Anti-ROR1 antibodies and uses thereof

Abstract

The present invention relates to an anti-ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1) antibody or an antigen-binding fragment thereof, a nucleic acid encoding the antibody, a recombinant expression vector comprising the nucleic acid, a host cell transfected with the recombinant expression vector, a method for producing the antibody or the antigen-binding fragment thereof, a bi / multispecific antibody comprising the antibody or the antigen-binding fragment thereof, an immune cell-engaging bi / multispecific antibody, an antibody-drug conjugate (ADC) in which the antibody or the antigen-binding fragment thereof is conjugated to a drug, a chimeric antigen receptor (CAR) comprising an scFv of the antibody as an antigen-binding site in the extracellular domain, an immune cell into which the chimeric antigen receptor has been introduced, a combination therapy composition comprising the antibody or the antigen-binding fragment thereof, a composition for preventing or treating cancer, and a method for preventing or treating cancer.

Description

[Technical Field]

[0001] The present invention relates to an anti-ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1) antibody or an antigen-binding fragment thereof, a nucleic acid encoding the antibody, a recombinant expression vector comprising the nucleic acid, a host cell transfected with the recombinant expression vector, a method for producing the antibody or the antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or the antigen-binding fragment thereof, an immune cell engaging bispecific or multispecific antibody comprising an scFv of the antibody and a second binding domain comprising one or more scFvs of antibodies that bind to an immune cell-activating antigen, an antibody-drug conjugate (ADC) in which the antibody or the antigen-binding fragment thereof is conjugated to a drug, a chimeric antigen receptor (CAR) comprising the scFv of the antibody as an antigen-binding site in the extracellular domain, an immune cell into which the chimeric antigen receptor has been introduced, a combination therapy composition comprising the antibody or the antigen-binding fragment thereof, or the immune cell, a composition for preventing or treating cancer, and a method for preventing or treating cancer.

[0002] [Background technology]

[0003] ROR (Receptor Tyrosine Kinase Like Orphan Receptor) is a representative group of membrane potential RTK (Receptor Tyrosine Kinase) receptors that exert diverse effects on cellular activities. The ROR family consists of ROR1 and ROR2, and the amino acid sequence similarity between the two proteins reaches approximately 60%. The ROR family consists of Ig, cysteine-rich, and kringle domains extracellularly, and tyrosine kinase, Ser / Thr-rich, and proline-rich domains internally.

[0004] ROR1 is expressed during embryogenesis and influences various early cellular activities. However, its expression gradually declines as adult tissue formation progresses. ROR1 is also expressed at intermediate stages of normal B cell maturation, but is not expressed in mature B and T cells or monocytes. However, ROR1 has been shown to be overexpressed in various tumor cells and is considered a fetal gene in malignant tumors. It has been reported that ROR1 activates non-canonical Wnt signaling by acting as a Wnt5a receptor, leading to cancer cell proliferation and metastasis. Therefore, ROR1 has emerged as a potential target for anti-cancer antibody therapeutics. Specifically, ROR1 was shown to be overexpressed in chronic lymphocytic leukemia (CLL), prompting active research to identify ROR1 overexpression in various cancers (Klein et al., 2001, J. Exp. Med 194:1625). As a result, ROR1 overexpression was confirmed not only in various blood cancers other than chronic lymphocytic leukemia (CLL) (acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL)), but also in solid cancers such as breast cancer, cervical cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), and brain cancer.

[0005] Initially, ROR1 was classified as a pseudokinase and was somewhat underestimated. However, overexpression of ROR1 in the above-mentioned cancers is known to be closely associated with cancer patient survival and cancer metastasis, highlighting the need for the development of effective therapeutic agents. International Patent WO2016 / 172726 A1 discloses an anti-ROR1 monoclonal antibody and its use. This antibody exhibits ROR1-specific properties and binds to and kills ROR1-overexpressing CLL cells, suggesting the potential for the development of antibody-based therapeutic agents that selectively recognize ROR1.

[0006] Because anti-ROR1 antibodies have different properties, they can be developed into a variety of anti-cancer antibodies tailored to the cancer in which the antigen is expressed. Targeting cancers with a high recurrence rate, such as CLL, can fulfill the unmet need for anti-cancer antibodies (Choi et al., 2018, Cell Stem Cell 22, 951-959). Cancer-specific expression of ROR1 is not only observed in simple cancer cells, but also in cancer stem cells and stemness, which helps suppress tumor metastasis and recurrence. Considering this expression in a variety of cancer-related cells, there are countless possibilities for developing diverse therapeutic agents based on existing antibodies.

[0007] Under these technical backgrounds, the present invention was accomplished by not only discovering antibodies specific to ROR1, but also selecting antibodies that more selectively recognize ROR1 on the surface of patient-derived cells, and confirming antibodies with superior anti-cancer efficacy and targeting accuracy compared to currently known therapeutic agents under development.

[0008]

[0009] Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a novel antibody against ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1) or an antigen-binding fragment thereof.

[0011] Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0012] It is yet another object of the present invention to provide a recombinant expression vector containing the nucleic acid or a host cell transfected with the recombinant expression vector.

[0013] It is still another object of the present invention to provide a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to ROR1.

[0014] It is yet another object of the present invention to provide a bi / multispecific antibody or an immune cell-engaging bi / multispecific antibody comprising the above antibody or an antigen-binding fragment thereof.

[0015] It is yet another object of the present invention to provide an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is conjugated to a drug.

[0016] A further object of the present invention is to provide a chimeric antigen receptor (CAR) comprising the scFv of the antibody as an antigen-binding site in the extracellular domain, an immune cell into which the chimeric antigen receptor has been introduced, and a composition for combined therapy comprising the immune cell.

[0017] It is yet another object of the present invention to provide a composition for preventing or treating cancer, or a method for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof, the bispecific or multispecific antibody, the antibody-drug conjugate, the chimeric antigen receptor, or the immune cell.

[0018] It is yet another object of the present invention to provide use of the antibody or antigen-binding fragment thereof, the bispecific or multispecific antibody, the antibody-drug conjugate, the chimeric antigen receptor, or the immune cell for the prevention or treatment of cancer.

[0019] It is yet another object of the present invention to provide use of the antibody or antigen-binding fragment thereof, the bispecific or multispecific antibody, the antibody-drug conjugate, the chimeric antigen receptor, or the immune cell for the manufacture of a medicament for the prevention or treatment of cancer.

[0020] [Means for solving the problem]

[0021] To achieve the above object, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1), including:

[0022] a heavy chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 16;

[0023] a heavy chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 41, 184, and 185;

[0024] a heavy chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 42 to 65;

[0025] a light chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 66 to 86;

[0026] A light chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 87 to 102, 186, and 187; and

[0027] A light chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 103 to SEQ ID NO: 121.

[0028] The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0029] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0030] The present invention also provides a host cell transfected with the recombinant expression vector.

[0031] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to ROR1, comprising the steps of culturing host cells to produce an antibody; and isolating and purifying the produced antibody.

[0032] The present invention also provides a bi / multispecific antibody or an immune cell-engaging bi / multispecific antibody comprising the above antibody or an antigen-binding fragment thereof.

[0033] The present invention also provides an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is conjugated to a drug.

[0034] The present invention also provides a chimeric antigen receptor (CAR) comprising an extracellular domain containing an antigen-binding site, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is an scFv of the antibody.

[0035] The present invention also provides an immune cell comprising the chimeric antigen receptor (CAR).

[0036] The present invention also provides a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof, the bispecific or multispecific antibody, the antibody-drug conjugate, the chimeric antigen receptor, or the immune cell.

[0037] The present invention also provides use of the antibody or antigen-binding fragment thereof, the bispecific or multispecific antibody, the antibody-drug conjugate, the chimeric antigen receptor, or the immune cell for the prevention or treatment of cancer.

[0038] The present invention also provides use of the antibody or antigen-binding fragment thereof, the bispecific or multispecific antibody, the antibody-drug conjugate, the chimeric antigen receptor, or the immune cell for the manufacture of a medicament for the prevention or treatment of cancer.

[0039] [Brief explanation of the drawings]

[0040] [Figure 1] FIG. 1 shows the results of analyzing the ROR1 binding properties of clones through ELISA.

[0041] [Figure 2] FIG. 1 shows the results of confirming that anti-ROR1 antibodies cross-link to human ROR1 and mouse ROR1.

[0042] [Figure 3] FIG. 1 shows the results of analyzing anti-ROR1 antibody clones that bind to each domain through ELISA.

[0043] [Figure 4] FIG. 1 shows the results of measuring the ROR1 expression level of each cell used in an example of the present invention.

[0044] [Figure 5] FIG. 1 shows the results of analyzing the binding ability of each antibody used in the examples of the present invention to the ROR1 antigen expressed in cells derived from lung cancer patient AMB-LC-0003T.

[0045] [Figure 6] FIG. 1 shows the results of analyzing the binding ability of each antibody used in the examples of the present invention to the ROR1 antigen expressed in the Jeko-1 cell line.

[0046] [Figure 7] FIG. 1 shows the results of cellular internalization analysis of antibodies used in the examples of the present invention.

[0047] [Figure 8] FIG. 1 shows the structure of an anti-ROR1 antibody-drug conjugate.

[0048] [Figure 9] FIG. 1 shows the results of purity analysis of antibody-drug conjugates used in the examples of the present invention.

[0049] [Figure 10] FIG. 1 shows the results of analyzing the average number of drug molecules conjugated per antibody molecule in antibody-drug conjugates used in the examples of the present invention.

[0050] [Figure 11] FIG. 1 shows the results of ELISA analysis of ROR1 binding of antibody-drug conjugates used in the examples of the present invention.

[0051] [Figure 12] FIG. 1 shows the results of in vitro toxicity evaluation of antibody-drug conjugates used in the examples of the present invention against patient-derived cells.

[0052] [Figure 13] FIG. 1 shows the results of in vitro toxicity evaluation of the antibody-drug conjugate used in the examples of the present invention on patient-derived cells, confirming that the antibody-drug conjugate exhibits significantly increased efficacy compared to existing antibody-drug conjugates.

[0053]

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0056]

[0057] Anti-ROR1 antibody

[0058] In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), comprising: a heavy chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 16; a heavy chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 41, 184, and 185; a heavy chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 42 to 65; a light chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 66 to 86; a light chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 87 to 102, 186, and 187; and a light chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 103 to 121.

[0059] As used herein, the term "antibody" refers to an anti-ROR1 antibody that specifically binds to ROR1. The scope of the present invention includes not only intact antibody forms that specifically bind to ROR1, but also antigen-binding fragments of the antibody molecules.

[0060] An intact antibody is a structure having two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond.

[0061] As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof that includes a variable region domain VH containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, and three constant region domains CH1, CH2, and CH3. As used herein, the term "light chain" refers to a full-length light chain and fragments thereof that includes a variable region domain VL containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, and a constant region domain CL.

[0062] The whole antibody includes subtypes of IgA, IgD, IgE, IgM, and IgG, and in particular, IgG includes IgG1, IgG2, IgG3, and IgG4. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0063] Antigen-binding fragments of antibodies or antibody fragments refer to fragments that retain antigen-binding function and include Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure comprising light and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is produced when the cysteine residues in the hinge region of Fab' form disulfide bonds.

[0064] Fv is the smallest antibody fragment containing only the heavy-chain variable region and the light-chain variable region. In a two-chain Fv, the heavy-chain variable region and the light-chain variable region are linked non-covalently. In a single-chain Fv (scFv), the heavy-chain variable region and the light-chain variable region are generally linked covalently via a peptide linker or directly at the C-terminus, and can form a dimer structure like a two-chain Fv. Such antibody fragments can be produced using protease enzymes (e.g., Fab can be obtained by limited digestion of an intact antibody with papain, or F(ab')2 can be obtained by digestion with pepsin) or by genetic engineering.

[0065] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region is a dimer of one heavy-chain variable domain and one light-chain variable domain.

[0066] The "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments generally contain a pair of Fab' fragments covalently linked by hinge region cysteines located at the C-terminus of the Fab' fragments.

[0067] "Single-chain Fv (scFv)" antibody fragments are structures consisting of a single polypeptide chain comprising the VH and VL domains of an antibody. A polypeptide linker between the VH and VL domains can further be included which enables the scFv to form the desired structure for antigen binding.

[0068] In one embodiment, antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFv, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of each of the foregoing antibodies.

[0069] The heavy chain constant region can be selected from any one of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) isotypes. For example, the constant region can be gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region can be kappa or lambda type.

[0070] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific because they are directed against a single antigenic site. In contrast to conventional (polyclonal) antibodies, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0071] "Epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes are usually composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0072] The "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (acceptor antibody) in which residues from a hypervariable region of the acceptor are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate possessing the desired specificity, affinity, and capacity.

[0073] The "human antibody" is a molecule derived from human immunoglobulin, and means that the entire amino acid sequence constituting the antibody, including the complementarity determining region and structural region, is composed of human immunoglobulin.

[0074] These include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chains are identical to or homologous to corresponding sequences in antibodies derived from yet another species or belonging to yet another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0075] The "variable region" of an antibody used in the present invention refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the heavy chain variable domain, and VL refers to the light chain variable domain.

[0076] "Complementary determining region (CDR)" refers to the amino acid residues of an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3.

[0077] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 17, and a heavy chain CDR3 of SEQ ID NO: 42; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 66, a light chain CDR2 of SEQ ID NO: 87, and a light chain CDR3 of SEQ ID NO: 103;

[0078] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 18, and a heavy chain CDR3 of SEQ ID NO: 43; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 67, a light chain CDR2 of SEQ ID NO: 88, and a light chain CDR3 of SEQ ID NO: 104;

[0079] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 19, and a heavy chain CDR3 of SEQ ID NO: 44; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 67, a light chain CDR2 of SEQ ID NO: 88, and a light chain CDR3 of SEQ ID NO: 104;

[0080] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 20, and a heavy chain CDR3 of SEQ ID NO: 45; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 68, a light chain CDR2 of SEQ ID NO: 89, and a light chain CDR3 of SEQ ID NO: 105;

[0081] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 18, and a heavy chain CDR3 of SEQ ID NO: 43; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 69, a light chain CDR2 of SEQ ID NO: 90, and a light chain CDR3 of SEQ ID NO: 106;

[0082] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 21, and a heavy chain CDR3 of SEQ ID NO: 46; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 67, a light chain CDR2 of SEQ ID NO: 88, and a light chain CDR3 of SEQ ID NO: 104;

[0083] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 6, a heavy chain CDR2 of SEQ ID NO: 22, and a heavy chain CDR3 of SEQ ID NO: 47; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 70, a light chain CDR2 of SEQ ID NO: 91, and a light chain CDR3 of SEQ ID NO: 107;

[0084] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 5, a heavy chain CDR2 of SEQ ID NO: 17, and a heavy chain CDR3 of SEQ ID NO: 48; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 67, a light chain CDR2 of SEQ ID NO: 88, and a light chain CDR3 of SEQ ID NO: 108;

[0085] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 23, and a heavy chain CDR3 of SEQ ID NO: 49; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 71, a light chain CDR2 of SEQ ID NO: 92, and a light chain CDR3 of SEQ ID NO: 109;

[0086] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 24, and a heavy chain CDR3 of SEQ ID NO: 50; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 72, a light chain CDR2 of SEQ ID NO: 90, and a light chain CDR3 of SEQ ID NO: 109;

[0087] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 25, and a heavy chain CDR3 of SEQ ID NO: 47; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 70, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 108;

[0088] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 8, a heavy chain CDR2 of SEQ ID NO: 26, and a heavy chain CDR3 of SEQ ID NO: 51; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 73, a light chain CDR2 of SEQ ID NO: 90, and a light chain CDR3 of SEQ ID NO: 110;

[0089] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 9, a heavy chain CDR2 of SEQ ID NO: 27, and a heavy chain CDR3 of SEQ ID NO: 52; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 74, a light chain CDR2 of SEQ ID NO: 94, and a light chain CDR3 of SEQ ID NO: 111;

[0090] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 28, and a heavy chain CDR3 of SEQ ID NO: 50; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 75, a light chain CDR2 of SEQ ID NO: 92, and a light chain CDR3 of SEQ ID NO: 112;

[0091] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 29, and a heavy chain CDR3 of SEQ ID NO: 53; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 76, a light chain CDR2 of SEQ ID NO: 91, and a light chain CDR3 of SEQ ID NO: 113;

[0092] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 30, and a heavy chain CDR3 of SEQ ID NO: 54; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 77, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 114;

[0093] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 12, a heavy chain CDR2 of SEQ ID NO: 31, and a heavy chain CDR3 of SEQ ID NO: 55; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 78, a light chain CDR2 of SEQ ID NO: 95, and a light chain CDR3 of SEQ ID NO: 115;

[0094] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 32, and a heavy chain CDR3 of SEQ ID NO: 56; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 79, a light chain CDR2 of SEQ ID NO: 96, and a light chain CDR3 of SEQ ID NO: 116;

[0095] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 33, and a heavy chain CDR3 of SEQ ID NO: 57; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 80, a light chain CDR2 of SEQ ID NO: 97, and a light chain CDR3 of SEQ ID NO: 114;

[0096] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 34, and a heavy chain CDR3 of SEQ ID NO: 58; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 81, a light chain CDR2 of SEQ ID NO: 98, and a light chain CDR3 of SEQ ID NO: 103;

[0097] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 14, a heavy chain CDR2 of SEQ ID NO: 35, and a heavy chain CDR3 of SEQ ID NO: 59; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 82, a light chain CDR2 of SEQ ID NO: 99, and a light chain CDR3 of SEQ ID NO: 106;

[0098] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 15, a heavy chain CDR2 of SEQ ID NO: 36, and a heavy chain CDR3 of SEQ ID NO: 60; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 76, a light chain CDR2 of SEQ ID NO: 91, and a light chain CDR3 of SEQ ID NO: 117;

[0099] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 37, and a heavy chain CDR3 of SEQ ID NO: 61; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 76, a light chain CDR2 of SEQ ID NO: 93, and a light chain CDR3 of SEQ ID NO: 118;

[0100] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 23, and a heavy chain CDR3 of SEQ ID NO: 49; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 71, a light chain CDR2 of SEQ ID NO: 92, and a light chain CDR3 of SEQ ID NO: 109;

[0101] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 38, and a heavy chain CDR3 of SEQ ID NO: 62; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 83, a light chain CDR2 of SEQ ID NO: 100, and a light chain CDR3 of SEQ ID NO: 109;

[0102] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 23, and a heavy chain CDR3 of SEQ ID NO: 49; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 71, a light chain CDR2 of SEQ ID NO: 92, and a light chain CDR3 of SEQ ID NO: 109;

[0103] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 23, and a heavy chain CDR3 of SEQ ID NO: 49; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 71, a light chain CDR2 of SEQ ID NO: 92, and a light chain CDR3 of SEQ ID NO: 119;

[0104] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 63; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 101, and a light chain CDR3 of SEQ ID NO: 120;

[0105] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 63; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 101, and a light chain CDR3 of SEQ ID NO: 109;

[0106] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 40, and a heavy chain CDR3 of SEQ ID NO: 64; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 85, a light chain CDR2 of SEQ ID NO: 97, and a light chain CDR3 of SEQ ID NO: 108;

[0107] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 16, a heavy chain CDR2 of SEQ ID NO: 41, and a heavy chain CDR3 of SEQ ID NO: 65; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 86, a light chain CDR2 of SEQ ID NO: 102, and a light chain CDR3 of SEQ ID NO: 121;

[0108] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 184, and a heavy chain CDR3 of SEQ ID NO: 63; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 101, and a light chain CDR3 of SEQ ID NO: 109;

[0109] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 185, and a heavy chain CDR3 of SEQ ID NO: 63; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 101, and a light chain CDR3 of SEQ ID NO: 109;

[0110] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 63; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 186, and a light chain CDR3 of SEQ ID NO: 109;

[0111] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 63; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 187, and a light chain CDR3 of SEQ ID NO: 109;

[0112] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 39, and a heavy chain CDR3 of SEQ ID NO: 63; a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 101, and a light chain CDR3 of SEQ ID NO: 110; or

[0113] A heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 185, and a heavy chain CDR3 of SEQ ID NO: 63; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 84, a light chain CDR2 of SEQ ID NO: 187, and a light chain CDR3 of SEQ ID NO: 110.

[0114] "Framework regions (FR)" are those variable domain residues other than the CDR residues. Each variable domain typically has four FRs: FR1, FR2, FR3 and FR4.

[0115] The binding affinity of the anti-ROR1 antibody to ROR1 is 10 -5 M~10 -12 For example, the binding affinity of an anti-ROR1 antibody to ROR1 is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 It's M.

[0116] The antibody or antigen-binding fragment thereof that binds to ROR1 may comprise a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 122 to 152, and 188 to 193. The antibody or antigen-binding fragment thereof that binds to ROR1 may comprise a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153 to 183, and 194 to 199.

[0117] In a specific embodiment of the present invention, the following may be included:

[0118] a heavy chain variable region of SEQ ID NO: 122 and a light chain variable region of SEQ ID NO: 153;

[0119] a heavy chain variable region of SEQ ID NO: 123 and a light chain variable region of SEQ ID NO: 154;

[0120] a heavy chain variable region of SEQ ID NO: 124 and a light chain variable region of SEQ ID NO: 155;

[0121] a heavy chain variable region of SEQ ID NO: 125 and a light chain variable region of SEQ ID NO: 156;

[0122] a heavy chain variable region of SEQ ID NO: 126 and a light chain variable region of SEQ ID NO: 157;

[0123] a heavy chain variable region of SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 158;

[0124] a heavy chain variable region of SEQ ID NO: 128 and a light chain variable region of SEQ ID NO: 159;

[0125] a heavy chain variable region of SEQ ID NO: 129 and a light chain variable region of SEQ ID NO: 160;

[0126] a heavy chain variable region of SEQ ID NO: 130 and a light chain variable region of SEQ ID NO: 161;

[0127] a heavy chain variable region of SEQ ID NO: 131 and a light chain variable region of SEQ ID NO: 162;

[0128] a heavy chain variable region of SEQ ID NO: 132 and a light chain variable region of SEQ ID NO: 163;

[0129] a heavy chain variable region of SEQ ID NO: 133 and a light chain variable region of SEQ ID NO: 164;

[0130] a heavy chain variable region of SEQ ID NO: 134 and a light chain variable region of SEQ ID NO: 165;

[0131] a heavy chain variable region of SEQ ID NO: 135 and a light chain variable region of SEQ ID NO: 166;

[0132] a heavy chain variable region of SEQ ID NO: 136 and a light chain variable region of SEQ ID NO: 167;

[0133] a heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 168;

[0134] a heavy chain variable region of SEQ ID NO: 138 and a light chain variable region of SEQ ID NO: 169;

[0135] a heavy chain variable region of SEQ ID NO: 139 and a light chain variable region of SEQ ID NO: 170;

[0136] a heavy chain variable region of SEQ ID NO: 140 and a light chain variable region of SEQ ID NO: 171;

[0137] a heavy chain variable region of SEQ ID NO: 141 and a light chain variable region of SEQ ID NO: 172;

[0138] a heavy chain variable region of SEQ ID NO: 142 and a light chain variable region of SEQ ID NO: 173;

[0139] a heavy chain variable region of SEQ ID NO: 143 and a light chain variable region of SEQ ID NO: 174;

[0140] a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 175;

[0141] a heavy chain variable region of SEQ ID NO: 145 and a light chain variable region of SEQ ID NO: 176;

[0142] a heavy chain variable region of SEQ ID NO: 146 and a light chain variable region of SEQ ID NO: 177;

[0143] a heavy chain variable region of SEQ ID NO: 147 and a light chain variable region of SEQ ID NO: 178;

[0144] a heavy chain variable region of SEQ ID NO: 148 and a light chain variable region of SEQ ID NO: 179;

[0145] a heavy chain variable region of SEQ ID NO: 149 and a light chain variable region of SEQ ID NO: 180;

[0146] a heavy chain variable region of SEQ ID NO: 150 and a light chain variable region of SEQ ID NO: 181;

[0147] a heavy chain variable region of SEQ ID NO: 151 and a light chain variable region of SEQ ID NO: 182;

[0148] a heavy chain variable region of SEQ ID NO: 152 and a light chain variable region of SEQ ID NO: 183;

[0149] a heavy chain variable region of SEQ ID NO: 188 and a light chain variable region of SEQ ID NO: 194;

[0150] a heavy chain variable region of SEQ ID NO: 189 and a light chain variable region of SEQ ID NO: 195;

[0151] a heavy chain variable region of SEQ ID NO: 190 and a light chain variable region of SEQ ID NO: 196;

[0152] a heavy chain variable region of SEQ ID NO: 191 and a light chain variable region of SEQ ID NO: 197;

[0153] the heavy chain variable region of SEQ ID NO: 192 and the light chain variable region of SEQ ID NO: 198; or

[0154] The heavy chain variable region of SEQ ID NO: 193 and the light chain variable region of SEQ ID NO: 199.

[0155]

[0156] scFv

[0157] scFv is an antibody fragment, which is a single polypeptide chain comprising the VH and VL domains of an antibody, and may further comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0158] In one embodiment, a single-chain Fv (scFv) comprising antibody VH and VL domains may be linked via a linker, where a heavy-chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 122 to 152 and 188 to 193 may be linked via a linker to a light-chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153 to 183 and 194 to 199.

[0159] In a specific embodiment of the present invention, the following may be included:

[0160] a heavy chain variable region of SEQ ID NO: 122 and a light chain variable region of SEQ ID NO: 153;

[0161] a heavy chain variable region of SEQ ID NO: 123 and a light chain variable region of SEQ ID NO: 154;

[0162] a heavy chain variable region of SEQ ID NO: 124 and a light chain variable region of SEQ ID NO: 155;

[0163] a heavy chain variable region of SEQ ID NO: 125 and a light chain variable region of SEQ ID NO: 156;

[0164] a heavy chain variable region of SEQ ID NO: 126 and a light chain variable region of SEQ ID NO: 157;

[0165] a heavy chain variable region of SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 158;

[0166] a heavy chain variable region of SEQ ID NO: 128 and a light chain variable region of SEQ ID NO: 159;

[0167] a heavy chain variable region of SEQ ID NO: 129 and a light chain variable region of SEQ ID NO: 160;

[0168] a heavy chain variable region of SEQ ID NO: 130 and a light chain variable region of SEQ ID NO: 161;

[0169] a heavy chain variable region of SEQ ID NO: 131 and a light chain variable region of SEQ ID NO: 162;

[0170] a heavy chain variable region of SEQ ID NO: 132 and a light chain variable region of SEQ ID NO: 163;

[0171] a heavy chain variable region of SEQ ID NO: 133 and a light chain variable region of SEQ ID NO: 164;

[0172] a heavy chain variable region of SEQ ID NO: 134 and a light chain variable region of SEQ ID NO: 165;

[0173] a heavy chain variable region of SEQ ID NO: 135 and a light chain variable region of SEQ ID NO: 166;

[0174] a heavy chain variable region of SEQ ID NO: 136 and a light chain variable region of SEQ ID NO: 167;

[0175] a heavy chain variable region of SEQ ID NO: 137 and a light chain variable region of SEQ ID NO: 168;

[0176] a heavy chain variable region of SEQ ID NO: 138 and a light chain variable region of SEQ ID NO: 169;

[0177] a heavy chain variable region of SEQ ID NO: 139 and a light chain variable region of SEQ ID NO: 170;

[0178] a heavy chain variable region of SEQ ID NO: 140 and a light chain variable region of SEQ ID NO: 171;

[0179] a heavy chain variable region of SEQ ID NO: 141 and a light chain variable region of SEQ ID NO: 172;

[0180] a heavy chain variable region of SEQ ID NO: 142 and a light chain variable region of SEQ ID NO: 173;

[0181] a heavy chain variable region of SEQ ID NO: 143 and a light chain variable region of SEQ ID NO: 174;

[0182] a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 175;

[0183] a heavy chain variable region of SEQ ID NO: 145 and a light chain variable region of SEQ ID NO: 176;

[0184] a heavy chain variable region of SEQ ID NO: 146 and a light chain variable region of SEQ ID NO: 177;

[0185] a heavy chain variable region of SEQ ID NO: 147 and a light chain variable region of SEQ ID NO: 178;

[0186] a heavy chain variable region of SEQ ID NO: 148 and a light chain variable region of SEQ ID NO: 179;

[0187] a heavy chain variable region of SEQ ID NO: 149 and a light chain variable region of SEQ ID NO: 180;

[0188] a heavy chain variable region of SEQ ID NO: 150 and a light chain variable region of SEQ ID NO: 181;

[0189] a heavy chain variable region of SEQ ID NO: 151 and a light chain variable region of SEQ ID NO: 182;

[0190] a heavy chain variable region of SEQ ID NO: 152 and a light chain variable region of SEQ ID NO: 183;

[0191] a heavy chain variable region of SEQ ID NO: 188 and a light chain variable region of SEQ ID NO: 194;

[0192] a heavy chain variable region of SEQ ID NO: 189 and a light chain variable region of SEQ ID NO: 195;

[0193] a heavy chain variable region of SEQ ID NO: 190 and a light chain variable region of SEQ ID NO: 196;

[0194] a heavy chain variable region of SEQ ID NO: 191 and a light chain variable region of SEQ ID NO: 197;

[0195] the heavy chain variable region of SEQ ID NO: 192 and the light chain variable region of SEQ ID NO: 198; or

[0196] The heavy chain variable region of SEQ ID NO: 193 and the light chain variable region of SEQ ID NO: 199.

[0197] The linker may be a peptide linker and may have a length of about 10 aa to 25 aa. For example, the linker may include hydrophilic amino acids such as glycine and / or serine, but is not limited thereto.

[0198] Specifically, the linker may be, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), but the linker may be, for example, (G n S) m (n and m each may be 1 to 10.) Specifically, the linker may contain GGGGS, and may be, for example, GGGGSGGGGSGGGGS of SEQ ID NO: 200 repeated three times.

[0199] "Phage display" is a technique in which mutant polypeptides are displayed on the surface of phages, e.g., filamentous phage particles, as fusion proteins with at least a portion of a coat protein. The utility of phage display lies in the fact that large libraries of randomized protein variants can be quickly and efficiently sorted to identify sequences that bind with high affinity to a target antigen. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides for those with specific binding properties.

[0200] Phage display technology has provided a powerful tool for generating and selecting novel proteins that bind to specific ligands (e.g., antigens). Phage display technology can be used to generate large libraries of protein variants and rapidly sort sequences that bind to target antigens with high affinity. Nucleic acids encoding mutant polypeptides are fused to nucleic acid sequences encoding viral coat proteins, such as the gene III protein or gene VIII protein. Monovalent phage display systems have been developed in which nucleic acid sequences encoding proteins or polypeptides are fused to nucleic acid sequences encoding portions of the gene III protein. In monovalent phage display systems, the gene fusion is expressed at low levels, while the wild-type gene III protein is also expressed, maintaining particle infectivity.

[0201] Demonstrating expression of peptides on the surface of filamentous phage and expression of functional antibody fragments in the periplasm of E. coli is key in developing antibody phage display libraries. Libraries of antibodies or antigen-binding polypeptides have been produced in a number of ways, for example, by altering a single gene by inserting random DNA sequences or by cloning related gene sequences. Libraries can be screened for expression of antibodies or antigen-binding proteins with desired characteristics.

[0202] Phage display technology offers several advantages over conventional hybridoma and recombinant methods for producing antibodies with desired characteristics. Such technology allows for the generation of large antibody libraries with diverse sequences in a short period of time without the use of animals. Hybridoma production and humanized antibody production can require several months of production time. Furthermore, because no immunization is required, phage antibody libraries can generate antibodies against antigens that are toxic or have low antigenicity. Furthermore, phage antibody libraries can be used to generate and identify novel therapeutic antibodies.

[0203] Techniques are available for generating human antibodies from immunized or non-immunized humans, germline sequence, or naive B cell Ig repertoires using phage display libraries. Various lymphoid tissues can be used to generate naive or non-immunized antigen-binding libraries.

[0204] Techniques for identifying and isolating high-affinity antibodies from phage display libraries are important for isolating novel therapeutic antibodies. Isolating high-affinity antibodies from libraries can depend on the size of the library, the efficiency of production in bacterial cells, and the diversity of the library. Library size can be reduced by improper folding of the antibody or antigen-binding protein and inefficient production due to the presence of stop codons. Expression in bacterial cells can be suppressed if the antibody or antigen-binding domain does not fold properly. Expression can be improved by mutating residues at the variable / constant interface or selected CDR residues relative to each other. The sequence of the scaffold region is one factor in providing proper folding when generating antibody phage libraries in bacterial cells.

[0205] In high-affinity antibody isolation, it is important to generate diverse libraries of antibodies or antigen-binding proteins. The CDR3 region has been shown to frequently participate in antigen binding. The CDR3 region on the heavy chain is highly diverse in size, sequence, and structural conformation, and can therefore be used to generate diverse libraries.

[0206] Diversity can also be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position, which may generate a greater variety of variant antibody sequences and increase the chances of identifying novel antibodies.

[0207] The antibodies or antibody fragments of the present invention can include not only the sequences of the anti-ROR1 antibodies of the present invention described herein, but also their biological equivalents, as long as they can specifically recognize ROR1. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side chain substitutions reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0208] Considering the above-mentioned biologically equivalent variants, the antibodies of the present invention or nucleic acid molecules encoding them are also understood to include sequences that exhibit substantial identity to the sequences set forth in SEQ ID NOs. The term "substantial identity" refers to sequences that exhibit at least 90% homology, most preferably at least 95%, 96% or more, 97% or more, 98% or more, or 99% or more when the sequences are aligned to maximize correspondence and analyzed using algorithms commonly known in the art. Alignment methods for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed at www.ncbi.nlm.nih.gov / BLAST / . Methods for sequence homology comparison using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0209] Based on this, the antibodies or antigen-binding fragments thereof of the present invention can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the sequences explicitly set forth herein or to the entirety of the sequences. Such homology can be determined by sequence comparison and / or alignment using methods known in the art. For example, percent sequence homology of the nucleic acids or proteins of the present invention can be determined using sequence comparison algorithms (e.g., BLAST or BLAST 2.0), passive alignment, or visual inspection.

[0210] In another aspect, the present invention relates to a nucleic acid encoding the antibody or antigen-binding fragment thereof. The nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention can be isolated and used to recombinantly produce the antibody or antigen-binding fragment thereof.

[0211] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic building blocks of nucleic acids, include not only natural nucleotides but also analogs with modified sugar or base moieties. The nucleic acid sequences encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0212] The DNA encoding the antibody can be easily isolated or synthesized using standard molecular biology techniques (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the heavy and light chains of the antibody), and the nucleic acid can be isolated and inserted into a replicable vector for further cloning (DNA amplification) or further expression. Based on this, in yet another aspect, the present invention relates to a recombinant expression vector comprising the nucleic acid.

[0213] The term "vector" as used herein refers to a means for expressing a gene of interest in a host cell, and includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. Components of a vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, a transcription termination sequence, and an antibody-encoding nucleic acid operably linked to the promoter and transcription termination sequence.

[0214] "Operably linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0215] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of transcription, and a transcription / transcription termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and they generally have a polyadenylation sequence as a transcription termination sequence.

[0216] In some cases, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Qiagen, USA).

[0217] The vectors contain antibiotic resistance genes commonly used in the art as selection markers, such as genes for resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0218] In yet another aspect, the present invention relates to a host cell transformed with the recombinant vector. The host cell used to produce the antibody of the present invention may be, but is not limited to, a prokaryote, yeast, or higher eukaryote cell.

[0219] Prokaryotic host cells such as strains of Bacillus, including Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0220] However, of greatest interest are animal cells, and examples of useful host cell lines can include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0221] In yet another aspect, the present invention relates to a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, the method comprising: culturing the host cell to produce an antibody; and isolating and purifying the produced antibody.

[0222] The host cells can be cultured in various media. Commercially available media can be used as the culture medium without limitation. Any other necessary supplements known to those skilled in the art may be included at appropriate concentrations. The culture conditions, such as temperature, pH, etc., are already used with the host cells selected for expression and will be apparent to those skilled in the art.

[0223] The antibody or antigen-binding fragment thereof can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and the resulting product can be purified using, for example, affinity chromatography. Additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can also be used.

[0224]

[0225] Bispecific or multispecific antibodies

[0226] In another aspect, the present invention relates to a bi- or multispecific antibody comprising the above-described antibody or an antigen-binding fragment thereof.

[0227] A bispecific antibody refers to an antibody that has the ability to bind to or compete with more than one target, and refers to a form in which antibodies that have the ability to bind to or compete with two different targets are bound, or an antibody in which an antibody that has the ability to bind to one target is bound to a substance that has the ability to compete with another target.

[0228] A multispecific antibody refers to an antibody that has binding specificities for at least three or more different antigens. Multispecific antibodies can include trispecific or higher-specific antibodies, such as trispecific antibodies, tetraspecific antibodies, or antibodies that target more than one target.

[0229] Bispecific or multispecific antibodies can be classified into scFv-based antibodies, Fab-based antibodies, IgG-based antibodies, etc. Bispecific or multispecific antibodies can simultaneously suppress or amplify two or more signals, which can be more effective than suppressing / amplifying a single signal, and can also be administered at a lower dose than when treating each signal with a separate signal suppressor, and can suppress / amplify two or more signals at the same time and space.

[0230] Methods for producing bispecific or multispecific antibodies are widely known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy chain / light chain pairs, where the two or more heavy chains have different specificities.

[0231] In the case of scFv-based bispecific or multispecific antibodies, diabodies can be produced by combining the VL and VH of different scFvs with each other to produce hybrid scFvs in a heterodimeric form; tandem ScFvs can be produced by linking different scFvs together; heterodimeric miniantibodies can be produced by expressing the CH1 and CL of Fab at the termini of each scFv; and heterodimeric scFv minibodies can be produced by substituting some amino acids in the CH3 domain, which is the homodimeric domain of Fc, to change it to a "knob-into-hole" heterodimeric structure and expressing these modified CH3 domains at the termini of different scFvs.

[0232] Fab-based bispecific or multispecific antibodies can be prepared in heterodimeric form by combining individual Fab's against specific antigens using disulfide bonds or intermediates. They can be prepared with two antigen binding valencies by expressing scFvs against different antigens at the heavy or light chain termini of a specific Fab, or with four antigen binding valencies in homodimeric form by inserting a hinge region between the Fab and scFv. Dual-targeting bibodies with three antigen binding valencies can be prepared by fusing scFvs against different antigens to the light and heavy chain termini of a Fab. Tri-targeting bibodies with three antigen binding valencies can also be prepared by chemically conjugating three different Fabs.

[0233] In the case of IgG-based bispecific or multispecific antibodies, Trion Pharma has produced hybrid hybridomas, or quadromas, by crossbreeding mouse and rat hybridomas to produce bispecific antibodies. Bispecific antibodies can also be produced in a "holes and knobs" configuration, where the light chain is shared but certain amino acids in the Fc CH3 homodimer domain are modified for different heavy chains. In addition to heterodimeric bispecific antibodies, two different scFvs can be fused to the constant domains of IgG in place of the light and heavy chain variable domains, producing a homodimeric (scFv)4-IgG. ImClone reported the creation of a bispecific antibody based on IMC-1C11, a chimeric monoclonal antibody against human VEGFR-2, by fusing only a single variable domain against mouse platelet-derived growth factor receptor-α to the amino terminus of the light chain of this antibody. Furthermore, using the dimerization and docking domain (DDD) of the protein kinase A (PKA) R subunit and the anchoring domain of PKA, a so-called "dock and lock (DNL)" method can be used to create antibodies with multiple antigen binding valencies against CD20.

[0234] A wide variety of recombinant antibody formats have been developed, including bispecific or multispecific antibodies that are bivalent or more than bivalent, trivalent or more than tetravalent, including, for example, the bivalent or more than trivalent or more than tetravalent antibodies described in International Patent Application Publication Nos. WO2001 / 077342, WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, WO2010 / 112193, WO2010 / 115589, WO2010 / 136172, WO2010 / 145792, WO2010 / 145793 and WO2011 / 117330. A more than bivalent, more than trivalent, or more than tetravalent antibody indicates that two or more binding domains, three or more binding domains, or four or more binding domains are present in the antibody molecule, respectively.

[0235] In a specific embodiment, the bispecific or multispecific antibodies of the present invention comprise the anti-ROR1 antibody or antigen-binding fragment thereof, specifically an IgG whole antibody or a fragment thereof, such as a single-chain F V , V H Domain and / or V L It may be contained in the form of a domain, Fab or (Fab)2.

[0236] In addition, antibodies that bind to targets different from the antibody targeting ROR1, for example, antibodies that target one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO, specifically, IgG whole antibodies or fragments thereof, for example, single-chain F V , V H Domain and / or V LIt may be contained in the form of a domain, Fab or (Fab)2.

[0237] Through the bi- or multispecific antibodies of the present invention, additional binding specificities induced or mediated by other targets besides ROR1 can be ensured.

[0238] For example, a bispecific antibody of the present invention may simultaneously target ROR1 and one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

[0239] For example, a multispecific antibody of the present invention can simultaneously target ROR1 and two or more proteins selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

[0240]

[0241] Immune Cell Engaging Bispecific or Multispecific Antibodies

[0242] In yet another aspect, the present invention relates to an immune cell-engaging bispecific or multispecific antibody comprising an scFv of an antibody and a second binding domain comprising one or more scFvs of an antibody that binds to an immune cell-activating antigen.

[0243] The immune cell-engaging bispecific or multispecific antibody transiently induces a cytolytic synapse between cytotoxic T cells and cancer target cells, resulting in the release of toxins.

[0244] In one embodiment, the immune cells may be one or more selected from the group consisting of T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells.

[0245] In one embodiment, the immune cell activation antigen can be selected from, for example, the following, and antibodies binding thereto can act as immune cell engagers:

[0246] T cell activation antigens are CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226;

[0247] NK cell activation antigens include NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160;

[0248] B cell activation antigens are OX40, CD40, or CD70;

[0249] the macrophage-activating antigen is a CD2 agonist, CD40, CD70, a TCR (Toll-like receptor) agonist, CD47, STING, or OX40L; or

[0250] The dendritic cell activating antigen is a CD2 agonist, OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist, or STING agonist.

[0251] Immune cell engagers are specifically described in US Patent Application Publication No. 2017 / 0368169, which is incorporated herein by reference.

[0252] Specifically, the immune cell-engaging bispecific or multispecific antibody comprises tandem scFvs and is capable of binding to the following antigen and to a surface antigen on cancer cells, the surface antigen on cancer cells being ROR1, which is targeted by the antibody of the present invention:

[0253] CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226;

[0254] NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160;

[0255] OX40, CD40, or CD70;

[0256] a CD2 agonist, CD40, CD70, a TCR (Toll-like receptor) agonist, CD47, STING, or OX40L; or

[0257] CD2 agonist, OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist, or STING agonist.

[0258] The immune cell-engaging bispecific or multispecific antibody may, for example, comprise a structure of the form VL(ROR1)-VH(ROR1)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(ROR1)-VL(ROR1)-VH(CD3 or CD16A)-VL(CD3 or CD16A), VH(CD3 or CD16A)-VL(CD3 or CD16A)-VH(ROR1)-VL(ROR1), or VH(CD3 or CD16A)-VL(CD3 or CD16A)-VL(ROR1)-VH(ROR1).

[0259] The scFv comprises, for example, a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 122 to 152, and 188 to 193, and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153 to 183, and 194 to 199, and the heavy chain variable region and light chain variable region may be linked by a linker.

[0260] The linker may be a peptide linker and may have a length of about 10 aa to 25 aa. For example, the linker may include hydrophilic amino acids such as glycine and / or serine.

[0261] The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n, or (GnS)m (n and m are each 1 to 10), and the linker may be, for example, (GnS)m (n and m are each 1 to 10). Specifically, the linker may include GGGGS, and may be, for example, GGGGSGGGSGGGGGS of SEQ ID NO: 200 repeated three times.

[0262] Exemplary immune cell-engaging bispecific or multispecific antibodies include blinatumomab (Amgen), which binds to CD3 and CD19; solitomab (Amgen), which binds to CD3 and EpCAM; MEDI 565 (MedImmune, Amgen), which binds to CD3 and CEA; and BAY2010112 (Bayer, Amgen), which binds to CD3 and PSMA. Exemplary DARTs include MGD006 (Macrogenics), which binds to CD3 and CD123; and MGD007 (Macrogenics), which binds to CD3 and gpA33. Exemplary TandAbs include AFM11 (Affimed Therapeutics), which binds to CD3 and CD19; and AFM13 (Affimed Therapeutics), which binds to CD30 and CD16A.

[0263]

[0264] Antibody-drug conjugates (ADCs)

[0265] In yet another aspect, the present invention relates to an antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof is conjugated to a drug.

[0266] In antibody-drug conjugates, the anti-cancer drug must be stably bound to the antibody before being delivered to the target cancer cells. Once delivered to the target, the drug must be released from the antibody and induce target cell death. To achieve this, the drug must be stably bound to the antibody and have sufficient cytotoxicity to induce target cell death when released from the target cells.

[0267] In one embodiment, the antibody may be conjugated to a drug via a linker, which is a linking moiety between the anti-ROR1 antibody and the drug and is cleavable under intracellular conditions, i.e., allows the drug to be released from the antibody in the intracellular environment, reflects the long half-life of the antibody, ensures that the antibody is stable in systemic circulation, and the bond between the linker and the drug does not affect the stability or pharmacokinetics of the antibody.

[0268] The linker may include, for example, a cleavable linker or a non-cleavable linker. In the case of a cleavable linker, like a peptide linker, it can be cleaved by an intracellular peptidase or protease enzyme, for example, a lysosomal or endosomal protease, and in the case of a non-cleavable linker, for example, a thioether linker, the drug can be released after the antibody is non-selectively degraded by intracellular hydrolysis.

[0269] In one embodiment, the cleavable linker may comprise a peptide linker. The peptide linker has a length of at least two amino acids. For example, the cleavable linker may comprise a dipeptide of Val-Cit, Val-Ala, Val-Cit, Phe-Leu, or Gly-Phe-Leu-Gly. Examples of linkers are specifically described in International Patent Application Publication No. WO2004 / 010957, which is incorporated herein by reference.

[0270] The antibody-drug conjugate forms an ADC-antigen complex by binding of the antibody domain of the ADC to an antigen on the target cancer cell, and is then internalized inside the cancer cell via the endosomal-lysosomal pathway, where the intracellular release of the cytotoxic drug is regulated by the internal environment of the endosome / lysosome.

[0271] In one embodiment, the cleavable linker may be pH-sensitive and susceptible to hydrolysis at a specific pH value. Generally, a pH-sensitive linker refers to a linker that can be hydrolyzed under acidic conditions. For example, the linker may be an acid-labile linker that can be hydrolyzed in the lysosome, such as a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, or ketal.

[0272] In other embodiments, the linker may be cleaved under reducing conditions, such as a disulfide linker. Various disulfide bonds can be formed using N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-3-(2-pyridyldithio)butyrate (SPDB), and N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene (SMPT). These disulfide linkers can be degraded by disulfide exchange with the thiol of intracellular glutathione.

[0273] The drug and / or drug-linker can be randomly conjugated through a lysine of the antibody or through a cysteine that is exposed upon reduction of the disulfide bond chain. In some cases, the linker-drug can be attached through a cysteine present in a genetically engineered tag, e.g., a peptide or protein. The genetically engineered tag, e.g., a peptide or protein, can contain an amino acid motif that can be recognized by isoprenoid transferase, for example. The peptide or protein can have a deletion at the carboxyl terminus of the peptide or protein, or a spacer unit can be added to the carboxyl (C) terminus of the peptide or protein via a covalent bond.

[0274] The peptide or protein can be linked to the amino acid motif either directly or via a covalent bond to a spacer unit, which consists of 1 to 20 amino acids, preferably a glycine unit.

[0275] The isoprenoid transferase may be, for example, farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase). FTase and GGTase I can recognize the CAAX motif of Chemical Formula 1, while GGTase II can recognize the XXCC, XCXC, or CXX motif (where C is cysteine, A is an aliphatic amino acid, and X is an amino acid that determines the substrate specificity of the isoprenoid transferase).

[0276] In yet another embodiment, the linker may comprise a β-glucuronide linker that is recognized and hydrolyzed by β-glucuronidase, which is abundantly present in lysosomes or overexpressed in some tumor cells. Unlike peptide linkers, β-glucuronide linkers have high hydrophilicity, which has the advantage of increasing the solubility of antibody-drug conjugates when conjugated with highly hydrophobic drugs.

[0277] In this regard, β-glucuronide linkers disclosed in International Patent Application Publication No. WO2015 / 182984 can be used, for example, β-glucuronide linkers containing a self-immolative group, the disclosure of which is incorporated by reference.

[0278] In some cases, the linker may be, for example, a non-cleavable linker, in which the drug is released only through intracellular antibody hydrolysis, producing, for example, an amino acid-linker-drug conjugate. Such linkers may be thioether groups or maleimidocaproyl groups, and can maintain stability in the blood.

[0279] According to one embodiment of the present invention, the linker-drug may be randomly attached through cysteines exposed upon reduction of the disulfide bond chain of the antibody, or may be attached by introducing an antibody-terminal binding peptide having the sequence GGGGGGCVIM.

[0280] The drug (including D in formula (1)) is a compound that exhibits a pharmacological effect and can be conjugated to an antibody, and specifically can be a chemotherapeutic agent, a toxin, a microRNA (miRNA), a siRNA, a shRNA, or a radioisotope. The chemotherapeutic agent can be, for example, a cytotoxic agent or an immunosuppressant. Specifically, it can include chemotherapeutic agents that function as microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators. It can also include immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, antiparasitic agents, or combinations thereof.

[0281] Such drugs include, for example, maytansinoids, auristatins (including MMAE, MMAF), aminopterin, actinomycin, bleomycin, tallysomycin, camptothecin, N8-acetylspermidine, 1-(2chloroethyl)-1,2-dimethylsulfonylhydrazide, esperamicin, etoposide, 6-mercaptopurine, dolastatins, trichothecenes, calicheamicin, taxol, taxanes, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L -asparaginase (L-asparaginase), mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustardmustard, cytoxan, etoposide, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase nase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C C), trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytosine arabinosidearabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrelin A, interferon-α, interferon-γ, tumor necrosis factor factor, gemcitabine, velcade, revamid, thalamid, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2 B2), peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, thapsigargin, nucleases, and toxins derived from bacteria, animals, and plants, but are not limited thereto.

[0282] In some cases, the drug can include one or more nucleophilic groups selected from the group consisting of amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form covalent bonds with electrophilic groups on linkers and linker reagents.

[0283] In a specific example of the present invention, an ADC was prepared by linking an antibody or antigen-binding fragment thereof of the present invention to a drug, such as auristatin (MMAE), via an MC-vc-PAB linker. Such an ADC was confirmed to exhibit the desired cytotoxicity.

[0284]

[0285] Chimeric antigen receptor (CAR)

[0286] In another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising an extracellular domain containing an antigen-binding site, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is an scFv of the antibody.

[0287] A chimeric antigen receptor (CAR) is a synthetic construct designed to induce an immune response against a target antigen and cells expressing that antigen. CARs contain an extracellular domain, a transmembrane domain, and an intracellular signaling domain. Cancer cells can be killed by introducing a gene encoding a receptor that recognizes a cancer cell surface antigen specifically expressed on the surface of cancer cells into immune cells. Immune cells containing a receptor that binds to an antigen specifically expressed on cancer cells can elicit an immune response targeting only cancer cells. The CAR contains the scFv of the anti-ROR1 antibody of the present invention as the antigen recognition site in the extracellular domain.

[0288] First-generation CARs comprise an extracellular domain containing an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain, and only CD3ζ is used as the signaling domain. However, they have problems with their therapeutic effect on cancer being weak and short-lasting. Such first-generation CARs are specifically described in U.S. Patent No. 6,319,494, which is incorporated herein by reference.

[0289] Second-generation CARs have been constructed by combining a costimulatory domain (CD28 or CD137 / 4-1BB) with CD3ζ to enhance immune cell reactivity. Compared to first-generation CARs, the number of CAR-bearing immune cells remaining in the body is significantly increased. While second-generation CARs use a single costimulatory domain, third-generation CARs use two or more costimulatory domains. To achieve in vivo expansion and persistence of CAR-bearing immune cells, the costimulatory domain can be combined with 4-1BB, CD28, or OX40. Second-generation CARs are specifically described in U.S. Patent Nos. 7,741,465, 7,446,190, or 9,212,229, and third-generation CARs are specifically described in U.S. Patent No. 8,822,647, both of which are incorporated herein by reference.

[0290] Fourth generation CARs contain additional genes encoding cytokines such as IL-12 or IL-15, allowing for the additional expression of cytokine CAR-based immune proteins, and fifth generation CARs further contain an interleukin receptor chain, e.g., IL-2Rβ, for immune cell enhancement. Fourth generation CARs are described in U.S. Patent No. 10,316,102, and fifth generation CARs are specifically described in U.S. Patent No. 10,336,810, both of which are incorporated herein by reference.

[0291] In one embodiment, the antigen-binding site of the extracellular domain is an antibody scFv. In an scFv comprising antibody VH and VL domains, the VH and VL domains may be linked via a linker. A heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 122 to 152 and 188 to 193 may be linked via a linker to a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153 to 183 and 194 to 199.

[0292] The linker may be a peptide linker and may have a length of about 10 aa to 25 aa. For example, the linker may include hydrophilic amino acids such as glycine and / or serine.

[0293] The linker may be, for example, (GS) n , (GGS) n , (GSGGS) n or (G n S) m (n and m are each 1 to 10), but the linker may be, for example, (G n S) m (n and m each may be 1 to 10.) Specifically, the linker may contain GGGGS, and may be, for example, GGGGSGGGGSGGGGS of SEQ ID NO: 200 repeated three times.

[0294] The transmembrane domain can be derived from natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can include the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or ICOS. If the transmembrane domain is synthetic, it can contain hydrophobic residues such as leucine and valine, or peptides containing phenylalanine, tryptophan, and valine at each end. A short oligo- or polypeptide linker of 2 to 10 amino acids in length can form a bond between the transmembrane domain and the CAR cytoplasmic signaling domain. A glycine-serine peptide can be used as the linker.

[0295] The signaling domain can induce activation of the normal effector function of immune cells in which the CAR is located, for example, by inducing cytolytic activation or helper activation through cytokine secretion. The signaling domain can include a truncated fragment of the intracellular signaling domain sufficient to transduce an effector function signal.

[0296] The signaling domain may include the cytoplasm of a T cell receptor (TCR) and a co-receptor that act in concert to initiate signaling after antigen receptor engagement.

[0297] It is also known that signals generated through the TCR alone are insufficient for complete T cell activation, and that costimulatory signals are required. Thus, T cell activation can involve initiating antigen-dependent primary activation through the TCR and acting in an antigen-dependent manner to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing primary cytoplasmic signaling sequences include TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0298] In some cases, the cytoplasmic domain of the CAR may include a CD3 zeta chain portion and a costimulatory signal transduction region. The costimulatory signal transduction region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. For example, it may include a ligand that specifically binds to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. The cytoplasmic signal transduction sequence within the CAR cytoplasmic signal transduction region may be linked via a peptide linker containing 2 to 10 amino acids, for example, glycine-serine.

[0299] In another aspect, the present invention relates to an immune cell into which the chimeric antigen receptor (CAR) has been introduced.

[0300] The immune cells can induce immunity and induce the desired cancer therapeutic effect, and may be selected from the group consisting of, but not limited to, T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells.

[0301] The antibody other than the above-mentioned antibody may be, for example, an antibody or antigen-binding fragment thereof that targets one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

[0302]

[0303] therapeutic composition

[0304] In yet another aspect, the present invention relates to a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.

[0305] The present invention may be, for example, a pharmaceutical composition for preventing or treating cancer, comprising: (a) a pharmaceutically effective amount of an antibody against ROR1 or its antigen-binding fragment thereof according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or immune cells comprising the chimeric antigen receptor; and (b) a pharmaceutically acceptable carrier. The present invention may also be a method for preventing or treating cancer, comprising the step of administering to a cancer patient the antibody against ROR1 or its antigen-binding fragment thereof according to the present invention, a bi- or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or immune cells comprising the chimeric antigen receptor.

[0306] "Prevention" means any action of administering the composition of the present invention to inhibit the growth of cancer or delay its progression, and "treatment" means inhibiting the development of cancer, reducing tumors, or eliminating cancer.

[0307] The cancer includes, for example, Hodgkin's lymphoma, non-Hodgkin's lymphoma (e.g., B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia), acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, multiple myeloma, or acute lymphocytic leukemia.

[0308] The cancers include, for example, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, anal cancer, uterine cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, Kaposi's sarcoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, brain stem glioma, pituitary adenocarcinoma, epidermoid carcinoma, carcinoma of the cervix squamous cell carcinoma, fallopian tube carcinoma, endometrial carcinoma, vaginal carcinoma, soft tissue sarcoma, urethral cancer, vulvar carcinoma, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvis carcinoma, spinal tumor, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, metastatic lesions of each of the above cancers, or a combination of the above cancers.

[0309] The cancer can be, for example, glioma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, or cervical cancer.

[0310] The pharmaceutically acceptable carriers contained in the compositions of the present invention are those commonly used in pharmaceutical formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the compositions of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0311] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, intradermal administration, topical administration, intranasal administration, intradermal administration, intrarectal administration, etc.

[0312] Since proteins or peptides are digested during oral administration, oral compositions must be formulated to coat or protect the active agent from degradation in the stomach. Pharmaceutical compositions can be administered by any device that allows the active agent to be delivered to target cells.

[0313] The appropriate dosage of the composition of the present invention varies depending on various factors, such as the formulation method, administration method, the patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity, and an ordinarily skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention is 0.0001 mg / kg to 100 mg / kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient for the prevention or treatment of cancer or autoimmune disease.

[0314] The pharmaceutical compositions of the present invention may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients in a manner readily practiced by those skilled in the art to which the invention pertains, and may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally contain a dispersing agent or stabilizer.

[0315]

[0316] Treatment method

[0317] In another aspect, the present invention relates to a composition for treating cancer, comprising the antibody or antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a chimeric antigen receptor, or an immune cell comprising the chimeric antigen receptor.

[0318] The present invention also relates to a method for treating cancer, comprising administering the antibody or antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a chimeric antigen receptor, or an immune cell comprising the chimeric antigen receptor.

[0319] In yet another aspect, the present invention relates to use of the antibody or antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a chimeric antigen receptor, or an immune cell comprising the chimeric antigen receptor for the prevention or treatment of cancer.

[0320] In yet another aspect, the present invention relates to use of the antibody or antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof, an antibody-drug conjugate, a chimeric antigen receptor, or an immune cell comprising the chimeric antigen receptor, for the manufacture of a medicament for the prevention or treatment of cancer.

[0321]

[0322] Combination treatment

[0323] The present invention relates to a composition for combined therapy comprising immune cells and a drug other than an anti-ROR1 antibody.

[0324] In one embodiment, the drug other than an anti-ROR1 antibody can include a chemotherapeutic agent or an antibody other than an anti-ROR1 antibody.

[0325] The drugs include maytansinoids, auristatins (including MMAE and MMAF), aminopterin, actinomycin, bleomycin, tallysomycin, camptothecin, N8-acetylspermidine, 1-(2chloroethyl)-1,2-dimethylsulfonylhydrazide, esperamicin, etoposide, 6-mercaptopurine, dolastatins, trichothecenes, calicheamicin, taxol, taxanes, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-aspartate, benzodiazepine, benzocaine ... L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustardmustard, cytoxan, etoposide, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase nase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C C), trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytosine arabinosidearabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrelin A, interferon-α, interferon-γ, tumor necrosis factor The inhibitor may be one or more selected from the group consisting of benzodiazepine, benzocaine, benzodiazepine, benzocaine-1, benzocaine-2, benzocaine-3, benzocaine-4, benzocaine-5, benzocaine-6, benzocaine-7, benzocaine-8, benzocaine-9, benzocaine-10, benzocaine-11, benzocaine-12, benzocaine-13, benzocaine-14, benzocaine-15, benzocaine-16, benzocaine-17, benzocaine-18, benzocaine-19, benzocaine-20, benzocaine-21, benzocaine-22, benzocaine-23, benzocaine-24, benzocaine-25, benzocaine-26, benzocaine-27, benzocaine-28, benzocaine-29, benzocaine-30, benzocaine-31, benzocaine-32, benzocaine-33, benzocaine-34, benzocaine-35, benzocaine-36, benzocaine-37, benzocaine-38, benzocaine-39, benzocaine-40, benzocaine-41, benzocaine-42, benzocaine-43, benzocaine-44, benzocaine-45, benzocaine-46, benzocaine-47, benzocaine-48, benzocaine-49, benzocaine-50, benzocaine-51, benzocaine-52, benzocaine-53, benzocaine-54, benzocaine-55, benzocaine-55, benzocaine-55, benzocaine-56, benzocaine-57, benzocaine-58, benzocaine-59, benzo

[0326] In one embodiment, the antibody other than the anti-ROR1 antibody may be, for example, an antibody or antigen-binding fragment thereof targeting one or more selected from the group consisting of PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, and MARCO.

[0327] The present invention relates to a combination therapeutic composition comprising an antibody or antigen-binding fragment thereof and one or more selected from the group consisting of:

[0328] (i) Immune cells;

[0329] (ii) an immune cell comprising an antigen receptor (CAR) containing an scFv against an antibody other than an anti-ROR1 antibody as an extracellular domain; and

[0330] (iii) Immune checkpoint inhibitors.

[0331]

[0332] The present invention also relates to a combination therapeutic composition comprising the immune cell-engaging bispecific or multispecific antibody and one or more selected from the group consisting of:

[0333] (i) Immune cells;

[0334] (ii) an immune cell comprising an antigen receptor (CAR) containing an scFv fragment against an antibody other than an anti-ROR1 antibody as an extracellular domain; and

[0335] (iii) Immune checkpoint inhibitors.

[0336] The immune cells are capable of immunotherapy, e.g., inducing immunity and inducing a desired cancer treatment effect, and may be selected from the group consisting of, but not limited to, T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells.

[0337] The antibody other than the anti-ROR1 antibody is an antibody that targets a target other than ROR1, and may be, for example, an antibody or antigen-binding fragment thereof that binds to LAG3, TIM3, CD137 (4-1BB), VISTA, CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, CD200R, transferrin receptor, c-Met, EGFR, HER2, KDR, PDGFRa, NRP1, or MARCO, but is not limited thereto.

[0338] The immune checkpoint inhibitor refers to a preparation capable of inducing T cell activation by blocking T cell inhibitory signals at the site where antigen-presenting cells (APCs) and immune cells, such as T cells, meet. The immune checkpoint inhibitor may be, for example, a drug targeting PD-1, PD-L1, BTLA, CTLA-4, VISTA, LAG3, TIM3, CD137 (4-1BB), CD258 (LIGHT), TIGIT, CD134 (OX40), CD28, CD278 (ICOS), CD27, CD154 (CD40L), CD357 (GITR), CD30, DR3, CD226 (DNAM1), CD96, CD200, or CD200R, but is not limited thereto.

[0339] The first and second components to be administered in combination may be administered simultaneously. Alternatively, the first and second components to be administered in combination may be administered separately at a certain time interval. The second component may be administered separately before or after the administration of the first component of the combination.

[0340]

[0341] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0342] [Example]

[0343] Example 1: Preparation of phage library (scFv)

[0344] To recover the previously constructed human-derived synthetic scFv library genes in phage form, six sub-libraries ((1) Bai, Xuelian & Kim, Jihye & Kang, Seungmin & Kim, Wankyu & Shim, Hyunbo. (2015). A Novel Human scFv Library with Non-Combinatorial Synthetic CDR Diversity. PloS one.10.e0141045.10.1371 / journal.pone.0141045., (2) Yang, Hye & Kang, Kyung & TCW, Julia & Shim, Hyunbo. (2009). Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. Molecules and cells.27.225-35.10.1007 / s10059-009-0028-9.) Samples were cultured in 400 ml of culture medium (SB / ampicillin / 2% glucose) for 2 hours. OD 600When the absorbance at 1000 x g reached 0.5-0.7, the cells were centrifuged at 5000 x g for 20 minutes. The supernatant was removed, and 10 plaque-forming units (pfu) of helper phage (VCSM13) were added to 400 ml of secondary culture medium (SB / ampicillin) and cultured again for 1 hour. Kanamycin antibiotic (the antibiotic gene introduced into the helper phage) was then added at a concentration of 70 μg / ml, and the cells were cultured with shaking at 30°C and 250 rpm for 16 hours to allow the phage library to be produced outside the host cells. The culture was then centrifuged, and PEG8000 (polyethylene glycol 8000) and NaCl were added to the supernatant. The recombinant phage was precipitated with stirring at 4°C for 2 hours. The mixture was centrifuged at 10,000 g and 4° C. for 30 minutes, and the precipitated pellet was suspended in PBS. The mixture was then centrifuged at 15,000 g and 4° C. for 30 minutes, and PBS was added to the precipitated phage pellet to recover the phage library. The concentration of the amplified sublibrary was calculated as the number of colonies generated by infecting TG1 cells with diluted phages and culturing them on LB / ampicillin solid culture medium.

[0345]

[0346] Example 2: Selection of anti-ROR1 specific antibodies (scFv) using biopanning

[0347] Panning was performed to select human antibodies that specifically bind to ROR1. Biopanning was performed using ROR1-Fc and ROR1-His proteins and patient-derived cells as follows. The recombinant antigens used were human ROR1 Fc protein (R&D Systems, 9490-RO, Recombinant Human ROR1 Fc Chimera Protein, CF) and human ROR1 His protein (Sino Biological, 13968-H08H, ROR1 Protein, Human, Recombinant (ECD, His Tag)). The patient-derived cell line, LC-074T, overexpressing ROR1, owned by AimedBio, was used.

[0348] Antigen immobilization biopanning: Human ROR1 Fc protein and negative Fc protein at concentrations of 5 μg / ml to 10 μg / ml were coated onto a 96-well plate for 16 hours at 4°C, and then blocked with 3% skim milk. After emptying the plate, the library (approximately 2.0 × 10 13 The phage were placed on a plate coated with negative Fc protein (pfu) and incubated at room temperature for 30 minutes. This step removes phages that bind to proteins other than human ROR1 from the antibody phage library and prevents nonspecific binding to proteins other than ROR1. The phages that did not bind to the negative Fc protein were collected and allowed to bind to a plate coated with human ROR1 Fc for 1 hour. After washing five to nine times with PBST (Phosphate buffered saline-0.05% Tween 20) solution to remove nonspecific binding, the phage were eluted with IgG elution buffer (Thermo Scientific, 21028, Pierce TM Phage antibodies specific to human ROR1 were collected using IgG Elution Buffer (pH 2.0). This was repeated three times in total, and the results of antigen-immobilized biopanning are shown in Table 1.

[0349] Bead-based biopanning: Biotinylated human ROR1 protein was attached to magnetic beads, and bead panning was performed repeatedly. Streptavidin-conjugated magnetic beads (Invitrogen, 11206D, Dynabeads) were used. TM The human ROR1 protein was biotinylated using a biotinylation kit (abcam, ab201795, Biotin Conjugation Kit (Fast, Type A)-Lightning-Link®) using Dynabeads. TM 100 μl of M-280 streptavidin beads were washed with PBST (phosphate buffered saline-0.1% Tween 20) and PBS using a magnetic bead separator, and then incubated with 50 nM to 100 nM biotinylated human ROR1 protein at room temperature for 30 minutes. The library was then prepared to a concentration of approximately 1.0 × 10 pfu and blocked with 3% nonfat dry milk. The blocked library was mixed with the pre-reacted biotinylated human ROR1-bead conjugate and incubated for 2 hours at room temperature. The library bound to the human ROR1 beads was then recovered using a magnetic bead separator and washed 7 to 12 times with PBST (phosphate buffered saline-0.1% Tween 20) and PBS. The beads were then eluted with IgG elution buffer (Thermo Scientific, 21028, Pierce). TM The phage antibodies bound to the biotinylated human ROR1-beads were eluted using IgG Elution Buffer (pH 2.0). This procedure was repeated three times. The results of the bead-based biopanning are shown in Table 2.

[0350] Biopanning using patient-derived cells: Phage antibodies obtained from antigen-fixed biopanning and bead-based biopanning were applied to Jurkat cells and allowed to bind for 1 hour at 4°C. The supernatant that did not bind to cells derived from ROR1-overexpressing patients was collected. This step removes phages that bind to cell membrane proteins other than ROR1 with the phage antibodies and prevents nonspecific binding of non-ROR1 proteins. The collected supernatant was applied to LC-074T cells (1.5x10^6) derived from ROR1 patients and allowed to bind for 1 hour at 4°C. To remove phages that did not bind to the patient-derived cells, the cells were transferred to a 15ml conical tube and centrifuged at 1,000g for 3 minutes to separate the cells. The cells were then washed 3-5 times with 3ml of cold PBS. The IgG elution buffer (Thermo Scientific, 21028, Pierce) was then added. TMThe cells were then centrifuged at 1,000 g for 3 minutes, and 50 μl of lysis buffer was added to the cells, followed by incubation on ice for 10 minutes. Cell debris was then removed by centrifugation at 12,000 rpm for 5 minutes. The supernatant containing the intracellular phage particles and the phage particles released from the cell surface were then added to culture medium (SB) containing previously grown TG1 cells. The phage particles were then allowed to infect TG1 cells by incubation at 37°C and 120 rpm for 1 hour. The cells were then plated onto LB / ampicillin solid medium, and the remaining solution was centrifuged at 4,000 rpm for 10 minutes. The precipitated TG1 cells were plated onto 15 cm of LB / ampicillin solid medium and incubated. 5 ml of SB culture medium (50% glycerol) was then added to recover individual colonies, which were then stored at -80°C. Next, 50 μl of the stored phage solution from the previous panning run was collected and used to amplify the phage particles. After incubation, helper phage was added, and the collected phage particles were isolated by PEG precipitation and used in the next panning run. This was repeated two times, and the results of biopanning using patient-derived cells are shown in Table 3. A third round of panning was performed using the phage antibodies obtained from patient-derived cells as recombinant proteins, and the results are shown in Table 4. It was confirmed that the proportion of phage particles after panning increased with each round compared to before panning.

[0351] [Table 1]

[0352] [Table 2]

[0353] [Table 3]

[0354] [Table 4]

[0355]

[0356] Example 3: Affinity ELISA screening and sequence analysis of anti-ROR1 specific antibodies (scFv)

[0357] ScFv screening was performed to select monoclonal antibodies that specifically bind to ROR1 from the phages recovered from the final round of panning. Each colony from the final round of panning was picked and inoculated into a 96-well plate containing 200 μl of SB / ampicillin culture medium and cultured at 37°C for 2–3 hours. To induce scFv-pIII protein expression, each well was treated with a final concentration of 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) and cultured overnight at 30°C. The cultured plate was centrifuged at 3,000 rpm for 15 minutes to remove the supernatant. To recover phage particles, 40 μl of TES (50 mM Tris, 1 mM EDTA, 20% sucrose, pH 8.0) solution was added per well and the cells were lysed at room temperature for 30 minutes. Thereafter, the cells were treated with 60 μl of 0.2× TES solution and left at 4° C. for 2 hours to lyse the cells, and then the plate was centrifuged at 3,000 rpm for 15 minutes to collect the supernatant.

[0358] The supernatant was added to each well of a 96-well plate coated with human ROR1, mouse ROR1, or negative Fc protein, and allowed to bind at room temperature for 2 hours. The plate was then washed four times with PBST and distilled water. The plate was then incubated at room temperature for 1 hour with an anti-HA antibody conjugated with HRP, which can bind to the HA tag, and washed six times with PBST and distilled water. TMB substrate solution (Thermo Scientific, 34029, 1-Step TMAfter color development with the addition of Ultra TMB-ELISA Substrate Solution, the color reaction was stopped with a stop solution (Invitrogen, SS04, ELISA Stop Solution), and the absorbance at OD 450 nm was measured. Thirty antibody clones that bind to human ROR1 or mouse ROR1 were selected through ELISA. The CDR sequences of each antibody are shown in Table 5, and the amino acid sequences of the heavy and light chain variable regions are shown in Table 6.

[0359]

[0360]

[0361]

[0362] [Table 5] TIFF2025526427000007.tif255165TIFF2025526427000008.tif118166

[0363]

[0364]

[0365]

[0366]

[0367] [Table 6] TIFF2025526427000010.tif255170TIFF2025526427000011.tif129170

[0368]

[0369] Example 4: Binding domain analysis of anti-ROR1 specific antibodies (scFv)

[0370] ELISA was performed to analyze the domains using the scFvs of the 30 antibody clones selected above. Expression vectors capable of expressing the domains with the antigens used for binding domain analysis were constructed, and the proteins were expressed and purified. For production, residues corresponding to amino acids 1 to 542 or 165 to 395 of the ROR1 amino acid sequence represented by UniProt ID: Q01973 were used. A gene block encoding the extracellular domain of ROR1 was constructed. The 3' end of the gene was ligated to a His tag. The gene was introduced into a pcDNA3.3 vector, and the vector was isolated. The protein was expressed and purified using transient transfection. After culturing for 5 days at 8% CO2, 37°C, and 130 rpm, the protein was purified from the cell culture supernatant. The culture medium was passed through a column (GE Healthcare, 17-5438-01, MabSelect TM The expressed antibody was then passed through a column containing IgG elution buffer (Thermo Scientific, 21028, Pierce). TM After elution with IgG Elution Buffer (pH 2.0), the eluted antibody fraction was concentrated by buffer exchange with PBS (pH 7.4) using an Amicon Ultra 30 kDa tube (Merck Millipore, UFC903024, Amicon® Ultra-15 Centrifugal Filter Unit). The purified domains were quantified using absorbance at 280 nm and the extinction coefficient.

[0371] Human ROR1 immunoglobulin domain (Human ROR1-Immunoglobulin domain, Acrobiosystems, RO1-H5221, Human / Cynomolgus / Rhesus macaque ROR1 (39-151, Ig-like domain) Protein, His Tag), human ROR1 frizzled domain (human ROR1-Frizzled domain, Acrobiosystems, RO1-H5222, Human / Cynomolgus / Rhesus macaque ROR1 (165-305, Frizzled domain) Protein, His Tag), human ROR1 kringle domain (human ROR1-Krigle, Acrobiosystems, RO1-H5223, Human / Cynomolgus / Rhesus macaque ROR1 (308-395, Kringle domain) Protein, His Tag) 96-well plates were coated with 2 μg / mL of human ROR1 immunoglobulin and frizzled domain (human ROR1-Immunoglobulin-Frizzled domain), and human ROR1 frizzled and kringle domain (human ROR1-Frizzled-Kringle domain) at 4°C for 16 hours, followed by blocking with 3% nonfat dry milk. Each scFv was then treated and incubated for 2 hours, followed by washing four times with PBST and distilled water. Subsequently, the plates were incubated for 1 hour at room temperature with an anti-HA antibody conjugated with HRP (Roche, 12013819001, Anti-HA-Peroxidase, High Affinity) that binds to the HA tag, followed by washing six times with PBST and distilled water. TMB substrate solution (Thermo Scientific, 34029, 1-Step TMAfter color development with the addition of Ultra TMB-ELISA Substrate Solution, the color reaction was stopped with a stop solution (Invitrogen, SS04, ELISA Stop Solution), and the absorbance at OD 450 nm was measured. The scFv binding to each domain was analyzed via ELISA. The binding characteristics of each clone, distinct from the epitope domain, were analyzed as shown in Figure 1.

[0372]

[0373] Example 5: Mammalian cell expression and purification of anti-ROR1 antibodies

[0374] Each clone obtained in the examples was cloned and produced in the form of a monoclonal antibody in the form of a complete immunoglobulin (IgG). To construct a heavy chain expression vector, DNA encoding the heavy chain, including the heavy chain variable and constant regions, was cloned into the pOptivec vector. To construct a light chain expression vector, DNA encoding the light chain, including the light chain variable and constant regions, was cloned into the pcDNA3.3 vector.

[0375] Proteins were expressed and purified using the light and heavy chain expression vectors via transient transfection. Expi293 expression medium (Gibco, A1435101, Expi293 TM Expi293F suspension cells (Gibco, A14527, Expi293F) grown in suspension in Expi293F Expression Medium TM Cells) were transfected with plasmids and Opti-MEMI (Gibco, 31985070, Opti-MEM TM I Reduced Serum Medium), ExpiFectamine293 (Gibco, 100014995, ExpiFectamine TMAfter culturing for 5 days at 8% CO2, 37°C, and 130 rpm, the protein was purified from the cell culture supernatant. The culture medium was then loaded onto a column (GE healthcare, 17-5438-01, MabSelect TM The expressed antibody was then passed through a column containing IgG elution buffer (Thermo Scientific, 21028, Pierce). TM After elution with IgG Elution Buffer (pH 2.0), the eluted antibody fraction was concentrated by buffer exchange with PBS (pH 7.4) using an Amicon Ultra 30 kDa tube (Merck Millipore, UFC903024, Amicon® Ultra-15 Centrifugal Filter Unit). The purified anti-ROR1 antibody was quantified using absorbance at 280 nm and the extinction coefficient.

[0376]

[0377] Example 6: Engineering improved biophysical properties for anti-ROR1 antibodies

[0378] Antibody optimization was performed to remove unwanted PTM sites from the antibodies obtained in the above examples. Optimization was performed based on the P015042v1 antibody sequence, which showed excellent efficacy in the in vitro experiments. Among the antibody sequences, sequences that may undergo deamidation and isomerization under stressed conditions during and after production due to unwanted PTMs were identified: HC 55G, LC S51, and LC G95A. According to existing literature, substituting N or D in a sequence susceptible to deamidation and isomerization significantly impairs antibody affinity (Patel, CN, Bauer, SP, Davies, J., Durbin, JD, Shiyanova, TL, Zhang, K., & Tang, JX (2016). N+1 engineering of an aspartate isomerization hotspot in the complementarity-determining region of a monoclonal antibody. Journal of Pharmaceutical Sciences, 105(2), 512-518. https: / / doi.org / 10.1016 / s0022-3549(15)00185-9). Therefore, we substituted the sequence after N or D in the sequence and performed optimization.

[0379] The above sequences were substituted through rational design, and the antibody affinity was measured. First, HC G55 was substituted with V (P015042v1-1) and K (P015042v1-2). Next, LC S51 was substituted with A (P015042v1-3) and K (P015042v1-4), and LC G95 was substituted with A (P015042v1-5). New variants (P015042v1-1 to P015042v1-5) were produced, and their affinities were measured. It was determined that the best substitutions were K for the HC G55 sequence, and K and A for the LC S51 and LC G95, respectively. Therefore, P015042v1-6, which contains all three of these sequence variations, was constructed.

[0380] The CDRs and heavy and light chain variable region sequences of each modified antibody are shown in Tables 7 and 8.

[0381] [Table 7]

[0382] [Table 8]

[0383]

[0384] Example 7: Analysis of ROR1-specific binding ability of anti-ROR1 antibodies (ELISA)

[0385] ELISA was performed to analyze the specific binding ability of the IgG antibodies of each clone selected in the above example to the antigen.

[0386] Human ROR1 and mouse ROR1 proteins were coated onto 96-well plates at 2 μg / mL for 16 hours at 4°C, followed by blocking with 3% nonfat dry milk. Subsequently, antibodies were added at concentrations of 300 nM, 60 nM, 12 nM, 2.4 nM, 0.48 nM, 0.096 nM, and 0.0192 nM, followed by incubation for 1 hour. After washing with PBST and distilled water, the plates were incubated with a chlorine-derived HRP-conjugated anti-human antibody (Invitrogen, 31482, Goat anti-Human IgGF(ab')2 Secondary Antibody, HRP) for 1 hour at room temperature, followed by washing again with PBST and distilled water. TMB substrate solution (Thermo Scientific, 34029, 1-Step TMAfter color development with the addition of Ultra TMB-ELISA Substrate Solution, the color reaction was stopped with a stop solution (Invitrogen, SS04, ELISA Stop Solution), and the absorbance at OD 450 nm was measured. The present anti-ROR1 antibody and its modified antibody with improved biological and physical properties were confirmed to cross-link to human ROR1 and mouse ROR1 (Figure 2).

[0387]

[0388] Example 8: Analysis of ROR1-specific binding ability of anti-ROR1 antibodies (SPR)

[0389] To quantitatively analyze the binding strength of the anti-ROR1 antibody to ROR1, surface plasmon resonance (SPR) was performed using a Biacore 3000 instrument (GE Healthcare).

[0390] Using the amine coupling method, human ROR1 or mouse ROR1 was diluted in 10 mM sodium acetate, pH 4.5, and immobilized on a CM5 sensor chip (GE Healthcare) at 300 response units (RU). Remaining activated moieties on the sensor chip surface were deactivated by adding 1 M ethanolamine-HCl (pH 8.5). Anti-ROR1 antibodies of the present invention were injected onto the antibody protein immobilized on the CM5 sensor chip at concentrations of 300 nM, 150 nM, 75 nM, 37.5 nM, 18.75 nM, 9.375 nM, and 4.6875 nM for 180 seconds (Ka), followed by a 180-second separation step (Kd) at the same flow rate to investigate the KD binding sensorgram. As a result, as shown in Figure 2, the anti-ROR1 antibody exhibited a sensorgram that specifically bound to human ROR1 and mouse ROR1, and the final KD values through Ka and Kd values (1:1 Langmuir 1:1 kinetics) are shown in Tables 9 and 10.

[0391] [Table 9]

[0392] [Table 10]

[0393]

[0394] Example 9: ROR1 domain mapping of anti-ROR1 antibodies

[0395] ELISA was performed to analyze the binding domains using IgG antibodies of the selected clones.

[0396] Human ROR1 immunoglobulin domain (Human ROR1-Immunoglobulin domain, Acrobiosystems, RO1-H5221, Human / Cynomolgus / Rhesus macaque ROR1 (39-151, Ig-like domain) Protein, His Tag), human ROR1 frizzled domain (human ROR1-Frizzled domain, Acrobiosystems, RO1-H5222, Human / Cynomolgus / Rhesus macaque ROR1 (165-305, Frizzled domain) Protein, His Tag), human ROR1 kringle domain (human ROR1-Krigle, Acrobiosystems, RO1-H5223, Human / Cynomolgus / Rhesus macaque ROR1 (308-395, Kringle domain) Protein, His Tag) 96-well plates were coated with 2 μg / mL of human ROR1 immunoglobulin and frizzled domain (human ROR1-Immunoglobulin-Frizzled domain), and human ROR1 frizzled and kringle domain (human ROR1-Frizzled-Kringle domain) at 4°C for 16 hours and then blocked with 3% nonfat dry milk. The plates were then treated with each antibody at concentrations of 300 nM, 60 nM, 12 nM, 2.4 nM, 0.48 nM, 0.096 nM, and 0.0192 nM and incubated for 1 hour. After washing with PBST and distilled water, the plates were incubated with a chlorine-derived HRP-conjugated anti-human antibody (Invitrogen, 31482, Goat anti-Human IgGF(ab')2 Secondary Antibody, HRP) for 1 hour at room temperature and then washed again with PBST and distilled water. TMB substrate solution (Thermo Scientific, 34029, 1-Step TMAfter color development with Ultra TMB-ELISA Substrate Solution, the color reaction was stopped with a stop solution (Invitrogen, SS04, ELISA Stop Solution), and the absorbance at OD 450 nm was measured. Anti-ROR1 antibody clones binding to each domain were analyzed by ELISA (Figure 3).

[0397]

[0398] Example 10: Quantitative analysis of ROR1 receptor expression by cell type

[0399] T-47D, Jeko-1 (abnormal cell lines), AMB-BT-0024T, AMB-BT-0016T, AMB-BT-0013T, AMB-LC-0002T, AMB-LC-0003T, and KUC-OC21-025T (abnormal patient-derived cells) were each 2 × 10 5 Cells were dispensed into a 96-well plate. Mouse IgG1-PE isotype (Invitrogen, 12-4714-82) and ROR1-2A2-PE (Biolegend, 357804) were added at 100 nM per well. After 30 minutes of incubation at 4°C, Quantibrite PE beads (BD, 340495) were added to 0.5 mL of PBS and flow cytometry was performed using a BD FACSAria flow cytometer. The beads were used to establish a baseline for ROR1 expression. The amount of ROR1 expressed in each cell treated with mouse isotype and ROR1-2A2-PE antibodies was compared with the beads to quantify the number of ROR1 receptors expressed per cell.

[0400] The human lymphoma cell line Jeko-1 showed high ROR1 expression, and among patient-derived cells (PDXC), the lung cancer-derived cells AMB-LC-0003T showed the highest ROR1 expression (Figure 4).

[0401] [Table 11]

[0402]

[0403] Example 11: Measurement of specific binding ability of anti-ROR1 antibodies to cell surface-expressed ROR1

[0404] Before anti-ROR1 antibodies can be used for therapeutic purposes, it is very important to first confirm whether they bind to antigens expressed on the cell surface. The cell-binding ability of the anti-ROR1 antibodies developed by the inventors was measured using FACS in the Jeko-1 cell line, which has been confirmed to have high ROR1 expression, and cells derived from the AMB-LC-0003T patient. Cells were plated at 1 x 10 per well in a 96-well plate. 5 The cells were dispensed at a ratio of cells / 100 μl of FACS buffer solution.

[0405] Cell binding assays were performed using the anti-ROR1 candidate antibodies P015004, P015042, P015043, and P015044, as well as P015042v1, which was obtained through improved biological and physical properties, and its variant P015042v1-6. Each antibody was diluted 10-fold from 100 nM to 10 nM, 1 nM, 100 pM, 10 pM, and 1 pM. The cells were incubated at 4°C for 30 minutes and then washed twice. PE-labeled goat anti-human IgG Fc PE (Thermofisher; 12-4998-82) was used as the secondary antibody, diluted 1:100 in FACS buffer, and 100 μl was dispensed into each well. The incubation was repeated at 4°C for 30 minutes and then washed twice. Finally, 100 μl of FACS buffer solution was dispensed into each well, mixed thoroughly with a pipette, and then flow cytometry was performed using a NovoCyte flow cytometer (Agilent). Analysis was performed using GraphPad Prism 9.3.1, and EC values were calculated using a log(agonist) vs. response-variable slope nonlinear graph model. 50 The value was derived.

[0406] Cell binding results showed higher MFI (Mean Fluorescence Intensity) in cells derived from patient AMB-LC-0003T, which had the highest ROR1 expression, than in the Jeko-1 cell line. Antibodies that showed sub-nanomolar EC50 values in both cell types included P015042 and its modified sequences, P015042v1 and P015042v1-6, and P015044, which also showed a nanomolar EC50 value. Other anti-ROR1 clones, P015004 and P015043, also showed EC50 values of <10 nM, demonstrating good cell binding ability (Figures 5 and 6).

[0407] [Table 12]

[0408] [Table 13]

[0409]

[0410] Example 12: Cellular internalization analysis of anti-ROR1 antibodies

[0411] To develop an antibody-drug conjugate, the anti-ROR1 antibody must not only bind to the ROR1 antigen present on the cell, but also, upon internalization, the conjugate drug must react with the ROR1 antigen as it penetrates into the cell. To analyze this, internalization was confirmed by detecting the ROR1 antigen remaining on the cell surface at various time points. Jeko-1 cells were cultured in duplicate at 1 x 10 per well. 5Cells were then added to the plate at 100 μl per well, and plates were prepared for each time point: 0 min, 10 min, 30 min, 1 h, 2 h, and 4 h after the start of cellular internalization. The ROR1 receptor remaining on the cell surface without antibody internalization was examined. Each cell was treated with 1 nM of each antibody and incubated for 30 min at 4°C. After 2 min of centrifugation at 1100×g and two washes, the remaining plates, except for the plate at 0 min, were mixed with 100 μl of FACS buffer and incubated at 37°C and 5% CO2. The plate at 0 min was immediately incubated with goat anti-human IgG Fc PE (Thermofisher, 12-4998-82) diluted 1:100 and 100 μl was dispensed per well for approximately 30 min at 4°C. After washing twice, flow cytometry was performed using a Novocyte flow cytometer (Agilent). The remaining ROR1 expression in the Jeko-1 cells was confirmed in the same manner for the other plates at different times. Each value was normalized based on the ROR1 expression level detected at 0 minutes and analyzed using the [inhibitor] vs. normalized response-variable slope nonlinear graph model in GraphPad Prism 9.3.1.

[0412] Each antibody clone showed different cellular internalization levels, and most antibodies were confirmed to be continuously internalized even after 4 hours. P015042v1-6, an engineered version of the P015042 antibody with improved biological and physical properties, showed a similar level of cellular internalization to MAC's UC-961 antibody, which exhibited the highest level of cellular internalization (Figure 7).

[0413]

[0414] Example 13: Preparation of anti-ROR1 antibody-drug conjugates

[0415] A general method for producing the antibody-drug conjugate of the present invention or its production intermediates will be described below, with the names and alternative symbols of the compounds included in each reaction scheme (Figure 8).

[0416] The antibody-drug conjugate represented by formula (1), in which the antibody (Y) and the drug-linker (LP) structure are linked via a thioether, can be produced, for example, by the following method.

[0417] [Number(1)]

[0418] X(1)+LP(2)→X-LP(3)

[0419] The exemplified drug-linker (LP) used is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin E (vc-PAB-MMAE; CCC(C)C(C(CC(=O)N1CCCC1C(C(C)C(=O)NC(C)C(C2=CC=CC=C2)O)OC)N(C)C(=O)C(C(C)C)NC(=O)C(C(C)C)N(C)C(=O)OCC3=CC=C(C=C3)NC(=O)C(CCCNC(=O)N)NC(=O)C(C(C)C)NC(=O)CCCCCN4C(=O)C=CC4=O), where X is a monoclonal antibody of IgG1 type having a sulfhydryl group.

[0420] Through reaction between the maleimidyl group of LP and the sulfhydryl group of X, an antibody-drug conjugate can be generated.

[0421] Antibody (X) having sulfhydryl groups can be obtained by the method described below: The antibody can be reacted with a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reduce the disulfide bonds in the hinge region of the antibody to form sulfhydryl groups.

[0422] Specifically, antibody (Y) is reacted with 5 mM TCEP (2-20 molar equivalents) as a reducing agent per antibody intracellular disulfide in a buffer containing 5 mM ethylenediaminetetraacetic acid (EDTA), a representative chelating agent, to produce antibody (X) with sulfhydryl groups resulting from partial or complete reduction of the antibody intracellular disulfides. The intracellular disulfide reduction reaction described above was carried out at 37°C for 2 hours. The buffer used in this example was a phosphate buffer solution (pH 7.4) containing saline, which was the same as that used in antibody production. The intracellular disulfide reduction reaction described above was carried out at 37°C for 2 hours.

[0423] After the reaction was completed, the sulfhydryl-containing antibody (X) solution was added to an Amicon Ultra (30 kDa, Millipore Co.) container and centrifuged (3950 G for 10-20 minutes) using an Eppendorf centrifuge (5810 R) to remove impurities such as TCEP and EDTA. The buffer was then exchanged with a phosphate buffer solution (pH 7.4) containing saline. This process was repeated three times.

[0424] After removing impurities through buffer exchange, the sulfhydryl-bearing antibody (X) was collected in an Amicon Ultra (30 kDa, Millipore Co.) container and reacted with 5-15 molar equivalents of 10 mM drug-linker (LP) at room temperature for 2 hours. Because the drug-linker (LP) can be deformed by light, this reaction was carried out in the dark. To increase the solubility of the compound, the drug-linker (LP) was dissolved in dimethylacetamide (DMA), a typical organic solvent. Approximately 10-20% v / v DMA was added to the buffer solution containing the sulfhydryl-bearing antibody (X).

[0425] The antibody-drug conjugation reaction can be terminated by inactivating the reactivity of unreacted drug-linkers (LPs) with a thiol-containing reagent. The thiol-containing reagent used in this example was N-acetyl-L-cysteine (NAC). Specifically, the reaction was terminated by adding 5 molar equivalents of NAC to the antibody-drug conjugation reaction mixture and storing it at room temperature for 30 minutes.

[0426] To remove the remaining drug-linker and NAC, the antibody-drug reaction mixture was added to an Amicon Ultra (30 kDa, Millipore Co.) vessel and centrifuged (3950 xg for 10-20 minutes) using an Eppendorf centrifuge 5810 R to exchange the buffer solution for a phosphate buffer solution (pH 7.4) containing saline. This process was repeated three times. Finally, the antibody-drug conjugate was recovered from the filter membrane of the Amicon Ultra (30 kDa, Millipore Co.) vessel.

[0427]

[0428] Example 14: Concentration analysis of anti-ROR1 antibody-drug conjugates

[0429] The concentration of antibody in the antibody-drug conjugate was measured using a spectrophotometer (Thermofisher Scientific NanoDrop 8000).

[0430]

[0431] Example 15: Purity analysis of anti-ROR1 antibody-drug conjugates

[0432] The purity of the antibody-drug conjugate was determined by size-exclusion high-performance liquid chromatography (SEC-HPLC) analysis using the method described below. The antibody-drug conjugate sample was prepared to a concentration of 0.5 mg / mL in a total volume of 25 μL. SEC-HPLC analysis was performed under the following conditions:

[0433] [Table 14]

[0434] The purity of the antibody-drug conjugate was determined by comparing the retention time of each peak in the antibody-drug conjugate chromatogram with that of a size marker (Gel filtration standard, Bio-Rad, 1511901). Since size exclusion chromatography detects larger molecules first, the peaks that appear earlier than the antibody peak (150 kDa) represent polymer aggregates that may be formed due to the hydrophobicity of the drug, while the peaks that appear later represent the conjugated and then separated drug. The purity of the antibody-drug conjugate itself was calculated by converting the area of the peak corresponding to 150 kDa in the entire chromatogram into a percentage.

[0435]

[0436] Example 16: Analysis of the average number of drug molecules conjugated per antibody molecule of anti-ROR1 antibody-drug conjugates (DAR analysis)

[0437] The average number of drug molecules conjugated per antibody molecule (Drug-to-Antibody Ratio, DAR) of antibody-drug conjugates was measured by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC) analysis using the method described below. As a pretreatment for HIC-HPLC analysis, the antibody-drug conjugate sample used for analysis was prepared to a concentration of 0.5 mg / mL and a total volume of 30 μL. HIC-HPLC analysis was performed under the following conditions.

[0438] [Table 15]

[0439] [Table 16]

[0440] Compared to the same unconjugated antibody, an antibody-drug conjugate exhibits higher hydrophobicity in proportion to the number of conjugated drug molecules, resulting in a longer retention time. A total of four disulfide bonds exist within an antibody, and two drugs can be conjugated to each disulfide bond. Therefore, up to five peaks (DAR 0, DAR 2, DAR 4, DAR 6, and DAR 8) can be observed in the HIC-HPLC chromatogram of an antibody-drug conjugate. The peaks that appear sequentially after the retention time of the unconjugated antibody (DAR 0) were assigned the following order: DAR 2, DAR 4, DAR 6, and DAR 8. Once the DAR assignment for each peak was completed, the average number of drug molecules conjugated per antibody molecule was calculated using the following formula:

[0441] TIFF2025526427000022.tif17170

[0442]

[0443] Example 17: Antigen binding ability analysis of anti-ROR1 antibody-drug conjugates

[0444] Affinity analysis of antibody-drug conjugates is performed to assess their comparability to the parent antibody and to determine whether there are any potential loss of affinity that may result from manufacturing processes such as disulfide bond reduction to generate antibodies with sulfhydryl groups and drug-linker conjugation. The analysis was performed using enzyme-linked immunosorbent assay (ELISA) as described below.

[0445] 2 μg / ml human and mouse ROR1 antigen (Sino Biological) was coated onto a 96-well plate (Costar 3590, Corning) in 50 μL aliquots per well. After shaking off the coated ROR1 antigen, 200 μL of 3% milk was added to each coated well for blocking. After 1 hour, the 3% milk was shaken off, and the parent antibody and antibody-drug conjugate were serially diluted 7 times, 1:5, starting from a maximum concentration of 300 nM, and 50 μL of each was added to the designated wells. After 1 hour of primary antigen-antibody binding, the plate was washed with 0.05% v / v surfactant (PBST) and distilled water (three times each). Fab-HRP was then diluted 1:3000 in 3% milk and 50 μL of each was added to each well. After the secondary binding reaction was carried out for 1 hour, the plate was washed with 0.05% PBST and triple-distilled water (three times each). After removing the remaining water from the plate, 50 μL of TMB (34029, Thermofisher) was dispensed into each well and allowed to react for 3 to 5 minutes. Finally, to terminate the reaction, 50 μL of stop solution (SS04, Thermofisher) was dispensed into each well.

[0446] [Table 17]

[0447] When the antigen-binding ability of the antibody-drug conjugate was 80% or more of that of the monoclonal antibody, the antibody-drug conjugate was considered to have similar antigen-binding ability.

[0448]

[0449] The three anti-ROR1 antibody-drug conjugates (P015004-vc-PAB-MMAE, P015042-vc-PAB-MMAE, and P015044-vc-PAB-MMAE) prepared as described above were evaluated using the antibody-drug conjugate analysis and QC methods described above, and the results are summarized in Table 18 below.

[0450] [Table 18]

[0451] Figure 9 shows the results of a purity analysis of the antibody-drug conjugates used in the examples of the present invention, Figure 10 shows the results of an analysis of the average number of drug molecules conjugated per antibody molecule of the antibody-drug conjugates used in the examples of the present invention, and Figure 11 shows the results of an ELISA analysis of the ROR1 binding of the antibody-drug conjugates used in the examples of the present invention.

[0452]

[0453] Example 18: In vitro cytotoxicity evaluation of anti-ROR1 antibody-drug conjugates

[0454] The toxicity of the anti-ROR1 candidate antibody-drug conjugates mentioned above was evaluated as anticancer drugs using AMB-LC-0003T, a cell line known to have high ROR1 expression and derived from a lung cancer patient, and AMB-LC-0002T, which has very low ROR1 expression.

[0455] Lung cancer patient-derived cells, AMB-LC-0003T and AMB-LC-0002T, were plated in triplicate in 40 μl of M10018 (Aimedbio) media at 500 cells per well in ultra-low attachment, U-bottom 384-well clear plates (S-bio, #MS-9384UZ). After centrifugation at 250 g for 2 minutes, the plates were incubated at 37°C with 5% CO for 24 hours. After incubation, each antibody-drug conjugate was diluted 3-fold from 500 nM to 0.314 pM. The plates were then incubated again at 37°C with 5% CO for 6 days.

[0456] After a total of 7 days of reaction, each cell formed spheroids in the 384-well plate, and images of the spheroids in each well were captured using the Operetta CLS (PerkinElmer) high-content screening system. To maintain the spheroid morphology of each cell in each well, no washing was performed before analysis. The captured images were converted to volume through additional analysis, and the spheroid volume values for each concentration were normalized based on the spheroid treated with the lowest drug concentration. Graphs were created using GraphPad Prism 9.3.1, with log (inhibitor) vs. normalized response-variable slope, and IC values were calculated. 50 The values were derived (Figure 12).

[0457] [Table 19]

[0458] [Table 20]

[0459] The P015042-vc-PAB-MMAE antibody-drug conjugate described above showed excellent IC in AMB-LC-0003T, which has high ROR1 expression. 50 In AMB-LC-0002T cells, which are derived from a patient with low ROR1 expression, the IC value was 100 nM or higher. 50 It was confirmed that the antibody-drug conjugate exhibited only ROR1-specific effects while retaining its potency. Compared to UC961-vc-PAB-MMAE, the antibody-drug conjugate disclosed in US Patent No. 10,335,496 B2, it exhibited approximately 8-fold higher relative potency, suggesting the ROR1-specific anti-cancer efficacy of the antibody-drug conjugate (Figure 13).

[0460] [Industrial Applicability]

[0461] The anti-ROR1 antibody or antigen-binding fragment thereof according to the present invention exhibits superior binding ability to ROR1 compared to existing anti-ROR1 antibodies, and can be usefully used in the prevention or treatment of target tumors or cancers.

[0462]

[0463] Although the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

[0464] [Sequence List Free Text]

[0465] Electronic file attached.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to ROR1 (Receptor Tyrosine Kinase Like Orphan Receptor 1), including: a heavy chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16; A heavy chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 17 to 41, 184, and 185; a heavy chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 42 to 65; a light chain CDR1 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 66 to 86; a light chain CDR2 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 87 to 102, 186, and 187; and A light chain CDR3 comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 103 to SEQ ID NO:

121.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 122 to 152, and 188 to 193.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153 to 183, and 194 to 199.

4. 2. The antibody or antigen-binding fragment thereof of claim 1, including single-chain Fv (scFv), single-chain antibody, Fab, F(ab'), and disulfide-linked Fvs (sdFv).

5. The scFv comprises a heavy chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 122 to 152, and 188 to 193, and a light chain variable region comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 153 to 183, and 194 to 199, linked via a linker. The antibody or antigen-binding fragment thereof according to claim 4.

6. The linker may be ｎ S) ｍ (n and m are each 1 to 10).

7. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A recombinant expression vector comprising the nucleic acid of claim 7.

9. A host cell transfected with the recombinant expression vector of claim 8.

10. 10. The host cell of claim 9, which is COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

11. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to ROR1, comprising: culturing the host cell of claim 9 to produce the antibody; and isolating and purifying the produced antibody.

12. A bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

13. An immune cell-engaging bispecific or multispecific antibody comprising an scFv of the antibody according to any one of claims 1 to 6 and a second binding domain comprising one or more scFvs of an antibody that binds to an immune cell-activating antigen.

14. An antibody-drug conjugate (ADC) in which the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 is conjugated to a drug.

15. The drugs include maytansinoids, auristatins (including MMAE and MMAF), aminopterin, actinomycin, bleomycin, tallysomycin, camptothecin, N8-acetylspermidine, 1-(2chloroethyl)-1,2-dimethylsulfonylhydrazide, esperamicin, etoposide, 6-mercaptopurine, dolastatin, trichothecenes, calicheamicin, taxol, taxanes, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-aspartate, benzodiazepine, benzocaine, benzophenone, benzocaine ... L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustardmustard), cytoxan, etoposide, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase nase), vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, decarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, crisnatol, mitomycin C C), trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethylenyl-1-beta-dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytosine arabinosidearabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrelin A A), interferon-α, interferon-γ, tumor necrosis factor, gemcitabine, velcade, levalmid, salamide, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2 15. The antibody-drug conjugate of claim 14, wherein the drug is at least one selected from the group consisting of peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, thapsigargin, nucleases, and toxins derived from bacteria, animals, or plants.

16. The antibody-drug conjugate of claim 14, wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker.

17. The antibody-drug conjugate of claim 16, wherein the linker is a cleavable linker or a non-cleavable linker.

18. The antibody-drug conjugate of claim 17, wherein the cleavable linker is an acid-labile linker, a disulfide linker, a peptide linker, or a beta-glucuronide linker, and the non-cleavable linker comprises a thioether group or a maleimidocaproyl group.

19. The antibody-drug conjugate of claim 16, wherein the linker is bound to a cysteine residue exposed upon reduction of a disulfide bond of the antibody or a cysteine residue present in a tag bound to the antibody.

20. A chimeric antigen receptor (CAR) comprising an extracellular domain having an antigen-binding site, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding site of the extracellular domain is an scFv of the antibody according to any one of claims 1 to 6.

21. An immune cell into which the chimeric antigen receptor (CAR) according to claim 20 has been introduced.

22. The immune cell according to claim 21, which is at least one selected from the group consisting of T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor-infiltrating lymphocytes (TIL), and dendritic cells.

23. A composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a bispecific or multispecific antibody comprising said antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising said antibody or antigen-binding fragment thereof, or an immune cell comprising said chimeric antigen receptor.

24. A method for preventing or treating cancer, comprising the step of administering the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, a bispecific or multispecific antibody comprising said antibody or antigen-binding fragment thereof, an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof, a chimeric antigen receptor comprising said antibody or antigen-binding fragment thereof, or an immune cell comprising said chimeric antigen receptor.

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