity after in vivo administration is reduced.

[0535] Example 3. ROR1xB7-H3 bispecific antibodies and antibody-drug conjugates (ADCs) containing the same

[0536] 3.1. Design and construction of improved anti-ROR1 / anti-B7-H3 bispecific antibodies

[0537] Based on the anti-ROR1 antibody clone BA6M11 selected in Example 1 and the anti-B7-H3 antibody clone B5 disclosed in International Publication WO2019-226017A1, an anti-ROR1 / anti-B7-H3 bispecific antibody containing both anti-ROR1 antibody and anti-B7-H3 antibody was prepared.

[0538] The bispecific antibody is designed in an IgG-scFv fusion format, in which the scFv antibody fragment targeting one of the antigens (ROR1 or B7-H3) is fused to the C-terminus (heavy chain C-terminus) of a full-length IgG antibody targeting the other antigen. In this example, a bispecific antibody comprising an anti-ROR1 IgG antibody and a B5 scFv linked to the C-terminus of each heavy chain of the anti-ROR1 IgG antibody was constructed.

[0539] In order to construct the anti-ROR1 / anti-B7-H3 bispecific antibody, DNA fragment 1 containing a nucleotide sequence encoding the heavy chain of the anti-ROR1 IgG antibody (BA6M11) was inserted into pcDNA3.4 (Invitrogen, A14697; plasmid 1), and DNA fragment 2 containing a nucleotide sequence encoding the light chain of the IgG antibody in the anti-ROR1 / anti-B7-H3 bispecific antibody was inserted into pcDNA3.4 (Invitrogen, A14697; plasmid 2). Subsequently, DNA fragment 3 encoding scFv was fused to a portion of DNA fragment 1 corresponding to the C-terminus of the IgG antibody Fc region in plasmid 1 using DNA fragment 4 encoding a peptide linker consisting of 20 amino acids in length (GGGGS) 3 (SEQ ID NO: 120) to construct a vector for expressing the bispecific antibody.

[0540] In addition, the anti-B7-H3 scFv (designated as B5) is in the form of a light chain variable region (VL) C-terminus and a heavy chain variable region (VH) C-terminus are each fused to VL103-VH44 (VL103: VL with a G(Gly)→C(Cys) mutation introduced at position 103; VH44: VH with a G→C mutation introduced at position 44) via a linker ((GGGGS)4) (SEQ ID NO: 114). These mutations stabilize the scFv and enable disulfide bond formation.

[0541] The amino acid sequence of the bispecific antibody (BA6M11xB5) thus prepared is listed in Table 20 below.

[0542] Table 20

[0543]

[0544]

[0545] (Linker 1: connection between BA6M11 (IgG) and B5 (scFv) (the C-terminus of the heavy chain in BA6M11 is connected to the N-terminus of the VL in B5);

[0546] Linker 2: Connection between VL and VH in B5 (scFv) (C-terminus of VL connected to N-terminus of VH)

[0547] The encoding nucleic acid sequence of the bispecific antibody (BA6M11xB5) is as follows:

[0548] [Heavy chain_BA6M11xB5] (SEQ ID NO: 122)

[0549]

[0550]

[0551] [Light chain_BA6M11] (SEQ ID NO: 123)

[0552]

[0553] According to the manufacturer's protocol, the coding DNA of the bispecific antibody cloned by transient expression was used ExpiCHO system (Thermo Fisher). The culture medium containing the expressed antibody was clarified by centrifugation and filtration to remove the suspension, and the antibody was purified using the affinity chromatography of MabSelect Sure resin and the size exclusion chromatography with Superdex200 resin. The purity of the purified antibody was analyzed using the HPLC with TSKgel SuperSW3000 column, and it was found that the purity exceeded 95%.

[0554] 3.2. Stability evaluation of anti-ROR1 / B7-H3 bispecific antibodies comprising improved anti-ROR1 clones

[0555] (1) Analysis of binding specificity of anti-ROR1 / B7-H3 bispecific antibody under stress conditions (ELISA)

[0556] As a control group, the BA6xB5 bispecific antibody comprising the parental cloned anti-ROR1 antibody BA6 without removal of the PTM hotspot was used, and for the experimental group, the BA6M11xB5 bispecific antibody was used, and the antibody-antigen binding affinity to human ROR1 and human B7H3 recombinant proteins was evaluated using an ELISA-based solution binding assay after storing both samples at 4°C (normal conditions) and 40°C (stress conditions) for two weeks. Specifically, 96-well microtiter plates (Nunc-ImmunoPlates, NUNC) were coated with human ROR1 protein (ROR1-His; Sino Biological, catalog number 13968-H08H) and human B7-H3 protein (B7H3-His; Sino Biological, catalog number 11188-H08H) at a concentration of 1 μg / ml in PBS solution at 4°C for 16 hours, and then the plates were blocked with 1% (v / v) BSA (bovine serum albumin) at 37°C for 2 hours to prevent nonspecific binding.

[0557] Then, the BA6xB5 and BA6M11xB5 bispecific antibodies stored for two weeks were Figure 7a-7dThe concentrations listed in were added to microtiter plates, and their binding capacity was analyzed by ELISA as follows. Specifically, after incubation at 37°C for 2 hours, the plate was washed 5 times with PBS containing 0.05% (v / v) Tween 20, and then the HRP-conjugated Fc polyclonal antibody reagent (Pierce, catalog number 31413) was diluted in 1% (v / v) BSA at a ratio of 1:30,000 and added to the washed microtiter plates, incubated at 37°C for 1 hour to allow the reaction, and then used to detect the bound bispecific antibodies. The reaction was developed using TMB (tetramethylbenzidine, Sigma, T0440). The enzymatic reaction was stopped with 0.5mol / L sulfuric acid, and the absorbance was measured at 450nm and 650nm using a microplate reader (Molecular Devices).

[0558] The results obtained are shown in Figure 7a-7d In. From Figure 7a-7d It can be seen that after evaluating the antigen-antibody binding capacity (ELISA; protein binding) using human ROR1 protein and human B7-H3 protein, it was confirmed that there was no significant difference in binding capacity between samples stored at 4°C and 40°C for two weeks. These results show that BA6M11xB5 maintains its potency while showing improved physicochemical properties in terms of its developability compared to BA6xB5. Ultimately, it was confirmed that the BA6M11 clone not only showed comparable binding to WT, but also had significantly improved developability through PTM engineering.

[0559] (2) Developability analysis of anti-ROR1 / B7-H3 bispecific antibodies

[0560] A BA6M11 clone whose PTM propensity was eliminated by PTM engineering from BA6 was selected, and a BA6M11xB5 bispecific antibody and a BA6xB5 bispecific antibody comprising BA6M11 were produced to examine the increased stability under stress conditions.

[0561] More specifically, after storing the BA6xB5 bispecific antibody and the BA6M11xB5 bispecific antibody at 4°C (normal conditions) and 40°C (stress conditions) for two weeks, each sample was analyzed, such as SE-HPLC, icIEF and LC / MS, to check the purity and impurity content. In addition, in order to determine whether changes in substance content also affect efficacy, an antigen-antibody binding ability analysis (ELISA; refer to Example 3.2 (1)) was performed.

[0562] The results obtained are shown in Table 21 below.

[0563] Table 21

[0564]

[0565] (*N / A: Not applicable)

[0566] As shown in Table 21, when stored at 4 ℃ and 40 ℃, BA6M11xB5 clones maintain high levels of antibody purity, with no significant difference compared to BA6xB5. With regard to charge variants, BA6M11xB5 clones show improvements that are superior to BA6xB5 clones, wherein the basic variant peak associated with the formation of succinimide is reduced, and the analysis using LC-MS also reveals that, compared to BA6xB5, BA6M11xB5 does not show an increase in succinimide in Fd and LC under 40 ℃ stress conditions, and does not form a fragment that appears to be cut in the Fd region. This reduction of charge variants means that the stability of antibody quality and the reliability of the purification process increase, as discussed in Example 1.3. It is also shown that when administered to humans, bispecific antibodies are expected to maintain low immunogenicity and valency.

[0567] 3.3. Analysis of binding specificity of anti-ROR1 / B7-H3 bispecific antibody to ROR1 and B7-H3 expressed on the cell surface (FACS)

[0568] To evaluate the cell binding ability of the anti-ROR1 / B7-H3 bispecific antibody, cells expressing ROR1 and B7-H3 were used to compare the cell binding ability of the bispecific antibody with that of the monospecific antibody.

[0569] As a cell line for comparison of cell binding ability, a CHO-huROR1-huB7-H3 cell line stably transfected with human ROR1 and human B7-H3 genes to artificially overexpress human ROR1 and human B7-H3 proteins (obtained by transfecting a ROR1-C HO recombinant cell line (BPS Bioscience) with a human B7-H3 gene (Origene) and selecting single cells with the desired gene insertion using G418) was used, and analyzed using a flow cytometer (LSRF ortessa X-20, BD Biosciences). Specifically, after dissociating and washing the overexpressing cell line with PBS, the number of cells was counted, and the cells were plated at 2×10 5The density of cells / well was inoculated into a V-bottom 96-well plate (96-well Plate-RV, Bioneer, 910D09). The antibody diluted 4 times from 100nM in a 1% BSA solution was added to the centrifuged cells at 100μL / well and incubated at 4°C for 1 hour. After 1 hour, the cells were washed twice with the same buffer and then incubated with FITC-labeled Fc-specific antibodies (goat anti-human IgG-FITC antibodies produced in goats, Sigma, F9512) diluted 500 times in 1% BSA at 100μL / well at 4°C for 1 hour. After the reaction, the cells were washed twice with the same buffer, resuspended in 100μL PBS, and analyzed using a FACS instrument. A total of 10,000 cells were detected for each measurement, and the data were analyzed using FlowJo software. The fold change value on the graph is represented by dividing the MFI (mean fluorescence intensity) of the experimental group treated with the antibody by the MFI of the control group treated only with the secondary antibody.

[0570] like Figure 8 As shown, the anti-ROR1 / B7-H3 bispecific antibody having more antigen binding sites showed higher binding affinity to cell lines expressing both ROR1 and B7-H3 compared to the monospecific antibody.

[0571] These results confirm that the anti-ROR1 / B7-H3 bispecific antibodies provided herein exhibit excellent binding affinity by simultaneously targeting two antigens. This enhanced binding ability exhibits excellent selectivity for cells expressing both targets (antigens) compared to cells expressing a single target (antigen). This feature is consistent with the goal of reducing toxicity and increasing efficacy through selectivity, which is an important element in the design of cancer treatment methods.

[0572] Comparison of cellular internalization between monospecific and bispecific antibodies

[0573] To compare the cellular internalization rate of the anti-ROR1 / B7-H3 bispecific antibody, cells expressing both ROR1 and B7-H3 were used to assess the internalization of the bispecific antibody. FabFluor-pHRed antibody labeling reagent (Sartorius, 4722) was used for the experiment. CHO-huROR1-huB7-H3 cells (the same cell line as used in Example 3.3) were cultured at 1×10 4 The cells were seeded at a density of 10 cells / mL in a 96-well plate (96-well transparent flat-bottom TC-treated culture microplate, Falcon, 353072) and cultured for 24 hours. FabFluor-pH Red antibody labeling reagents were mixed in a 1:1 molar ratio and added to the plate containing cells after incubation at 37°C for 15 min. The plate was then placed in a real-time cell analysis system (IncubCyte S3, Sartorius) and analyzed by software for phase contrast and fluorescence at 1-hour intervals. Fig. 9 As shown, an increase in the fluorescence signal over time was observed, confirming the internalization process of the antibody into the cell and reaching the lysosome.

[0574] like Fig. 9 As shown, the anti-ROR1 / B7-H3 bispecific antibody with more antigen-binding sites increased the fluorescence signal more rapidly in the cell line expressing both ROR1 and B7-H3 than the monospecific antibody, indicating that the bispecific antibody is internalized into cells more efficiently than the monospecific antibody due to its ability to simultaneously target two antigens.

[0575] These results indicate that the anti-ROR1 / B7-H3 bispecific antibodies provided herein promote efficient cellular internalization by simultaneously targeting two antigens.

[0576] Based on the described results, it is expected that developing bispecific antibodies into ADCs (antibody-drug conjugates) can selectively enhance the efficacy of ADCs in cells expressing both targets (antigens). This feature was further studied and confirmed in the subsequent examples.

[0577] 3.5. Manufacturing of anti-ROR1 / anti-B7-H3 bispecific antibody ADC

[0578] (1) Manufacturing of bispecific antibody ADC

[0579] In order to conjugate the drug to the amino acid position 205 of the antibody light chain, the valine (Valine) at the Kabat position 205 in the constant region of the antibody light chain is mutated to cysteine ​​(Cysteine) (V205C), and dithiothreitol (DTT) or a similar reducing agent is reacted to generate a thiol group at the antibody light chain V205C, and the antibody is conjugated to the drug by thiosuccinimide formation via Michael addition. Specifically, the antibody is prepared to a concentration greater than 5 mg / ml by ultrafiltration / diafiltration (UF / DF), 1M tris (hydroxymethyl) aminomethane (Tris-HCl) (pH8.8) and 500mM ethylenediaminetetraacetic acid (EDTA) are added, and the final concentration of the antibody is adjusted to 5 mg / ml, 75mM Tris-HCl and 2mM EDTA. 100mM dithiothreitol (DTT) was added to the prepared antibody at a molar ratio of 1:20 antibody: DTT, and reacted at 25°C for 16.5 hours to separate the free cysteine ​​linked by a disulfide bond on the cysteine ​​at 205 of the antibody light chain. After this process (called "decapping"), the decapped antibody was separated using cation exchange chromatography (CEX). The reaction product was eluted with SPHP-B buffer [50mM tris (hydroxymethyl) aminomethane (Tris-HCl), pH7.5, 0.5M sodium chloride] on a Hitrap SPHP column (GE Healthcare) equilibrated with SPHP-A buffer [10mM succinate, pH5.0]. For the reconstitution of the uncapped antibody, the antibody to be oxidized was prepared in 75mM tris (hydroxymethyl) aminomethane (pH 7.5) using 1M Tris-HCl, and dehydroascorbic acid (DHAA, oxidized vitamin C) was added at an antibody: DHAA ratio of 1: 20, and reoxidized for 2 hours in the dark at 25°C. Then, the reoxidized antibody was separated using cation exchange chromatography (CEX) and eluted with SPHP-C buffer [10mM succinate pH 5.0, 0.5M sodium chloride]. The purified antibody was concentrated to more than 5mg / ml by ultrafiltration / diafiltration (UF / DF). To produce antibody-drug conjugates, antibodies were prepared in 1M tris (hydroxymethyl) aminomethane (Tris-HCl, pH 7.0) to a final concentration of 5mg / ml, and drugs were added at an antibody: drug molar ratio of 1: 10, and reacted at 25°C for 16.5 hours. Antibody-drug conjugates were separated using cation exchange chromatography (CEX) and eluted with SPHP-C buffer. To isolate only the DAR2 antibody-drug conjugate, hydrophobic interaction chromatography (HIC) was used. For this purpose, 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% 2-propanol].The antibody was exchanged into HS buffer [20 mM histidine, pH 6.0, 240 mM sucrose] by ultrafiltration / diafiltration (UF / DF) to remove 2-propanol and finally obtain the antibody-drug conjugate, the drug used was pyrrolobenzoazepine (PBD), and the obtained antibody was named 'Antibody (V205C)-T-PBD'.

[0580] (2) Purity and DAR measurement of prepared bispecific antibody ADC

[0581] The purity of the manufactured ADC was measured using size exclusion high performance liquid chromatography (SE-HPLC). The measurement was performed using an Agilent 1200 series HPLC instrument equipped with a Tosoh TSKgel G3000SWxl column (Tosoh Bioscience), and the elution position and area under the curve (AUC) of each sample were compared to determine the purity of each sample. The main peak purity of the ADC exceeded 99.0% (see Table 23 and Figures 10a to 10c ).

[0582] The drug-to-antibody ratio (DAR) of ADC was determined using a liquid chromatography-mass spectrometry (LC / MS) method. To remove N-glycans, 1 unit of PNGaseF (NEB) / 100 μg ADC (in PBS) was added at a concentration of 1 mg / ml and incubated at 37°C for 15 hours. The Waters UPLC Class I instrument coupled to the Waters Synapt G2-S LC / MS system was equipped with an Acquity UPLCBEH200 SEC 1.7 μm (4.6*150 mm) column and equilibrated with a mobile phase consisting of 30% (v / v) acetonitrile, 0.1% (v / v) formic acid, and 0.05% trifluoroacetic acid (TFA). 5 μg of the sample with N-glycan removed was loaded and analyzed by LC / MS. The DAR (drug-to-antibody ratio) of ADC was measured by LC / MS to confirm that each antibody molecule was conjugated with 2 drugs (see Tables 22, 23, and Figures 11a to 11c ).

[0583] Table 22

[0584] ADC purity(%) DAR BA6(M11 / V205C)xB5-T-PBD 99.63 2.0 BA6(M11 / V205C)-T-PBD 99.54 2.0 B5(V205C)-T-PBD 99.57 2.0

[0585] Table 23

[0586]

[0587] (3) Ligand binding activity test using bispecific antibody ADC

[0588] The ligand binding affinity of BA6M11 (V205C) -T-PBD monospecific antibody and BA6xB5 (M11) (V205C) -T-PBD bispecific antibody ADC to human ROR1 recombinant protein was evaluated using an ELISA-based solution binding assay. Specifically, 96-well microtiter plates (Nunc-Immuno Plates, NUNC) were coated with ROR1 protein (ROR1-His; Sino Biological, catalog number 13968-H08H) at a concentration of 1 μg / ml in PBS solution at 4°C for 16 hours and blocked with 1% (v / v) BSA (bovine serum albumin) for 2 hours at 37°C to prevent nonspecific binding.

[0589] Then, BA6M11(V205C)-T-PBD monospecific antibody and BA6xB5(M11)(V205C)-T-PBD bispecific antibody were used to Fig.12 The concentrations listed in were added to microtiter plates and their binding capacity was analyzed by ELISA as follows: After incubation at 37°C for 2 hours, the plates were washed 5 times with PBS containing 0.05% (v / v) Tween 20, and then HRP-conjugated Fc polyclonal antibody reagent (Pierce, catalog number 31413) diluted 1:30,000 in 1% (v / v) BSA was added to the washed microtiter plates, incubated at 37°C for 1 hour to allow the reaction to proceed, and used to detect the bound bispecific antibodies. The reaction was developed using TMB (tetramethylbenzidine, Sigma, T0440). The enzymatic 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 Devices).

[0590] Fig.12 The results shown in the Figures show that the ligand binding ability of the prepared BA6xB5(M11)(V205C)-T-PBD bispecific antibody to human ROR1 protein is comparable to that of the prepared BA6M11(V205C)-T-PBD monospecific antibody. This confirms that the excellent ROR1 antigen binding ability of BA6M11 is retained even after being manufactured into a bispecific antibody ADC format.

[0591] Comparative evaluation of cytotoxicity of bispecific antibody ADCs

[0592] The anti-cancer cell death efficacy of ADCs made using the bispecific antibodies of the present disclosure was compared with that of monospecific antibody ADCs. Cancer cell lines Calu-3 (KCLB, catalog number 30055) and Calu-6 (ATCC, catalog number HTB-56) expressing both ROR1 and B7-H3 were used, and the cell death ability was compared by administering ADC to the cells and measuring the metabolic activity of the cells. In order to determine whether the effect of the bispecific antibody is simply a combination of monospecific antibody targets, a control group treated with both anti-ROR1 monospecific antibody and anti-B7-H3 monospecific antibody (combination treatment, indicated by "+") was also evaluated for comparison. Cancer cell lines were cultured using the recommended culture medium. 5×10 3 Cells / well were inoculated into a 96-well culture plate with a volume of 50 μl and cultured for 4-6 hours, and then 50 μl of ADC diluted at various concentrations was distributed into the 96-well plate containing cells. Then, the cells were cultured at 37°C, 5% CO2 for about 6 days. The number of viable cells was quantified using WST-8 (Dojindo, CK04).

[0593] Fig.13 Depicted are the cytotoxic results of monospecific or bispecific antibodies against cancer cell lines, using B5 as an anti-B7-H3 antibody and BA6M11 as an anti-ROR1 antibody. It was observed that in cancer cell lines (Calu-3, Calu-6) expressing both ROR1 and B7-H3, the cytotoxicity of the bispecific antibody (BA6M11xB5) ADC was greater than the combined cytotoxicity of the monospecific antibody (BA6M11, B5) ADC. On the contrary, in the KATOIII cell line (ATCC, catalog number HTB-103) that hardly expresses ROR1 and B7-H3, the ADC exhibited minimal cytotoxicity, and there was no significant difference in cytotoxicity between the monospecific antibody ADC and the bispecific antibody ADC. This indicates that the enhanced cytotoxicity of the bispecific antibody ADC compared to the monospecific antibody ADC is antigen-dependent. The results confirm that treatment with bispecific antibodies has higher cytotoxicity than the combination of monospecific antibodies, confirming that the excellent cytotoxicity of the bispecific antibody ADC disclosed herein is not just the effect of the monospecific antibody combination.

[0594] Although some embodiments have been described herein, it should be understood by those skilled in the art that these embodiments are given by way of illustration only, and that various modifications, changes and variations may be made without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention shall be limited only by the appended claims and their equivalents.

Claims

1. An anti-ROR1 antibody or an 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.

2. The anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1, comprising 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.

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

5. The pharmaceutical composition according to claim 4, wherein The cancer expresses ROR1. 6 . A composition for detecting ROR1, comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 or 2. 7 . A composition for diagnosing cancer, comprising the anti-ROR1 antibody or antigen-binding fragment thereof according to claim 1 or 2 .

8. The composition according to claim 7, wherein The cancer expresses ROR1.

9. An anti-ROR1 / anti-4-1BB bispecific antibody, comprising: an anti-ROR1 antibody or an antigen-binding fragment thereof; and Anti-4-1BB antibody or antigen-binding fragment thereof, in, The anti-ROR1 antibody or its antigen-binding fragment comprises: 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.

10. The anti-ROR1 / anti-4-1BB bispecific antibody according to claim 9, wherein The anti-ROR1 antibody or antigen-binding fragment thereof comprises 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.

11. The anti-ROR1 / anti-4-1BB bispecific antibody according to claim 9 or 10, wherein The anti-4-1BB antibody or antigen-binding fragment thereof comprises: 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; 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.

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

13. The anti-ROR1 / anti-4-1BB bispecific antibody according to any one of claims 9 to 12, wherein The anti-4-1BB antibody or antigen-binding fragment thereof comprises: 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 The light chain variable region comprises the amino acid sequence of SEQ ID NO:42, 43, 44, 45, 46 or 47.

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 its antigen-binding fragment according to any one of claims 9 to 13 and a pharmaceutically acceptable excipient.

16. The pharmaceutical composition according to claim 15, wherein The cancer expresses ROR1.

17. An anti-ROR1 / anti-B7-H3 bispecific antibody, comprising: an anti-ROR1 antibody or an antigen-binding fragment thereof; and an anti-B7-H3 antibody or an antigen-binding fragment thereof, in, The anti-ROR1 antibody or its antigen-binding fragment comprises: 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.

18. The anti-ROR1 / anti-B7-H3 bispecific antibody according to claim 17, wherein The anti-ROR1 antibody or antigen-binding fragment thereof comprises 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.

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

20. The anti-ROR1 / anti-B7-H3 bispecific antibody according to any one of claims 16 to 19, wherein The anti-B7-H3 antibody or antigen-binding fragment thereof comprises: (i) a heavy chain complementarity determining region selected from the group consisting of: (a) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 58, (b) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 49, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 59, (c) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 60, (d) a HCDR1 comprising the amino acid sequence of SEQ ID NO:48, a HCDR2 comprising the amino acid sequence of SEQ ID NO:55, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:61, and (e) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 62, and (f) a HCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 57, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 60; and (ii) a light chain complementarity determining region selected from the group consisting of: (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) LCDR1 comprising the amino acid sequence of SEQ ID NO:67, LCDR2 comprising the amino acid sequence of SEQ ID NO:72, and 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.

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

22. A nucleic acid molecule encoding the anti-ROR1 / anti-B7-H3 bispecific antibody or antigen-binding fragment thereof according to 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 according to claim 23, wherein The cancer expresses either or both of ROR1 and B7-H3. 25 . An antibody-drug conjugate, 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 cytotoxic drug linked thereto.

26. The antibody-drug conjugate according to claim 25, wherein The antibody-drug conjugate has a structure of antibody-linker-drug.

27. The antibody-drug conjugate according to claim 25 or 26, wherein The cytotoxic drug is an anticancer agent.

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

29. The pharmaceutical composition according to claim 28, wherein The cancer expresses either or both of ROR1 and B7-H3.

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