Anti-ROR1 antibodies and uses thereof

Abstract

The present invention relates to: a receptor tyrosine kinase-like orphan receptor 1 (ROR1) antibody or an antigen-binding fragment thereof; a nucleic acid encoding the same; a recombinant expression vector carrying the nucleic acid; a host cell transfected with the recombinant expression vector; a method for preparing the antibody or the antigen-binding fragment thereof; a bispecific or multispecific antibody carrying the antibody or the antigen binding fragment thereof; the immune cells are connected with bispecific or multispecific antibodies; an antibody-drug conjugate (ADC), wherein the antibody or antigen-binding fragment thereof binds to a drug; a chimeric antigen receptor (CAR) comprising the scFv of the antibody as the antigen binding site of the extracellular domain; an immune cell having a chimeric antigen receptor introduced therein; a composition for combination therapy including the antibody or an antigen-binding fragment thereof; a pharmaceutical 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 or the antigen-binding fragment thereof; 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 and a second binding domain, wherein the scFv comprises the scFv of the antibody, and the second binding domain comprises at least one scFv of an antibody that binds to an immune cell activation antigen; an antibody-drug conjugate (ADC), wherein the antibody or the antigen-binding fragment thereof is coupled to a drug; a chimeric antigen receptor (CAR) comprising the scFv of the antibody as an antigen-binding site of the extracellular domain; an immune cell into which the chimeric antigen receptor is introduced; a composition for combined therapy comprising the antibody or the antigen-binding fragment thereof or the immune cell; a composition for preventing or treating cancer comprising the antibody or the antigen-binding fragment thereof or the immune cell, and a method for preventing or treating cancer comprising the antibody or the antigen-binding fragment thereof or the immune cell. Background Art

[0002] ROR (receptor tyrosine kinase-like orphan receptor) is a representative group of transmembrane RTK (receptor tyrosine kinase) receptors that have different effects on the overall activity of the cell. The ROR family consists of ROR1 and ROR2, and the amino acid sequence similarity of the two proteins is about 60%. The ROR family includes Ig, cysteine-rich and kringle domains outside the cell, and tyrosine kinase, Ser / Thr-rich and proline-rich domains inside the cell.

[0003] ROR1 is expressed during embryonic development and affects various activities of early cells, but its expression gradually decreases as adult tissue formation progresses. In addition, ROR1 is expressed in the intermediate stage of normal B cell maturation, but not in mature B cells, T cells or monocytes. However, ROR1 is known to be overexpressed in various tumor cells and is therefore considered a fetal gene for malignant tumors. It has been reported that ROR1 can act as a receptor for Wnt5a, activate non-classical Wnt signals, and lead to cancer cell proliferation and metastasis. Therefore, ROR1 has begun to become a representative target for anti-cancer antibody therapy. Specifically, since ROR1 was found to be overexpressed in chronic lymphocytic leukemia (CLL), whether ROR1 is overexpressed in various cancers has been thoroughly studied (Klein et al., 2001, J. Exp. Med 194; 1625). Thus, ROR1 overexpression has been demonstrated not only in chronic lymphocytic leukemia (CLL), but also in a variety of blood cancers (acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma (DLBCL)), as well as in solid tumors such as breast cancer, cervical cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), and brain cancer.

[0004] Although ROR1 was initially classified as a pseudokinase and underestimated, it is known that overexpression of ROR1 in the above cancer types is closely related to the survival of cancer patients and cancer metastasis, so it is necessary to develop effective therapeutic drugs. International Patent No. WO 2016 / 172726 A1 discloses an anti-ROR1 monoclonal antibody and its use. The antibody shows ROR1-specific properties and can bind to and kill CLL cells in which ROR1 is overexpressed, indicating that it is possible to develop an antibody-based therapeutic agent that selectively recognizes ROR1.

[0005] Since anti-ROR1 antibodies have different properties, it is possible to develop various anti-cancer antibodies according to the type of cancer in which the antigen is expressed. By targeting cancer types with high recurrence rates, such as CLL, the unmet demand for anti-cancer antibodies can be met (Choi et al., 2018, Cell Stem Cell 22, 951-959). The cancer-specific expression of ROR1 occurs not only in cancer cells, but also in cancer stem cells or stem cells, which helps to inhibit tumor metastasis or recurrence. Considering its expression in various cancer-related cells, the possibility of developing various therapeutic drugs based on existing antibodies is innumerable.

[0006] In this technical context, the inventors not only discovered antibodies with specificity for ROR1, but also selected antibodies that can more selectively recognize ROR1 on the surface of patient-derived cells, and identified antibodies with higher anti-cancer efficacy and targeting accuracy than conventional known therapeutic drugs under development, thereby completing the present invention. Summary of the invention

[0007] The object of the present invention is to provide a novel antibody or antigen-binding fragment thereof against ROR1 (receptor tyrosine kinase-like orphan receptor 1).

[0008] Another object of the present invention is to provide a nucleic acid encoding the antibody or its antigen-binding fragment.

[0009] Another object of the present invention is to provide a recombinant expression vector comprising the nucleic acid or a host cell transfected with the recombinant expression vector.

[0010] Another object of the present invention is to provide a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to ROR1.

[0011] Another object of the present invention is to provide a bispecific or multispecific antibody or an immune cell engaging bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof.

[0012] Yet another object of the present invention is to provide an antibody-drug conjugate (ADC), wherein the antibody or antigen-binding fragment thereof is conjugated to a drug.

[0013] Another object of the present invention is to provide a chimeric antigen receptor (CAR) comprising the scFv of the antibody as the extracellular domain antigen binding site, an immune cell into which the chimeric antigen receptor is introduced, and a composition comprising the immune cell for combined therapy.

[0014] Another object of the present invention is to provide a composition for preventing or treating cancer or a method for preventing or treating cancer, comprising the antibody or its antigen-binding fragment, the bispecific or multispecific antibody, the antibody-drug conjugate, the chimeric antigen receptor or the immune cell.

[0015] Another object of the present invention is 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 preventing or treating cancer.

[0016] Another object of the present invention is 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 in the manufacture of a medicament for preventing or treating cancer.

[0017] In order 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), comprising:

[0018] a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16;

[0019] a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 41, SEQ ID NO: 184, and SEQ ID NO: 185;

[0020] a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO:42 to SEQ ID NO:65;

[0021] a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 66 to SEQ ID NO: 86;

[0022] a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 87 to SEQ ID NO: 102, SEQ ID NO: 186, and SEQ ID NO: 187, and

[0023] A light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 103 to SEQ ID NO: 121.

[0024] The present invention also provides a nucleic acid encoding the antibody or the antigen-binding fragment thereof.

[0025] The invention also provides a recombinant expression vector comprising the nucleic acid.

[0026] The invention also provides a host cell transfected with the recombinant expression vector.

[0027] The present invention also provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, comprising producing the antibody by culturing host cells; and separating and purifying the produced antibody.

[0028] The present invention also provides a bispecific or multispecific antibody or an immune cell-engaging bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof.

[0029] The present invention also provides an antibody-drug conjugate (ADC), wherein the antibody or antigen-binding fragment thereof is conjugated to a drug.

[0030] The present invention also provides a chimeric antigen receptor (CAR), which includes an extracellular domain containing an antigen binding site, a transmembrane domain and an intracellular signal transduction domain, wherein the antigen binding site of the extracellular domain is the scFv of the antibody.

[0031] The present invention also provides an immune cell comprising a chimeric antigen receptor (CAR).

[0032] The present invention also provides a composition for preventing or treating cancer, comprising the antibody or its antigen-binding fragment, the bispecific or multispecific antibody, the antibody-drug conjugate, the chimeric antigen receptor or the immune cell.

[0033] 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 preventing or treating cancer.

[0034] 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 in the manufacture of a drug for preventing or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shown are the results of analyzing the clone-dependent ROR1 binding properties by ELISA.

[0036] Figure 2 The results showing the cross-linking of the anti-ROR1 antibody to human ROR1 and mouse ROR1 are shown.

[0037] Figure 3 Shown are the results of ELISA analysis of anti-ROR1 antibody clones binding to individual domains.

[0038] Figure 4 The results of measuring the ROR1 expression level of cells used in Examples of the present invention are shown.

[0039] Figure 5 The results of analysis of the ability of the antibodies used in the Examples of the present invention to bind to the ROR1 antigen expressed in lung cancer cells derived from an AMB-LC-0003T patient are shown.

[0040] Figure 6 The results of analysis of the ability of the antibodies used in the Examples of the present invention to bind to the ROR1 antigen expressed in the Jeko-1 cell line are shown.

[0041] Figure 7 The results of cellular internalization analysis of the antibodies used in Examples of the present invention are shown.

[0042] Figure 8 The structure of the anti-ROR1 antibody-drug conjugate is shown.

[0043] Fig. 9 The results of purity analysis of the antibody-drug conjugates used in the Examples of the present invention are shown.

[0044] Fig.10 The results of analysis of the average number of drug molecules conjugated per antibody molecule of the antibody-drug conjugates used in Examples of the present invention are shown.

[0045] Fig.11 The ELISA test results of the antibody-drug conjugate used in the examples of the present invention binding to ROR1 are shown.

[0046] Fig.12 The results of in vitro toxicity evaluation of the antibody-drug conjugates used in the examples of the present invention on patient-derived cells are shown.

[0047] Fig.13 The results of in vitro toxicity evaluation of the antibody-drug conjugates used in the examples of the present invention on patient-derived cells are shown, demonstrating that the therapeutic efficacy is greatly improved compared to traditional antibody-drug conjugates. DETAILED DESCRIPTION

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the terms used herein are well known and typical in the art.

[0049] Anti-ROR1 Antibody

[0050] Aspects of the present invention relate to an antibody or antigen-binding fragment thereof that specifically binds to ROR1 (receptor tyrosine kinase-like orphan receptor 1), the antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16; a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 41, SEQ ID NO: 184 and SEQ ID NO: 185; a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 42 to SEQ ID NO: 65; a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 66 to SEQ ID NO: 86; a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 87 to SEQ ID NO: 102, SEQ ID NO: 186 and SEQ ID NO: 187; a light chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 103 to SEQ ID NO: At least one amino acid sequence in the group consisting of IDNO:121.

[0051] 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 antibody molecules.

[0052] The structure of a complete antibody has two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by disulfide bonds.

[0053] As used herein, the term "heavy chain" is understood to include full-length heavy chains and fragments thereof, including a variable region domain VH and three constant region domains CH1, CH2 and CH3, wherein the variable region domain VH includes an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen. In addition, the term "light chain" used herein is understood to include full-length light chains and fragments thereof, including a variable region domain VL and a constant region domain CL, wherein the variable region domain VL includes an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen.

[0054] Whole antibodies include subtypes of IgA, IgD, IgE, IgM, and IgG, and in particular, IgG includes IgG1, IgG2, IgG3, and IgG4. Heavy chain constant regions include gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2) subtypes. Light chain constant regions include kappa (κ) type and lambda (λ) type.

[0055] The antigen-binding fragment of an antibody or an antibody fragment is a fragment having an antigen-binding function, and includes Fab, F(ab'), F(ab')2 and Fv. Among antibody fragments, Fab has a structure comprising light chain and heavy chain variable regions, a light chain constant region and a first heavy chain constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that Fab' has a hinge region including at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 domain. F(ab')2 is generated by disulfide bonds between cysteine ​​residues in the hinge region of Fab'.

[0056] Fv corresponds to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. Double-chain Fv is configured so that the heavy chain variable region and the light chain variable region are connected by non-covalent bonds, and single-chain Fv (scFv) is configured so that the heavy chain variable region and the light chain variable region are usually connected by a peptide linker therebetween with a covalent bond, or directly connected at the C-terminus to form a dimer structure identical to the double-chain Fv. Such antibody fragments can be obtained using proteolytic enzymes (e.g., Fab can be obtained by restrictive cleavage of the whole antibody with papain, and F(ab')2 can be obtained by cleavage with pepsin), or can be constructed by genetic recombination technology.

[0057] The "Fv" fragment is an antibody fragment that contains the recognition and binding sites of an intact antibody. This region is a dimer of one heavy chain variable domain and one light chain variable domain connected together.

[0058] The "Fab" fragment includes the variable and constant regions of the light chain and the variable and first constant region (CH1) of the heavy chain. F(ab')2 antibody fragments typically include a pair of Fab' fragments covalently linked by cysteine ​​in the hinge region at the C-terminus of the Fab' fragments.

[0059] "Single-chain Fv (scFv)" antibody fragments are constructs composed of a single polypeptide chain comprising the VH domain and VL domain of an antibody. A polypeptide linker may also be included between the VH domain and the VL domain to allow the scFv to form an ideal antigen binding structure.

[0060] In one embodiment, examples of 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 Fv (sdFV), anti-idiotypic (anti-Id) antibodies, epitope-binding fragments of these antibodies, etc.

[0061] The heavy chain constant region may be any one selected from isotypes such as gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε). For example, the constant region is γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), or γ4 (IgG4). The light chain constant region may be κ type or λ type.

[0062] Monoclonal antibody is the antibody obtained from substantially homogeneous antibody population, wherein the individual antibodies constituting the population are identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibody has a high degree of specificity and is directed against a single antigenic site. Different from typical (polyclonal) antibodies (generally including different antibodies directed against different determinants (epitopes)), each monoclonal antibody is directed against a single determinant on the antigen.

[0063] The term "epitope" refers to a protein determinant to which an antibody can specifically bind. An epitope usually consists of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally has specific three-dimensional structural features and specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that the binding of the former is lost in the presence of denaturing solvents, while the binding of the latter is not lost.

[0064] "Humanized" forms of non-human (e.g., mouse) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity.

[0065] "Human antibody" is a molecule derived from human immunoglobulin, which means that the amino acid sequence constituting the antibody, including the complementarity determining regions and framework regions, is entirely composed of human immunoglobulin.

[0066] A portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chain or chains are identical or homologous to the corresponding sequence in antibodies derived from other species or belonging to other antibody classes or subclasses, including "chimeric" antibodies (immunoglobulins), as well as fragments of such antibodies that exhibit the desired biological activity.

[0067] As used herein, an antibody "variable region" is a light or heavy chain portion of an antibody molecule including the amino acid sequence of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain, and VL refers to the variable domain of the light chain.

[0068] "Complementarity determining region" (CDR) refers to the amino acid residues of an antibody variable domain that are essential for antigen binding. Each variable domain typically has three CDRs, which are designated CDR1, CDR2, and CDR3.

[0069] 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; and 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,

[0070] 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; and 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,

[0071] 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; and 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,

[0072] 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; and 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,

[0073] 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; and 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,

[0074] 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; and 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,

[0075] 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; and 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,

[0076] 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; and 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,

[0077] 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; and 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,

[0078] 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; and 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,

[0079] 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; and 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,

[0080] 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; and 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,

[0081] 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; and 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,

[0082] 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; and 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,

[0083] 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; and 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,

[0084] 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; and 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,

[0085] 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; and 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,

[0086] 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; and 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,

[0087] 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; and 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,

[0088] 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; and 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,

[0089] 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; and 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,

[0090] 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; and 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,

[0091] 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; and 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,

[0092] 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; and 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,

[0093] 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; and 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,

[0094] 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; and 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,

[0095] 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; and 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,

[0096] 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; and 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,

[0097] 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; and 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,

[0098] 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; and 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,

[0099] 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; and 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,

[0100] 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; and 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,

[0101] 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: 101, 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: 10, a heavy chain CDR2 of SEQ ID NO: 39, 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: 186, 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: 10, a heavy chain CDR2 of SEQ ID NO: 39, 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: 109,

[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; and 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

[0105] The heavy chain variable region includes the heavy chain CDR1 of SEQ ID NO:10, the heavy chain CDR2 of SEQ ID NO:185 and the heavy chain CDR3 of SEQ ID NO:63; and the light chain variable region includes the light chain CDR1 of SEQ ID NO:84, the light chain CDR2 of SEQ ID NO:187 and the light chain CDR3 of SEQ ID NO:110.

[0106] "Framework regions" (FRs) are the variable domain residues other than the CDR residues. Each variable domain generally has four FRs, designated FR1, FR2, FR3, and FR4.

[0107] The binding affinity of anti-ROR1 antibody to ROR1 is 10 -5 M to 10 -12 For example, the binding affinity of anti-ROR1 antibody to ROR1 is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 - 11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10M to 10 -11 M, 10 -5 M to 10 - 10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M.

[0108] The antibody or antigen-binding fragment thereof that binds to ROR1 may comprise a heavy chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 122 to SEQ ID NO: 152 and SEQ ID NO: 188 to SEQ ID NO: 193. The antibody or antigen-binding fragment thereof that binds to ROR1 may comprise a light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 153 to SEQ ID NO: 183 and SEQ ID NO: 194 to SEQ ID NO: 199.

[0109] In a specific embodiment according to the present invention, the antibody or antigen-binding fragment thereof may comprise:

[0110] The heavy chain variable region of SEQ ID NO: 122 and the light chain variable region of SEQ ID NO: 153;

[0111] The heavy chain variable region of SEQ ID NO: 123 and the light chain variable region of SEQ ID NO: 154;

[0112] The heavy chain variable region of SEQ ID NO: 124 and the light chain variable region of SEQ ID NO: 155;

[0113] The heavy chain variable region of SEQ ID NO: 125 and the light chain variable region of SEQ ID NO: 156;

[0114] The heavy chain variable region of SEQ ID NO: 126 and the light chain variable region of SEQ ID NO: 157;

[0115] The heavy chain variable region of SEQ ID NO: 127 and the light chain variable region of SEQ ID NO: 158;

[0116] The heavy chain variable region of SEQ ID NO: 128 and the light chain variable region of SEQ ID NO: 159;

[0117] The heavy chain variable region of SEQ ID NO: 129 and the light chain variable region of SEQ ID NO: 160;

[0118] The heavy chain variable region of SEQ ID NO: 130 and the light chain variable region of SEQ ID NO: 161;

[0119] The heavy chain variable region of SEQ ID NO: 131 and the light chain variable region of SEQ ID NO: 162;

[0120] The heavy chain variable region of SEQ ID NO: 132 and the light chain variable region of SEQ ID NO: 163;

[0121] The heavy chain variable region of SEQ ID NO: 133 and the light chain variable region of SEQ ID NO: 164;

[0122] The heavy chain variable region of SEQ ID NO: 134 and the light chain variable region of SEQ ID NO: 165;

[0123] The heavy chain variable region of SEQ ID NO: 135 and the light chain variable region of SEQ ID NO: 166;

[0124] The heavy chain variable region of SEQ ID NO: 136 and the light chain variable region of SEQ ID NO: 167;

[0125] The heavy chain variable region of SEQ ID NO: 137 and the light chain variable region of SEQ ID NO: 168;

[0126] The heavy chain variable region of SEQ ID NO: 138 and the light chain variable region of SEQ ID NO: 169;

[0127] The heavy chain variable region of SEQ ID NO: 139 and the light chain variable region of SEQ ID NO: 170;

[0128] The heavy chain variable region of SEQ ID NO: 140 and the light chain variable region of SEQ ID NO: 171;

[0129] The heavy chain variable region of SEQ ID NO: 141 and the light chain variable region of SEQ ID NO: 172;

[0130] The heavy chain variable region of SEQ ID NO: 142 and the light chain variable region of SEQ ID NO: 173;

[0131] The heavy chain variable region of SEQ ID NO: 143 and the light chain variable region of SEQ ID NO: 174;

[0132] The heavy chain variable region of SEQ ID NO: 144 and the light chain variable region of SEQ ID NO: 175;

[0133] The heavy chain variable region of SEQ ID NO: 145 and the light chain variable region of SEQ ID NO: 176;

[0134] The heavy chain variable region of SEQ ID NO: 146 and the light chain variable region of SEQ ID NO: 177;

[0135] The heavy chain variable region of SEQ ID NO: 147 and the light chain variable region of SEQ ID NO: 178;

[0136] The heavy chain variable region of SEQ ID NO: 148 and the light chain variable region of SEQ ID NO: 179;

[0137] The heavy chain variable region of SEQ ID NO: 149 and the light chain variable region of SEQ ID NO: 180;

[0138] The heavy chain variable region of SEQ ID NO: 150 and the light chain variable region of SEQ ID NO: 181;

[0139] The heavy chain variable region of SEQ ID NO: 151 and the light chain variable region of SEQ ID NO: 182;

[0140] The heavy chain variable region of SEQ ID NO: 152 and the light chain variable region of SEQ ID NO: 183;

[0141] The heavy chain variable region of SEQ ID NO: 188 and the light chain variable region of SEQ ID NO: 194;

[0142] The heavy chain variable region of SEQ ID NO: 189 and the light chain variable region of SEQ ID NO: 195;

[0143] The heavy chain variable region of SEQ ID NO: 190 and the light chain variable region of SEQ ID NO: 196;

[0144] The heavy chain variable region of SEQ ID NO: 191 and the light chain variable region of SEQ ID NO: 197;

[0145] The heavy chain variable region of SEQ ID NO: 192 and the light chain variable region of SEQ ID NO: 198; or

[0146] The heavy chain variable region of SEQ ID NO:193 and the light chain variable region of SEQ ID NO:199.

[0147] scFv

[0148] scFv is an antibody fragment, which consists of a single polypeptide chain comprising the VH and VL domains of an antibody. A polypeptide linker may be additionally included between the VH and VL domains to allow scFv to form an ideal antigen binding structure.

[0149] In one embodiment, in a single-chain Fv (scFv) comprising the VH and VL domains of an antibody, the VH and VL domains may be connected via a linker. A heavy chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 122 to SEQ ID NO: 152 and SEQ ID NO: 188 to SEQ ID NO: 193 may be connected via a linker to a light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 153 to SEQ ID NO: 183 and SEQ ID NO: 194 to SEQ ID NO: 199.

[0150] In a specific embodiment according to the present invention, the scFv may comprise:

[0151] The heavy chain variable region of SEQ ID NO: 122 and the light chain variable region of SEQ ID NO: 153;

[0152] The heavy chain variable region of SEQ ID NO: 123 and the light chain variable region of SEQ ID NO: 154;

[0153] The heavy chain variable region of SEQ ID NO: 124 and the light chain variable region of SEQ ID NO: 155;

[0154] The heavy chain variable region of SEQ ID NO: 125 and the light chain variable region of SEQ ID NO: 156;

[0155] The heavy chain variable region of SEQ ID NO: 126 and the light chain variable region of SEQ ID NO: 157;

[0156] The heavy chain variable region of SEQ ID NO: 127 and the light chain variable region of SEQ ID NO: 158;

[0157] The heavy chain variable region of SEQ ID NO: 128 and the light chain variable region of SEQ ID NO: 159;

[0158] The heavy chain variable region of SEQ ID NO: 129 and the light chain variable region of SEQ ID NO: 160;

[0159] The heavy chain variable region of SEQ ID NO: 130 and the light chain variable region of SEQ ID NO: 161;

[0160] The heavy chain variable region of SEQ ID NO: 131 and the light chain variable region of SEQ ID NO: 162;

[0161] The heavy chain variable region of SEQ ID NO: 132 and the light chain variable region of SEQ ID NO: 163;

[0162] The heavy chain variable region of SEQ ID NO: 133 and the light chain variable region of SEQ ID NO: 164;

[0163] The heavy chain variable region of SEQ ID NO: 134 and the light chain variable region of SEQ ID NO: 165;

[0164] The heavy chain variable region of SEQ ID NO: 135 and the light chain variable region of SEQ ID NO: 166;

[0165] The heavy chain variable region of SEQ ID NO: 136 and the light chain variable region of SEQ ID NO: 167;

[0166] The heavy chain variable region of SEQ ID NO: 137 and the light chain variable region of SEQ ID NO: 168;

[0167] The heavy chain variable region of SEQ ID NO: 138 and the light chain variable region of SEQ ID NO: 169;

[0168] The heavy chain variable region of SEQ ID NO: 139 and the light chain variable region of SEQ ID NO: 170;

[0169] The heavy chain variable region of SEQ ID NO: 140 and the light chain variable region of SEQ ID NO: 171;

[0170] The heavy chain variable region of SEQ ID NO: 141 and the light chain variable region of SEQ ID NO: 172;

[0171] The heavy chain variable region of SEQ ID NO: 142 and the light chain variable region of SEQ ID NO: 173;

[0172] The heavy chain variable region of SEQ ID NO: 143 and the light chain variable region of SEQ ID NO: 174;

[0173] The heavy chain variable region of SEQ ID NO: 144 and the light chain variable region of SEQ ID NO: 175;

[0174] The heavy chain variable region of SEQ ID NO: 145 and the light chain variable region of SEQ ID NO: 176;

[0175] The heavy chain variable region of SEQ ID NO: 146 and the light chain variable region of SEQ ID NO: 177;

[0176] The heavy chain variable region of SEQ ID NO: 147 and the light chain variable region of SEQ ID NO: 178;

[0177] The heavy chain variable region of SEQ ID NO: 148 and the light chain variable region of SEQ ID NO: 179;

[0178] The heavy chain variable region of SEQ ID NO: 149 and the light chain variable region of SEQ ID NO: 180;

[0179] The heavy chain variable region of SEQ ID NO: 150 and the light chain variable region of SEQ ID NO: 181;

[0180] The heavy chain variable region of SEQ ID NO: 151 and the light chain variable region of SEQ ID NO: 182;

[0181] The heavy chain variable region of SEQ ID NO: 152 and the light chain variable region of SEQ ID NO: 183;

[0182] The heavy chain variable region of SEQ ID NO: 188 and the light chain variable region of SEQ ID NO: 194;

[0183] The heavy chain variable region of SEQ ID NO: 189 and the light chain variable region of SEQ ID NO: 195;

[0184] The heavy chain variable region of SEQ ID NO: 190 and the light chain variable region of SEQ ID NO: 196;

[0185] The heavy chain variable region of SEQ ID NO: 191 and the light chain variable region of SEQ ID NO: 197;

[0186] The heavy chain variable region of SEQ ID NO: 192 and the light chain variable region of SEQ ID NO: 198; or

[0187] The heavy chain variable region of SEQ ID NO:193 and the light chain variable region of SEQ ID NO:199.

[0188] The linker may be a peptide linker and may have a length of about 10-25 aa.Examples of linkers may include, but are not limited to, hydrophilic amino acids such as glycine and / or serine.

[0189] Specifically, the linker may comprise, for example, (GS) n 、(GGS) n 、(GSGGS) n or (GnS) m (wherein n and m are each 1 to 10), and the linker can be, for example, (G n S) m (wherein n and m are each 1 to 10.) Specifically, the linker may include GGGGS, such as GGGGSGGGGSGGGGS of SEQ ID NO: 200 repeated three times.

[0190] "Phage display" is a technique for displaying variant polypeptides as fusion proteins with at least part of an envelope protein on the surface of a bacteriophage (e.g., a filamentous phage particle). The usefulness of phage display is that it can rapidly and efficiently sort through large libraries of random protein variants for sequences that bind to a target antigen with high affinity. Displaying peptide and protein libraries on phage has been used to screen millions of polypeptides to identify polypeptides with specific binding properties.

[0191] Phage display technology provides a powerful tool for producing and screening novel proteins combined with specific ligands (e.g., antigens). By using phage display technology, a large library of protein variants can be constructed, and sequences combined with target antigens with high affinity can be quickly sorted. The nucleic acid encoding variant polypeptides merges with the nucleic acid sequence encoding viral envelope proteins such as gene III protein or gene VIII protein. Monovalent phage display systems have been developed, in which the nucleic acid sequence encoding protein or polypeptide merges with the nucleic acid sequence encoding a part of gene III protein. In monovalent phage display systems, gene fusion is expressed at a low level, and wild-type gene III protein is also expressed, thereby maintaining particle infectivity.

[0192] Demonstration of peptide expression on the surface of filamentous phage and expression of functional antibody fragments in the periplasm of Escherichia coli is important for developing antibody phage display libraries. Libraries of antibodies or antigen-binding polypeptides have been constructed in a variety of ways, such as by changing a single gene via insertion of a random DNA sequence or by cloning a family of related genes. The library can be screened to express antibodies or antigen-binding proteins with desired properties.

[0193] Phage display technology has several advantages over conventional hybridoma and recombinant methods in producing antibodies with desired characteristics. This technology allows the generation of large antibody libraries with different sequences in a short period of time without the use of animals. The production of hybridoma or humanized antibodies may take several months of production time. In addition, since no immunization is required, phage antibody libraries can even be used to produce antibodies against toxic or low immunogenic antigens. Phage antibody libraries can also be used to generate and identify new therapeutic antibodies.

[0194] Techniques for generating human antibodies using phage display libraries from immune or non-immune humans, germline sequences, or naive B cell Ig repertoires can be used. Naive or non-immune antigen binding libraries can be constructed using various lymphoid tissues.

[0195] The technology that can identify and isolate high-affinity antibodies from phage display libraries is very important for isolating new therapeutic antibodies. The isolation of high-affinity antibodies from the library can depend on the size of the library, the production efficiency in bacterial cells, and the diversity of the library. Due to the presence of stop codons and improper folding of antibodies or antigen-binding proteins, the production efficiency is low and the size of the library is reduced. If the antibody or antigen-binding domain is not properly folded, expression in bacterial cells may be inhibited. Expression can be improved by alternating mutations of residues on the variable / constant interface surface or selected CDR residues. When constructing an antibody phage library in bacterial cells, the sequence of the framework region is an element that provides correct folding.

[0196] In the isolation of high affinity antibodies, it is very important to construct different libraries of antibodies or antigen binding proteins. The CDR3 region is often found to be involved in antigen binding. Since the CDR3 region on the heavy chain varies greatly in size, sequence and structural morphology, various libraries can be constructed using the same method.

[0197] In addition, diversity can be generated by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position. The use of all 20 amino acids can lead to the generation of variant antibody sequences with high diversity and increase the chance of identifying new antibodies.

[0198] The antibodies or antibody fragments according to the present invention may include not only the sequence of the anti-ROR1 antibody of the present invention described herein, but also its biological equivalents to the extent that it can specifically recognize ROR1. For example, the amino acid sequence of the antibody may be additionally modified 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. Amino acid variation is based on the relative similarity of amino acid side chain substituents in terms of, for example, hydrophobicity, hydrophilicity, charge, size, etc. From the analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues, alanine, glycine and serine are similar in size, and phenylalanine, tryptophan and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine and histidine can be regarded as biological functional equivalents, alanine, glycine and serine can be regarded as biological functional equivalents, and phenylalanine, tryptophan and tyrosine can be regarded as biological functional equivalents.

[0199] In view of the above-mentioned variation with equivalent biological activity, the antibody of the present invention or the nucleic acid molecule encoding it should be understood to include a sequence that shows basic identity with the sequence listed in the sequence number. When the sequence of the present invention is compared with any other sequence to correspond to each other as much as possible, and when the compared sequence is analyzed using an algorithm commonly used in the art, "basic identity" means that the sequence shows at least 90% homology, most preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. Alignment methods for sequence comparison are known in the art. NCBI Basic Local Alignment Search Tool (BLAST) can be accessed by NCBI and other places, and can be used in combination with sequencing programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. BLAST is available at www.ncbi.nlm.nih.gov / BLAST / . Methods for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0200] Based on this, antibody or its Fab according to the present invention can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the specific sequence listed herein or all sequences.This homology can use methods known in the art to carry out sequence comparison and / or comparison to determine.For example, the percentage sequence homology of nucleic acid or protein according to the present invention can be determined using sequence comparison algorithm (i.e. BLAST or BLAST 2.0), manual comparison or visual estimation.

[0201] In another aspect, the present invention relates to nucleic acids encoding the antibodies or antigen-binding fragments thereof. Antibodies or antigen-binding fragments thereof can be recombinantly produced by isolating nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present invention.

[0202] The meaning of "nucleic acid" broadly covers DNA (gDNA and cDNA) and RNA molecules as nucleotides of the basic building blocks of nucleic acids, which include not only natural nucleotides but also analogs with modified sugar or base regions. The sequence of the nucleic acid encoding the heavy chain variable region and light chain variable region of the present invention can be modified. Such modifications include the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0203] DNA encoding the antibody can be easily isolated or synthesized using conventional molecular biological techniques (e.g., using oligonucleotide probes that specifically bind to the DNA encoding the heavy and light chains of the antibody). The nucleic acid is isolated and inserted into a replicable vector for further cloning (DNA amplification) or further expression. Based on this, on the other hand, the present invention relates to a recombinant expression vector comprising the nucleic acid.

[0204] As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, including plasmid vectors, cosmid vectors, viral vectors, such as phage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. The components of the vector generally include, but are not limited to, one or more selected from the following: a signal sequence, an origin of replication, at least one antibiotic resistance marker gene, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the antibody is operably linked to the promoter and the transcription termination sequence.

[0205] "Operably linked" means a functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence or a transcriptional regulator binding site array) and a different nucleic acid sequence, so that the control sequence plays a role in controlling the transcription and / or translation of the different nucleic acid sequence.

[0206] When prokaryotic cells are used as hosts, a strong promoter capable of initiating 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 or T7 promoter), a ribosome binding site for initiating translation, and a transcription / translation termination sequence are generally included. In addition, for example, when a eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter, a β-actin promoter, a human hemoglobin promoter, or a human creatine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5k promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter) can be used, and a polyadenylation sequence is usually used as a transcription termination sequence.

[0207] In some cases, the vector can be fused to another sequence to facilitate purification of the antibody expressed therefrom. Examples of sequences fused thereto include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (six histidine; Qiagen, USA).

[0208] The vector includes an antibiotic resistance gene commonly used in the art as a selection marker, for example, a gene conferring resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin or tetracycline.

[0209] In another aspect, the present invention relates to a host cell transfected with a recombinant expression vector. The host cell used to produce the antibody of the present invention can be, but is not limited to, a prokaryotic cell, a yeast cell, and a higher eukaryotic cell.

[0210] Prokaryotic host cells such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, as strains belonging to the genus Bacillus, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used.

[0211] Here, the interest in animal cells is the greatest, 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, and HT1080.

[0212] In another aspect, the present invention relates to a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to ROR1, comprising producing the antibody by culturing host cells; and isolating and purifying the produced antibody.

[0213] Host cells can be cultured in a variety of culture media. Any commercially available culture media can be used as culture media without limitation. All other necessary supplements known to those skilled in the art may be included and the concentrations are appropriate. Culture conditions, such as temperature, pH, etc., have been used with the host cells selected for expression, which will be apparent to those skilled in the art.

[0214] To recover the antibody or antigen-binding fragment thereof, impurities can be removed by, for example, centrifugation or ultrafiltration, and the resulting product can be purified using, for example, affinity chromatography, etc. Other additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc., can be used.

[0215] Bispecific or multispecific antibodies

[0216] In yet another aspect, the present invention relates to a bispecific or multispecific antibody comprising the antibody or antigen-binding fragment thereof.

[0217] A bispecific antibody refers to an antibody that has the ability to bind to or antagonize one or more targets and is configured so that antibodies having the ability to bind to or antagonize two different targets are combined, or refers to an antibody in which an antibody having the ability to bind to one target is combined with a substance having the ability to antagonize another target.

[0218] Multispecific antibodies are antibodies that have binding specificity for at least three different antigens. Multispecific antibodies may include trispecific antibodies or higher specific antibodies, such as trispecific antibodies, tetraspecific antibodies, or antibodies targeting more targets.

[0219] Antibodies belonging to bispecific antibodies or multispecific antibodies can be divided into scFv-based antibodies, Fab-based antibodies, and IgG-based antibodies. Bispecific or multispecific antibodies can inhibit or amplify two or more signals at the same time, and therefore can be more effective than inhibiting / amplifying a single signal. Compared with the case of treating the signals with respective signal inhibitors, low-dose administration is possible, and two or more signals can be inhibited / amplified at the same time and space.

[0220] Methods for producing bispecific or multispecific antibodies are well known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy chain / light chain pairs under conditions where the two or more heavy chains have different specificities.

[0221] For scFv-based bispecific or multispecific antibodies, diantibodies can be produced by combining the VL and VH of different scFvs to produce a heterodimeric hybrid scFv, or by connecting different scFvs to each other to produce a tandem scFv. Heterodimeric miniantibodies can also be produced by expressing the CH1 and CL of Fab at the end of each scFv. Miniantibodies in the form of heterodimeric scFvs can be produced by replacing certain amino acids in the CH3 domain, which is the homodimeric domain of Fc, to change it into a heterodimeric structure of a "knob-in-hole" form, and expressing the altered CH3 domain at different ends of each scFv.

[0222] Fab-based bispecific antibodies or multispecific antibodies can be produced in the form of heterodimeric Fab by binding Fab' fragments against specific antigens to each other using disulfide bonds or mediators, and can be produced in the form of having two antigen valencies by expressing scFvs against different antigens at the end of the heavy chain or light chain of a specific Fab, or in the form of homodimers with four antigen valencies by providing a hinge region between Fab and scFv. In addition, it is also possible to provide a dual-targeting bibody with three antigen valencies by fusing scFvs against different antigens to the light chain and heavy chain ends of Fab, and to provide a three-targeting bibody with three antigen valencies by fusing different scFvs to the light chain and heavy chain ends of Fab.

[0223] For bispecific or multispecific antibodies based on IgG, Trion Pharma has disclosed a method for producing bispecific antibodies, i.e., hybridizing mouse and rat hybridomas again to form hybridized hybridomas, so-called quadromas. In addition, bispecific antibodies can be produced in a heterodimeric form, i.e., a so-called "hole and knob" form, by changing some amino acids of the CH3 homodimer domain of Fc for different heavy chains while sharing the light chain portion. In addition to heterodimeric bispecific antibodies, homodimers (scFv) 4-IgG can also be produced by fusing two different scFvs to the constant domains of the IgG light and heavy chains instead of the variable domains and expressing them therein. In addition, ImClone has reported the production of a bispecific antibody based on IMC-1C11. IMC-1C11 is a chimeric monoclonal antibody against VEGFR-2 that only fuses the univariate domain of mouse platelet-derived growth factor receptor-α to the amino terminus of the light chain of the antibody. In addition, the so-called "dock and lock (DNL) approach" using the dimerization and docking domain (DDD) of the protein kinase A (PKA) R subunit and the anchoring domain of PKA can generate antibodies with various antigenic valencies against CD20.

[0224] A variety of recombinant antibody forms have been developed, including at least bivalent, trivalent or tetravalent bispecific or multispecific antibodies. For example, 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 disclose at least at least bivalent, trivalent or tetravalent antibodies. At least bivalent, trivalent or tetravalent antibodies represent that there are two or more binding domains, three or more binding domains or four or more binding domains in the antibody molecule, respectively.

[0225] In a specific embodiment, the bispecific antibody or multispecific antibody according to the present invention may comprise an anti-ROR1 antibody or antigen-binding fragment in the form of an IgG complete antibody or a fragment thereof, for example, a single chain Fv, V H Domain and / or V L domain, Fab or (Fab)2.

[0226] In addition, antibodies that bind to targets different from antibodies targeting ROR1, for example, antibodies targeting at least one 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, can be included in the form of IgG complete antibodies or fragments thereof, for example, single chain Fv, V H Domain and / or V L domain, Fab or (Fab)2.

[0227] By means of the bispecific antibody or multispecific antibody according to the present invention, additional binding specificities induced or mediated by targets other than ROR1 can be obtained.

[0228] For example, the bispecific antibody according to the present invention can simultaneously target ROR1 and at least one 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.

[0229] For example, the multispecific antibody according to the present invention can simultaneously target ROR1 and two 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.

[0230] Immune cell engagement with bispecific or multispecific antibodies

[0231] In another aspect, the invention relates to an immune cell engaging bispecific or multispecific antibody comprising an scFv of an antibody and an scFv consisting of a second binding domain comprising at least one scFv of an antibody that binds to an immune cell activating antigen.

[0232] Immune cell binding to bispecific or multispecific antibodies transiently induces a cytolytic synapse between cytotoxic T cells and cancer target cells, thereby releasing toxins.

[0233] In one embodiment, the immune cell may be 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.

[0234] In one embodiment, the immune cell activation antigen can be selected from the following, and the antibody binding thereto can serve as an immune cell engager:

[0235] T cell activation antigens such as CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226;

[0236] NK cell activating antigens such as 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;

[0237] B cell activation antigens such as OX40, CD40, or CD70;

[0238] Macrophage activating antigens such as CD2 agonists, CD40, CD70, TCR (Toll-like receptor) agonists, CD47, STING, or OX40L; and

[0239] Dendritic cell activating antigens such as CD2 agonists, OX40, OX40L, 41BB agonists, TCR agonists, CD47 agonists or STING agonists.

[0240] Immune cell binding agents are described in detail in U.S. Patent Application Publication No. 2017 / 0368169, which is incorporated herein by reference.

[0241] Specifically, the immune cell engaging bispecific antibody or multispecific antibody may comprise a tandem scFv and may bind to the following antigens and surface antigens on cancer cells, wherein the surface antigen on cancer cells is ROR1 targeted by the antibody according to the present invention:

[0242] CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226;

[0243] 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;

[0244] OX40, CD40, or CD70;

[0245] CD2 agonist, CD40, CD70, TCR (toll-like receptor) agonist, CD47, STING or OX40L; or

[0246] CD2 agonist, OX40, OX40L, 41BB agonist, TCR agonist, CD47 agonist or STING agonist.

[0247] Immune cell engaging bispecific antibodies or multispecific antibodies can, for example, include structures 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).

[0248] For example, the scFv may include a heavy chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 122 to SEQ ID NO: 152 and SEQ ID NO: 188 to SEQ ID NO: 193 and a light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 153 to SEQ ID NO: 183 and SEQ ID NO: 194 to SEQ ID NO: 199, and the heavy chain variable region and the light chain variable region may be connected via a linker.

[0249] The linker may be a peptide linker and may have a length of about 10-25 aa. Examples of linkers may include hydrophilic amino acids, such as glycine and / or serine.

[0250] The linker may include, for example, (GS)n, (GGS)n, (GSGGS)n or (GnS)m (wherein n and m are 1 to 10, respectively), but the linker may be, for example, (G n S) m (wherein n and m are 1 to 10, respectively). Specifically, the linker may include GGGGS, such as GGGGSGGGGSGGGGS of SEQ ID NO: 200 repeated three times.

[0251] Examples of immune cell-engaging bispecific or multispecific antibodies may include: rantumomab (Amgen) that binds to CD3 and CD19; solitomab (Amgen) that binds to CD3 and EpCAM; MEDI 565 (MedImmune, Amgen) that binds to CD3 and CEA; and BAY2010112 (Bayer, Amgen) that binds to CD3 and PSMA. Examples of DARTs include MGD006 (Macrogenics) that binds to CD3 and CD123 and MGD007 (Macrogenics) that binds to CD3 and gpA33. Examples of TandAbs may include AFM11 (Affimed Therapeutics) that binds to CD3 and CD19 and AFM13 (Affimed Therapeutics) that binds to CD30 and CD16A.

[0252] Antibody-drug conjugates (ADCs)

[0253] In other aspects, the invention relates to an antibody-drug conjugate (ADC), wherein the antibody or antigen-binding fragment thereof is conjugated to a drug.

[0254] Anti-cancer-drug conjugates require that the anti-cancer drug be stably attached to the antibody until the anti-cancer drug is delivered to the target cancer cells. The drug delivered to the target should be released from the antibody, thereby inducing the death of the target cells. To this end, the drug should be stably bound to the antibody and should have sufficient cytotoxicity to induce the death of the target cells when released from the target cells.

[0255] In one embodiment, the antibody can be conjugated to the drug via a linker. The linker is a site that connects the anti-ROR1 antibody to the drug and allows the drug to be released from the antibody in a cleavable form under intracellular conditions (i.e., in the intracellular environment). In order to reflect the long half-life of the antibody, the linker should be stable during the systemic circulation of the antibody, and the combination of the linker and the drug should not affect the stability and pharmacokinetics of the antibody.

[0256] For example, the linker can include a cleavable linker or a non-cleavable linker. For a cleavable linker, such as a peptide linker, it can be cleaved by intracellular peptidases or proteases such as lysosomal or endosomal proteases, while for a non-cleavable linker, such as a thioether linker, the antibody can be non-selectively degraded by intracellular hydrolysis, and then the drug can be released.

[0257] In one embodiment, the cleavable linker may include a peptide linker. The peptide linker has a length of at least two amino acids. The cleavable linker may include a dipeptide of Val-Cit, Val-Ala, Val-Cit or Phe-Leu or Gly-Phe-Leu-Gly. Examples of linkers are described in detail in International Patent Application Publication No. WO2004 / 010957, which may be incorporated herein by reference.

[0258] For antibody-drug conjugates, the antibody region of the ADC binds to the antigen of the target cancer cell to form an ADC-antigen complex, which is then internalized into the cancer cell through the endosomal-lysosomal pathway. In this case, the intracellular release of the cytotoxic drug is controlled by the internal environment of the endosome / lysosome.

[0259] In one embodiment, the cleavable linker is pH sensitive and can be susceptible to hydrolysis at a predetermined pH. In general, a pH sensitive linker means that it can be hydrolyzed under acidic conditions. Examples of acid-labile linkers that can be hydrolyzed in lysosomes include hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.

[0260] In another embodiment, the linker can also be cleaved under reducing conditions and can include, for example, a disulfide linker. Various disulfide bonds can be formed using the following: SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene). This disulfide linker can be degraded by disulfide exchange with the thiol of intracellular glutathione.

[0261] Drugs and / or drug-linkers can be randomly conjugated through the lysine of the antibody, or can be conjugated through the cysteine ​​exposed during the reduction of the disulfide bond chain. In some cases, the linker-drug can be combined by the cysteine ​​present in the genetic engineering tag (e.g., peptide or protein). The genetic engineering tag (e.g., peptide or protein) can include an amino acid motif that can be recognized by, for example, isoprenoid transferase. The peptide or protein has a deletion at the carboxyl terminal of the peptide or protein, or the covalent addition of a spacer unit at the carboxyl (C) terminal of the peptide or protein.

[0262] The peptide or protein can be covalently linked to the amino acid motif directly, or can be covalently linked to the amino acid motif through the use of a spacer unit. The amino acid spacer unit consists of 1 to 20 amino acids, and the glycine unit is particularly preferred.

[0263] The isoprenoid transferase can be, for example, farnesyltransferase (FTase, farnesyl protein transferase) or geranylgeranyltransferase (GGTase), and FTase and GGTase I can recognize the CAAX motif in the above formula 1, while GGTase II can recognize XXCC, XCXC or CXX motif (wherein C is cysteine, A is an aliphatic amino acid, and X is an amino acid that determines the substrate specificity of the isoprenoid transferase).

[0264] In another embodiment, the linker may include a β-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase (β-glucuronidase), which is abundantly present in lysosomes or overexpressed in some tumor cells. Unlike peptide linkers, this linker is highly hydrophilic and can improve the solubility of the antibody-drug conjugate when combined with a highly hydrophobic drug.

[0265] In this regard, the β-glucuronic acid linkers disclosed in International Patent Application Publication No. WO 2015 / 182984, for example, β-glucuronic acid linkers containing a thiol group, may be used, the above document being incorporated herein by reference.

[0266] In some cases, the linker can be, for example, a non-cleavable linker, and the drug is released by only one step of antibody hydrolysis in the cell to produce, for example, an amino acid-linker-drug complex. This type of linker can be a thioether group or a maleimidocaproyl group and can remain stable in the blood.

[0267] According to an embodiment of the present invention, the linker-drug may be randomly bound through cysteine ​​exposed when the disulfide bond chains of the antibody are reduced, or the linker-drug may be bound by introducing an antibody terminal binding peptide having the sequence GGGGGGGCVIM.

[0268] Drugs (including D in chemical formula (1)) are agents that exhibit pharmacological effects and can bind to antibodies, and specific examples thereof may include chemotherapeutic agents, toxins, microRNA (miRNA), siRNA, shRNA, and radioisotopes. Chemotherapeutic agents may be, for example, cytotoxic agents or immunosuppressants. Specifically, chemotherapeutic agents that can be used as microtubule inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators may be included. In addition, immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, anthelmintics, or combinations thereof may also be included.

[0269] For example, the drug may be, but is not limited to, at least one selected from the group consisting of: mytansinoid, auristatin (including MMAE and MMAF), aminopterin, actinomycin, bleomycin, talisomycin, camptothecin, N8-acetyl spermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazine, esperamycin, etoposide, 6-mercaptopurine, dolastatin, trichothemene, hecene), calicheamicin, taxol, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cyclophosphamide, etoposide, 5- Fluorouracil, CNU (bischloroethyl nitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, decarbazine, etoposide, teniposide de), topotecan, 9-aminocamptothecin, crisnatol, mitomycin C, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (arabinoside),C), cytosinearabinoside, fludarabine, tamoxifen, raloxifene, megestrol acetate, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A A), interferon-α, interferon-γ, tumor necrosis factor, gemcitabine, Velcade, Revlimid, thalidomide, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, thapsigargin, nucleases, and toxins of bacterial, animal, or plant origin.

[0270] In some cases, the drug may include at least one nucleophilic group selected from the group consisting of amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide, each of which is capable of reacting with electrophilic groups on linkers and linker reagents to form a covalent bond.

[0271] In a specific embodiment according to the present invention, an antibody or antigen-binding fragment thereof according to the present invention is linked to a drug (such as auristatin (MMAE)) via a MC-vc-PAB linker to produce an ADC. It has been demonstrated that such an ADC exhibits desirable cytotoxicity.

[0272] Chimeric Antigen Receptor (CAR)

[0273] In another aspect, the present invention relates to a chimeric antigen receptor (CAR), comprising an extracellular domain comprising an antigen binding site, a transmembrane domain and an intracellular signal transduction domain, wherein the antigen binding site of the extracellular domain is the scFv of the antibody.

[0274] Chimeric antigen receptor (CAR) is a synthetic construct designed to induce an immune response to a target antigen and cells expressing the antigen. CAR comprises an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain. Cancer cells can be killed by introducing genes encoding receptors that recognize cancer cell surface antigens into immune cells, and the cancer cell surface antigens are specifically expressed on the surface of cancer cells. By including immune cells that bind to receptors specifically expressed in cancer cells, an immune response can be induced by targeting only cancer cells. CAR comprises an scFv of an anti-ROR1 antibody according to the present invention as an antigen recognition site in the extracellular domain.

[0275] The first generation of CAR includes an extracellular domain (including an antigen recognition site specifically expressed in cancer cells), a transmembrane domain, and an intracellular signal transduction domain, wherein only CD3ζ is used as a signal transduction domain, but there is a problem that the therapeutic effect on cancer is not significant and the duration is short. This first generation of CAR is described in detail in U.S. Patent No. 6,319,494, which is incorporated herein by reference.

[0276] In order to improve the responsiveness to immune cells, the second generation CAR is made by binding the costimulatory domain (CD28 or CD137 / 4-1BB) to CD3ζ. Compared with the first generation CAR, the second generation CAR greatly increases the number of CAR-containing immune cells remaining in the body. The second generation CAR uses a costimulatory domain, while the third generation CAR uses two or more costimulatory domains. In order to achieve the expansion and continuation of immune cells including CAR in vivo, the costimulatory domain can be combined with 4-1BB, CD28 or OX40. The second generation CAR is specifically described in U.S. Patent Nos. 7,741,465, 7,446,190 or 9,212,229, and the third generation CAR is specifically described in U.S. Patent No. 8,822,647, which are all incorporated herein by reference.

[0277] The fourth generation CAR includes additional genes encoding cytokines such as IL-12 or IL-15, allowing additional expression of immune proteins based on CAR cytokines, and the fifth generation CAR further includes an interleukin receptor chain, such as IL-2Rβ, to enhance immune cells. The fourth generation CAR is specifically described in U.S. Patent No. 10,316,102, and the fifth generation CAR is specifically described in U.S. Patent No. 10,336,810, which are all incorporated herein by reference.

[0278] In one embodiment, the antigen binding site of the extracellular domain is an scFv of an antibody. In an scFv comprising the VH and VL domains of an antibody, the VH and VL domains may be connected by a linker. A heavy chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 122 to SEQ ID NO: 152 and SEQ ID NO: 188 to SEQ ID NO: 193 may be connected to a light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 153 to SEQ ID NO: 183 and SEQ ID NO: 194 to SEQ ID NO: 199 via a linker.

[0279] The linker may be a peptide linker and may have a length of about 10-25 aa. Examples of linkers may include hydrophilic amino acids, such as glycine and / or serine.

[0280] The linker may comprise, for example, (GS) n 、(GGS) n 、(GSGGS) n or (G n S) m (wherein n and m are each 1 to 10), and the linker can be, for example, (G n S) m (wherein n and m are each 1 to 10.) Specifically, the linker may include GGGGS, such as GGGGSGGGGSGGGGS of SEQ ID NO: 200 repeated three times.

[0281] The transmembrane domain can be derived from a natural or synthetic source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. The transmembrane domain can include α, β or ζ chains of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS. When the transmembrane domain is synthesized, it can include hydrophobic residues such as leucine and valine, or can include phenylalanine, tryptophan and valine peptides at each end. By a short oligopeptide linker or polypeptide linker with a length of 2 to 10 amino acids, a key can be formed between the transmembrane domain and the cytoplasmic signaling domain of CAR. Glycine-serine peptides can be used as linkers.

[0282] The signal transduction domain can induce the activation of the normal effector function of the immune cell where the CAR is located. For example, the signal transduction domain can induce cytolytic activation or auxiliary (helper) activation by secreting cytokines. The signal transduction domain can include a truncated fragment of the intracellular signal transduction domain sufficient to transduce the effector function signal.

[0283] The signal transduction domain may comprise the cytoplasmic portion of a T cell receptor (TCR) and a co-receptor that interact upon antigen receptor engagement to initiate signal transduction.

[0284] The signal generated by a single TCR is known to be generally insufficient to fully activate T cells, and costimulatory signals may also be required. Therefore, T cell activation may involve starting antigen-dependent primary activation via TCR, and play a role in an antigen-dependent manner to provide secondary or costimulatory signals. The initial cytoplasm signal transduction sequence is to stimulate or regulate the initial activation of the TCR complex in an inhibitory manner. The initial cytoplasm signal transduction sequence that works in a stimulating manner may contain a signal transduction motif that is referred to as an immunoreceptor tyrosine-based activation motif or ITAM. The example of the ITAM containing the initial cytoplasm signal transduction sequence can include TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b and CD66d.

[0285] In some cases, the cytoplasmic domain of CAR may include CD3ζ chain portion and costimulatory signal region.Costimulatory signal transduction region may be a part of the CAR of the intracellular domain comprising costimulatory molecules. Its example may include ligands specifically bound 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, etc. The cytoplasmic signal transduction sequence in the cytoplasmic signal transduction part of CAR can be connected via a peptide linker (e.g., glycine-serine) comprising 2 to 10 amino acids.

[0286] In yet another aspect, the present invention relates to an immune cell into which a chimeric antigen receptor (CAR) is introduced.

[0287] The immune cell is capable of inducing immunity to obtain the desired cancer treatment effect, and for example, can be 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, but is not limited thereto.

[0288] Antibodies other than the antibodies described above may be antibodies or antigen-binding fragments thereof targeting at least one selected from the group consisting of, for example, 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.

[0289] Therapeutic agent composition

[0290] In another aspect, the present invention relates to a composition for preventing or treating cancer, which contains the antibody or its antigen-binding fragment, a bispecific or multispecific antibody comprising the antibody or its antigen-binding fragment, an antibody-drug conjugate comprising the antibody or its antigen-binding fragment, a chimeric antigen receptor comprising the antibody or its antigen-binding fragment, or an immune cell comprising the chimeric antigen receptor.

[0291] For example, the present invention may provide a pharmaceutical composition for preventing or treating cancer, comprising (a) a pharmaceutically effective amount of an anti-ROR1 antibody or an antigen-binding fragment thereof according to the present invention, a bispecific or multispecific antibody comprising the antibody or its antigen-binding fragment, an antibody-drug conjugate comprising the antibody or its antigen-binding fragment, a chimeric antigen receptor comprising the antibody or its antigen-binding fragment, or an immune cell comprising the chimeric antigen receptor; and (b) a pharmaceutically acceptable carrier. In addition, the present invention may provide a method for preventing or treating cancer, comprising administering to a cancer patient an anti-ROR1 antibody or an antigen-binding fragment thereof according to the present invention, a bispecific or multispecific antibody comprising the antibody or its antigen-binding fragment, an antibody-drug conjugate comprising the antibody or its antigen-binding fragment, a chimeric antigen receptor comprising the antibody or its antigen-binding fragment, or an immune cell comprising a chimeric antigen receptor.

[0292] The term "prevention" refers to any action to inhibit cancer growth or slow cancer progression by administering a composition according to the present invention, and the term "treatment" refers to inhibiting cancer development, reducing a tumor, or removing cancer.

[0293] Examples of such cancers include Hodgkin lymphoma, non-Hodgkin 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 lymphoma, lymphoplasmacytic lymphoma, and hairy cell leukemia), acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, and acute lymphocytic leukemia.

[0294] Examples of cancer include ovarian cancer, colorectal cancer, stomach cancer, testicular cancer, anal cancer, uterine cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, Kaposi's sarcoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, cervical cancer, brain stem glioma, pituitary adenocarcinoma, epithelial cancer, cervical squamous cell carcinoma, fallopian tube cancer, endometrial cancer, vaginal cancer, soft tissue sarcoma, urethral cancer, vulvar cancer, penile cancer, bladder cancer, kidney cancer or ureter cancer, renal pelvis cancer, spinal cord tumors, central nervous system (CNS) tumors, primary CNS lymphomas, angiogenic tumors, metastatic lesions thereof, or combinations thereof.

[0295] The cancer can be, for example, glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, or cervical cancer.

[0296] The pharmaceutically acceptable carrier included in the composition according to the present invention comprises those commonly used in preparation, and its example includes but is not limited to lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate and mineral oil.In addition to the above-mentioned components, the composition of the present invention can also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives etc.

[0297] The pharmaceutical composition of the present invention may be administered orally or parenterally, and parenteral administration may include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration.

[0298] When administered orally, since proteins or peptides are digestible, oral compositions must be formulated to coat the active agent to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device that allows the active material to be transported to the target cells.

[0299] The suitable dosage of the composition according to the present invention may vary due to factors such as formulation method, mode of administration, patient age, weight, sex, morbidity, food, administration time, route of administration, excretion rate and reaction sensitivity, and an experienced doctor can easily determine and prescribe the effective dose for the desired treatment or prevention. For example, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to prevent or treat cancer or autoimmune diseases.

[0300] The pharmaceutical composition of the present invention can be prepared in unit dosage form, or it can be prepared in a multi-dose container by using a pharmaceutically acceptable carrier and / or excipient according to methods that are easily mastered by those skilled in the art. Here, the preparation can be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or can be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and can additionally include a dispersant or stabilizer.

[0301] Treatment

[0302] In a further aspect, the present invention relates to a composition for treating cancer, which composition comprises an antibody or an antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or the antigen-binding fragment thereof, the antibody-drug conjugate, the chimeric antigen receptor or an immune cell comprising the chimeric antigen receptor.

[0303] In addition, the present invention also relates to a method for treating cancer, comprising administering an antibody or an antigen-binding fragment thereof, a bispecific or multispecific antibody comprising the antibody or the antigen-binding fragment thereof, the antibody-drug conjugate, the chimeric antigen receptor, or an immune cell comprising the chimeric antigen receptor.

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

[0305] Another aspect of the present invention relates to the use of the antibody or its antigen-binding fragment, the bispecific or multispecific antibody comprising the antibody or its antigen-binding fragment, the antibody-drug conjugate, the chimeric antigen receptor or the immune cell comprising the chimeric antigen receptor in the manufacture of a medicament for preventing or treating cancer.

[0306] Combination therapy

[0307] The present invention relates to a composition for combined therapy, which comprises immune cells and a drug other than an anti-ROR1 antibody.

[0308] In one embodiment, the drug other than an anti-ROR1 antibody may include a chemotherapeutic agent or an antibody other than an anti-ROR1 antibody.

[0309] The drug can be at least one selected from the group consisting of maytansine, auristatin (including MMAE and MMAF), aminopterin, actinomycin, bleomycin, talithromycin, camptothecin, N8-acetyl spermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazine, esperamicin, etoposide, 6-mercaptopurine, dolastatin, trichothecenes, calicheamicin, taxol, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine Bazine, procarbazine, topotecan, nitrogen mustard, cyclophosphamide, etoposide, 5-fluorouracil, CNU (bischloroethyl nitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, trioxalic acid, dacarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, clenatol, mitomycin C, trimetrexate, mycophenolic acid, thiazolidinone, ribavirin, EICAR (5-ethynyl-1-β-D-ribofuranosyl imidazole-4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (arabinoside) C), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol acetate, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, gemcitabine, Velcade, Revlimid, thalidomide, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, doxycycline, mitoxantrone, verapamil, thapsigargin, nucleases, and toxins derived from bacteria, animals, or plants.

[0310] In one embodiment, an antibody other than an anti-ROR1 antibody may be an antibody or an antigen-binding fragment thereof targeting at least one selected from the group consisting of: for example, 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.

[0311] The present invention relates to a composition for combined therapy, comprising the antibody or antigen-binding fragment thereof and at least one selected from the group consisting of:

[0312] (i) immune cells;

[0313] (ii) an immune cell comprising a chimeric antigen receptor (CAR) comprising, as an extracellular domain, a scFv fragment of an antibody other than an anti-ROR1 antibody; and

[0314] (iii) Immune checkpoint inhibitors.

[0315] Furthermore, the present invention relates to a composition for combination therapy, comprising an immune cell engaging bispecific or multispecific antibody and at least one selected from the group consisting of:

[0316] (i) immune cells;

[0317] (ii) an immune cell comprising a chimeric antigen receptor (CAR) comprising, as an extracellular domain, a scFv fragment of an antibody other than an anti-ROR1 antibody; and

[0318] (iii) Immune checkpoint inhibitors.

[0319] Immune cells are capable of inducing immunotherapy, for example, obtaining immunity for a desired cancer treatment effect, and may be selected from the group consisting of, for example, T cells, NK cells, cytokine-induced killer cells (CIK), activated cytotoxic T lymphocytes (CTL), macrophages, tumor infiltrating lymphocytes (TIL), and dendritic cells, but are not limited thereto.

[0320] Antibodies other than anti-ROR1 antibodies are antibodies targeting targets other than ROR1, and may be antibodies or antigen-binding fragments thereof that bind to, for example, 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 are not limited thereto.

[0321] An immune checkpoint inhibitor is a drug that can induce 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. An immune checkpoint inhibitor can be a drug that targets: for example, 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.

[0322] The first and second components to be co-administered may be administered simultaneously. Alternatively, the first and second components to be co-administered may be administered separately within a predetermined time interval. Among the components to be co-administered, the second component may be administered separately before or after the administration of the first component.

[0323] A better understanding of the present invention can be obtained through the following examples. These examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention, which is obvious to those of ordinary skill in the art.

[0324] Example 1: Preparation of phage library (scFv)

[0325] In order to recover the phage vectors having the genes of the synthetic scFv library of human origin constructed according to the conventional method in the form of phage, six sublibrary samples ((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. 10e0141045 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.) were cultured in 400 ml of culture medium (SB / ampicillin / 2% glucose) for 2 hours. 600 When the absorbance reached 0.5-0.7, the supernatant was removed by centrifugation at 5,000 g for 20 minutes, and then 10 12 pfu (plaque forming unit) of helper phage (VCSM13) was added to 400 ml of secondary culture medium (SB / penicillin) and cultured for another hour. Thereafter, kanamycin antibiotic (antibiotic gene introduced into helper phage) was added at a concentration of 70 μg / ml, and then shake-cultured at 30°C and 250 rpm for 16 hours to produce the phage library outside the host cell. Thereafter, PEG 8000 (polyethylene glycol 8000) and NaCl were added to the supernatant obtained by centrifugation of the culture, and stirred at 4°C for 2 hours to precipitate the recombinant phage. The phage library was recovered by centrifugation at 10,000g and 4°C for 30 minutes, PBS was added to suspend the precipitated pellet, centrifuged at 15,000g and 4°C for 30 minutes, and then PBS was added to the precipitated phage pellet. The concentration of the amplified sublibrary was calculated as the number of colonies generated by diluting the recovered phage, infecting TG1 cells with it, and culturing them on LB / ampicillin solid medium.

[0326] Example 2: Selection of anti-ROR1 specific antibodies (scFv) using biopanning

[0327] Panning was performed to select human antibodies that specifically bind to ROR1. The following biopanning was performed using ROR1-Fc protein, ROR1-His protein, and patient-derived cells. Human ROR1 Fc protein (R&D Systems, 9490-RO, recombinant human ROR1 Fc Chimera protein, CF) and human ROR1His protein (Sino Biological, 13968-H08H, ROR1 protein, human, recombinant (ECD, His tag)) were used as recombinant antigens, and LC-074T overexpressing ROR1 owned by Aimedbio was used as patient-derived cells.

[0328] Antigen immobilization biopanning: Coat 96-well plates with 5 to 10 μg / ml human ROR1 Fc protein and negative Fc protein at 4°C for 16 hours, then block with 3% skim milk. After clearing the plate, add the library to the plate coated with (approximately 2.0x10 13 pfu) negative Fc protein plate, and react at room temperature for 30 minutes. This step is to remove phages that bind to proteins other than human ROR1 protein from the antibody phage library to prevent non-specific binding to proteins other than ROR1 protein. Recover phages that are not bound to negative Fc proteins and allow them to bind to the 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 non-specific binding, use IgG elution buffer (Thermo Scientific, 21028, Pierce TM IgG elution buffer, pH 2.0) was used to recover phage antibodies specific for human ROR1. A total of three rounds were performed, and the results of antigen-immobilized biopanning are shown in Table 1 below.

[0329] Bead-based biopanning: Magnetic bead panning was repeated by attaching magnetic beads to biotinylated human ROR1 protein. Streptavidin-conjugated magnetic beads (Invitrogen, 11206D, Dynabeads TM M-280 streptavidin) and used a biotinylation kit (Abcam, ab201795, Biotin Link Kit (Fast, Type A) -Lightning- ) Human ROR1 protein was biotinylated. 100 μl of Dynabeads were washed with PBST (phosphate buffered saline-0.1% Tween 20) solution and PBS solution using a magnetic bead separator. TM M-280 streptavidin beads were added and reacted with 50-100 nM biotinylated human ROR1 protein for 30 minutes at room temperature. Afterwards, the library was collected to approximately 1.0x1013 pfu, and then blocked with 3% skim milk. The blocked library and pre-reacted biotinylated human ROR1-bead conjugate were mixed and reacted at room temperature for 2 hours. Thereafter, the library bound to human ROR1 beads was recovered with a magnetic bead separator and washed 7-12 times with PBST (phosphate buffered saline-0.1% Tween 20) solution and PBS solution, and then eluted with IgG elution buffer (Thermo Scientific, 21028, Pierce TM Phage antibodies bound to biotinylated human ROR1 beads were eluted with IgG elution buffer, pH 2.0. A total of three rounds were performed, and the results of bead-based biopanning are shown in Table 2 below.

[0330] Biopanning using patient-derived cells: Jurkat cells were treated with phage antibodies obtained from antigen-immobilized biopanning and bead-based biopanning and allowed to bind for 1 hour at 4°C, and then the supernatant that was not bound to patient-derived cells overexpressing ROR1 was recovered. This step is to remove phages that bind to cell membrane proteins other than ROR1 from the phage antibodies to prevent nonspecific binding to cells other than ROR1. LC-074T (1.5x10^6), which are ROR1 patient-derived cells, was treated with the recovered supernatant and allowed to bind for 1 hour at 4°C. Thereafter, in order to remove phages that were not bound to patient-derived cells, they were transferred to a 15 ml conical tube, and then centrifuged at 1,000g for 3 minutes to separate the cells, and then rinsed three to five times with 3 ml of cold PBS. Thereafter, IgG elution buffer (ThermoScientific, 21028, Pierce TMIgG elution buffer, pH 2.0) was placed on ice for 10 minutes to separate the phages on the cell surface from the cell surface. After centrifugation at 1,000g for 3 minutes, 50 μl of lysis buffer was added to the cells, and then placed on ice for 10 minutes to lyse the cells. The cells were centrifuged at 12,000rpm for 5 minutes to separate cell debris, and then the supernatant containing phage particles in the cells and the phages separated from the cell surface were added to the medium (SB) containing pre-grown TG1, and then cultured at 37°C and 120rpm for 1 hour to infect TG1 cells with phage particles. Thereafter, the cells were plated on LB / ampicillin solid medium, the remaining solution was centrifuged at 4,000rpm for 10 minutes, and the precipitated TG1 was plated on 15cm of LB / ampicillin solid medium, and then cultured. Then, 5ml of SB medium (50% glycerol) was added to recover the colonies and store (-80°C). Then, to repeat the panning round, 50 μl of the stored phage solution from the previous round was taken for phage particle amplification. After incubation, the phage particles recovered by adding helper phage were separated by PEG precipitation and used in the same manner for the next round of panning. Two rounds were performed, and the results of biopanning using patient-derived cells are shown in Table 3 below. Three rounds were performed using phage antibodies obtained from patient-derived cells as recombinant proteins, and the results are shown in Table 4 below. The results confirmed that as the rounds continued, the proportion of phage particles after panning increased compared to before panning.

[0331] [Table 1]

[0332] Round Input (cfu / ml) Output (cfu / ml) Recovery ratio (output / input) 1 2.33E+13 1.67E+06 7.17E-08 2 1.81E+13 7.67E+06 4.24E-07 3 1.73E+13 3.59E+07 1.08E-06

[0333] [Table 2]

[0334] Round Input (cfu / ml) Output (cfu / ml) Recovery ratio (output / input) 1 4.13E+12 1.65E+06 3.98E-07 2 8.70E+12 8.19E+05 9.41E-08 3 1.65E+12 6.67E+06 4.04E-06

[0335] [Table 3]

[0336] Round Input (cfu / ml) Output (cfu / ml) Recovery ratio (output / input) 1 5.36E+12 5.15E+06 9.61E-07 2 3.06E+12 3.73E+06 1.22E-06

[0337] [Table 4]

[0338] Round Input (cfu / ml) Output (cfu / ml) Recovery ratio (output / input) 1 1.15E+13 3.59E+05 3.12E-08 2 1.37E+13 5.26E+07 3.84E-06 3 1.32E+12 5.19E+07 3.93E-05

[0339] Example 3: Affinity ELISA screening and sequence analysis of anti-ROR1 specific antibodies (scFv)

[0340] We performed ScFv screening to screen monoclonal antibodies that specifically bind to ROR1 from the phages recovered in the last round of panning. Colonies were extracted from the last round of panning, inoculated into 96-well plates containing 200 μl of SB / ampicillin medium, and then cultured at 37°C for 2-3 hours. After that, to induce the expression of scFv-pIII protein, each well was treated with IPTG (isopropyl β-D-1-thiogalactopyranoside) at a final concentration of 1 mM and cultured overnight at 30°C. The culture plate was centrifuged at 3,000 rpm for 15 minutes to remove the supernatant, and then 40 μl of TES (50 mM Tris, 1 mM EDTA, 20% sucrose, pH 8.0) solution was added to each well to recover phage particles, and the cells were lysed at room temperature for 30 minutes. Then, the cells were treated with 60 μl of 0.2X TES solution and placed at 4°C for 2 hours for lysis, and then the plate was centrifuged at 3,000 rpm for 15 minutes to collect the supernatant.

[0341] The supernatant was added to each well of a 96-well plate coated with human ROR1, mouse ROR1, and negative Fc protein, and then bound for 2 hours at room temperature and washed four times with PBST and distilled water. HRP-conjugated anti-HA antibody capable of binding to the HA tag was used to bind for 1 hour at room temperature and then washed six times with PBST and distilled water. TM After color development with Ultra TMB-ELISA substrate solution), the color reaction was stopped with stop solution (Invitrogen, SS04, ELISA stop solution), and the absorbance was measured at OD 450nm. 30 antibody clones that bind to human ROR1 or mouse ROR1 were screened by ELISA method, and the CDR sequence of each antibody is shown in Table 5 below, and the amino acid sequences of the heavy chain and light chain variable regions are shown in Table 6 below.

[0342] [Table 5]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348] [Table 6]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356] Example 4: Analysis of the binding domain of anti-ROR1 specific antibody (scFv)

[0357] ELISA analysis was performed using scFvs of the above 30 selected antibody clones to analyze the domain. An expression vector capable of expressing a domain that binds to an antigen for binding domain analysis was constructed, followed by protein expression and purification. For production, residues corresponding to amino acids 1 to 542 or 165 to 395 of the ROR1 amino acid sequence indicated by UniProtID: Q01973 were used. A gene block encoding the extracellular domain of ROR1 was constructed. The 3' end of the gene was conjugated to a His tag. The vector was obtained by introducing the gene into a pcDNA3.3 vector. The protein was expressed and purified using transient transfection. After culturing for 5 days under 8% CO2, 37°C and 130rpm, the protein was purified from the cell culture supernatant. The culture fluid was passed through a column (GE Healthcare, 17-5438-01, MabSelect TM SuRe) to allow the expressed antibody to bind to the column. IgG elution buffer (Thermo Scientific, 21028, Pierce TM After elution with IgG elution buffer, pH 2.0, the cells were washed with AmiconUltra 30 kDa tubes (Merck Millipore, UFC903024, The eluted antibody fractions were concentrated by buffer exchange with PBS (pH 7.4) using a 50 μl Ultra-15 centrifugal filter device. The purified domains were quantified using absorbance at 280 nm and extinction coefficient.

[0358] 96-well plates were coated with 2 μg / ml of each of the following for 16 h at 4°C and then blocked with 3% skim milk: human ROR1-immunoglobulin domain (ACROBiosystems, RO1-H5221, human / cynomolgus / rhesus monkey ROR1 (39-151, Ig-like domain) protein, His tag), human ROR1-frizzled domain (ACROBiosystems, RO1-H5222, human / cynomolgus / rhesus monkey ROR1 (165-305, Frizzled domain ( domain) protein, His tag) and human ROR1-kringle domain (ACROBiosystems, RO1-H5223, human / cynomolgus monkey / macaque ROR1 (308-395, kringle domain) protein, His tag), human ROR1-immunoglobulin-curled domain and human ROR1-curled-kringle domain. Then, the plate was treated with each scFv and reacted for 2 hours, and then washed four times with PBST and distilled water. After binding for 1 hour at room temperature using an HRP-conjugated anti-HA antibody (Roche, 12013819001, Anti-HA-Peroxidase, High Affinity) that can bind to the HA tag, it was washed six times with PBST and distilled water. After adding TMB substrate solution (Thermo Scientific, 34029, 1-Step TM After color development with Ultra TMB-ELISA substrate solution), the color reaction was stopped with stop solution (Invitrogen, SS04, ELISA stop solution) and the absorbance was measured at OD 450nm. The binding of scFv to each domain was analyzed by ELISA. Figure 1 As shown, the epitope domains and binding properties of individual clones were analyzed differently.

[0359] Example 5: Mammalian cell expression and purification of anti-ROR1 antibodies

[0360] Each clone obtained in the embodiment of the cloned and produced to produce a complete immunoglobulin (IgG) monoclonal antibody in the form of cloned. In order to construct a heavy chain expression vector, the DNA encoding the heavy chain (including the heavy chain variable region and the constant region) was cloned into the pOptivec vector. In addition, in order to construct a vector expressing the light chain, the DNA encoding the light chain (including the light chain variable region and the light chain constant region) was cloned into the pcDNA3.3 vector.

[0361] Proteins were expressed and purified by transient transfection using light and heavy chain expression vectors. TMI reduced serum medium) and ExpiFectamine293 (Gibco, 100014995, ExpiFectamine TM 293) were transfected into Expi293 expression medium (Gibco, A1435101, Expi293 TM Expi293F suspension cells (Gibco, A14527, Expi293F TM After culturing for 5 days under 8% CO2, 37°C and 130 rpm, the protein was purified from the cell culture supernatant. The culture fluid was passed through a column (GE Healthcare, 17-5438-01, MabSelect TM SuRe) to allow the expressed antibody to bind to the column. IgG elution buffer (Thermo Scientific, 21028, Pierce TM After elution with IgG elution buffer, pH 2.0, the cells were washed with AmiconUltra 30 kDa tubes (Merck Millipore, UFC903024, The eluted antibody fraction was concentrated by buffer exchange with PBS (pH 7.4) using an Ultra-15 centrifugal filter device. The purified anti-ROR1 antibody was quantified using absorbance at 280 nm and extinction coefficient.

[0362] Example 6: Engineering for improving the biological and physical properties of anti-ROR1 antibodies

[0363] Antibody optimization was performed to remove unwanted PTM sites in the antibodies obtained in the above examples. Optimization was performed based on the P015042v1 antibody sequence that showed excellent efficacy in in vitro experimental results. In the antibody sequence, sequences that are prone to deamidation and isomerization due to undesirable PTMs during and after production were found: HC 55G, LC S51, and LC G95A. In existing papers, we already know that replacing N or D in sequences that are prone to deamidation and isomerization will significantly weaken the affinity of the antibody (Patel, CN, Bauer, SP, Davis, J., Durbin, JD, Shiyanova, TL, Zhang, K., & Tang, JX (2016). N+1 engineering of anaspartate isomerization hotspot in the complete entarity-determining region of a monoclonal antibody. Journal of Pharmace utical Sciences, 105 (2), 512-518. https: / / doi.org / 10.1016 / s0022-3549(15)00185-9 ). Therefore, optimization is performed by replacing the sequence following N or D in the sequence.

[0364] Through rational design, the above sequences were replaced one by one to measure the antibody affinity. First, HC G55 was replaced by V (P015042v1-1) and K (P015042v1-2). In addition, LC S51 was replaced by A (P015042v1-3) and K (P015042v1-4), and LC G95 was replaced by A (P015042v1-5). Based on the results of the generation of new variants (P015042v1-1 to P015042v1-5) and the measurement of their affinity, it was judged that the HC G55 sequence was best replaced by K, and LC S51 and LC G95 were best replaced by K and A, respectively. Therefore, P015042v1-6 with all three sequence modifications was constructed.

[0365] The CDR and heavy and light chain variable region sequences of the modified antibodies are shown in Tables 7 and 8 below.

[0366] [Table 7]

[0367]

[0368]

[0369] [Table 8]

[0370]

[0371]

[0372]

[0373] Example 7: Analysis of the specific binding ability of anti-ROR1 antibodies to ROR1 (ELISA)

[0374] ELISA was performed using the IgG antibody of each clone selected in the above examples to analyze the specific binding ability to the antigen.

[0375] A 96-well plate was coated with each of human ROR1 protein and mouse ROR1 protein at a concentration of 2 μg / ml at 4°C for 16 hours, and then blocked with 3% skim milk. Then, the plate was treated with each antibody at concentrations of 300nM, 60nM, 12nM, 2.4nM, 0.48nM, 0.096nM and 0.0192nM, and reacted for 1 hour. After washing with PBST and distilled water, a goat-derived HRP-conjugated anti-human antibody (Invitrogen, 31482, goat anti-human IgG F(ab')2 secondary antibody, HRP) was used for binding at room temperature for 1 hour, and then washed again with PBST and distilled water. After adding TMB substrate solution (Thermo Scientific, 34029, 1-Step TM After color development with Ultra TMB-ELISA substrate solution), the color development reaction was stopped with stop solution (Invitrogen, SS04, ELISA stop solution), and the absorbance was measured at OD 450nm. It was confirmed that the anti-ROR1 antibody of the present invention and the modified antibody with improved biological and physical properties cross-linked with human ROR1 and mouse ROR1 ( Figure 2 ).

[0376] Example 8: Analysis of the specific binding ability of anti-ROR1 antibodies to ROR1 (SPR)

[0377] In order to more quantitatively analyze the ability of the anti-ROR1 antibody to bind to ROR1, SPR (surface plasmon resonance) analysis was performed using a Biacore 3000 instrument (GE Healthcare).

[0378] Using the amine coupling method, human ROR1 or mouse ROR1 was diluted with 10 mM sodium acetate at pH 4.5 and immobilized on a CM5 sensor chip (GE Healthcare) at 300 response units (RU). 1 M ethanolamine-HCl (pH 8.5) was added to inactivate the activated portion remaining on the surface of the sensor chip. The anti-ROR1 antibody of the present invention was injected into the antibody protein immobilized on the CM5 sensor chip (Ka) at concentrations of 300, 150, 75, 37.5, 18.75, 9.375 and 4.6875 nM for 180 seconds and then separated at the same flow rate for another 180 seconds (Kd) to analyze the KD binding sensorgram. Therefore, as Figure 2 As shown, the anti-ROR1 antibodies exhibited sensorgrams that specifically bound to human ROR1 and mouse ROR1, and the final KD values ​​derived from the Ka values ​​and Kd values ​​(1:1 Langmuir 1:1 kinetics) are shown in Tables 9 and 10 below.

[0379] [Table 9]

[0380]

[0381] [Table 10]

[0382]

[0383] Example 9: ROR1 domain map of anti-ROR1 antibodies

[0384] ELISA analysis was performed using IgG antibodies against selected clones to analyze the binding domains.

[0385] 96-well plates were coated with 2 μg / ml of each of the following for 16 h at 4°C and then blocked with 3% skim milk: human ROR1-immunoglobulin domain (ACROBiosystems, RO1-H5221, human / cynomolgus / rhesus monkey ROR1 (39-151, Ig-like domain) protein, His tag), human ROR1-frizzled domain (ACROBiosystems, RO1-H5222, human / cynomolgus / rhesus monkey ROR1 (165-305, Frizzled domain ( domain) protein, His tag) and human ROR1-kringle domain (ACROBiosystems, RO1-H5223, human / cynomolgus / macaque ROR1 (308-395, kringle domain) protein, His tag), human ROR1-immunoglobulin-coiled domain and human ROR1-coiled-kringle domain. Then, the plate was treated with each antibody at concentrations of 300nM, 60nM, 12nM, 2.4nM, 0.48nM, 0.096nM and 0.0192nM and reacted for 1 hour. After washing with PBST and distilled water, goat-derived HRP-conjugated anti-human antibody (Invitrogen, 31482, goat anti-human IgG F(ab')2 secondary antibody, HRP) was used for binding at room temperature for 1 hour, and then washed again with PBST and distilled water. After adding TMB substrate solution (Thermo Scientific, 34029, 1-Step TM After color development with Ultra TMB-ELISA substrate solution), the color reaction was stopped with stop solution (Invitrogen, SS04, ELISA stop solution) and the absorbance was measured at OD 450nm. The anti-ROR1 antibody clones ( Figure 3 ).

[0386] Example 10: Quantitative analysis of ROR1 receptor expression by cell type

[0387] Each of T-47D, Jeko-1 (abnormal cell line), AMB-BT-0024T, AMB-BT-0016T, AMB-BT-0013T, AMB-LC-0002T, AMB-LC-0003T, and KUC-OC21-025T (abnormal patient-derived cells) was cultured at 2×10 5The number of cells was distributed in duplicate in a 96-well plate. Each of 100nM mouse IgG1-PE isotype (Invitrogen, 12-4714-82) and ROR1-2A2-PE (BioLegend, 357804) was added to each well. The cells were incubated at 4°C for 30 minutes, and then Quantibrite PE beads (BD, 340495) were added to 0.5ml of PBS, and then flow cytometry was performed using a BD FACSAria flow cytometer. The reference point for ROR1 expression was set using beads, and the expression level of ROR1 in cells treated with mouse isotypes and ROR1-2A2-PE antibodies was compared with the beads, and the number of ROR1 receptors expressed was quantified according to different cell types.

[0388] The human lymphoma cell line Jeko-1 showed high ROR1 expression, while among patient-derived cells (PDXC), patient-derived lung cancer cells AMB-LC-0003T showed the highest ROR1 expression ( Figure 4 ).

[0389] [Table 11]

[0390]

[0391] Example 11: Measurement of the specific binding ability of anti-ROR1 antibodies to ROR1 expressed on the cell surface

[0392] Before using anti-ROR1 antibodies therapeutically, it is very important to determine whether the antibodies can 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 Jeko-1 cell line and AMB-LC-0003T patient-derived cells that have been confirmed to have high ROR1 expression. Each cell type was cultured at 1x10 5 cells / 100 μl FACS buffer were dispensed into 96-well plates.

[0393] Cell binding ability was analyzed using anti-ROR1 candidate antibodies P015004, P015042, P015043, P015044, P015042v1 and one of their modified substances P015042v1-6 with improved biological and physical properties. Each antibody was serially diluted 10-fold from 100nM to 10nM, 1nM, 100pM, 10pM and 1pM, and cells were treated with it. After reacting at 4°C for 30 minutes, wash twice. PE-labeled goat anti-human IgG FcPE (ThermoFisher; 12-4998-82) was diluted 1:100 with FACS buffer as a secondary antibody, and 100μl of it was distributed per well. React again at 4°C for 30 minutes and wash twice. Finally, 100 μl of FACS buffer was dispensed into each well and mixed by pipette before flow cytometry was performed using a NovoCyte flow cytometer (Agilent). GraphPad Prism 9.3.1 was used for analysis, and the EC values ​​were derived using a log(agonist) vs. response-variable slope nonlinear plot model. 50 value.

[0394] Based on the results of cell binding capacity, AMB-LC-0003T patient-derived cells, which had the highest expression of ROR1, showed a higher MFI (mean fluorescence intensity) than the Jeko-1 cell line. Subnanomolar EC was shown in both cell types. 50 The antibodies were P015042, P015042v1, and P015042v1-6, which were obtained by additional modifications to their sequences, and P015044 also showed nanomolar EC 50 Other anti-ROR1 clones such as P015004 and P015043 also showed EC values ​​<10 nM 50 , thus showing good cell binding ability ( Figure 5 and 6 ).

[0395] [Table 12]

[0396] Analysis results of the ability to bind to ROR1 antigen expressed in lung cancer cells derived from AMB-LC-0003T patients

[0397] Hit <![CDATA[EC 50 (nM)]]> P015004 4.60 P015042 0.15 P015042v1 0.16 P015043 5.30 P015044 1.78

[0398] [Table 13]

[0399] Analysis of antibody binding ability to ROR1 antigen expressed in Jeko-1 cell line

[0400] Hit <![CDATA[EC 50 (nM)]]> P015004 8.16 P015042 0.081 P015042v1 0.014 P015042v1-6 0.011 P015043 1.70 P015044 0.13

[0401] Example 12: Analysis of cellular internalization of anti-ROR1 antibodies

[0402] In order to develop antibody-drug conjugates, the anti-ROR1 antibody must not only bind to the ROR1 antigen in the cell, but also be internalized into the cell so that the drug in the conjugate can penetrate into the cell and elicit a response. To analyze this, internalization was confirmed by detecting the residual ROR1 antigen on the cell surface at different time points. 5 Jeko-1 cells were distributed in duplicate at an amount of 10 cells / 100 μl, and plates were prepared at 0 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours and 4 hours after the start of cell internalization, and ROR1 receptors that were not internalized by the antibody but still remained on the cell surface were confirmed. The cells were treated with 1 nM of each antibody and then reacted at 4°C for 30 minutes. After centrifugation at 1,100g for 2 minutes, two washes were performed. Except for the 0-minute plate, the cells in the remaining plates were mixed with 100 μl of FACS buffer and reacted at 37°C and 5% CO2. At 0 minutes in the plate, goat anti-human IgG Fc PE (ThermoFisher, 12-4998-82) was diluted at a ratio of 1:100, 100 μl was distributed to each well, and then reacted at 4°C for about 30 minutes. After washing twice, flow cytometry was performed using a NovoCyte flow cytometer (Agilent), and other plates were treated in the same manner at various times to confirm that ROR1 was still expressed in Jeko-1 cells. Values ​​were normalized based on the ROR1 expression level confirmed at 0 minutes and analyzed using the [Inhibitor] vs. Normalized Response-Variable Slope Nonlinear Plot Model in GraphPad Prism 9.3.1.

[0403] Internalization varied for each antibody clone, and most antibodies continued to be internalized after 4 hours. P015042v1-6, which was engineered to improve the biological and physical properties of the P015042 antibody, showed similar levels of internalization to Merck's UC-961 antibody, which had the best internalization ( Figure 7 ).

[0404] Example 13: Production of anti-ROR1 antibody-drug conjugates

[0405] This article describes a general method for producing antibody-drug conjugates or intermediates thereof according to the present invention. The compound names and abbreviations contained in each reaction formula are shown and described ( Figure 8 ).

[0406] The antibody-drug conjugate represented by formula (1) in which the antibody (Y) and the drug-linker (LP) structure are linked via thioether can be produced, for example, by the following method.

[0407] [Formula 1]

[0408] X (1) + LP (2) → X-LP (3)

[0409] The drug-linker (LP) used was maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl monomethyl auristatin E (vc-PAB-MMAE; CCC(C)C(C(CC(=O)N1CCCC1C(C(C)C(=O)NC(C)C(C2=CC=CC=C2)O)OC)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), and X was an IgG1 monoclonal antibody with a thiol group.

[0410] By reaction between the maleimide group of LP and the thiol group of X, an antibody-drug conjugate can be produced.

[0411] The antibody (X) having a thiol group can be obtained according to the following method: The antibody can be reacted with a reducing agent such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to thereby reduce the disulfide bonds in the hinge region of the antibody, thereby forming a thiol group.

[0412] Specifically, 2 to 20 molar equivalents of 5mM TCEP are used as a reducing agent for each disulfide bond in the antibody, and reacted with the antibody (Y) in a buffer containing 5mM ethylenediaminetetraacetic acid (EDTA), a representative chelating agent, to produce an antibody (X) having a sulfhydryl group, wherein the disulfide bonds in the antibody are partially or completely reduced. The disulfide bond reduction reaction in the above antibody is carried out at 37° C. for 2 hours. The buffer used is the same as the phosphate buffered saline (pH 7.4) used in the antibody production. The disulfide bond reduction reaction in the above antibody is carried out at 37° C. for 2 hours.

[0413] In order to remove impurities such as TCEP and EDTA remaining after the reaction is completed, the antibody (X) solution with a thiol group is added to Amicon Ultra (30 kDa, Millipore Co.), and then centrifuged (at 3950G for 10 to 20 minutes) using a centrifuge (Eppendorf centrifuge 5810R), and the buffer is exchanged with phosphate buffered saline (pH 7.4). This process is repeated three times.

[0414] The antibody (X) having a thiol group, from which impurities were removed by buffer exchange, was recovered from Amicon Ultra (30 kDa, Millipore Co.) and reacted with 5 to 15 molar equivalents of 10 mM drug-linker (LP) at room temperature for 2 hours. Since the drug-linker (LP) may change under the influence of light, the reaction was carried out in the dark. In order to increase the solubility of the compound, the drug-linker (LP) was dissolved in a representative organic solvent, dimethylacetamide (DMA). About 10% to 20% v / v of DMA was added to the buffer containing the antibody (X) having a thiol group.

[0415] The antibody-drug conjugation reaction can be terminated by inactivating the reactivity of the unreacted drug-linker (LP) with a thiol-containing reagent. The thiol-containing reagent used is N-acetyl-L-cysteine ​​(NAC). Specifically, the reaction can be terminated by adding 5 molar equivalents of NAC to the antibody-drug conjugation reaction solution and keeping it at room temperature for 30 minutes.

[0416] In order to remove the residual drug-linker and NAC, the antibody-drug reaction solution was added to Amicon Ultra (30 kDa, Millipore Co.), and then centrifuged (at 3950G for 10 to 20 minutes) using a centrifuge (Eppendorf centrifuge 5810R), and the buffer was exchanged with phosphate buffered saline (pH 7.4). This process was repeated three times. Finally, the antibody-drug conjugate was recovered from the filter membrane of Amicon Ultra (30 kDa, Millipore Co.).

[0417] Example 14: Analysis of concentration of anti-ROR1 antibody-drug conjugates

[0418] The antibody concentration in the antibody-drug conjugate was measured using a spectrophotometer (ThermoFisher Scientific NanoDrop 8000).

[0419] Example 15: Analysis of the Purity of Anti-ROR1 Antibody-Drug Conjugates

[0420] The purity of the antibody-drug conjugate was measured by size exclusion high performance liquid chromatography (SEC-HPLC) analysis using the following method. As a pretreatment for analysis, the antibody-drug conjugate sample was prepared at a concentration of 0.5 mg / ml and a total volume of 25 μl. The SEC-HPLC analysis was performed under the following conditions.

[0421] [Table 14]

[0422] SEC-HPLC system configuration

[0423]

[0424] The purity of the antibody-drug conjugate is measured by comparing and contrasting the retention time of each peak appearing in the antibody-drug conjugate chromatogram with the retention time of each peak of the size marker (Gel Filtration Standard, Bio-Rad, 1511901). In size exclusion chromatography, larger molecules are detected first, so the peak that appears first based on the antibody peak (150 kDa) is analyzed as a polymer aggregate generated due to the hydrophobicity of the drug, and the subsequent peaks are analyzed as drugs separated after conjugation. The purity of the antibody-drug conjugate itself is calculated by converting the area of ​​the peak corresponding to 150 kDa in the entire chromatogram into a percentage.

[0425] Example 16: Analysis of the average number of drug molecules conjugated per antibody molecule of anti-ROR1 antibody-drug conjugates (DAR analysis)

[0426] The following method was used to measure the average number of drug molecules conjugated per antibody molecule in the antibody-drug conjugate (drug-antibody ratio, DAR) by hydrophobic interaction high performance liquid chromatography (HIC-HPLC). As a pretreatment for HIC-HPLC analysis, the antibody-drug conjugate samples for analysis were prepared at a concentration of 0.5 mg / ml and a total volume of 30 μl. HIC-HPLC analysis was performed under the following conditions.

[0427] [Table 15]

[0428] HIC-HPLC system configuration

[0429]

[0430] [Table 16]

[0431] HIC-HPLC gradient program

[0432] Time (min) % Mobile phase A % Mobile phase B 0 100 0 0.3 100 0 20 0 100 20.1 100 0 30 100 0

[0433] Compared with the same drug-unconjugated antibody, the antibody-drug conjugate shows a higher hydrophobicity that is proportional to the number of conjugated drug molecules, and therefore has a longer retention time. There are a total of four disulfide bonds in the antibody, and each disulfide bond can couple two drugs. 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 the antibody-drug conjugate. The peaks that appear successively after the retention time of the drug unconjugated antibody (DAR 0) are named in the order of DAR 2, DAR 4, DAR 6 and DAR 8 above. Once the DAR distribution of each peak is completed, the number of drug molecules conjugated on average per antibody molecule is calculated using the following formula.

[0434]

[0435] Example 17: Analysis of the binding ability of anti-ROR1 antibody-drug conjugates to antigens

[0436] Affinity analysis of antibody-drug conjugates is used to assess equivalence to the parent antibody and also to determine whether processes such as disulfide bond reduction and drug-linker conjugation compromise potential affinity when generating antibodies with sulfhydryl groups. This analysis is performed using the following enzyme-linked immunosorbent assay (ELISA).

[0437] 96-well plates (Costar 3590, Corning) were coated by dispensing 50 μl of 2 μg / ml of human or mouse-derived ROR1 antigen (Sino Biological) per well. After shaking off the coated ROR1 antigen, 200 μl of 3% skim milk was dispensed through each coated well for blocking. After 1 hour, 3% skim milk was shaken off, and the parent antibody and antibody-drug conjugate were serially diluted seven times at a ratio of 1 / 5 from the highest concentration of 300 nM, and then 50 μl was dispensed in the designated wells. After the first antigen-antibody binding for 1 hour, the plate was washed with 0.05% v / v surfactant (PBST) and distilled water (three times each). Then, Fab-HRP was diluted in 3% skim milk at a ratio of 1:3000, and 50 μl was dispensed per well. After 1 hour of the secondary binding reaction, the plate was washed with 0.05% PBST and triple distilled water (three times each). After removing the residual water on the plate, 50 μl of TMB (34029, ThermoFisher) was dispensed into each well and reacted for 3 to 5 minutes. Finally, 50 μl of stop solution (SS04, ThermoFisher) was added into each well to stop the reaction.

[0438] [Table 17]

[0439] Affinity Analyzer and Conditions

[0440] Analyzer Infinite M200 Pro (Tecan) Analysis wavelength 450nm Data processing GraphPad Prism 9

[0441] When the binding ability of the antibody-drug conjugate to the antigen is 80% or more compared to the monoclonal antibody, the antibody-drug conjugate is judged to have similar binding ability to the antigen.

[0442] According to the above antibody-drug conjugate analysis and QC method, the three anti-ROR1 antibody-drug conjugates thus prepared (P015004-vc-PAB-MMAE, P015042-vc-PAB-MMAE and P015044-vc-PAB-MMAE) were evaluated, and the results are summarized in Table 18 below.

[0443] [Table 18]

[0444] Anti-ROR1 Antibody-Drug Conjugate Analysis and QC

[0445]

[0446] Fig. 9 The results of the purity analysis of the antibody-drug conjugates used in the examples of the present invention are shown. Fig.10 The results of the 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 are shown, and Fig.11 The results of ELISA for ROR1 binding of the antibody-drug conjugates used in the Examples of the present invention are shown.

[0447] Example 18: In vitro cytotoxicity assessment of anti-ROR1 antibody-drug conjugates

[0448] The toxicity of the above anti-ROR1 candidate antibody-drug conjugates as anticancer drugs was evaluated in patient-derived lung cancer cells AMB-LC-0003T, which are known to have high ROR1 expression, and AMB-LC-0002T, which have very low ROR1 expression.

[0449] Patient-derived lung cancer cells AMB-LC-0003T and AMB-LC-0002T were distributed in triplicate in each ultra-low adsorption U-bottom 384-well transparent plate (S-Bio, #MS-9384UZ) at 500 cells / well in 40 μl of M10018 (Aimedbio) culture medium. After centrifugation at 250 g for 2 minutes, the reaction was carried out at 37 ° C and 5% CO2 for 24 hours. After that, each antibody-drug conjugate was serially diluted 3 times from 500nM to 0.314pM and treated with it. The plate was reacted again for 6 days at 37 ° C and 5% CO2.

[0450] After a total of 7 days of reaction, spheroids were formed from cells in 384-well plates, so images of spheroids in each well were captured using OperettaCLS (PerkinElmer) according to the high content screening method. In order to maintain the spheroid form of cells in each well, no washing was performed before analysis. Through additional analysis, the captured images were converted to volume, and the spheroid volume values ​​by concentration were normalized based on the spheroids treated with the lowest drug concentration. GraphPad Prism9.3.1 was used to create graphs with log(inhibitor) vs. normalized response-variable slope, and IC 50 value( Fig.12 ).

[0451] [Table 19]

[0452]

[0453] [Table 20]

[0454]

[0455] The above P015042-vc-PAB-MMAE antibody-drug conjugate showed excellent IC in AMB-LC-0003T with high ROR1 expression 50 (2.30 nM), while in AMB-LC-0002T cells derived from patients with low ROR1 expression, only IC 50 The relative potency is about 8 times that of the antibody-drug conjugate UC961-vc-PAB-MMAE disclosed in U.S. Pat. No. 10,335,496B2, and thus it is expected to exert ROR1-specific anticancer efficacy as an antibody-drug conjugate ( Fig.13 ).

[0456] Industrial Applicability

[0457] Compared with existing anti-ROR1 antibodies, the anti-ROR1 antibodies or antigen-binding fragments thereof of the present invention may exhibit stronger binding ability to ROR1 and may be effectively used for the prevention or treatment of desired tumors or cancers.

[0458] The specific parts of the present invention have been described in detail above, and it is obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the essential scope of the present invention is defined by the appended claims and their equivalents.

[0459] Sequence table custom text

[0460] Attach electronic file.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to ROR1 (receptor tyrosine kinase-like orphan receptor 1), comprising: a heavy chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16; a heavy chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 41, SEQ ID NO: 184, and SEQ ID NO: 185; a heavy chain CDR3 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO:42 to SEQ ID NO:65; a light chain CDR1 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 66 to SEQ ID NO: 86; a light chain CDR2 comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 87 to SEQ ID NO: 102, SEQ ID NO: 186, and SEQ ID NO: 187, and A light chain CDR3 comprising at least one amino acid sequence 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, wherein It comprises a heavy chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 122 to SEQ ID NO: 152 and SEQ ID NO: 188 to SEQ ID NO:

193.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein It comprises a light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 153 to SEQ ID NO: 183 and SEQ ID NO: 194 to SEQ ID NO:

199.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein Contains single-chain Fv (scFv), single-chain antibody, Fab, F(ab') or disulfide-linked Fv (sdFv).

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein The scFv comprises a heavy chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 122 to SEQ ID NO: 152 and SEQ ID NO: 188 to SEQ ID NO: 193 and a light chain variable region comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 153 to SEQ ID NO: 183 and SEQ ID NO: 194 to SEQ ID NO: 199, connected via a linker.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein The connector comprises (G n S) m (wherein 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 according to claim 7.

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

10. The host cell according to claim 9, wherein The host cell 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 an antigen-binding fragment thereof that specifically binds to ROR1, the method comprising: Producing the antibody by culturing the host cell according to claim 9; and The produced antibodies are isolated and purified.

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 a scFv of an antibody according to any one of claims 1 to 6, and a scFv consisting of a second binding domain, wherein the second binding domain comprises a scFv of at least one antibody that binds to an immune cell activating antigen.

14. An antibody-drug conjugate (ADC), wherein: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 is conjugated to a drug.

15. The antibody-drug conjugate of claim 14, wherein the drug is selected from at least one of the group consisting of maytansine, auristatin (including MMAE and MMAF), aminopterin, actinomycin, bleomycin, talithromycin, camptothecin, N8-acetyl spermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonyl hydrazine, esperamicin, etoposide, 6-mercaptopurine, dolastatin, trichothecenes, calicheamicin, taxol, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, duocarmycin, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, Platinum, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cyclophosphamide, etoposide, 5-fluorouracil, CNU (bischloroethyl nitrosourea), irinotecan, camptothecin, bleomycin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinorelbine, chlorambucil, melphalan, carmustine, lomustine, busulfan, trioxalic acid, dacarbazine, etoposide, teniposide, topotecan, 9-aminocamptothecin, clenatol, mitomycin C, trimetrexate, mycophenolic acid, thiazolidinone, ribavirin, EICAR (5-ethynyl-1-β-D-ribofuranosyl imidazole-4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (arabinoside) C), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol acetate, goserelin, leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, gemcitabine, Velcade, Revlimid, thalidomide, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, doxycycline, mitoxantrone, verapamil, thapsigargin, nucleases, and toxins derived from bacteria, animals, or plants.

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

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

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

19. The antibody-drug conjugate according to claim 16, wherein The linker is conjugated to a cysteine ​​residue exposed during the reduction of disulfide bonds of the antibody, or to a cysteine ​​residue present in a tag bound to the antibody.

20. A chimeric antigen receptor (CAR), comprising an extracellular domain comprising an antigen binding site, a transmembrane domain, and an intracellular signaling domain, wherein: The antigen binding site of the extracellular domain is a 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 is introduced.

22. The immune cell according to claim 21, wherein The immune cell 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 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.

24. A method for preventing or treating cancer, comprising administering the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, 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.

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